Enzymatic Glycosylation Strategies in the Production of Bioactive Compounds

Ci a ion: And eu, A.; ´
Co o i´c, M.;
Ga cia-Sanz, C.; San os, A.S.;
Mili oje i´c, A.; O ega-Nie o, C.;
Ma eo, C.; Bezb adica, D.; Palomo,
J.M. Enzyma ic Glycosyla ion
S a egies in he P oduc ion o
Bioac i e Compounds. Ca alys s 2023,
13, 1359. h ps://doi.o g/10.3390/
ca al13101359
Academic Edi o : E angelos Topakas
Recei ed: 28 Augus 2023
Re ised: 28 Sep embe 2023
Accep ed: 1 Oc obe 2023
Published: 11 Oc obe 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
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4.0/).
ca alys s
Re iew
Enzyma ic Glycosyla ion S a egies in he P oduc ion o
Bioac i e Compounds
Alicia And eu 1, Ma ija ´
Co o i´c 2, Ca la Ga cia-Sanz 1, A. So ia San os 1, Ana Mili oje i´c 2,
Cla a O ega-Nie o 1, Cesa Ma eo 1,* , Dejan Bezb adica 2,* and Jose M. Palomo 1,*
1Ins i u o de Ca álisis y Pe oleoquímica (ICP), CSIC, Ma ie Cu ie 2, 28049 Mad id, Spain;
[email p o ec ed] (A.A.); c.ga [email p o ec ed] (C.G.-S.); [email p o ec ed] (A.S.S.);
[email p o ec ed] (C.O.-N.)
2Depa men o Biochemical Enginee ing and Bio echnology, Facul y o Technology and Me allu gy,
Uni e si y o Belg ade, Ka negije a 4, 11000 Belg ade, Se bia; [email p o ec ed] (M. ´
C.);
[email p o ec ed] (A.M.)
*Co espondence: [email p o ec ed] (C.M.); [email p o ec ed] (D.B.); [email p o ec ed] (J.M.P.)
Abs ac :
Enzyma ic glycosyla ion is a e sa ile and sus ainable bio echnological app oach ha plays
a pi o al ole in he p oduc ion o bioac i e compounds. This p ocess in ol es he enzyma ic ans e
o suga moie ies on o a ious accep o molecules, such as small molecules, pep ides, o p o eins,
esul ing in he syn hesis o glycosides. These glycosides o en exhibi enhanced bioac i i y, imp o ed
solubili y, and enhanced s abili y, making hem aluable in pha maceu icals, nu aceu icals, and he
ood indus y. This e iew explo es he di e se enzyma ic glycosyla ion s a egies employed in he
syn hesis o bioac i e compounds. I highligh s he enzyma ic ca alys s in ol ed, including glycosyl-
ans e ases, glycosidases, glycophospho ylases, and glycosyn hases. I conside s he ad an ages
and disad an ages o hese bioca alys s in he s e eoselec i e and egioselec i e syn hesis o di e en
ypes o glycosyla ed molecules, phenolic and alipha ic alcohols, oligosaccha ides, polysaccha ides,
glycode i a i es, glycopep ides, and glycop o eins wi h a clea ocus on ood and pha maceu ical
chemis y. Fu he mo e, he e iew ou lines a ious sou ces o suga dono s, ac i a ed glycosides,
and suga nucleo ides, as well as he u iliza ion o enginee ed enzymes and mic oo ganisms o
glycosyla ion eac ions. The ad an ages o enzyma ic glycosyla ion, including i s high egioselec-
i i y, s e eoselec i i y, and sus ainabili y, a e emphasized. The e o e, hese app oaches combining
he use o di e en ca aly ic sys ems, he imp o emen o ools such as immobiliza ion echnology
o chemical o gene ic modi ica ion o imp o e he glycosyla ion p ocess, could be use ul ools in
con inuous bio echnological ad ancemen s.
Keywo ds: enzymes; glycosyla ion; oligosaccha ides; glycode i a i es
1. In oduc ion
The e a e many compounds desc ibed in he li e a u e wi h good biological ac i i y
such as an ioxidan s, an i-in lamma o ies, o di e en d ugs used in di e en p ocesses
and wi h good applicabili y in di e en ields such as pha macology, swee ene s, e c.
Howe e , in many cases, hese compounds a e abso bed in he gas oin es inal ac and
me abolized e y quickly in he bloods eam and especially in he li e . In o he cases,
he p oblem is ha since mos o hem a e highly hyd ophobic, hei solubili y in aqueous
medium is e y low, so he concen a ions in blood a e e y small, causing a dec ease
in he biological ac i i y [
1
]. Thus, in na u e, mos o he compounds wi h biological
ac i i y a e associa ed wi h suga esidues. This helps in di e en biological p ocesses
such as he main enance o cell in eg i y, in cell ecogni ion, o in de ense mechanisms a
he molecula le el [
2
–
5
]. Imi a ing he na u e o glycosyla ion is a e y e ec i e p ocess
o ans o m na u al p oduc s, and in many cases enhances o modi ies hei biological
ac i i y a e al e ing hei physical, chemical, and biological p ope ies [
6
]. Conside ing
Ca alys s 2023,13, 1359. h ps://doi.o g/10.3390/ca al13101359 h ps://www.mdpi.com/jou nal/ca alys s
Ca alys s 2023,13, 1359 2 o 28
his, on many occasions, he de i a iza ion o he hyd oxyla ed compounds wi h simple
suga s o wi h oligosaccha ide chains is in e es ing o con empla e. Depending on he
ype o linkage o med, in many cases hese compounds a e less hyd olyzable, and hei
me abolism occu s in he colon and is caused by he gu mic obio a. In addi ion, suga
de i a iza ion makes he compounds mo e soluble in an aqueous medium, inc easing
hei concen a ion in blood. A he cen e o any enzyma ic glycode i a iza ion s a egy
is a glycosidic bond- o ming enzyme. A numbe o ac o s mus be aken in o accoun
when selec ing a sui able ca alys , in pa icula (i) p ac ical access o he co esponding
subs a es (and ca alys ); (ii) abili y o he ca alys o ac on a wide ange o subs a es; and
(iii) e iciency o glycosidic bond o ma ion.
In o ganic chemis y, na u al glycosidases (GH) can be used o ca alyze di e en
syn he ic p ocesses. Al hough glycosidases use o be classi ied acco ding o he ype o
glycosidic bond o med o hyd olyzed by hei ca aly ic ac i i y (galac osidases, glucosi-
dases, e c.), hey a e usually g ouped in o wo main g oups, such as glycosidases and
ansglycosidases. Glycosidases a e able o o m glycosidic bonds by e e se hyd olysis,
wi h equilib ium shi . T ans e ases a e essen ial enzymes in ol ed in he glycosyla ion
o bioac i e compounds, playing a pi o al ole in modi ying he s uc u e and unc ion o
hese molecules. These glycosyl ans e ases (GT) enzymes acili a e he ans e o speci ic
suga moie ies om ac i a ed dono molecules on o accep o compounds, such as p o eins,
pep ides, o small molecules. The glycosyla ion p ocess, ca alyzed by ans e ases, leads o
he o ma ion o glycoconjuga es, which o en exhibi enhanced bioac i i y, imp o ed solu-
bili y, and inc eased s abili y. The di e si y o ans e ases, including glycosyl ans e ases,
sul o ans e ases, and sialyl ans e ases, among o he s, enables he gene a ion o a wide
ange o glycoconjuga es wi h a ying glycan s uc u es and unc ions [7].
In addi ion o he mul i ude o enzymes ha exis in na u e, in ecen yea s, di e en
mu an enzymes ha e been de eloped ha ha e had hei hyd oly ic capaci y elimina ed,
g ea ly imp o ing hei syn hesis yields [
8
]. Finally, in some cases, he e a e desc ibed
p ocesses in which di e en cell lines ha e been used o ca alyze eac ions in ol ing
glycosidase enzymes [9].
In he p esen e iew, di e en p ocesses o g ea in e es in syn hesis using he
di e en ca aly ic sys ems desc ibed abo e will be add essed, as well as he di e en
applica ions whe e hese enzyma ic p ocesses can be employed (Figu e 1).
Ca alys s 2023, 13, x 2 o 28
a e al e ing hei physical, chemical, and biological p ope ies [6]. Conside ing his, on
many occasions, he de i a iza ion o he hyd oxyla ed compounds wi h simple suga s o
wi h oligosaccha ide chains is in e es ing o con empla e. Depending on he ype o link-
age o med, in many cases hese compounds a e less hyd olyzable, and hei me abolism
occu s in he colon and is caused by he gu mic obio a. In addi ion, suga de i a iza ion
makes he compounds mo e soluble in an aqueous medium, inc easing hei concen a-
ion in blood. A he cen e o any enzyma ic glycode i a iza ion s a egy is a glycosidic
bond- o ming enzyme. A numbe o ac o s mus be aken in o accoun when selec ing a
sui able ca alys , in pa icula (i) p ac ical access o he co esponding subs a es (and ca -
alys ); (ii) abili y o he ca alys o ac on a wide ange o subs a es; and (iii) e iciency o
glycosidic bond o ma ion.
In o ganic chemis y, na u al glycosidases (GH) can be used o ca alyze di e en syn-
he ic p ocesses. Al hough glycosidases use o be classi ied acco ding o he ype o gly-
cosidic bond o med o hyd olyzed by hei ca aly ic ac i i y (galac osidases, gluco-
sidases, e c.), hey a e usually g ouped in o wo main g oups, such as glycosidases and
ansglycosidases. Glycosidases a e able o o m glycosidic bonds by e e se hyd olysis,
wi h equilib ium shi . T ans e ases a e essen ial enzymes in ol ed in he glycosyla ion
o bioac i e compounds, playing a pi o al ole in modi ying he s uc u e and unc ion o
hese molecules. These glycosyl ans e ases (GT) enzymes acili a e he ans e o speci ic
suga moie ies om ac i a ed dono molecules on o accep o compounds, such as p o-
eins, pep ides, o small molecules. The glycosyla ion p ocess, ca alyzed by ans e ases,
leads o he o ma ion o glycoconjuga es, which o en exhibi enhanced bioac i i y, im-
p o ed solubili y, and inc eased s abili y. The di e si y o ans e ases, including glyco-
syl ans e ases, sul o ans e ases, and sialyl ans e ases, among o he s, enables he gen-
e a ion o a wide ange o glycoconjuga es wi h a ying glycan s uc u es and unc ions
[7].
In addi ion o he mul i ude o enzymes ha exis in na u e, in ecen yea s, di e en
mu an enzymes ha e been de eloped ha ha e had hei hyd oly ic capaci y elimina ed,
g ea ly imp o ing hei syn hesis yields [8]. Finally, in some cases, he e a e desc ibed
p ocesses in which di e en cell lines ha e been used o ca alyze eac ions in ol ing gly-
cosidase enzymes [9].
In he p esen e iew, di e en p ocesses o g ea in e es in syn hesis using he di -
e en ca aly ic sys ems desc ibed abo e will be add essed, as well as he di e en appli-
ca ions whe e hese enzyma ic p ocesses can be employed (Figu e 1).
Figu e 1. Concep scheme o enzyma ic glycosyla ion o bioac i e compounds and applica ions.
Figu e 1. Concep scheme o enzyma ic glycosyla ion o bioac i e compounds and applica ions.
Ca alys s 2023,13, 1359 3 o 28
F om his s a egy, i is in e es ing o emphasize he syn hesis o de i a i es o phenolic
compounds because o hei high in e es due o hei , in many cases, good biological ac i -
i y and hei low solubili y. This makes hem excellen candida es o hei glycosyla ion
due o he ad an ages p o ided by such ea men , as men ioned abo e.
2. Enzyma ic Glycosyla ion o Phenolic Compounds
Phenolic compounds a e an impo an class o chemicals con aining one o mo e
hyd oxyl g oups bounded di ec ly o an a oma ic hyd oca bon g oup [
10
]. Conside -
ing hei chemical s uc u es, phenolic compounds can be di ided in o se e al classes,
such as la onoids, phenolic acids, annins, couma ins, quinones, s ilbens, and lignans
(Figu e 2) [
11
]. They possess a ious bene icial bioac i i ies including an ioxidan , an-
ia he ogenic, an i-in lamma o y, and an imic obial p ope ies, which make hem gene ally
in e es ing in a ious scien i ic a eas [
12
–
14
]. Howe e , despi e nume ous biological ac-
i i ies, hei wide applica ion is limi ed due o hei low solubili y and s abili y in wa e ,
since hey a e easily deg aded by ligh i adia ion in aqueous solu ion [
10
]. Glycosyla ion
o phenolic compound seems o be a use ul ool o enhance hei solubili y and s abili y in
wa e , as well as o imp o e hei biological and pha macological p ope ies, such as y osi-
nase inhibi o y ac i i y, b owning- esis an ac i i y, and an i umo p ope ies, by inc easing
hei bioa ailabili y o , some imes, by dec easing he oxici y and side e ec s [6,10,15].
Ca alys s2023,13,x 3o 28


F om hiss a egy,i isin e es ing oemphasize hesyn hesiso de i a i eso phe‐
noliccompoundsbecauseo  hei highin e es due o hei ,inmanycases,goodbiological
ac i i yand hei lowsolubili y.Thismakes hemexcellen candida es o  hei glycosyl‐
a iondue o head an agesp o idedbysuch ea men ,asmen ionedabo e.
2.Enzyma icGlycosyla iono PhenolicCompounds
Phenoliccompoundsa eanimpo an classo chemicalscon ainingoneo mo ehy‐
d oxylg oupsboundeddi ec ly oana oma ichyd oca bong oup[10].Conside ing hei 
chemicals uc u es,phenoliccompoundscanbedi idedin ose e alclasses,suchas la‐
onoids,phenolicacids, annins,couma ins,quinones,s ilbens,andlignans(Figu e2)
[11].Theypossess a iousbene icialbioac i i iesincludingan ioxidan ,an ia he ogenic,
an i‐in lamma o y,andan imic obialp ope ies,whichmake hemgene allyin e es ing
in a iousscien i ica eas[12–14].Howe e ,despi enume ousbiologicalac i i ies, hei 
wide applica ionislimi eddue o hei lowsolubili yands abili yinwa e ,since hey
a eeasilydeg adedbyligh i adia ioninaqueoussolu ion[10].Glycosyla iono phenolic
compoundseems obeause ul ool oenhance hei solubili yands abili yinwa e ,as
wellas oimp o e hei biologicalandpha macologicalp ope ies,suchas y osinasein‐
hibi o yac i i y,b owning‐ esis an ac i i y,andan i umo p ope ies,byinc easing
hei bioa ailabili yo ,some imes,bydec easing he oxici yandsidee ec s[6,10,15].

