© 2025 The Au ho (s). Published by he Royal Socie y o Chemis y Ma e . Ad ., 2025, 6, 5303–5309 | 5303
Ci e his: Ma e . Ad ., 2025,
6, 5303
Bioca aly ic applica ion and s uc u al elucida ion
o obus bac e ial p o ein nanocages†
Ognjen Pec
´anac, ‡
a
Alexande Belyy, ‡*
b
Ca e ina Ma in,
a
Rosalie Kleissen,
a
Ma co W. F aaije *
c
and Nikola Lonc
ˇa *
a
Encapsulins, bac e ial p o ein nanocompa men s, ha e eme ged as p omising pla o ms o enhancing
bioca alys s abili y. This s udy p esen s he iden i ica ion and s uc u al cha ac e iza ion o wo
encapsulins: A h oEnc om A h obac e sp. SLBN-53 and Dend oEnc om Dend ospo obac e
que cicolus. Bo h bac e ial encapsulins we e success ully o e exp essed in Esche ichia coli and pu i ied.
C yo-elec on mic oscopy e ealed ha A h oEnc assembles in o a 20 nm T= 1 icosahed al capsid
composed o 60 subuni s, wi h i s s uc u e de e mined a 2.9 Å esolu ion, whe eas Dend oEnc o ms a
40 nm T= 4 icosahed al complex wi h 240 subuni s, esol ed a 3.4 Å esolu ion. Bo h s uc u es exhibi
he cha ac e is ic HK97 phage-like old. To explo e hei unc ional po en ial, he encapsulins we e used
o pack wo dis inc enzymes: CyanoPOX, a heme-con aining pe oxidase, and PTDH-mFMO, a usion
enzyme combining phosphi e dehyd ogenase wi h a la in-con aining monooxygenase. Dend oEnc
packed wi h CyanoPOX exhibi ed enhanced p o eoly ic s abili y, effec i ely shielding i s ca go om
chymo ypsin deg ada ion. Ac i i y assays wi h ABTS and guaiacol con i med ha encapsula ed
CyanoPOX e ained enzyma ic unc ion wi hin Dend oEnc. Finally, mu a ing he po e o Dend oEnc
esul ed in a i e old educ ion in ac i i y, demons a ing he po en ial o uning subs a e diffusion. This
s udy ad ances ou unde s anding o encapsulin di e si y and highligh s hei po en ial as e sa ile
pla o ms o enzyme s abiliza ion, bioca aly ic and o he bio echnological applica ions.
In oduc ion
Compa men aliza ion is a undamen al cha ac e is ic o all
cells. I enables he seg ega ion and coo dina ion o physiological
unc ions in a egula ed and o de ly manne .
1
To mee hese
di e se equi emen s, cells ha e e ol ed mechanisms ha include
lipid- and p o ein-based o ganelles.
2
Euka yo ic cells a e p ima ily
known o hei eliance on lipid-based o ganelles, whe eas p o-
ka yo ic cells p edominan ly employ p o ein-based compa men-
aliza ion s a egies.
3,4
In he pas , bac e ial mic ocompa men s
ha e been ecognized as he p ima y class o p o ein-based
o ganelles in bac e ia.
4
These la ge p o ein shells, o en exhibi ing
a diame e la ge han 100 nm, encapsula e and o ganize speci ic
me abolic enzymes wi hin a selec i ely pe meable p o ein shell.
This a angemen enhances eac ion efficiency, seques e s oxic
in e media es, and enables he de elopmen o new me abolic
pa hways in di e se bac e ial species.
4,5
A li le o e a decade ago, a
second ex ensi e class o bac e ial p o ein nanocompa men s
known as encapsulins was cha ac e ized.
6
Encapsulins a e obus
icosahed al p o ein s uc u es o med in i o h ough he sel -
assembly o 60 o 240 p o ome subuni s ha exhibi he HK97
(Hong Kong 97) phage-like old.
7
S uc u al analyses e ealed ha
encapsulin p o ome s a e composed o h ee highly conse ed
domains: he axial domain (A-domain), which o ms he co e o
he subuni , he pe iphe al domain (P-domain), in ol ed in in e -
subuni in e ac ions, and he ex ended loop (E-loop), which con-
ibu es o capsid assembly and s abili y.
6,8
Encapsulins ypically
ange om24 o42nmindiame e wi hdiffe en iangula ion
numbe s (T=1,T=3,o T=4).
9
A dis inc i e cha ac e is ic o
encapsulins is hei capabili y o encapsula e a ge ed ca go p o-
eins, o en enzymes.
10
This ac ion is acili a ed h ough a selec i e
loading mechanism media ed by sho a ge ing pep ides loca ed
a he N- o C- e minus o a ca go p o ein.
11
Genes ha encode
ca go p o eins and encapsulin a e o ganized in a co- egula ed
ope on ha de ines he encapsulin sys em.
12,13
The a ionale
go e ning he selec i e encapsula ion o speci ic ca go p o eins
wi hin bac e ial mic ocompa men s emains unclea . P edomi-
nan classes o ca go p o eins iden i ied wi hin encapsulins
include dye-decolo izing pe oxidases (DyPs), e i in-like p o eins,
a
GECCO Bio ech, Ze nikepa k 6, 9747 AN G oningen, The Ne he lands.