Figu e2.Chemicals uc u eso  hemainclasseso phenoliccompounds.
Ex ac iono phenolicglycosides omna u alsou cesiscomplexanduneconomical,
whileachemicalapp oach o  hei syn hesis equi es oomanys epso p o ec ion,ac i‐
a ion,coupling,anddep o ec ion[10].On heo he hand,due o heenzyme’s egio‐
ands e eoselec i i yandmild eac ioncondi ions,enzyma icglycosyla ionisconside ed
a e ya ac i e,en i onmen ally iendlys a egy o ob aining hese aluablecom‐
pounds[16].Toda e,i hasbeenp o en ha enzymessuchasglycosyl ans e ases(EC2.4)
andglycosidases(EC3.2.1)canca alyze heglycosyla iono phenoliccompounds[10].In
his e iew, hemos  ep esen a i e ecen  esea chon hebio ans o ma iono phenolic
compounds,usingpu eenzymeso wholemic obialcells ocon e glycosyla ionpo en‐
ial,asbioca alys swillbediscussed.
2.1.Glycosyl ans e ase
Glycosyl ans e ases(GTs)a easubclasso enzymes ha ca alyze he ans e o a
suga  esidue om a ioussuga dono s oa a ie yo impo an biomoleculesasaccep‐
o sincludingmono‐,di‐,o oligo‐ca bohyd a es,glycans,lipids,pep ides,andnume ous
Figu e 2. Chemical s uc u es o he main classes o phenolic compounds.
Ex ac ion o phenolic glycosides om na u al sou ces is complex and uneconomical,
while a chemical app oach o hei syn hesis equi es oo many s eps o p o ec ion, ac i a-
ion, coupling, and dep o ec ion [
10
]. On he o he hand, due o he enzyme’s egio- and
s e eoselec i i y and mild eac ion condi ions, enzyma ic glycosyla ion is conside ed a e y
a ac i e, en i onmen ally iendly s a egy o ob aining hese aluable compounds [
16
].
To da e, i has been p o en ha enzymes such as glycosyl ans e ases (EC2.4) and gly-
cosidases (EC3.2.1) can ca alyze he glycosyla ion o phenolic compounds [
10
]. In his
e iew, he mos ep esen a i e ecen esea ch on he bio ans o ma ion o phenolic com-
pounds, using pu e enzymes o whole mic obial cells o con e glycosyla ion po en ial, as
bioca alys s will be discussed.
2.1. Glycosyl ans e ase
Glycosyl ans e ases (GTs) a e a subclass o enzymes ha ca alyze he ans e o a
suga esidue om a ious suga dono s o a a ie y o impo an biomolecules as accep o s
including mono-, di-, o oligo-ca bohyd a es, glycans, lipids, pep ides, and nume ous o he
small molecules [
17
]. The e o e, hey se e c i ical oles in oligosaccha ide, polysaccha ide,
Ca alys s 2023,13, 1359 4 o 28
and glycoconjuga e biosyn hesis, as well as p o ein glycosyla ion and he syn hesis o
aluable na u al p oduc s [
17
]. Based on hei ca aly ic p ope ies, glycosyl ans e ases a e
classi ied in o wo g oups: Leloi and non-Leloi enzymes [10].
2.1.1. Leloi Glycosyl ans e ase
In i o
syn hesis and modi ica ion o glycans is ca alyzed by Leloi glycosyl ans-
e ases. Due o a s ic speci ici y and high e iciency in glycosyla ion o a ious na u al
accep o s using ac i a ed glycosyl dono s (e.g., nucleo ide-suga conjuga es, lipid phos-
pha e suga s, and phospha e suga s), hese enzymes a e widely used o he syn hesis
o phenolic glycoconjuga es [
10
]. A summa y o di e en examples has been included in
Table 1.
Feng and co-wo ke s iden i ied phenolic glycosyl ans e ase MhGT1 om he ila-
men ous ungus Muco hiemalis which exhibi ed a p omising capabili y o egio- and
s e eospeci ic O-glycosyla ion o di e en phenolic compounds [
18
]. By examining 93
compound lib a ies o phenols om T adi ional Chinese Medicinal he bs as subs a es,
hey e ealed ha MhGT1 exhibi ed e y b oad subs a e speci ici y and ca alyzed he
glycosyla ion o 72 s uc u ally di e se d ug-like sca olds and s e ols using u idine diphos-
pha e (UDP) glucose as a suga dono . In e es ingly, MhGT1 showed high egiospeci ici y
owa ds compounds bea ing p enyl moie ies and we e mo e ac i e wi h p enyla ed phe-
nols as subs a es compa ing o hei non-p enyla ed analogues, which was explained
by he la ge ac i e ca i y o MhGT1 consis ing o hyd ophobic and cha ged amino acid
esidues [
18
]. This is pa icula ly impo an , ha ing in mind ha p enyla ed phenolics
show be e bioac i i ies compa ed o hei phenolic p ecu so s, including ca dio-, neu o-,
and os eop o ec ion, as well as an ioxidan and an i-diabe ic ac i i y [19].
Table 1. Example o di e en Leloi glycosyl ans e ases.
Enzyme Glycosyl Accep o Glycosyl
Dono Condi ions/P oduc s Yield Re s.
glycosyl ans e ase
MhGT1 om he
ilamen ous ungus
Muco hiemalis
glycycouma in and
93 phenolic
subs a es
UDP Glu
30–40 ◦C, pH 8.0–9.0
enzyme ac i i y
enhanced by
Ca2+, Mg2+ and Ba2+ ions
a e age con e sion
89% o subs a es
con aining p enyl
g oups
[18]
UDP
glycosyl ans e ases
UGT58A1 om
Absidia coe ulea
UGT59A1 om
Rhizopus japonicas
13 di e en phenolic
accep o s including
lignans, la onoids,
an h aquinon,
s ilbene,
benzophenone,
cu cuminoid,
xan hones, and
couma in
UDP Glu
UDP GluA
50 mM T is-HCl pH 8–9,
5 mM MgCl2, 400 µM UDP-Glc,
200 µM magnolol, and 200 µg o
p o ein, 45 ◦C, 2 h.
Mg2+, Mn2+, Ca2+, Ba2+
inc ease ac i i y
Ni
2+
, Co
2+
and Cu
2+
, dec ease ac i i y
94% [20]
UDP
glycosyl ans e ase,
Bs-PUGT om
B. sub ilis PI18,
(cloned and
exp essed in E. coli
BL21 (DE3)
and pu i ied)
y osol, cinnamic
alcohol, anillin,
e ulic acid, and
ca eic acid
ehalose,
suc ose, lac ose.
mal ose, s a ch,
glucose
pH 8.0, 30 ◦C, 25 g/L suc ose, 10 mM
Ca2+, and 0.5 g/L ca eic acid
78.3% wi h ca eic acid
as accep o [21]
UDP-
glucosyl ans e ase
om Beau e ia
bassiana ATCC 7159
exp essed in E. coli
que ce in
es e a ol, cu cumin
and zea alenone
UDP Glc
2.5 mM que ce in, 2 mM suga dono ,
1 mM MgCl2, 20 mM T is–HCl pH 8
and 0.125 mg/mL o pu i ied
ecombinan BbGT p o ein, 35 ◦C
Ca2+, Mg2+, and Mn2+ s imula e he
ac i i y o BbGT, while Zn2+ inhibi i .
i e monoglucosyla ed and one
diglucosyla ed que ce in de i a i es
by BbGT-ha bo ing E. coli
que ce in-7-O-β-d-
glucosides 0.34 mM om
0.83 mM que ce in in 24 h
by BbGT-ha bo ing
E. coli
que ce in-3-O-β-d-
glucoside 0.22mM om
0.41 mM que ce in in 12 h
by BbGT-ha bo ing
S. ce e isiae
[22]
Ca alys s 2023,13, 1359 5 o 28
In ano he s udy, Xie e al. iden i ied wo no el phenolic UDP glycosyl ans e ases
(PUGTs) in ungi, memb ane-bound p o eins UGT58A1 ( om Absidia coe ulea), and UGT59A1
( om Rhizopus japonicas), which can ans e suga moie ies om ac i e dono s o phenolic
accep o s and o m co esponding glycosides [
20
]. Whole cells o hese wo ungi we e
success ully applied o he glycosyla ion o wo phenolic compounds, magnolol and
honokiol, o ob ain he co esponding bioac i e glycosides wi h high yields [
23
], hence, he
au ho s used ecombinan enzymes om hese wo ungi and in es iga ed hei ca aly ic
p omiscui y, egioselec i i y, and s e eoselec i i y in he o ma ion o di e en phenolic
glycosides. The ac i i ies o bo h enzymes a e s ongly in luenced by he p esence o
a ious ca ions—Mg2+, Mn2+, Ca2+, Ba2+ inc ease ac i i y, Ni2+, Co2+, and Cu2+, dec ease
ac i i y, while wi h Zn2+ he glycosyla ion ac i i y was diminished [20].
As po en ial subs a es, 13 di e en phenolic accep o s wi h di e se s uc u es we e
es ed, including lignans, la onoids, an h aquinon, s ilbene, benzophenone, cu cuminoid,
xan hones, and couma in. Bo h enzymes we e able o ca alyze he glucosyla ion o mag-
nolol, honokiol, ch ysin, emodin, cu cumin, and h ee xan hones (1,3,6- ihyd oxyxan hone,
1,3,7- ihyd oxyxan hone, and 1,3,6,8- e ahyd oxyxan hone). UGT58A1 enabled highe con-
e sion a es o all sui able subs a es compa ed o UGT59A1. Addi ionally, se e al phenolic
subs a es— ise in, es e a ol, 2,4-dihyd oxy bezophenone, 1,3,8- ihyd oxylxan hone, and
7-hyd oxy-4-me hylcouma in—we e sui able glycosyl accep o s only o UGT58A1, in-
dica ing ha his enzyme possesses a pa icula ly high po en ial o be used as a ca alys
in he syn hesis o bioac i e glycosides. By using subs a es ha con ain mul iple nucle-
ophiles, single monoglucosyla ed p oduc s we e ob ained, sugges ing egioselec i i y o
UGT58A1. Fo example, ise in possesses ou phenolic hyd oxyl g oups, howe e a single
p oduc , ise in-3-O-
β
-glucopy anoside, was de ec ed and iden i ied, wi h a con e sion
deg ee o 94%.
Rega ding ole ance o suga dono s, ou o h ee es ed (UDP-Glc, UDP-GlcA, and
UDP-Gal), UGT58A1 was able o ecognize UDP-Glc and UDP-GlcA wi h magnolol as an
accep o [23].
Ano he phenolic UDP glycosyl ans e ase, Bs-PUGT om Bacillus sub ilis PI18, was
cloned and exp essed in Esche ichia coli BL21 (DE3) [
21
]. The pu i ied enzyme was es ed
wi h se e al na u ally occu ing phenolics such as y osol, cinnamic alcohol, anillin, e ulic
acid, hymol, cinnamic acid, and ca eic acid. Ob ained esul s p o ed i s high ac i i y
owa ds all examined subs a es excep in hymol and cinnamic acid. By using whole-cell
E. coli/Bs-PUGT as a ca alys and ca eic acid as a model subs a e in a p ocess assis ed
by 10 mM Ca
2+
ions, 78.3% mola yield o ca eic acid glucosides was accomplished a
op imized condi ions. The addi ion o me al ions p o ed o be a aluable ool o inc easing
he mola yield o ca eic acid glucosides and sho ening he eac ion ime.
A no el glycosyl ans e ase gene (BbGT) om Beau e ia bassiana ATCC 7159 was
disco e ed and he e ologously exp essed in E. coli by Ren e al. [
22
]. A e unc ional
cha ac e iza ion h ough
in i o
eac ions, i was shown ha he pu i ied enzyme was UDP-
glucosyl ans e ase, which could con e que ce in in o i e monoglucosyla ed de i a i es
and one diglucoside. Me al ions such as Ca
2+
, Mg
2+
, and Mn
2+
s imula e he ac i i y o
BbGT, while Zn
2+
show an inhibi i e e ec on he enzyma ic ac i i y o BbGT. Addi ionally,
he au ho s epo ed he b oad subs a e speci ici y o BbGT, since i was shown ha , in
addi ion o que ce in, his enzyme could also ca alyze he glucosyla ion o es e a ol,
cu cumin, and zea alenone. Mo eo e , conside ing ha whole-cell bio ans o ma ion
can p o ide UDP-glucose in si u, he
in i o
glucosyla ion o que ce in by BbGT was
also in es iga ed.
In addi ion o E. coli, di e en mic obial exp ession hos , including Saccha omyces
ce e isiae,Pseudomonas pu ida, and Pichia pas o is, we e used. In e es ingly, i was shown ha
BbGT exp essed in E. coli showed di e en p oduc p o iles
in i o
and
in i o
, since he
main p oduc o he
in i o
glucosyla ion o que ce in was que ce in-7-O-
β
-D-glucoside,
while diglucoside was no de ec ed. On he o he hand, he p oduc s o que ce in gluco-
syla ion also depended on he mic obial hos used, since he majo p oduc in P. pu ida,

Ca alys s 2023,13, 1359 6 o 28
and P. pas o is was he same as in E. coli, que ce in-7-O-
β
-D-glucoside, while he enzyme
dominan ly p oduced que ce in-3-O-β-D-glucoside in S. ce e isiae.
2.1.2. Non-Leloi Glycosyl ans e ase
Compa ed o Leloi glycosyl ans e ases, which equi e expensi e glycosyl dono s
and gene ally ole a e na u al accep o s, non-Leloi glycosyl ans e ases a e compa ible
wi h low-cos dono s and a wide ange o accep o s, which is e y impo an in e ms o
p oduc ion p ocess scale-up [
10
]. A summa y o di e en examples has been included in
Table 2.
Table 2. Examples o di e en non-Leloi glycosyl ans e ases.
Enzyme Glycosyl
Accep o Glycosyl Dono Condi ions/P oduc s Yield Re s.
cyclodex in
glucano ans e ase
(CGTase)
amylosuc ase om
Deinococcus
geo he malis
(DGAS)
isoque ci in (IQ) suc ose
Fo DGAS: 50 mg/mL suc ose,
1 mg/mL o isoque ci in, in
50 mM T is-HCl bu e pH-7 a
45 ◦C, 1 uni s/mL, 24 h
Fo CGTase: 50 mg/mL o soluble
s a ch, 1 mg/mL o isoque ci in,
in 50 mM ci a e phospha e bu e
pH7 a 70 ◦C, 1 uni s/mL, 24 h
P oduc s: IQ-glucoside,
IQ-diglucoside and
IQ- iglucoside
97% con e sion
wi h DGAS
75% con e sion
wi h CGTase
[24]
amylosuc ase om
Deinococcus
geo he malis
(DGAS)
15 di e en
la onoids, a ious
mono-, di- and i-
hyd oxy la ones
(HFVO) and -
hyd oxy la anones
(HFVA)
suc ose
200 mM o suc ose, 50 µM o
HFVO o HFVA, 1 uni /mL o
DGAS, 50 mM bu e
pH 5–8.8 a 45 ◦C
100% o
HFVAs and
HFVOs wi h
6-hyd oxyl g oup
50–60% o HFVAs
and HFVOs wi h
40-hyd oxyl g oup
[25]
dex ansuc ase
om Leuconos oc
mesen e oides
B-1299CB4
exp essed in E. coli
epigalloca echin
galla e (EGCG) suc ose Nine di e en eac ion p oduc s
we e pu i ied
91.43% con e sion
o EGCG [6]
cyclodex in glyco-
syl ans e ase om
The moanae obac e
sp.
epigalloca echin
galla e (EGCG)
hyd olyzed po a o
s a ch
EGCG (20 mM), hyd olyzed
po a o s a ch (20 mg/mL),
pa ially pu i ied To uzyme 3.0 L
(5% / ), 50 ◦C, 150 pm
P oduc s: EGCG-30-O-α-D-
glucopy anoside and
EGCG-7-O-α-D-glucopy anoside
58% EGCG
30-O-α-D-
glucopy anoside
13% EGCG
7-O-α-D-
glucopy anoside
[26]
cyclodex in glyco-
syl ans e ase
(CGTase) om
The moanae obac e
sp.
hespe e in soluble s a ch
hespe e in 15 mg/mL, soluble
s a ch 180 mg/mL, CGTase 10%
/ , bis(2-me hoxye hyl) e he
30% / , 10 mM sodium ci a e
bu e pH 5.0 60% / , 60 ◦C,
1000 pm
main p oduc : hespe e in-7-O-α-
glucopy anosid
4.1% [27]
Enzyma ic glycosyla ion o isoque ci in (IQ) using di e en glycosyl ans e ases,
comme cial cyclodex in glucano ans e ase (CGTase), and amylosuc ase om Deinococ-
cus geo he malis (DGAS) was in es iga ed by Rha e al. [
24
]. Bo h enzymes showed
signi ican ansglycosyla ion ac i i y and, as a p oduc , a eac ion mix u e o isoqueci in
de i a i es, including IQ-glucoside, IQ-diglucoside, and IQ- iglucoside, was syn hesized.
Ca alys s 2023,13, 1359 7 o 28
Howe e , se e al ad an ages o DGAS o e CGTase we e obse ed, including highe
p oduc i i y, simple p ocess, and ewe by-p oduc s. Highe biocon e sion was achie ed
wi h DGAS as a bioca alys , as well as 97% con e sion o IQ compa ed o 75% wi h CG-
Tase [
24
]. The mos impo an me i o DGAS applica ion is he highe yield o speci ic
isoque ci in- iglucoside, which is he mos bioa ailable o m among
α
-isoque ci in gluco-
sides. In he ollowing s udy, he same g oup in es iga ed he DGAS ca alyzed si e-speci ic
α
-glycosyla ion o 15 di e en la onoids, a ious mono-, di-, and i-hyd oxy la ones
(HFVO), as well as -hyd oxy la anones (HFVA) [
25
]. The egioselec i i y o he used
bioca alys was con i med, since i was shown ha in he 3–OH and 7–OH posi ions he
ansglycosyla ion o la onoids is negligible (Figu e 3), while DGAS showed s ong ans-
glycosyla ion eac i i y owa ds he 6–OH and 4
0
–OH posi ions o he mono-HFVOs and
-HFVAs wi h glucose eleased om suc ose as a suga dono . I was hypo hesized by
he au ho s ha his kind o -OH selec i i y is due o he speci ic in e ac ion be ween
he enzyme and subs a e, since he diphenyl p opane backbone o la onoids i s well
in o he ac i e pocke o he enzyme, so he 4
0−
OH and 6
−
OH axial posi ions in he
la onoid molecules we e eadily a ailable o ansglycosyla ion, while he 3–OH and
7–OH equa o ial posi ions we e inaccessible o ansglycosyla ion wi h DGAS.
Ca alys s2023,13,x 7o 28


speci icisoque ci in‐ iglucoside,whichis hemos bioa ailable o mamongα‐isoque ‐
ci inglucosides.In he ollowings udy, hesameg oupin es iga ed heDGASca alyzed
si e‐speci icα‐glycosyla iono 15di e en  la onoids, a iousmono‐,di‐,and i‐hy‐
d oxy la ones(HFVO),aswellas‐hyd oxy la anones(HFVA)[25].The egioselec i i y
o  heusedbioca alys wascon i med,sincei wasshown ha in he3–OHand7–OH
posi ions he ansglycosyla iono  la onoidsisnegligible(Figu e3),whileDGAS
showeds ong ansglycosyla ion eac i i y owa ds he6–OHand4′–OHposi ionso  he
mono‐HFVOsand‐HFVAswi hglucose eleased omsuc oseasasuga dono .I was
hypo hesizedby heau ho s ha  hiskindo ‐OHselec i i yisdue o hespeci icin e ‐
ac ionbe ween heenzymeandsubs a e,since hediphenylp opanebackboneo  la o‐
noids i swellin o heac i epocke o  heenzyme,so he4′−OHand6−OHaxialposi ions
in he la onoidmoleculeswe e eadilya ailable o  ansglycosyla ion,while he3–OH
and7–OHequa o ialposi ionswe einaccessible o  ansglycosyla ionwi hDGAS.

Figu e3.Regioselec i i yo amylosuc ase omDeinococcusgeo he malis(DGAS)in eac ion ohy‐
d oxy la onesandhyd oxy la anonesα‐glycosyla ion[25].
E icien syn hesiso no elepigalloca echingalla e‐glucosides(EGCG‐Gs)waspe ‐
o medbyusingdex ansuc ase omLeuconos ocmesen e oidesB‐1299CB4exp essedinE.
coliasaca alys andsuc oseassuga  esiduedono [6].The esponsesu aceme hodo‐
logicallyop imizedp ocessenabledanepigalloca echingalla e(EGCG)con e sionde‐
g eeo 91.43%.Ninedi e en  eac ionp oduc swe epu i iedand i eo  hemwe eiden‐
i iedasno elcompoundsbyNMRspec oscopy.No elcompoundshad45 o368‐ old
highe wa e solubili ycompa ed opa en moleculeEGCG.Allde i a i eshadlowe 
an ioxidan ac i i ycompa ed oEGCGandDPPH adicalsca engingcapaci ydec ease
washighe inmoleculeswi hmo eglucosyluni sa ached.Fu he mo e,7–OHg oup
glucosyla ionwasp o en oha ea unc ional oleinmush oom y osinaseinhibi o y
ac i i yaswellasinc easingb owning‐ esis an ac i i ies[6].
Inano he wo k, hesyn hesiso  a iousα‐glucosylde i a i eso EGCGwaspe ‐
o medinaqueous eac ionmediuma 50°Cbya ansglycosyla ion eac ionca alyzed
bycyclodex inglycosyl ans e ase omThe moanae obac e sp.usinghyd olyzedpo a o
s a chasaglucosyldono [26].Twomonoglucosideswe esyn hesizedas hemain eac‐
ionp oduc s,EGCG‐3′‐O‐α‐d‐glucopy anosideandEGCG‐7‐O‐α‐d‐glucopy anoside,
wi hcon e siondeg eeso 58%and13%, espec i ely(Figu e4).Thesame esea chg oup
in es iga ed hes e eo‐and egioselec i i yo cyclodex inglycosyl ans e ase(CGTase)
omThe moanae obac e sp.inaglycosyla iono hespe e inusing hesolubles a chasa
glucosyldono [27].Asp oduc s,se e alglucosideswe esyn hesized.Themainp oduc 
waspu i ied,ands uc u alanalysishasshown ha  heglucosyla iono  hehespe e in
akesplacea posi ionO‐7o  heA ing.Unde anop imized eac ioncondi ion,which
included hep esenceo 30%o bis(2‐me hoxye hyl)e he asaco‐sol en , hemaximum
concen a iono monoglucosidewasapp oxima ely2mM,ob aineda e 24ho  eac ion.
Figu e 3.
Regioselec i i y o amylosuc ase om Deinococcus geo he malis (DGAS) in eac ion o
hyd oxy la ones and hyd oxy la anones α-glycosyla ion [25].
E icien syn hesis o no el epigalloca echin galla e-glucosides (EGCG-Gs) was pe -
o med by using dex ansuc ase om Leuconos oc mesen e oides B-1299CB4 exp essed in
E. coli as a ca alys and suc ose as suga esidue dono [
6
]. The esponse su ace me hod-
ologically op imized p ocess enabled an epigalloca echin galla e (EGCG) con e sion deg ee
o 91.43%. Nine di e en eac ion p oduc s we e pu i ied and i e o hem we e iden i ied
as no el compounds by NMR spec oscopy. No el compounds had 45 o 368- old highe
wa e solubili y compa ed o pa en molecule EGCG. All de i a i es had lowe an ioxidan
ac i i y compa ed o EGCG and DPPH adical sca enging capaci y dec ease was highe
in molecules wi h mo e glucosyl uni s a ached. Fu he mo e, 7–OH g oup glucosyla ion
was p o en o ha e a unc ional ole in mush oom y osinase inhibi o y ac i i y as well as
inc easing b owning- esis an ac i i ies [6].
In ano he wo k, he syn hesis o a ious
α
-glucosyl de i a i es o EGCG was pe -
o med in aqueous eac ion medium a 50
◦
C by a ansglycosyla ion eac ion ca alyzed by
cyclodex in glycosyl ans e ase om The moanae obac e sp. using hyd olyzed po a o
s a ch as a glucosyl dono [
26
]. Two monoglucosides we e syn hesized as he main eac ion
p oduc s, EGCG-3
0
-O-
α
-d-glucopy anoside and EGCG-7-O-
α
-d-glucopy anoside, wi h
con e sion deg ees o 58% and 13%, espec i ely (Figu e 4). The same esea ch g oup
in es iga ed he s e eo- and egioselec i i y o cyclodex in glycosyl ans e ase (CGTase)
om The moanae obac e sp. in a glycosyla ion o hespe e in using he soluble s a ch as a
glucosyl dono [
27
]. As p oduc s, se e al glucosides we e syn hesized. The main p oduc
was pu i ied, and s uc u al analysis has shown ha he glucosyla ion o he hespe e in
akes place a posi ion O-7 o he A ing. Unde an op imized eac ion condi ion, which
Ca alys s 2023,13, 1359 8 o 28
included he p esence o 30% o bis(2-me hoxye hyl) e he as a co-sol en , he maximum
concen a ion o monoglucoside was app oxima ely 2 mM, ob ained a e 24 h o eac ion.
Al hough he con e sion yield o hespe e in was only 4.1%, his is he i s epo o using
he ee enzymes o
α
-glucosyla ion o hespe e in ins ead o whole cells. This was also
ex ended o ano he example o glycosyla ed phenolic compounds [28].
Ca alys s2023,13,x 8o 28


Al hough hecon e sionyieldo hespe e inwasonly4.1%, hisis he i s  epo o using
he eeenzymes o α‐glucosyla iono hespe e inins eado wholecells.Thiswasalso
ex ended oano he exampleo glycosyla edphenoliccompounds[28].