E-mail: n.lonca @gecco-bio ech.com
b
Memb ane Enzymology, Uni e si y o G oningen, Nijenbo gh 3, 9747 AG
G oningen, The Ne he lands. E-mail: [email p o ec ed]
c
Molecula Enzymology, Uni e si y o G oningen, Nijenbo gh 3, 9747 AG
G oningen, The Ne he lands. E-mail: m.w. aaij[email p o ec ed]
†Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: h ps://doi.o g/
10.1039/d5ma00268k
‡Equally con ibu ed.
Recei ed 23 d Ma ch 2025,
Accep ed 24 h June 2025
DOI: 10.1039/d5ma00268k
sc.li/ma e ials-ad ances
Ma e ials
Ad ances
PAPER
Open Access A icle. Published on 26 June 2025. Downloaded on 11/23/2025 1:23:21 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
View Jou nal
| View Issue
5304 | Ma e . Ad ., 2025, 6, 5303–5309 © 2025 The Au ho (s). Published by he Royal Socie y o Chemis y
and some me alloenzymes, sugges ing a po en ial unc ional
ela ionship be ween hese p o ein amilies and he encapsulin
sys em.
10,14
A common cha ac e is ic obse ed among he iden-
i ied ca go p o eins appea s o be hei in ol emen in edox
eac ions,
15
leading o he hypo hesis ha encapsulins may play
a c ucial ole in cellula de oxi ica ion mechanisms and oxida i e
s ess managemen .
16,17
The inhe en efficiency o encapsulins,
coupled wi h hei capaci y o accommoda e o eign ca go
p o eins,
18
makes hem p omising enzyme immobilize s wi h
b oad po en ial applica ions in ields o a ge ed he apeu ics,
19
accine pla o ms,
20
and nanoscale bio eac o s.
21
This s udy epo s on he s uc u al and unc ional p ope ies
o wo newly disco e ed bac e ial encapsulins, named Dend oEnc
and A h oEnc, om Dend ospo obac e que cicolus and A h obac-
e sp. SLB-53, espec i ely. Using c yo-elec on mic oscopy (c yo-
EM) and single-pa icle analysis we de e mined s uc u es o a 240-
subuni Dend oEnc and a 60-subuni A h oEnc, expanding ou
knowledge on s uc u al di e si y o encapsulins. We also demon-
s a e ha Dend oEnc effec i ely encapsula es a bac e ial lac o-
pe oxidase (CyanoPOX),
22
wi h u he e idence showing ha
CyanoPOX e ains i s ac i i y a e encapsula ion. No ably, p o eo-
ly ic deg ada ion s udies show ha once encapsula ed, CyanoPOX
exhibi s esis ance o chymo ypsin deg ada ion. These indings
sugges ha Dend oEnc has signi ican po en ial o s abilizing
and p o ec ing enzymes, offe ing exci ing p ospec s o i s applica-
ion in a ious bio echnological ields.
Resul s and discussion
Iden i ica ion o A h oEnc and Dend oEnc
To expand he di e si y o a ailable nanocompa men s o
bio echnological pu poses, we conduc ed a gene ic sea ch o
uncha ac e ized encapsulins using p e iously cha ac e ized
encapsulins om Mycolicibac e ium hassiacum (EncMh, WP_
005630281.1)
10
and Myxococcus xan hus (EncA, WP_026114001)
23
as e e ence sequences. This esul ed in he iden i ica ion o a
p edic ed p o ein (278 esidues) om D. que cicolus ha sha ed
47% sequence iden i y wi h EncA, and a p edic ed p o ein (265
esidues) om A. sp. SLBN-53 ha sha ed 83% sequence iden i y
wi h EncMh. These espec i e genes we e cloned in a pBAD ec o
o o e exp ession in E. coli. Success ul o e p oduc ion o bo h
p o eins in E. coli was con i med by SDS-PAGE analysis, which
e ealed p ominen bands a he expec ed molecula weigh s o
A h oEnc (28.5 kDa) and Dend oEnc (30 kDa) (Fig. S1, ESI†).
De elopmen o A h oEnc and Dend oEnc as p o ein
packaging sys ems
Exp ession and pu i ica ion p o ocols o bo h A hoEnc and
Dend oEnc we e success ully es ablished, yielding app oxi-
ma ely 150 mg o encapsulins pe li e o cul u e. To assess
hei po en ial o a ge ed p o ein encapsula ion, we in es i-
ga ed he packaging o wo dis inc enzymes: CyanoPOX and
PTDH-mFMO, which belong o diffe en enzyme classes and
equi e diffe en co ac o s. CyanoPOX, a heme-con aining pe -
oxidase om Cyanobac e ium sp. TDX16, exhibi s p ope ies
simila o bo ine lac ope oxidase,
22
while PTDH-mFMO is a
usion enzyme combining phosphi e dehyd ogenase (PTDH)
o co ac o egene a ion wi h a la in-con aining monooxygen-
ase om Me hylophaga sp. (mFMO), capable o oxidizing indole
o p oduce indigo dye.