Figu e4.Anillus a iono  heenzymeselec ione ec onEGCGglycosyla ion egioselec i i y.Re‐
ac ionca alyzedbycyclodex inglycosyl ans e ase omThe moanae obac e sp.
2.1.3.C‐Glycosyl ans e ases
Due o hei po en ialbene i s ohumanheal hand hei be e  esis ance ohyd ol‐
ysiscompa ed oo he  ypeso glycosides, la onoidC‐glycosidesha e ecen lya ac ed
inc easeda en ion[29].C‐glycosyl ans e ases(CGT)a e hemos impo an bioca alys 
used o  heenzyma icsyn hesiso  la onoidC‐glycosides,since heyha eshownhigh
e iciencyand egiospeci ici y(Table3)[30].
Hee al. oundano elC‐glycosyl ans e aseTcCGT1 om hemedicinalplan T ol‐
liuschinensis,which ep esen s he i s CGT oca alyze he8‐C‐glycosyla iono  la ones
(Figu e5)[30].Mo eo e , hisenzymecouldca alyze heC‐glycosyla iono 36s uc u ‐
allydi e en  la onoidsand o 12subs a es hecon e sionyieldwasabo e77%.Inad‐
di ion oC‐glycosyla ion, heau ho s epo ed ha  heTcCGT1showed hehighca aly ic
capabili ies o O‐glycosyla ion,sincei ca alyzed heO‐glycosyla iono 44subs a es,o 
which31we e la onoids,wi hcon e sionyieldso mo e han80% o 8subs a es.Mos 
in e es ingly,TcCGT1exhibi edbo hC‐andO‐glycosyla ionac i i y owa ds17sub‐
s a es,including a ious la onoids.A e in es iga iono  heenzymec ys als uc u e
and heca aly icmul i unc ionals uc u almechanism,i was e ealed ha  hela gesub‐
s a edi e si yo TcCGT1isenabledbyaspacioussuga accep o bindingpocke .Mo e‐
o e ,conduc iono  hesi e‐di ec edmu agenesisa I94EandG284Kenabled heO‐gly‐
cosyla ionac i i yo TcCGT1,whilesupp essing heC‐glycosyla ionac i i y[30].
Peie al.de elopedaone‐po  wo‐enzymesys em,whichp o idedane icien 
me hod o  hep oduc iono isoo ien inandiso i exin, la onoidglycosides,in ol ing
hecyclingand egene a iono cos lyUDP‐glucosein he eac ion[31].

Figu e 4.
An illus a ion o he enzyme selec ion e ec on EGCG glycosyla ion egioselec i i y.
Reac ion ca alyzed by cyclodex in glycosyl ans e ase om The moanae obac e sp.
2.1.3. C-Glycosyl ans e ases
Due o hei po en ial bene i s o human heal h and hei be e esis ance o hyd olysis
compa ed o o he ypes o glycosides, la onoid C-glycosides ha e ecen ly a ac ed
inc eased a en ion [
29
]. C-glycosyl ans e ases (CGT) a e he mos impo an bioca alys
used o he enzyma ic syn hesis o la onoid C-glycosides, since hey ha e shown high
e iciency and egiospeci ici y (Table 3) [30].
He e al. ound a no el C-glycosyl ans e ase TcCGT1 om he medicinal plan T ollius
chinensis, which ep esen s he i s CGT o ca alyze he 8-C-glycosyla ion o la ones
(Figu e 5) [
30
]. Mo eo e , his enzyme could ca alyze he C-glycosyla ion o 36 s uc u ally
di e en la onoids and o 12 subs a es he con e sion yield was abo e 77%. In addi ion o
C-glycosyla ion, he au ho s epo ed ha he TcCGT1 showed he high ca aly ic capabili ies
o O-glycosyla ion, since i ca alyzed he O-glycosyla ion o 44 subs a es, o which 31 we e
la onoids, wi h con e sion yields o mo e han 80% o 8 subs a es. Mos in e es ingly,
TcCGT1 exhibi ed bo h C- and O-glycosyla ion ac i i y owa ds 17 subs a es, including
a ious la onoids. A e in es iga ion o he enzyme c ys al s uc u e and he ca aly ic
mul i unc ional s uc u al mechanism, i was e ealed ha he la ge subs a e di e si y o
TcCGT1 is enabled by a spacious suga accep o binding pocke . Mo eo e , conduc ion o
he si e-di ec ed mu agenesis a I94E and G284K enabled he O-glycosyla ion ac i i y o
TcCGT1, while supp essing he C-glycosyla ion ac i i y [30].
Ca alys s 2023,13, 1359 9 o 28
Table 3. Examples o di e en C-glycosyl ans e ases.
Enzyme Glycosyl
Accep o Glycosyl Dono Condi ions/
P oduc s Yield Re s.
C-glycosyl ans e ase
TcCGT1 om
medicinal plan
T ollius chinensis
apigenin, lu eolina
and
36 mo e
s uc u ally
di e en
la onoids
UDP Glc
50 mm phospha e bu e pH 8
0.5 mM UDP–Glc, 0.2 mM
aglycone, 50 mg pu i ied
ecombinan TcCGT1, 30 ◦C
o 12 h
100% con e sion o
apigenin and
lu eolin
>77% o
12 subs a es
[30]
C-glucosyl ans e ase
(G 6CGT) om
Gen iana i lo a
exp essed in E. coli
BL21 combined wi h
Glycine max suc ose
syn hase (GmSUS)
lu eoilin and
apigenin UDP Glc
500 mM suc ose, 1.0 mM
lu eolin, 0.3 mM UDP and
50 mM phospha e bu e pH 7.5,
3% DMSO ( / ), 5 mU/mL
G 6CGT, and 20 mU/mL
GmSUS, 48 h a 45 ◦C
p oduc s: isoo ien in
and iso i exin
94.7% con e sion
o lu eolin
97.1% con e sion
o apigenin
[31]
Ca alys s2023,13,x 9o 28


Table3.Exampleso di e en C‐glycosyl ans e ases.
EnzymeGlycosyl
Accep o GlycosylDono Condi ions/
P oduc sYieldRe s.
C‐glycosyl ans e aseTcCGT1
ommedicinalplan T ollius
chinensis
apigenin,lu eolina
and
36mo es uc u ally
di e en  la onoids
UDPGlc
50mmphospha ebu e pH8
0.5mMUDP–Glc,0.2mM
aglycone,50mgpu i ied
ecombinan TcCGT1,30°C o 
12h
100%con e siono 
apigeninandlu eolin
>77% o 12subs a es
[30]
C‐glucosyl ans e ase(G 6CGT)
omGen iana i lo aexp essedin
E.coliBL21combinedwi hGlycine
maxsuc osesyn hase(GmSUS)
lu eoilinandapigeninUDPGlc
500mMsuc ose,1.0mM
lu eolin,0.3mMUDPand50
mMphospha ebu e pH7.5,
3%DMSO( / ),5mU/mL
G 6CGT,and20mU/mL
GmSUS,48ha 45°C
p oduc s:isoo ien inand
iso i exin
94.7%con e siono 
lu eolin
97.1%con e siono 
apigenin
[31]
Conside ing heunsa is ac o yp oduc i i yo isoo ien insyn hesisincons uc ed
ecombinan E.coli[32], heau ho sde elopeden i onmen allysa ee icien enzyma ic
in i oglycosyla ion[31](Figu e5).Theyha eusedC‐glucosyl ans e ase(G 6CGT)
omGen iana i lo aexp essedinE.coliBL21combinedwi hGlycinemaxsuc osesyn‐
hase(GmSUS) o  hep oduc iono isoo ien inandiso i exin.G CGTwasused o  egi‐
oselec i eglycosyla ion,sincei was epo ed ha  hisenzymecouldglycosyla ece ain
la onoidsa  heC‐6posi ion,whileGmSUSwasu ilized ocons uc  he egene a ion
sys emo UDP‐glucose.Byop imizingcoupled eac ioncondi ions, hehighmola con‐
e sionso p ecu so  la onoids,lu eolin(94.7%)andapigenin(97.1%),we eob ainedin
hesyn hesiso isoo ien inandiso i exin, espec i ely.

Figu e5.Regioselec i eglycosyla iono apigeninandlu eolinca alyzedby:(a)C‐glycosyl ans e ‐
ase ommedicinalplan T olliuschinensis(TcCGT1)[31]and(b)C‐glycosyl ans e ase omGen iana
i lo a(G 6CGT)[31].
2.2.Glycosidases
Glycosylhyd olases(GHs)o glycosidasesa eenzymeswi h hep ima y unc iono 
glycosidiclinkagehyd olysis,howe e  heycanalsop oducenewglycosidicbonds
h ougha ansglycosyla ion eac ionwhenal e na i enucleophilespa icipa easaccep‐
o s.Va ioussuga uni scouldbe ans e ed oa angeo di e en nucleophilicaccep‐
o s,includingphenoliccompounds,wi hacomple es e eoselec i i yand e yhigh e‐
gioselec i i y.Exampleso di e en glycosidasesa eshowninTable4.

Figu e 5.
Regioselec i e glycosyla ion o apigenin and lu eolin ca alyzed by: (
a
) C-glycosyl ans e ase
om medicinal plan T ollius chinensis (TcCGT1) [
31
] and (
b
) C-glycosyl ans e ase om Gen iana
i lo a (G 6CGT) [31].
Pei e al. de eloped a one-po wo-enzyme sys em, which p o ided an e icien me hod
o he p oduc ion o isoo ien in and iso i exin, la onoid glycosides, in ol ing he cycling
and egene a ion o cos ly UDP-glucose in he eac ion [31].
Conside ing he unsa is ac o y p oduc i i y o isoo ien in syn hesis in cons uc ed
ecombinan E. coli [
32
], he au ho s de eloped en i onmen ally sa e e icien enzyma ic
in i o
glycosyla ion [
31
] (Figu e 5). They ha e used C-glucosyl ans e ase (G 6CGT) om
Gen iana i lo a exp essed in E. coli BL21 combined wi h Glycine max suc ose syn hase (Gm-
SUS) o he p oduc ion o isoo ien in and iso i exin. G CGT was used o egioselec i e
glycosyla ion, since i was epo ed ha his enzyme could glycosyla e ce ain la onoids
a he C-6 posi ion, while GmSUS was u ilized o cons uc he egene a ion sys em o
UDP-glucose. By op imizing coupled eac ion condi ions, he high mola con e sions o
p ecu so la onoids, lu eolin (94.7%) and apigenin (97.1%), we e ob ained in he syn hesis
o isoo ien in and iso i exin, espec i ely.
2.2. Glycosidases
Glycosyl hyd olases (GHs) o glycosidases a e enzymes wi h he p ima y unc ion
o glycosidic linkage hyd olysis, howe e hey can also p oduce new glycosidic bonds
h ough a ansglycosyla ion eac ion when al e na i e nucleophiles pa icipa e as accep-
o s. Va ious suga uni s could be ans e ed o a ange o di e en nucleophilic accep o s,
including phenolic compounds, wi h a comple e s e eoselec i i y and e y high egioselec-
i i y. Examples o di e en glycosidases a e shown in Table 4.
Ca alys s 2023,13, 1359 16 o 28
Ca alys s2023,13,x 16o 28



Figu e13.Glucosidesp oducedby ansglycosyla iono EGCGwi h hesyn haseBGL‐1‐E521G
[28].
Ano he in e es ingglycosyn hasesa e ocusedonaβ‐1,3o β‐1,4speci ici y, o ex‐
ampleinglucansyn hesis[55,58,59].AHo deum ulga eE231Gsyn hasemedia edsel ‐
condensa iono α‐lamina ibiosyl luo ideand3‐ hio‐α‐lamina ibiosyl luo ide opoly‐
me swi hdi e en polyme iza iondeg ees.Mo eo e ,p oduc iono mixed‐linked1,3‐
1,4β‐glucans omdi‐, i‐,and e a‐saccha idedono shasbeenachie ed,whe eby un‐
ingo  heβ‐1,3andβ‐1,4linkage a iop oducedglucans ha dono occu inna u e.
Recen ly,p og esshasbeenmade owa ds hede elopmen o β‐1,3‐glucansyn‐
hasesemploying he mo‐ esis an β‐glucosidasesasna i eenzymes,wi h heinsi u o ‐
ma iono glycosyl o ma edono s,whichallowed heuseo bo h he luo idedono o 
anexogenous o ma enucleophile op oduceaβ‐1,3disaccha ide

[58,59].
On heo he hand, he ea ealsoglycosyn hasesde i ed omendo‐glycosidases.
These ypeso enzymesenable heuseo oligosaccha ideswi hdi e en deg eeso 
polyme iza ions oac asglycosyldono s.The i s glycosyn hase epo ed oe icien ly
p omo e hesel ‐condensa iono oligosaccha idedono sin opolysaccha ideswascon‐
s uc edbygene a ing heE197Amu an o  he e ainingcellulase,Cel7B,o Humicola
insolens(HiCel7B)(Figu e14)[60].