24
Fo a ge ed encapsula ion, a 30- esidue C- e minal pep ide
ag (PPPLPDSEPDREIPADDGSLGIGSLKGTRS), de i ed om a
na i e dye-decolo izing pe oxidase (DyP) o Mycolicibac e ium
hassiacum, was used o each ca go enzyme.
10
Co-exp ession o
encapsulins and ca go enzymes was achie ed using a wo-
plasmid exp ession sys em, esul ing in high exp ession le els
o bo h encapsulins and hei espec i e ca gos. The op imized
p ocess yielded app oxima ely 150 mg o each ca go-loaded
encapsulin pe li e o cul u e. SDS-PAGE analysis (Fig. S2, ESI†)
o size-exclusion ch oma og aphy (SEC) ac ions con i med suc-
cess ul encapsula ion, showing dis inc bands co esponding o
he encapsulin and ca go p o eins-CyanoPOX (B60 kDa) and
PTDH-mFMO (B100 kDa).
S uc u al elucida ion o A h oEnc and Dend oEnc
To deciphe he a chi ec u e o A h oEnc and Dend oEnc,
pu i ied samples we e subjec ed o single-pa icle c yo-EM
analysis. The s uc u e o emp y A h oEnc was de e mined a
2.9 Å esolu ion. The s uc u e e ealed a sphe ical capsid wi h a
diame e o 20 nm ha is composed o 60 iden ical subuni s,
o ganized wi h he T= 1 icosahed al symme y (Fig. 1A). Fu he
s uc u al analysis e ealed ha he A h oEnc monome adop s
he cha ac e is ic HK97 phage-like old, as desc ibed p e iously
7
(Fig. 1A). Elec os a ic and hyd ophobici y analysis o he encap-
sulin po e, o med by A-domains o 5 subuni s, e ealed a
sligh ly posi i e su ace cha ge on i s ou e side due o he
p esence o his idines and a ing o hyd ophobic y osines on
he inne side, po en ially egula ing po e unc ion and sub-
s a e selec i i y (Fig. 1C).
We hen p oceeded wi h s uc u al analysis o Dend oEnc
and de e mined i s s uc u e a he esolu ion o 3.4 Å using
single pa icle c yo-EM analysis. Ou ini ial hypo hesis, based
on sequence simila i y wi h p e iously s udied encapsulins,
sugges ed ha Dend oEnc o ms a 180-subuni s uc u e. How-
e e , c yo-EM e ealed ha Dend oEnc assembles in o a 40 nm
240-me complex wi h T= 4 icosahed al symme y (Fig. 1D).
Wi hin he asymme ic uni , ou subuni s exhibi mino
s uc u al diffe ences in he A domain and E-loop, allowing
hem o old in o he la ge T= 4 icosahed al assembly (Fig. 1E).
Due o i s s uc u al o ganiza ion, A-domains o Dend oEnc
o m wo ypes o po es, 5- old and 6- old, o ganized by 5 and 6
encapsulin subuni s, espec i ely (Fig. 1F). The 6- old po e,
o med by hyd ophilic aspa agines and h eonines and u he
s abilized by a ing o hyd ophobic alines and phenylalanines,
is nea ly closed, limi ing any anspo h ough he encapsulin
shell. In con as , he 5- old po e is mo e open and is composed
o hyd ophilic h eonines and aspa agines (Fig. 1G). In com-
pa ison o he analogical 5- old po e o A h oEnc (Fig. 1B), he
po e o Dend oEnc is 1 Å la ge in diame e (10 Å) and does no
ha e a hyd ophobic in e io (Fig. 1G). In e es ingly, Dend oEnc
o ms an addi ional iangula -shape po e a he in e ace o
Pape Ma e ials Ad ances
Open Access A icle. Published on 26 June 2025. Downloaded on 11/23/2025 1:23:21 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
© 2025 The Au ho (s). Published by he Royal Socie y o Chemis y Ma e . Ad ., 2025, 6, 5303–5309 | 5305
P-domains and E-loops o encapsulin subuni s (Fig. 1F). This
can be conside ed as a seconda y en y poin o subs a e.
Thus, Dend oEnc po es a e be e designed o acili a e he
anspo o hyd ophilic compounds, ende ing i mo e sui a-
ble o po en ial bio echnological applica ions.
P o eoly ic s abili y o Dend oEnc cons uc s
Some encapsulins ha e been demons a ed o p o ec ca go
p o eins om p o eoly ic deg ada ion.
10,25
To de e mine
whe he his applies o Dend oEnc, we e alua ed i s abili y o
shield ca go p o eins om p o ease ac i i y. Incuba ion o
emp y Dend oEnc and CyanoPOX-loaded Dend oEnc wi h chy-
mo ypsin, ollowed by SDS-PAGE analysis, demons a ed lim-
i ed p o eoly ic deg ada ion o he encapsulin shell (Fig. S3,
ESI†). Addi ionally, he encapsula ed CyanoPOX emained
in ac a e 24 hou s o chymo ypsin exposu e, whe eas non-
encapsula ed CyanoPOX was deg aded. These esul s con i m
he p o ec i e unc ion o Dend oEnc, u he alida ing i s
abili y o effec i ely encapsula e and sa egua d ca go p o eins.