Figu e14.Polysaccha idessyn hesizedca alyzedbyglycosyn hases.
Thispowe ulglycosyn haseca alyzes he ans e o α‐cellobiosylandα‐lac osyl
luo ides(CelFandLacF, espec i ely) oa a ie yo subs a es, esul ingin he o ma ion
o aβ‐1,4glycosidiclinkage(β‐1,4glycosyn hases).
4.2.Galac osyn hases
Func ionaloligosaccha idesandglycanssuchasgalac o‐N‐biose(GNB)andlac o‐N‐
biose(LNB)glycoconjuga esa eimpo an ca bohyd a esde i a i es ha a ep esen in
Figu e 13.
Glucosides p oduced by ansglycosyla ion o EGCG wi h he syn hase BGL-1-E521G [
28
].
Recen ly, p og ess has been made owa ds he de elopmen o
β
-1,3-glucan syn hases
employing he mo- esis an
β
-glucosidases as na i e enzymes, wi h he in si u o ma ion o
glycosyl o ma e dono s, which allowed he use o bo h he luo ide dono o an exogenous
o ma e nucleophile o p oduce a β-1,3 disaccha ide [58,59].
On he o he hand, he e a e also glycosyn hases de i ed om endo-glycosidases.
These ypes o enzymes enable he use o oligosaccha ides wi h di e en deg ees o poly-
me iza ions o ac as glycosyl dono s. The i s glycosyn hase epo ed o e icien ly p o-
mo e he sel -condensa ion o oligosaccha ide dono s in o polysaccha ides was cons uc ed
by gene a ing he E197A mu an o he e aining cellulase, Cel7B, o Humicola insolens
(HiCel7B) (Figu e 14) [60].
Ca alys s2023,13,x 16o 28



Figu e13.Glucosidesp oducedby ansglycosyla iono EGCGwi h hesyn haseBGL‐1‐E521G
[28].
Ano he in e es ingglycosyn hasesa e ocusedonaβ‐1,3o β‐1,4speci ici y, o ex‐
ampleinglucansyn hesis[55,58,59].AHo deum ulga eE231Gsyn hasemedia edsel ‐
condensa iono α‐lamina ibiosyl luo ideand3‐ hio‐α‐lamina ibiosyl luo ide opoly‐
me swi hdi e en polyme iza iondeg ees.Mo eo e ,p oduc iono mixed‐linked1,3‐
1,4β‐glucans omdi‐, i‐,and e a‐saccha idedono shasbeenachie ed,whe eby un‐
ingo  heβ‐1,3andβ‐1,4linkage a iop oducedglucans ha dono occu inna u e.
Recen ly,p og esshasbeenmade owa ds hede elopmen o β‐1,3‐glucansyn‐
hasesemploying he mo‐ esis an β‐glucosidasesasna i eenzymes,wi h heinsi u o ‐
ma iono glycosyl o ma edono s,whichallowed heuseo bo h he luo idedono o 
anexogenous o ma enucleophile op oduceaβ‐1,3disaccha ide

[58,59].
On heo he hand, he ea ealsoglycosyn hasesde i ed omendo‐glycosidases.
These ypeso enzymesenable heuseo oligosaccha ideswi hdi e en deg eeso 
polyme iza ions oac asglycosyldono s.The i s glycosyn hase epo ed oe icien ly
p omo e hesel ‐condensa iono oligosaccha idedono sin opolysaccha ideswascon‐
s uc edbygene a ing heE197Amu an o  he e ainingcellulase,Cel7B,o Humicola
insolens(HiCel7B)(Figu e14)[60].

Figu e14.Polysaccha idessyn hesizedca alyzedbyglycosyn hases.
Thispowe ulglycosyn haseca alyzes he ans e o α‐cellobiosylandα‐lac osyl
luo ides(CelFandLacF, espec i ely) oa a ie yo subs a es, esul ingin he o ma ion
o aβ‐1,4glycosidiclinkage(β‐1,4glycosyn hases).
4.2.Galac osyn hases
Func ionaloligosaccha idesandglycanssuchasgalac o‐N‐biose(GNB)andlac o‐N‐
biose(LNB)glycoconjuga esa eimpo an ca bohyd a esde i a i es ha a ep esen in
Figu e 14. Polysaccha ides syn hesized ca alyzed by glycosyn hases.
This powe ul glycosyn hase ca alyzes he ans e o
α
-cellobiosyl and
α
-lac osyl
luo ides (CelF and LacF, espec i ely) o a a ie y o subs a es, esul ing in he o ma ion
o a β-1,4 glycosidic linkage (β-1,4 glycosyn hases).
4.2. Galac osyn hases
Func ional oligosaccha ides and glycans such as galac o-N-biose (GNB) and lac o-N-
biose (LNB) glycoconjuga es a e impo an ca bohyd a es de i a i es ha a e p esen in a
wide scope o bioac i e compounds. Thus, s aigh o wa d access o his ype o sca olds
is c ucial, wi h e sa ile applica ions in medicinal chemis y and biology [61–66]. So, Y-W.
Kim e al. [
66
] s udied a new ou e o access D-Lac o- and D-Galac o-N-bioside glycans
(D-LNB and D-GNB, espec i ely) in ol ing an enzyma ic pa hway. In pa icula , glycosyn-
hases ha e been e ealed o be use ul in he p epa a ion o se e al oligosaccha ides and
o he glycoconjuga es [
67
–
72
], since hey possess ele an ansglycosyla ion ac i i y wi h
glycosyl luo ides o glycosyl azides wi h no appea ing hyd olysis, gi ing oppo uni y o

Ca alys s 2023,13, 1359 17 o 28
he syn hesis o galac osyl
β
-1,3-linked ans e p oduc s, such as D-LNB and D-GNB. The
au ho s desc ibed he syn hesis o a galac osyn hase de i ed om he glycoside hyd olase
(GH) amily 35
β
-galac osidase. Fo ha , a
β
-galac osidase om Bacillus ci culans mu an
(BgaC), whe e Ala, Gly, and Se we e inse ed as subs i u ions o ca aly ic nucleophiles,
was used o explo e he po en ial ca aly ic ac i i y when
α
-D-galac opy anosyl luo ide
(αGF) and 4-ni ophenyl β-d-glucopy anoside (pNβG) we e used as he suga dono and
accep o , espec i ely.
A e he eac ion comple ion, BgaC syn hase bea ing Ala and Se did no yield he
desi ed ans e p oduc s, leading only o hyd olysis o he suga dono (
α
GF). The one
bea ing Gly indeed gene a ed a ans e p oduc , and a e ca e ul LC-MS analysis by he
au ho s, he obse ed p oduc was a disaccha ide bea ing di e en glycosidic linkages
om pNβG [66].
To u he expand he selec i i y s udies using his BgaC-bea ing Gly, 18 di e en a yl
suga accep o s we e employed using
α
GF as he suga dono a 25
◦
C o 5h, wi h i e
glycosides being iden i ied as accep o s o his BgaC. Thus, hese i e accep o s we e hen
used o pe o m he ans e eac ion leading o 10 di e en p oduc s (1a–1e and 2a–2e)
bea ing he
β
-1,3-linkage, which was con i med by 13C-NMR (Figu e 15). Analysis by
HPLC and LC-MS also o e uled he o ma ion o isaccha ides [66].
Ca alys s2023,13,x 17o 28


awidescopeo bioac i ecompounds.Thus,s aigh o wa daccess o his ypeo sca ‐
oldsisc ucial,wi h e sa ileapplica ionsinmedicinalchemis yandbiology[61–66].So,
Y‐W.Kime al.[66]s udiedanew ou e oaccessD‐Lac o‐andD‐Galac o‐N‐biosidegly‐
cans(D‐LNBandD‐GNB, espec i ely)in ol inganenzyma icpa hway.Inpa icula ,
glycosyn hasesha ebeen e ealed obeuse ulin hep epa a iono se e aloligosaccha‐
idesando he glycoconjuga es[67–72],since heypossess ele an  ansglycosyla ion
ac i i ywi hglycosyl luo ideso glycosylazideswi hnoappea inghyd olysis,gi ing
oppo uni y o  hesyn hesiso galac osylβ‐1,3‐linked ans e p oduc s,suchasD‐LNB
andD‐GNB.Theau ho sdesc ibed hesyn hesiso agalac osyn hasede i ed om he
glycosidehyd olase(GH) amily35β‐galac osidase.Fo  ha ,aβ‐galac osidase omBa‐
cillusci culansmu an (BgaC),whe eAla,Gly,andSe we einse edassubs i u ions o 
ca aly icnucleophiles,wasused oexplo e hepo en ialca aly icac i i ywhenα‐D‐ga‐
lac opy anosyl luo ide(αGF)and4‐ni ophenylβ‐d‐glucopy anoside(pNβG)we eused
as hesuga dono andaccep o , espec i ely.
A e  he eac ioncomple ion,BgaCsyn hasebea ingAlaandSe didno yield he
desi ed ans e p oduc s,leadingonly ohyd olysiso  hesuga dono (αGF).Theone
bea ingGlyindeedgene a eda ans e p oduc ,anda e ca e ulLC‐MSanalysisby he
au ho s, heobse edp oduc wasadisaccha idebea ingdi e en glycosidiclinkages
ompNβG[66].
To u he expand heselec i i ys udiesusing hisBgaC‐bea ingGly,18di e en 
a ylsuga accep o swe eemployedusingαGFas hesuga dono a 25°C o 5h,wi h
i eglycosidesbeingiden i iedasaccep o s o  hisBgaC.Thus, hese i eaccep o swe e
henused ope o m he ans e  eac ionleading o10di e en p oduc s(1a–1eand2a–
2e)bea ing heβ‐1,3‐linkage,whichwascon i medby13C‐NMR(Figu e15).Analysisby
HPLCandLC‐MSalsoo e uled he o ma iono  isaccha ides[66].