Ac i i y o Dend oEnc CyanoPOX in he p esence o ABTS and
guaiacol
To e alua e whe he CyanoPOX e ained enzyma ic ac i i y
when encapsula ed wi hin Dend oEnc, wo known subs a es
o his bac e ial pe oxidase,
22
ABTS (2,20-azino-bis(3-e hyl-
benzo hiazoline-6-sul onic acid)) and guaiacol, we e es ed. Den-
d oEnc CyanoPOX exhibi ed ac i i y owa d bo h subs a es. A
10 mM, he ac i i y o ABTS and guaiacol was 0.02 U mg
1
and
0.59 10
3
Umg
1
espec i ely. No de ec able ac i i y was
obse ed in emp y Dend oEnc, con i ming ha he enzyma ic
ac i i y o igina ed solely om he encapsula ed CyanoPOX. These
esul s indica e ha he po es o Dend oEnc a e sufficien ly la ge
o allow diffusion o ela i ely bulky subs a es, such as ABTS,
enabling efficien enzyma ic ca alysis wi hin he nanocompa -
men . Table S4 and eqn (S1) (ESI†) ou line he calcula ion p oce-
du e used o de e mine speci ic ac i i y.
Enginee ing he 5- old po e o Dend oEnc
Wi h es ablished p o ocols o he ecombinan p oduc ion o
Dend oEnc ca ying speci ic ca go, we explo ed he effec s o
modi ying i s po es. S uc u al insigh s in o he Dend oEnc
po e a chi ec u e guided he design o a mu a ion aimed a
na owing o closing he po e. Using an encapsula ed enzyme
wi h subs a es o a ying sizes allowed us o assess he impac
o his modi ica ion on subs a e diffusion.
Speci ically, we a ge ed esidue N198, a key componen o
he 5- and 6- old po es a chi ec u e (Fig. 1G). By subs i u ing
N198 wi h yp ophan, a bulky amino acid, we aimed o es ic
Fig. 1 C yo-EM s uc u es o A h oEnc and Dend oEnc encapsulins. (A) C yo-EM econs uc ion o A h oEnc wi h T= 1 icosahed al symme y wi h 60
subuni s and a s uc u e o i s HK97-like old domain. (B) Schema ic o ganiza ion o A h oEnc and a close-up o i s 5- old po e. (C) Elec os a ic su ace
and hyd ophobici y o A h oEnc’s po e. (D) C yo-EM econs uc ion o Dend oEnc e ealing T= 4 icosahed al symme y wi h 240 subuni s. An
asymme ic uni is shown in colo s. (E) S uc u al alignmen o ou diffe en subuni s o Dend oEnc (in o ange, ed, g een and yellow) and a subuni o
A h oEnc (g ay). (F) Schema ic o ganiza ion o Dend oEnc and i s 5- old, 6- old and iangula -shaped po es. (G) S uc u al o ganiza ion o Dend oEnc’s
5- and 6- old po es.
Ma e ials Ad ances Pape
Open Access A icle. Published on 26 June 2025. Downloaded on 11/23/2025 1:23:21 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
5306 | Ma e . Ad ., 2025, 6, 5303–5309 © 2025 The Au ho (s). Published by he Royal Socie y o Chemis y
subs a e en y in o he encapsulin. Co-exp ession o he Den-
d oEnc N198W a ian wi h CyanoPOX success ully yielded
CyanoPOX-loaded Dend oEnc N198W. To assess he s uc u al
consequences o his mu a ion, we de e mined i s s uc u e o a
3.6 Å esolu ion using single-pa icle c yo-EM analysis, p o id-
ing insigh s in o he impac o po e enginee ing on encapsulin
a chi ec u e.
S uc u al analysis o he Dend oEnc N198W CyanoPOX
complex e ealed ha , simila o wild- ype Dend oEnc, he 6-
old po e emains ully closed wi h a igh hyd ophobic ba ie
(Fig. 2). Mo eo e , he in oduc ion o yp ophan a posi ion
N198 esul ed in a u he educ ion in he diame e o he 5-
old po e. The i e yp ophan esidues align p ecisely a he
po e, cons ic ing i s diame e o 5 Å and effec i ely limi ing
molecula passage in and ou o he encapsulin. Impo an ly,
he N198W mu a ion did no change he o e all a chi ec u e o
encapsulin, showing i s abili y o ole a e signi ican mu a ions
while p ese ing i s in eg i y, u he highligh ing i s po en ial
o enginee ing ailo ed molecula ga ing p ope ies.