Figu e15.Syn hesiso glycode i a i esca alyzedbyaBgaC‐Glygalac osyn hase.
Rega dingca aly ice iciency,BgaC‐Glywas e ealed ope o mbe e whenα‐con‐
igu edglycosideswe eused.Thus, heau ho swe eable ode elopanunp eceden ed
syn hesiso agalac osyn hasede i ed omglycosidehyd olase(GH) amily35β‐galac‐
osidase,whichwasable ogene a e ans e p oduc sbea ing hedesi edbea ingo  he
β‐1,3‐linkage.Fu he mo e,pa a‐ni ophenol‐αLNBandpa a‐ni ophenol‐αGNBwe eob‐
ainedinanup o98%yield[66].
Figu e 15. Syn hesis o glycode i a i es ca alyzed by a BgaC-Gly galac osyn hase.
Rega ding ca aly ic e iciency, BgaC-Gly was e ealed o pe o m be e when
α
-
con igu ed glycosides we e used. Thus, he au ho s we e able o de elop an unp ece-
den ed syn hesis o a galac osyn hase de i ed om glycoside hyd olase (GH) amily 35
β
-galac osidase, which was able o gene a e ans e p oduc s bea ing he desi ed bea ing
o he
β
-1,3-linkage. Fu he mo e, pa a-ni ophenol-
α
LNB and pa a-ni ophenol-
α
GNB
we e ob ained in an up o 98% yield [66].
Gi en he e sa ili y o
β
-galac osidase om Bacillus ci culans mu an (BgaC), P. Bo-
ja o áe al. [
73
] epo ed he syn he ic applica ion o hese mu an enzymes o he ans-
o ma ion o
α
-galac osyl luo ide (
α
GF) and
β
-galac osyl azide (
β
GN3)
α
-galac osyl o
es he glycosyn hase ac i i y. Those h ee di e en mu an s we e ob ained using selec i e
mu agenesis, ia ac i e si e modi ica ion o glycine, alanine, and h eonine, and hey we e
hen applied in he ans o ma ion. S ill, hese dono s we e no success ul and ins ead, wo
mu an s (bea ing glycine and h eonine) we e employed in he selec i e syn hesis o azido-
unc ionalized N-ace yllac osamine using p-ni ophenyl
β
-d-galac oside as a galac osyl
dono (Figu e 16).
Ca alys s 2023,13, 1359 18 o 28
Ca alys s2023,13,x 18o 28


Gi en he e sa ili yo β‐galac osidase omBacillusci culansmu an (BgaC),P.Boja‐
o áe al.[73] epo ed hesyn he icapplica iono  hesemu an enzymes o  he ans‐
o ma iono α‐galac osyl luo ide(αGF)andβ‐galac osylazide(βGN3)α‐galac osyl o
es  heglycosyn haseac i i y.Those h eedi e en mu an swe eob ainedusingselec‐
i emu agenesis, iaac i esi emodi ica ion oglycine,alanine,and h eonine,and hey
we e henappliedin he ans o ma ion.S ill, hesedono swe eno success ulandin‐
s ead, womu an s(bea ingglycineand h eonine)we eemployedin heselec i esyn‐
hesiso azido‐ unc ionalizedN‐ace yllac osamineusingp‐ni ophenylβ‐d‐galac osideas
agalac osyldono (Figu e16).

Figu e16.Syn hesiso azido‐ unc ionalizedN‐ace yllac osamine.
Fu he mo e, hep epa edmu an sunexpec edlys ill e ainedamino pa o  hei 
hyd oly icac i i y,whichcanjus i ywhyαGFandβGN3we eno success uldono s.The
au ho ss udied hisbeha io bymolecula docking.Thus, he esul spublishedby he
au ho shighligh  ha  heca aly icnucleophilemayno been i ely alid oallglyco‐
sidases,bu ins ead,s uc u alin e ac ionsin heac i esi eshouldbejudged[74,75].
Ino de  oachie e hesyn hesiso  aluableα‐galac osyloligosaccha ides[76,77],a
glycosyn hase om heBac e oides he aio aomic onglycosidehyd olase amily(GH)97
(BTsyn hase)wasusedincombina ionwi hβ‐galac osylazide(βGN3)andα‐galac osyl
luo ide(αGF)asdono s,andlac osewasusedasanaccep o wi h heassis anceo ex‐
e nalanions.In hiscase,asp e iouslyobse ed, he o ma ep o ed obe hebes assis‐
an  o a aining hedesi edoligosaccha idesin he ansglycosyla ion.E en houghin‐
hibi iono  hedono clea age eac ionisbe e pe o medby heazide hanby he o ‐
ma e,anaccumula iono βGN3wasobse edwhen hiswasused,wi halowyieldo  he
ans e p oduc .Tojus i y hese esul s, heau ho spe o medkine ics udies ha sug‐
ges ed he o ma iono acomplexbe ween heenzyme,βGN3,andlac ose,whichlimi ed
he ans e  eac ionin heazide‐ escued eac ion[75].
In hisway,GTsyn hasewasable op oduceα‐galac osides ia o ma e‐ escued
ansglycosyla ion,whichwasachie edina90%yieldusingxyloseo lac oseas heac‐
cep o ,wi hgalac osyl luo ideas hedono .
4.3.Fucosyn hases
Aspa agine‐linkedglycosyla ion,alsoknownasN‐glycosyla ion,isoneo  hemos 
p e alen p o einpos ‐ ansla ionalmodi ica ionsinmammalsandplaysakey olein
egula ing hein insicp ope iesandbiological unc ionso basicp o eins[76,77].Inpa ‐
icula ,co e ucosyla ionlinking16‐linked ucose o hedeepes aspa agine‐linkedN‐
ace ylglucosamine(GlcNAc)moie yinN‐glycansisanimpo an modi ica iono N‐gly‐
cop o eins.In iguinge idencesugges s ha co eglycop o ein ucosyla ion egula esdi‐
e secellula  unc ions.Fo ins ance,se e als udiesha eshown ha inc easedassocia‐
ionwi hco e ucosyla ioniso enassocia edwi h hede elopmen o cance [78–80].
Howe e , hesyn hesiso awell‐de ined ucosyla edco eglycop o eins uc u e e‐
mainsachallenging askdue o hecomplexi yo mul iphasechemicalsyn hesiso  he
Figu e 16. Syn hesis o azido- unc ionalized N-ace yllac osamine.
Fu he mo e, he p epa ed mu an s unexpec edly s ill e ained a mino pa o hei
hyd oly ic ac i i y, which can jus i y why
α
GF and
β
GN3 we e no success ul dono s. The
au ho s s udied his beha io by molecula docking. Thus, he esul s published by he
au ho s highligh ha he ca aly ic nucleophile may no be en i ely alid o all glycosidases,
bu ins ead, s uc u al in e ac ions in he ac i e si e should be judged [74,75].
In o de o achie e he syn hesis o aluable
α
-galac osyl oligosaccha ides [
76
,
77
],
a glycosyn hase om he Bac e oides he aio aomic on glycoside hyd olase amily (GH) 97
(BT syn hase) was used in combina ion wi h
β
-galac osyl azide (
β
GN3) and
α
-galac osyl
luo ide (
α
GF) as dono s, and lac ose was used as an accep o wi h he assis ance o ex e nal
anions. In his case, as p e iously obse ed, he o ma e p o ed o be he bes assis an o
a aining he desi ed oligosaccha ides in he ansglycosyla ion. E en hough inhibi ion
o he dono clea age eac ion is be e pe o med by he azide han by he o ma e, an
accumula ion o
β
GN3 was obse ed when his was used, wi h a low yield o he ans e
p oduc . To jus i y hese esul s, he au ho s pe o med kine ic s udies ha sugges ed he
o ma ion o a complex be ween he enzyme,
β
GN3, and lac ose, which limi ed he ans e
eac ion in he azide- escued eac ion [75].
In his way, GT syn hase was able o p oduce
α
-galac osides ia o ma e- escued
ansglycosyla ion, which was achie ed in a 90% yield using xylose o lac ose as he
accep o , wi h galac osyl luo ide as he dono .
4.3. Fucosyn hases
Aspa agine-linked glycosyla ion, also known as N-glycosyla ion, is one o he mos
p e alen p o ein pos - ansla ional modi ica ions in mammals and plays a key ole in
egula ing he in insic p ope ies and biological unc ions o basic p o eins [
76
,
77
]. In
pa icula , co e ucosyla ion linking 16-linked ucose o he deepes aspa agine-linked
N-ace ylglucosamine (GlcNAc) moie y in N-glycans is an impo an modi ica ion o N-
glycop o eins. In iguing e idence sugges s ha co e glycop o ein ucosyla ion egula es
di e se cellula unc ions. Fo ins ance, se e al s udies ha e shown ha inc eased associa-
ion wi h co e ucosyla ion is o en associa ed wi h he de elopmen o cance [78–80].
Howe e , he syn hesis o a well-de ined ucosyla ed co e glycop o ein s uc u e
emains a challenging ask due o he complexi y o mul iphase chemical syn hesis o
he inabili y o he biosyn he ic 16- ucosyl ans e ase (FUT8) o di ec ly ucosyla e ull-
sized ma u e N-glycans [
81
–
84
]. Fo his eason, a me hod o di ec ucosyla ion o in ac
glycopep ides and glycop o eins is highly desi able. Following his app oach, he in es-
iga ion g oup o med by Wang and cowo ke s [
85
] desc ibes he design and gene a ion
o a po en ial
α
1,6- ucosyn hase and ucoligase o di ec co e ucosyla ion o in ac N-
glycop o eins wi hou p oduc hyd olysis by using no el mu an s de i ed om Lac obacillus
casei α- ucosidase.
Fi s ly, hey c ea ed se e al mu an s o he L. casei 1,6- ucosidase glycosyn hase and
glycoligase and hey assessed he enzymes’ capaci y o co e ucosyla e a ange o accep o
subs a es (Figu e 17).
Ca alys s 2023,13, 1359 19 o 28
Ca alys s2023,13,x 19o 28


inabili yo  hebiosyn he ic16‐ ucosyl ans e ase(FUT8) odi ec ly ucosyla e ull‐sized
ma u eN‐glycans[81–84].Fo  his eason,ame hod o di ec  ucosyla iono in ac gly‐
copep idesandglycop o einsishighlydesi able.Following hisapp oach, hein es iga‐
iong oup o medbyWangandcowo ke s[85]desc ibes hedesignandgene a iono a
po en ialα1,6‐ ucosyn haseand ucoligase o di ec co e ucosyla iono in ac N‐glyco‐
p o einswi hou p oduc hyd olysisbyusingno elmu an sde i ed omLac obacillus
caseiα‐ ucosidase.
Fi s ly, heyc ea edse e almu an so  heL.casei1,6‐ ucosidaseglycosyn haseand
glycoligaseand heyassessed heenzymes’capaci y oco e ucosyla ea angeo accep o 
subs a es(Figu e17).

Figu e17.E alua iono  ucosidasemu an s o di ec co e ucosyla iono N‐glycans.Rep in ed
wi hpe mission om e 85.Copy igh 2017Ame icanChemicalSocie y.
Following heglycosyn haseconcep p oposedbyWi he sandco‐wo ke s[86], hey
pe o medsi e‐di ec edmu agenesisa  heiden i iednucleophilein heAl Cα1,6‐ uco‐
sidase,D200 ogene a eselec edmu an s,includingD200G,D200S,D200A,andD200T.
Simila  o his,speci icmu an sa  hepu a i egene icacid/base esidue,E274,suchas
E274A,E274S,E274G,andE274D,we ec ea ed op o idepo en ialglycoligases.Excep 
o E274D,noneo  hesemu an sshowedmo e han aceso  esidualhyd olysisac i i y
due omu a ionsa  hec i ical esidues.Inaddi ion, hiss udycon i med ha  heD200
esidueis henucleophileand ha  heE274 esidueismos likely hegene alacid/base.
The e o e, heyassessed hesyn hesizedmu an saspo en ialglycosyn haseso gly‐
coligases.Thus, hepo en ialglycosyla ionac i i yo  henucleophilicmu an s,including
D200G,D200S,D200A,andD200T,was es edusingbo hβ‐glycosylazideand heβ‐gly‐
cosyl luo ideas hedono subs a esandFmoc‐Asn(GlcNAc)‐OHas heaccep o sub‐
s a e(Figu e18).Noglycosyla ionp oduc swe eobse edinanyo  hecasess udied,
and his indingindica ed ha  henucleophilicmu an s es eddidno ac asaglycosyn‐
hase.Then, hey es ed heuseo α‐ ucosyl luo ideas hedono subs a e(Figu e18).
In e es ingly, heE274Amu an displayedgoodenzyma icac i i y o ans e a ucose
esidue o heGlcNAcmoie yo  heaccep o , esul ingindisaccha ideFucα1,6GlcNAc‐
Asnwi h egio‐ands e eospeci ici y(Figu e18).Simila ou comeswe ep oducedby he
o he  womu an s,E274GandE274S,whichwe elikewisee ec i eα1,6‐ ucosyla ionca ‐
alys s.
Figu e 17.
E alua ion o ucosidase mu an s o di ec co e ucosyla ion o N-glycans. Rep in ed wi h
pe mission om e . [85]. Copy igh 2017 Ame ican Chemical Socie y.
Following he glycosyn hase concep p oposed by Wi he s and co-wo ke s [
86
],
hey pe o med si e-di ec ed mu agenesis a he iden i ied nucleophile in he Al C
α
1,6-
ucosidase, D200 o gene a e selec ed mu an s, including D200G, D200S, D200A, and D200T.
Simila o his, speci ic mu an s a he pu a i e gene ic acid/base esidue, E274, such as
E274A, E274S, E274G, and E274D, we e c ea ed o p o ide po en ial glycoligases. Excep
o E274D, none o hese mu an s showed mo e han aces o esidual hyd olysis ac i i y
due o mu a ions a he c i ical esidues. In addi ion, his s udy con i med ha he D200
esidue is he nucleophile and ha he E274 esidue is mos likely he gene al acid/base.
The e o e, hey assessed he syn hesized mu an s as po en ial glycosyn hases o gly-
coligases. Thus, he po en ial glycosyla ion ac i i y o he nucleophilic mu an s, including
D200G, D200S, D200A, and D200T, was es ed using bo h
β
-glycosyl azide and he
β
-
glycosyl luo ide as he dono subs a es and Fmoc-Asn(GlcNAc)-OH as he accep o
subs a e (Figu e 18). No glycosyla ion p oduc s we e obse ed in any o he cases s udied,
and his inding indica ed ha he nucleophilic mu an s es ed did no ac as a glycosyn-
hase. Then, hey es ed he use o
α
- ucosyl luo ide as he dono subs a e (Figu e 18).
In e es ingly, he E274A mu an displayed good enzyma ic ac i i y o ans e a ucose
esidue o he GlcNAc moie y o he accep o , esul ing in disaccha ide Fuc
α
1,6GlcNAc-Asn
wi h egio- and s e eospeci ici y (Figu e 18). Simila ou comes we e p oduced by he o he
wo mu an s, E274G and E274S, which we e likewise e ec i e α1,6- ucosyla ion ca alys s.
In addi ion, hey ound ha he Al C mu an s (E274A, E274G, and E274S) only dis-
played educed ac i i y in he absence o he GlcNAc accep o . While he wild- ype Al C
could p omp ly hyd olyze he dono subs a e, i hyd olyzed - ucosyl luo ide slowly.
These indings collec i ely sugges ed ha he Al C mu a ions ep esen ed a class o dis-
inc O- ucoligase o co e ucosyla ion, able o u ilize inexpensi e syn he ic- ucosyl lu-
o ide as he dono subs a e a he han he p icey GDP- ucose as equi ed by he
α
1,6-
ucosyl ans e ase (FUT8).Simul aneously, hey es ed i he mu an s could also ucosyla e
he GlcNAc moie y in he se ing o di e en pep ides sequences. The Endo-F3 D165A
glycosyn hase may use he Fuc
α
1,6GlcNAc-pep ides as excellen accep o subs a es o
p oduce co e- ucosyla ed complex N-glycopep ides (Figu e 18). Finally, he au ho s demon-
s a e he po en ial applicabili y o ucoligase E274A o he si e-speci ic inco po a ion o a
co e ucose in he complex oligosaccha ide moie y o N-glycopep ides (Figu e 19).
Fu he mo e, expe imen al e idence e ealed ha in he glycoligase-ca alyzed uco-
syla ion, he ucose moie y was in ac added pa icula ly o he inne mos Asn-linked
GlcNAc moie y o he glycopep ide. This s a egy was success ully ex ended in he selec i e
glycosyla ion o p o eins and an ibodies [85].
Ca alys s 2023,13, 1359 20 o 28
Ca alys s2023,13,x 20o 28



Figu e18.T ansglycosyla ionwi h:(a)po en ialα‐Fucosyn hases;(b)α‐Fucoligases;(c)and(d)
Al Cα1,6‐ ucoligaseE274Amu an .Adap ed igu e om e .[85].
Inaddi ion, hey ound ha  heAl Cmu an s(E274A,E274G,andE274S)onlydis‐
played educedac i i yin heabsenceo  heGlcNAcaccep o .While hewild‐ ypeAl C
couldp omp lyhyd olyze hedono subs a e,i hyd olyzed‐ ucosyl luo ideslowly.
These indingscollec i elysugges ed ha  heAl Cmu a ions ep esen edaclasso dis‐
inc O‐ ucoligase o co e ucosyla ion,able ou ilizeinexpensi esyn he ic‐ ucosyl luo‐
ideas hedono subs a e a he  han hep iceyGDP‐ ucoseas equi edby heα1,6‐
ucosyl ans e ase(FUT8).Simul aneously, hey es edi  hemu an scouldalso ucosyla e
heGlcNAcmoie yin hese ingo di e en pep idessequences.TheEndo‐F3D165A
glycosyn hasemayuse heFucα1,6GlcNAc‐pep idesasexcellen accep o subs a es o
p oduceco e‐ ucosyla edcomplexN‐glycopep ides(Figu e18).Finally, heau ho s
demons a e hepo en ialapplicabili yo  ucoligaseE274A o  hesi e‐speci icinco po‐
a iono aco e ucosein hecomplexoligosaccha idemoie yo N‐glycopep ides(Figu e
19).

Figu e19.Fucoligase‐ca alyzeddi ec co e ucosyla iono N‐glycopep ides.Adap ed
igu e om e .[85].
Figu e 18.
T ansglycosyla ion wi h: (
a
) po en ial
α
-Fucosyn hases; (
b
)
α
-Fucoligases; (
c
) and (
d
) Al C
α1,6- ucoligase E274A mu an . Adap ed igu e om e . [85].
Ca alys s2023,13,x 20o 28



Figu e18.T ansglycosyla ionwi h:(a)po en ialα‐Fucosyn hases;(b)α‐Fucoligases;(c)and(d)
Al Cα1,6‐ ucoligaseE274Amu an .Adap ed igu e om e .[85].
Inaddi ion, hey ound ha  heAl Cmu an s(E274A,E274G,andE274S)onlydis‐
played educedac i i yin heabsenceo  heGlcNAcaccep o .While hewild‐ ypeAl C
couldp omp lyhyd olyze hedono subs a e,i hyd olyzed‐ ucosyl luo ideslowly.
These indingscollec i elysugges ed ha  heAl Cmu a ions ep esen edaclasso dis‐
inc O‐ ucoligase o co e ucosyla ion,able ou ilizeinexpensi esyn he ic‐ ucosyl luo‐
ideas hedono subs a e a he  han hep iceyGDP‐ ucoseas equi edby heα1,6‐
ucosyl ans e ase(FUT8).Simul aneously, hey es edi  hemu an scouldalso ucosyla e
heGlcNAcmoie yin hese ingo di e en pep idessequences.TheEndo‐F3D165A
glycosyn hasemayuse heFucα1,6GlcNAc‐pep idesasexcellen accep o subs a es o
p oduceco e‐ ucosyla edcomplexN‐glycopep ides(Figu e18).Finally, heau ho s
demons a e hepo en ialapplicabili yo  ucoligaseE274A o  hesi e‐speci icinco po‐
a iono aco e ucosein hecomplexoligosaccha idemoie yo N‐glycopep ides(Figu e
19).

Figu e19.Fucoligase‐ca alyzeddi ec co e ucosyla iono N‐glycopep ides.Adap ed
igu e om e .[85].
Figu e 19.
Fucoligase-ca alyzed di ec co e ucosyla ion o N-glycopep ides. Adap ed igu e om
e . [85].
4.4. Chi inases
Chi inases a e glycoside hyd olases (GH) ha ca alyze he hyd olysis o chi in gene a -
ing chi o-oligosaccha ides (COS). Chi in and chi osans, i s pa ially deace yla ed de i a es,
a e p esen in mos li ing o ganisms, including bac e ia, ungi, plan s, and animals [
87
,
88
]
and exhibi immunos imulan ac i i ies in mammals and plan s [
89
,
90
]. In addi ion, se e al
s udies ha e shown ha hei b eakdown p oduc s, COS, ha e an imic obial and an i umo
ac i i ies in animals, immunoenhancing e ec s in humans as die a y supplemen s
[91–95]
,
and disease p o ec i e esponses in plan s [
96
,
97
], which makes hem sui able o ag icul-
u al and medical applica ions [98–102]. Mos o hei biological ac i i ies equi e deg ees
o polyme iza ion la ge han he e asaccha ide [
89
]. Howe e , his is di icul o p oduce
in a chemical syn hesis due o he wa e insolubili y o he p oduc s, which becomes highe
as he deg ee o polyme iza ion (DP) inc eases. Thus, in he sea ch o bioac i e COS p o-
Ca alys s 2023,13, 1359 21 o 28
duc ion, enzyma ic syn hesis ep esen s a po en ial s a egy h ough he ansglycosyla ion
ac i i y o chi inases [103].
Fo his pu pose, se e al esea ch g oups ha e s udied he glycosyn hase ac i i y o
a ious chi inases o ob ain long-chain oligome s wi h po en ial biological applica ions.
Alsina e al. [
89
] ha e s udied he glycosyn hase-like ac i i y o six chi inases o he
glycosyl hyd olases amily 18 (GH18) o ob ain la ge oligome s o polyme s, which could
be mo e esis an . They selec ed ou endo-chi inases o a bac e ial and wo endo-chi inases
o an a chaeal o igin, and hen mu a ed he ca aly ic assis ing esidue o alanin. Thus,
he hyd olase ac i i y would be educed and an oxazoline de i a e could be p o ided,
which would ac as a dono subs a e o a condensa ion wi h an accep o , ca alyzing he
polyme iza ion eac ion (Figu e 20).
Ca alys s2023,13,x 21o 28


Fu he mo e,expe imen ale idence e ealed ha in heglycoligase‐ca alyzed uco‐
syla ion, he ucosemoie ywasin ac addedpa icula ly o heinne mos Asn‐linked
GlcNAcmoie yo  heglycopep ide.Thiss a egywassuccess ullyex endedin heselec‐
i eglycosyla iono p o einsandan ibodies[85].
4.4.Chi inases
Chi inasesa eglycosidehyd olases(GH) ha ca alyze hehyd olysiso chi ingen‐
e a ingchi o‐oligosaccha ides(COS).Chi inandchi osans,i spa iallydeace yla edde i‐
a es,a ep esen inmos li ingo ganisms,includingbac e ia, ungi,plan s,andanimals
[87,88]andexhibi immunos imulan ac i i iesinmammalsandplan s[89,90].Inaddi‐
ion,se e als udiesha eshown ha  hei b eakdownp oduc s,COS,ha ean imic obial
andan i umo ac i i iesinanimals,immunoenhancinge ec sinhumansasdie a ysup‐
plemen s[91–95],anddiseasep o ec i e esponsesinplan s[96,97],whichmakes hem
sui able o ag icul u alandmedicalapplica ions[98–102].Mos o  hei biologicalac i ‐
i ies equi edeg eeso polyme iza ionla ge  han he e asaccha ide[89].Howe e , his
isdi icul  op oduceinachemicalsyn hesisdue o hewa e insolubili yo  hep oduc s,
whichbecomeshighe as hedeg eeo polyme iza ion(DP)inc eases.Thus,in hesea ch
o bioac i eCOSp oduc ion,enzyma icsyn hesis ep esen sapo en ials a egy h ough
he ansglycosyla ionac i i yo chi inases[103].
Fo  hispu pose,se e al esea chg oupsha es udied heglycosyn haseac i i yo 
a iouschi inases oob ainlong‐chainoligome swi hpo en ialbiologicalapplica ions.
Alsinae al.[89]ha es udied heglycosyn hase‐likeac i i yo sixchi inaseso  he
glycosylhyd olases amily18(GH18) oob ainla ge oligome so polyme s,whichcould
bemo e esis an .Theyselec ed ou endo‐chi inaseso abac e ialand woendo‐chi‐
inaseso ana chaealo igin,and henmu a ed heca aly icassis ing esidue oalanin.
Thus, hehyd olaseac i i ywouldbe educedandanoxazolinede i a ecouldbep o‐
ided,whichwouldac asadono subs a e o acondensa ionwi hanaccep o ,ca alyz‐
ing hepolyme iza ion eac ion(Figu e20).
Theenzymesselec edwe eBacillusci culans,Se a iap o eamaculansChiD,Laceyella
pu idaChiA,Se a iama cescensChiC,Py ococcus u iosusChiB,andThe mococcusko‐
daka aensisChiA.Among hem,LpChiA,SmChiC,andP ChiBha eno  epo edTGac‐
i i yonchi ooligosaccha ide(COS)subs a es.