The ac i i y o he Dend oEnc N198W CyanoPOX mu an was
es ed using ABTS as a subs a e (0.004 U mg
1
), e ealing a 5- old
educ ion in ac i i y compa ed o he CyanoPOX packed in wild-
ype Dend oEnc. This dec ease aligns wi h s uc u al insigh s
showing ha he mu a ion educed he diame e o he 5- old po e. Finally, o in es iga e he olding o CyanoPOX wi hin Den-
d oEnc, we compa ed 2D class a e ages o emp y and ca go-
loaded Dend oEnc. This analysis enabled us o isualize he
densi y co esponding o he encapsula ed enzyme (Fig. 3),
p o iding u he con i ma ion o efficien ca go loading. How-
e e , he obse ed densi y appea ed bulky and uns uc u ed,
wi h no indica ion o a de ined symme ical posi ioning o he
ca go. Since he symme y o he ca go migh diffe om ha o
he encapsulin shell, we pe o med shell sub ac ion o analyse
he ca go alone. Fu he 2D classi ica ions and 3D e inemen s
did no e eal a clea s uc u al o ganiza ion, sugges ing ha
he encapsula ed enzymes do no adop a ixed symme ical
a angemen wi hin he encapsulin. These indings imply ha
CyanoPOX may emain in a lexible s a e inside Dend oEnc.
Ma e ials and me hods
Chemicals and ma e ials
The syn he ic genes encoding o Dend oEnc and A h oEnc
we e o de ed cloned in he pBAD ec o (ampicillin esis ance)
om Twis Bioscience. The syn he ic gene encoding o ca go
p o eins (CyanoPOX-ca go and PTDH-mFMO-ca go) we e
o de ed a IDT (In eg a ed DNA Technologies). To p epa e
Dend oEnc mu an , p ime s we e o de ed om Eu o ins geno-
mics. Supe dex 200 Inc ease 10/300 GL column was pu chased
om Cy i a. Chymo ypsin om bo ine panc eas and o he
chemicals we e pu chased om Sigma-Ald ich.
Cloning, p o ein co-exp ession and pu i ica ion
IDT ools we e used o codon op imize he sequences (Table S1,
ESI†) ha encode ca go enzymes (CyanoPOX-ca go and PTDH-
mFMO-ca go) ha bo ing a N- e minal 6His- ag and he
C- e minal a ge ing pep ide o enable hei exp ession in E. coli.
Fig. 2 C yo-EM s uc u e o he Dend oEnc N198W CyanoPOX po es.
The N198W mu a ion in oduced a hyd ophobic plug in he 6- old po e
and educed he size o he 5- old po e.
Fig. 3 2D class a e ages (abo e) o emp y Dend oEnc WT and Den-
d oEnc N198W CyanoPOX and indi idual encapsulins Dend oEnc N198W
CyanoPOX wi h and wi hou ca go (below).
Pape Ma e ials Ad ances
Open Access A icle. Published on 26 June 2025. Downloaded on 11/23/2025 1:23:21 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
© 2025 The Au ho (s). Published by he Royal Socie y o Chemis y Ma e . Ad ., 2025, 6, 5303–5309 | 5307
The a ge ing pep ide used in his wo k belongs o a DyP-
pe oxidase
10
om he same ope on as he encapsulin gene. These
syn he ic genes we e cloned in pET ec o (kanamycin esis ance)
using he Golden Ga e me hodology.
26
5mgo heca goand
encapsulin PCR mix u es we e added o 50 mLRbCl
2
compe en
E. coli BL21-AI cells. The cells we e le o incuba e on ice o
30 min ollowed by he hea shock o 45 seconds a 42 1C. A e
he hea shock, he cells we e placed on ice o ano he 3 minu es.
500 mL o SOC media was used o eco e he cells. They we e hen
placed in he he mal shake a 37 1C o 45 min, 700 pm. 100 mL
o he cell mix u e was sp ead on o he LB aga pla es con aining
100 mgmL
1
ampicillin and 50 mgmL
1
kanamycin. LB aga pla es
we e placed in o an incuba o a 37 1C o e nigh . O e nigh
cul u es we e g own in 5 mL LB medium ha was supplemen ed
wi h 100 mgmL
1
ampicillin and 50 mgmL
1
kanamycin and we e
incuba ed a 37 1C, 135 pm. Te i ic B o h medium (500 mL),
supplemen ed wi h 100 mgmL
1
ampicillin and 50 mgmL
1
kanamycin was used o he exp ession. Cul u es we e incuba ed
a 37 1C, 135 pm in baffled lasks un il he OD
600
eached B0.75.
Exp ession o ca go enzymes and encapsulins was induced
by adding IPTG (1 mM inal concen a ion) and L-a abinose
(0.02% inal concen a ion). 5-Aminole ulinic acid (1 mM, inal
concen a ion) and i on sul a e (1 mM, inal concen a ion)
we e added o he cul u es con aining CyanoPOX ca go in o de
o acili a e he biosyn hesis o heme. Cul u es we e incuba ed
a 24 1C a 135 pm o 48 hou s. They we e hen ha es ed by
cen i uga ion (3700 pm, 40 minu es, 4 1C). Cell pelle s we e
esuspended in 35 mL lysis buffe (50 mM KPi, 150 mM NaCl,
pH 7.5) and we e sonica ed on ice (5 s on 5 s off, 10 minu es,
70% ampli ude). Clea ed cell ee ex ac was ob ained by
cen i uga ion a 12000 pm o 45 minu es a 4 1C. Fo
p ecipi a ion o encapsulin-ca go cons uc s, equal olumes o
cell ee ex ac we e mixed on ice wi h equal olumes o 12%
PEG-8000, 50 mM KPi, pH 7.5, 2 M NaCl (used o A h oEnc
cons uc s) and 8% PEG-8000, 50 mM KPi pH 7.5, 2 M NaCl
(used o Dend oEnc cons uc s). The ob ained solu ions we e
incuba ed o 4 hou s a 4 1C ollowed by cen i uga ion a
12000 pm o 45 minu es a 4 1C. The ob ained pelle s we e
esuspended in 50 mM KPi, 150 mM NaCl, pH 7.5. The
esuspended mix u e was selec i ely loaded on o Ni Sepha ose
g a i y columns con aining 3 mL esin in o de o emo e he
excess unencapsula ed ca go-enzyme.
Size exclusion ch oma og aphy (SEC) was used wi h he in en-
ion o u he polishing encapsulins. A Supe dex 200 inc ease
10/300 GL column wi h 20 mM KPi, 150 mM NaCl, pH 7.5 was
used. Wa eleng hs we e se a 280 nm and 420 nm co esponding
o s anda d p o ein and ca go abso bance espec i ely. F ac ions
co esponding o he peak con aining bo h wa eleng hs we e
pulled oge he and he SDS-PAGE analysis was hen pe o med
o assess he pu i y o encapsulins (Fig. S2 and S3, ESI†).
C yo-EM, da a analysis, model building
3ml o encapsulins a concen a ions 3 o 6 mg mL
1
, we e
applied on o a eshly glow-discha ged coppe R2/1 300 mesh
g id (Quan i oil), blo ed o 3 s on bo h sides wi h blo ing
o ce 0 and plunge- ozen in liquid e hane using he Vi obo
Ma k IV sys em (The mo Fishe Scien i ic) a 13 1C and 100%
humidi y.
Da ase s we e collec ed using a Talos A c ica ansmission
elec on mic oscope (The mo Fishe Scien i ic) equipped wi h
an XFEG a 200 kV using he au oma ed da a-collec ion so -
wa e EPU e sion 2.7 (The mo Fishe Scien i ic). 2 images pe
hole wi h de ocus ange o 0.5 o 2.5 mm we e collec ed wi h
K3 de ec o (Ga an) ope a ed in supe esolu ion mode. Image
s acks wi h 50 ames we e collec ed wi h he o al exposu e
ime o 3.5 s and o al dose o 45 e
Å
2
.
Fo A h oEnc, 719 mic og aphs we e used o da a p oces-
sing. A e mo ion co ec ion and CTF es ima ion pe o med in
Relion e sion 3.1,
27
42 006 pa icles we e picked in c YOLO,
28
ex ac ed wi h box size 330 and pixel size 1.04 in Relion e sion
3.1, and impo ed in C yoSPARC. 20200 pa icles a e 2D
classi ica ion we e used o ab ini io 3D e inemen wi h simul a-
neous so ing in o 3 classes. One class wi h he la ges numbe o
pa icles, 15677, was used o non-uni o m 3D e inemen wi h
icosahed al (I) symme y. The econs uc ion and co esponding
pa icle alignmen s we e used o Bayesian polishing in Relion,
ollowed by inal non-uni o m 3D e inemen in C yoSPARC.
Fo WT Dend oEnc, 4498 mic og aphs we e used o da a
p ocessing. A e mo ion co ec ion and CTF es ima ion pe -
o med in Relion e sion 3.1, 108 615 pa icles we e picked in
c YOLO,
28
ex ac ed wi h box size 440 and pixel size 1.59 in
Relion e sion 3.1, and impo ed in C yoSPARC. 14526 pa icles
a e 2D classi ica ion we e used o ab ini io 3D e inemen wi h
simul aneous so ing in o 3 classes. One class wi h he la ges
numbe o pa icles, 6765, was used o non-uni o m 3D e ine-
men wi h icosahed al (I) symme y. These pa icles we e sub-
jec ed o one mo e ound o 3D e inemen wi h simul aneous
so ing in o 2 classes. The class wi h he la ges numbe o
pa icles, 4549, was used o non-uni o m 3D e inemen wi h
icosahed al (I) symme y. The econs uc ion and co esponding
pa icle alignmen s we e used o Bayesian polishing in Relion,
ollowed by inal non-uni o m 3D e inemen in C yoSPARC.
Finally, o Dend oEnc N198W, 8176 mic og aphs we e used
o da a p ocessing. A e mo ion co ec ion and CTF es ima ion
pe o med in Relion e sion 3.1, 35472 pa icles we e picked in
c YOLO, ex ac ed wi h box size 440 and pixel size 1.59 in Relion,
and impo ed in C yoSPARC. 15958 pa icles a e 2D classi ica-
ion we e used o ab ini io 3D e inemen wi h simul aneous
so ing in o 3 classes. One class wi h he la ges numbe o
pa icles, 10 561, was used o non-uni o m 3D e inemen wi h
icosahed al (I) symme y. The econs uc ion and co esponding
pa icle alignmen s we e used o Bayesian polishing in Relion,
ollowed by one mo e ab ini io 3D e inemen wi h simul aneous
so ing in o 3 classes. The class wi h 3997 pa icles was used o
he inal non-uni o m 3D e inemen . To demons a e encapsula-
ion o Dend oEnc wi h CyanoPOX, we pe o med pa icle sub-
ac ion in C yoSPARC using a mask co e ing he Dend oEnc shell.