Figu e20.Schemeillus a ing heglycosyn hase‐likeac i i yo GH18mu an chi inases oob ain
mac ooligome so chi osanusingDP5oxassubs a e.Rep in edwi hpe mission om e .[89].
Copy igh 2019Else ie .
Figu e 20.
Scheme illus a ing he glycosyn hase-like ac i i y o GH18 mu an chi inases o ob ain
mac ooligome s o chi osan using DP5ox as subs a e. Rep in ed wi h pe mission om e . [
89
].
Copy igh 2019 Else ie .
The enzymes selec ed we e Bacillus ci culans,Se a ia p o eamaculans ChiD, Laceyella
pu ida ChiA, Se a ia ma cescens ChiC, Py ococcus u iosus ChiB, and The mococcus
kodaka aensis ChiA. Among hem, LpChiA, SmChiC, and P ChiB ha e no epo ed TG
ac i i y on chi ooligosaccha ide (COS) subs a es.
In ela ion o hyd olase ac i i y, wo ypes o e asaccha ides we e used as subs a es,
wi h he inding ha all enzymes showed ac i i y, especially BcChiA,LpChiA and SmChiC.
Rega ding o glycosyn hase ac i i y, he subs a e used was pen aace ylchi open aose
oxazoline (DP5ox). A e wo hou s o eac ion, he au ho s ound ha a mix u e o COS
was gene a ed o all mu an enzymes, om DP5 o DP15, wi h DP10, DP7, and DP8 being
he main p oduc s. Howe e , due o esidual hyd olase ac i i y, a e 18 h o incuba ion
lowe han expec ed yields o he DP, >10 p oduc s we e ob ained as a esul o hyd olysis
and ansglycosyla ion eac ions. The bes esul was ob ained o TkChiA, wi h a yield o
55% o DP10 a e 18 h.
The me hod ollowed by he au ho s pe o ming a single mu a ion a he assis an
esidue in GH18 chi inases does no seem adequa e o ob ain COS wi h a la ge DP due
o he esidual p esence o hyd olase ac i i y in he mu an enzymes. Fu he mu a ions
would be needed o a oid he hyd olyza ion and enhance he glycosyn hase-like ac i i y.
In a new app oach, Alsina e al. [
104
] ocused on he s udy o he bac e ial enzyme
SpChiD. I had been shown ha a single mu a ion in he assis an esidue did no elimina e
he hyd olase ac i i y, so a di e en s a egy was es ed. Fo his pu pose, di e en ac i e

Ca alys s 2023,13, 1359 22 o 28
si es we e mu a ed o achie e a g ea e educ ion in he hyd olase ac i i y. In addi ion o
mu a ion D151A al eady being pe o med, o he s we e added based on p e ious s udies
and simula ion models ha would enhance TG and dec ease hyd olase ac i i ies.
A second gene a ion o ou mu an s wi h an addi ional mu a ion in each o hem
(S110G/D151A, G113S/D151A, F119A/D151A, and D149A/D151A) was pe o med. In
one o he mu an s (D149A/D151A), signi ican imp o emen s in he wan ed ac i i y
we e achie ed, ob aining DP10 as he majo i y p oduc wi h he bes a io and an insoluble
p oduc yield o 30%. This enzyme was selec ed o design a hi d gene a ion o new mu an s.
The iple mu an s had educed hyd olase ac i i y compa ed o he p e ious mu an s.
Glycosyn hase ac i i y also imp o ed, excep in wo o hem. A e 18 h, p ecipi a e yields
o 22 o 68% we e ob ained, wi h DP10 as he majo p oduc and ge ing he bes esul s o
Y154W/D149A/D151A and Y28A/D149A/D151A.
The chi inases enginee ed in his hi d gene a ion ha e be e oligome iza ion yields
han hose o a single mu a ion and hose o any epo ed GH18 ansglycosyla ing chi inase.
Thus, h ough a se ies o a ge ed mu a ions, i has been possible o design hyb ids
wi h be e glycosyl syn hase-like ac i i y, bu a u he educ ion in hyd olase ac i i y
would s ill be necessa y o he designed mu an s o ha e uly applicable ac i i y.
Following a simila s a egy, Ohnuma e al. [
105
], mu a ed GH19 chi inases om B yum
co ona um (BcChi-A) o ob ain chi in oligosaccha ides using success ully 4,6-dime hoxy-
1,3,5- iazin-2-yl α-chi obioside [DMT- α-(GlcNAc)2] as a dono subs a e (Figu e 21).
Ca alys s 2023, 13, x 23 o 28
Figu e 21. (GlcNAc)4 syn hesis ca alyzed by glycosyn hase de i ed om BcChi-A. Rep in ed wi h
pe mission om e . [105]. Copy igh © 2018, Ox o d Uni e si y P ess.
Single mu an s did no show glycosyn hase ac i i y wi h he subs a e a e 48 h o
eac ion. Howe e , he double mu an s (E70G/S102A and E70G/S102C) showed ac i i y
and (GlcNAc)4 was ob ained as he majo p oduc . Fo E70G/S102A, he p oduc ion yield
o (GlcNAc)4 was 22.4%. Small-chain oligosaccha ides we e also ob ained as seconda y
p oduc s, showing he p esence o esidual hyd olase ac i i y in he mu an s, al hough i
was much lowe compa ed o wild- ype enzymes.
Thus, he in oduc ion o well-s udied combined mu a ions in di e en glycosyl hy-
d olases, such as he chi inases used in he desc ibed s udies, can s ongly educe hyd o-
lase ac i i y and gene a e glycosyn hase-like ac i i y in he new mu an s, which allows us
o ob ain oligosaccha ides and long-chain glycoconjuga es om di e en subs a es.
5. Conclusions and Fu u e P ospec
This e iew a icle summa izes some o he mos ecen ad ances in enzyma ic gly-
cosyla ion p ocesses ocused on he ab ica ion o impo an bioac i e compounds. Ap-
plicabili y o glycosyla ion p ocess by glycosidases, glycosyl ans e ases, o he so-called
glycosyn hases ha e been desc ibed, along wi h he ad an ages o disad an ages o hese
enzymes in each p ocess.
Thus, he u u e p ospec s o enzyma ic glycosyla ion s a egies in he p oduc ion o
bioac i e compounds a e p omising and hold signi ican po en ial in se e al a eas:
(i) D ug de elopmen : Enzyma ic glycosyla ion can be used o enhance he pha maco-
kine ics and he apeu ic e icacy o d ugs. Fu u e de elopmen s may ocus on de-
signing glycosyla ion s a egies ha imp o e d ug a ge ing, bioa ailabili y, and e-
duce side e ec s. This app oach could lead o he c ea ion o mo e e ec i e and sa e
pha maceu icals.
(ii) P ecision medicine: Tailo ing glycosyla ion pa e ns in bioac i e compounds o
ma ch indi idual pa ien p o iles could become a eali y. This pe sonalized medicine
app oach may in ol e using enzyma ic glycosyla ion o p oduce glycoconjuga es
ha a e op imized o speci ic pa ien popula ions, po en ially imp o ing ea men
ou comes.
(iii) Immuno he apy: Glycosyla ion plays a c ucial ole in modula ing he immune e-
sponse. Enzyma ic glycosyla ion s a egies could be used o de elop glycoconjuga es
ha enhance he e icacy o immuno he apies, such as cance accines and monoclo-
nal an ibodies, by imp o ing hei in e ac ion wi h immune cells and educing im-
mune e asion.
Figu e 21.
(GlcNAc)4 syn hesis ca alyzed by glycosyn hase de i ed om BcChi-A. Rep in ed wi h
pe mission om e . [105]. Copy igh © 2018, Ox o d Uni e si y P ess.
Single and double mu an s we e c ea ed by changing he ca aly ic base (Glu70) and he
esidue Se 102, which ac s by ixing a wa e molecule. In a p e ious s udy, he au ho s had
demons a ed ha he single mu an s showed glycosyn hase ac i i y wi h
α
-(GlcNAc)2
luo ide as dono subs a e. In his pape , he au ho s es ed hese single mu an s and new
double mu an s wi h DMT-
α
-(GlcNAc)2 o ob ain (GlcNAc)4, which we e designed o
imp o e he ac i i y o he single ones.
Single mu an s did no show glycosyn hase ac i i y wi h he subs a e a e 48 h o
eac ion. Howe e , he double mu an s (E70G/S102A and E70G/S102C) showed ac i i y
and (GlcNAc)4 was ob ained as he majo p oduc . Fo E70G/S102A, he p oduc ion yield
o (GlcNAc)4 was 22.4%. Small-chain oligosaccha ides we e also ob ained as seconda y
p oduc s, showing he p esence o esidual hyd olase ac i i y in he mu an s, al hough i
was much lowe compa ed o wild- ype enzymes.
Thus, he in oduc ion o well-s udied combined mu a ions in di e en glycosyl hy-
d olases, such as he chi inases used in he desc ibed s udies, can s ongly educe hyd olase
ac i i y and gene a e glycosyn hase-like ac i i y in he new mu an s, which allows us o
ob ain oligosaccha ides and long-chain glycoconjuga es om di e en subs a es.
5. Conclusions and Fu u e P ospec
This e iew a icle summa izes some o he mos ecen ad ances in enzyma ic glycosy-
la ion p ocesses ocused on he ab ica ion o impo an bioac i e compounds. Applicabili y
Ca alys s 2023,13, 1359 23 o 28
o glycosyla ion p ocess by glycosidases, glycosyl ans e ases, o he so-called glycosyn-
hases ha e been desc ibed, along wi h he ad an ages o disad an ages o hese enzymes
in each p ocess.
Thus, he u u e p ospec s o enzyma ic glycosyla ion s a egies in he p oduc ion o
bioac i e compounds a e p omising and hold signi ican po en ial in se e al a eas:
(i)
D ug de elopmen : Enzyma ic glycosyla ion can be used o enhance he pha ma-
cokine ics and he apeu ic e icacy o d ugs. Fu u e de elopmen s may ocus on
designing glycosyla ion s a egies ha imp o e d ug a ge ing, bioa ailabili y, and
educe side e ec s. This app oach could lead o he c ea ion o mo e e ec i e and
sa e pha maceu icals.
(ii) P ecision medicine: Tailo ing glycosyla ion pa e ns in bioac i e compounds o ma ch
indi idual pa ien p o iles could become a eali y. This pe sonalized medicine ap-
p oach may in ol e using enzyma ic glycosyla ion o p oduce glycoconjuga es ha a e
op imized o speci ic pa ien popula ions, po en ially imp o ing ea men ou comes.
(iii)
Immuno he apy: Glycosyla ion plays a c ucial ole in modula ing he immune e-
sponse. Enzyma ic glycosyla ion s a egies could be used o de elop glycoconjuga es
ha enhance he e icacy o immuno he apies, such as cance accines and mono-
clonal an ibodies, by imp o ing hei in e ac ion wi h immune cells and educing
immune e asion.
(i )
Func ional oods and nu aceu icals: Enzyma ic glycosyla ion can be applied o
enhance he unc ional p ope ies o ood ing edien s and nu aceu icals. Fu u e
esea ch may ocus on p oducing glycoconjuga es ha o e imp o ed bioa ailabili y
o nu ien s, be e as e, and enhanced heal h bene i s.
( )
Bio echnology: Enzyma ic glycosyla ion is essen ial in he bio echnology indus y
o he p oduc ion o he apeu ic glycop o eins, accines, and glycolipids. Ongoing
ad ancemen s may lead o mo e e icien and scalable p ocesses, educing p oduc ion
cos s and imp o ing he quali y o biopha maceu icals.
( i)
Sus ainabili y: Enzyma ic glycosyla ion is conside ed an en i onmen ally iendly
app oach compa ed o chemical me hods. The u u e may see inc eased emphasis
on de eloping mo e sus ainable glycosyla ion p ocesses, using enewable eeds ocks,
and educing was e gene a ion.
( ii)
Glycoenginee ing: Ad ances in glycoenginee ing may enable he p ecise con ol
o glycosyla ion pa e ns, allowing o he c ea ion o bioac i e compounds wi h
de ined glycan s uc u es. This could lead o he de elopmen o no el he apeu ics
and diagnos ics.
( iii)
Enzyme enginee ing: Con inued esea ch in enzyme enginee ing may esul in he
disco e y o design o mo e e icien and obus glycosyl ans e ases and o he glyco-
syla ion enzymes, expanding he ange o subs a es and glycan s uc u es ha can
be syn hesized.
In conclusion, enzyma ic glycosyla ion s a egies a e poised o play an inc easingly
impo an ole in he p oduc ion o bioac i e compounds, o e ing oppo uni ies o in-
no a ion in d ug de elopmen , pe sonalized medicine, and a ious indus ies. As ou
unde s anding o glycosyla ion mechanisms and enzyme capabili ies deepens, we can an-
icipa e exci ing de elopmen s ha will shape he u u e o bioac i e compound syn hesis
and applica ion.
Au ho Con ibu ions:
Concep ualiza ion, J.M.P., D.B. and C.M.; w i ing—o iginal d a p epa a ion,
A.A., C.G.-S., A.S.S., C.O.-N., M. ´
C., A.M., J.M.P., D.B. and C.M.; w i ing— e iew and edi ing, A.A.,
C.G.-S., A.S.S., C.O.-N., M. ´
C., A.M., J.M.P., D.B. and C.M.; supe ision, J.M.P. and D.B. All au ho s
ha e ead and ag eed o he published e sion o he manusc ip .
Ca alys s 2023,13, 1359 24 o 28
Funding:
The au ho s hank he unding om Spanish Na ional Resea ch Council (CSIC), Technolog-
ical De elopmen and Inno a ions o he Republic o Se bia (Con ac No. 451-03-47/2023-01/200135,
p og amme IDEAS, p ojec no. 7750109 (P In P Enzy) and Eu opean Commission, p ojec “Twin-
ning o in ensi ied enzyma ic p ocesses o p oduc ion o p ebio ic-con aining unc ional ood and
bioac i e cosme ics” g an no. 101060130, HORIZON-WIDERA-2021-ACCESS-02-01.
Acknowledgmen s:
The au ho s hank he suppo o he Spanish Na ional Resea ch Council (CSIC)
and Minis y o Science, Technological De elopmen and Inno a ions o he Republic o Se bia. The
au ho s also wish o acknowledge he unding om Eu opean Commission, p ojec “Twinning o
in ensi ied enzyma ic p ocesses o p oduc ion o p ebio ic-con aining unc ional ood and bioac i e
cosme ics “HORIZON-WIDERA-2021-ACCESS-02-01. The Au ho s would like o acknowledge
ne wo king suppo by he COST Ac ion CA18132 (GlycoNanoP obes).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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