B- ac o sha pened econs uc ions o inal e inemen s
we e used o model building. Alpha old 2
29
model p edic ions
o encapsulins we e i in o he expe imen al densi y in Isolde
30
and e ined in Phenix.
31
The s a is ics p esen ed in Table S3
(ESI†) was calcula ed by MolP obi y.
32
Ma e ials Ad ances Pape
Open Access A icle. Published on 26 June 2025. Downloaded on 11/23/2025 1:23:21 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
5308 | Ma e . Ad ., 2025, 6, 5303–5309 © 2025 The Au ho (s). Published by he Royal Socie y o Chemis y
P o eoly ic s abili y o Dend oEnc cons uc s
To assess p o eoly ic esis ance, pu i ied Dend oEnc encapsu-
lin ob ained a e SEC and non-encapsula ed CyanoPOX (bo h
a 1 mg mL
1
) we e es ed o p o eoly ic esis ance wi h chymo-
ypsin om bo ine panc eas. The p o ease s ock solu ion
(40 U mL
1
) was p epa ed by dissol ing bo ine chymo ypsin
in 100 mM T is-HCl buffe (pH 7.8) wi h 10 mM CaCl
2
.25mLo
1mgmL
1
encapsulin o CyanoPOX was mixed wi h 475 mLo
p o ease s ock solu ion. The diges ions we e pe o med a 50 1C
o 24 h. Samples we e analyzed by SDS PAGE.
Ac i i y o Dend oEnc CyanoPOX cons uc s
Reac ions wi h Dend oEnc CyanoPOX (1 mg mL
1
) and Den-
d oENC N198W CyanoPOX (1 mg mL
1
) we e pe o med a 25 1C.
Enzyma ic eac ion wi h guaiacol was es ed a 10 mM concen-
a ion in a 50 mM KPi, 150 mM NaCl, pH 7.5, while ABTS was
es ed a 10 mM concen a ion in 50 mM KPi, 150 mM NaCl, pH
5.0. 0.5 mM o H
2
O
2
was used in all o he eac ions. Fo ma ion o
oxidized p oduc s was moni o ed a diffe en wa eleng hs on he
BioTek Syne gy HTX mul i-mode mic opla e eade (ABTS e
420
=
36.0 mM
1
cm
1
and guaiacol e
470
=26.6mM
1
cm
1
).
22
Reac ion
a es we e de e mined om he linea egions o he eac ion
cu es.
Dend oEnc N198W mu an gene a ion
Dend oEnc mu a ion was pe o med using he QuickChange
me hod.
33
The 25 mL PCR mix con ained he ollowing compo-
nen s: 12.5 mL o P uUl a II Ho s a PCR Mas e Mix, 1 mLo
o wa d p ime (10 mM), 1 mL o e e se p ime (10 mM), 1 mLo
plasmid (100 ng mL
1
), 0.4 mL o DMSO, and MQ wa e o a inal
olume o 25 mL.
Conclusions
This s udy ad ances he unde s anding o bac e ial encapsulins
and hei po en ial applica ions in bioca alysis h ough he
iden i ica ion and cha ac e iza ion o wo no el sys ems:
A h oEnc om A h obac e sp. SLBN-53 and Dend oEnc om
Dend ospo obac e que cicolus. S uc u al analyses e ealed ha
A h oEnc assembles in o a 20 nm capsid wi h T= 1 icosahed al
symme y, while Dend oEnc o ms a la ge 40 nm s uc u e
wi h T= 4 icosahed al symme y. Func ional cha ac e iza ion
demons a ed ha Dend oEnc CyanoPOX exhibi s enzyma ic
ac i i y wi h ABTS and guaiacol subs a es. No ably, Dend oEnc
displayed esis ance o p o eoly ic deg ada ion and p o ec ed
encapsula ed enzymes du ing 24 hou s o exposu e o chymo-
ypsin. Fu he mo e, he N198W mu a ion in Dend oEnc,
designed o modula e po e size, educed subs a e accessibili y,
esul ing in a 5- old dec ease in enzyma ic ac i i y ela i e o
he wild ype. These indings expand he known di e si y o
encapsulin sys ems and highligh hei po en ial as obus
pla o ms o enzyme s abiliza ion and bioca alysis, offe ing
p omising possibili ies o u u e applica ions in nano eac o
design and bio echnology.
Au ho con ibu ions
M. W. F. and N. L. concei ed and supe ised expe imen s. A. B.
pe o med he s uc u al cha ac e iza ion. O. P., C. M. and R. K.
ca ied ou he iden i ica ion and biochemical cha ac e iza ion.
O. P., A. B., N. L. and M. W. F. con ibu ed in w i ing he
manusc ip and app o e he inal e sion o he manusc ip .
Con lic s o in e es
The e a e no con lic s o decla e.
Da a a ailabili y
Coo dina es o he c yo-EM s uc u es o A h oEnc, WT
Dend oEnc and Dend oEnc N198W ha e been deposi ed in
he Elec on Mic oscopy Da a Bank unde accession numbe s
EMD-52714, 52715 and 52716. The co esponding molecula
models ha e been deposi ed a he wwPDB wi h accession
codes PDB 9I8D, 9I8E and 9I8F. O he ele an da a suppo ing
he indings o his s udy a e ully included wi hin he main
a icle and i s ESI.†
Acknowledgemen s
O. P., M. W. F. and N. L. ecei ed unding om he Eu opean
Union’s Ho izon 2020 esea ch and inno a ion p og am unde
G an Ag eemen 101000607 (P ojec OXIPRO). We acknow-
ledge D Alessand o Bo e io o use ul discussions on encap-
sulin s uc u es. We hank A em S e senko, Roman Koning,
and F ank Faas o help wi h c yo-EM da a collec ion and
Michiel Pun e o main aining he compu ing clus e . C yo-
EM da a we e collec ed a he elec on mic oscopy acili ies o
he Uni e si y o G oningen and Leiden Uni e si y Medical
Cen e , nodes o The Ne he lands Elec on Mic oscopy In a-
s uc u e (NEMI).
Re e ences
1 J. A. Jones and T. W. Giessen, Bio echnol. Bioeng., 2021, 118,
491–505.
2 T. W. Giessen, Annu. Re . Biochem., 2022, 91, 1–19.
3 C. G eening and T. Li hgow, Na . Re . Mic obiol., 2020, 18,
677–689.
4 C. A. Ke eld, C. Aussigna gues, J. Za zycki, F. Cai and
M. Su e , Na . Re . Mic obiol., 2018, 16, 277–290.
5 N.W.Kennedy,C.E.Mills,T.M.Nichols,C.H.Ab ahamson
and D. Tullman-E cek, Cu . Opin. Mic obiol., 2021, 63, 36–42.
6 M. Su e , D. Boeh inge , S. Gu mann, S. Gu
¨n he , D. P angish ili,
M.J.Loessne ,K.O.S e e ,E.Webe -BanandN.Ban,Na . S uc .
Mol. Biol., 2008, 15, 939–947.
7 W. R. Wikoff, L. Liljas, R. L. Duda, H. Tsu u a, R. W.
Hend ix and J. E. Johnson, Science, 2000, 289, 2129–2133.
8 E. E en, N. R. Wa s, F. Mon ecinos and P. T. Wing ield,
Biomolecules, 2024, 14, 624.
Pape Ma e ials Ad ances
Open Access A icle. Published on 26 June 2025. Downloaded on 11/23/2025 1:23:21 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
© 2025 The Au ho (s). Published by he Royal Socie y o Chemis y Ma e . Ad ., 2025, 6, 5303–5309 | 5309
9 J. A. Jones, R. Benisch and T. W. Giessen, J. Ma e . Chem. B,
2023, 11, 4377–4388.
10 N. Lonc
ˇa , H. J. Rozeboom, L. E. F anken, M. C. A. S ua
and M. W. F aaije, Biochem. Biophys. Res. Commun., 2020,
529, 548–553.
11 T. W. Giessen, B. J. O lando, A. A. Ve degaal, M. G.
Chambe s, J. Ga dene , D. C. Bell, G. Bi ane, M. Liao and
P. A. Sil e , eLi e, 2019, 8, e46070.
12 T. W. Giessen and P. A. Sil e , Na . Mic obiol., 2017,
2, 17029.
13 R. J. Nichols, C. Cassidy-Ams u z, T. Chaija asphong and
D. F. Sa age, C i . Re . Biochem. Mol. Biol., 2017, 52, 583–594.
14 R. Rahmanpou and T. D. H. Bugg, FEBS J., 2013, 280,
2097–2104.
15 C. A. McHugh, J. Fon ana, D. Nemecek, N. Cheng, A. A.
Aksyuk, J. B. Heymann, D. C. Winkle , A. S. Lam, J. S. Wall,
A. C. S e en and E. Hoiczyk, EMBO J., 2014, 33, 1896–1911.
16 K. A. Lien, K. Dinshaw, R. J. Nichols, C. Cassidy-Ams u z,
M. Knigh and R. Singh, e al.,eLi e, 2021, 10, e74358.
17 J. M. Rod ı
´guez, C. Allende-Balles e o, J. J. L. M. Co nelissen
and J. R. Cas o
´n, Nanoma e ials, 2021, 11, 1467.
18 A. Tamu a, Y. Fuku ani, T. Takami, M. Fujii, Y. Nakaguchi
and Y. Mu akami, e al.,Bio echnol. Bioeng., 2015, 112,
13–20.
19 T. W. Giessen, Cu . Opin. Chem. Biol., 2016, 34, 1–10.
20 P. Lagou e, C. Mignon, G. S ad hagen, S. Po isopon, S. Donna ,
J.Mas ,A.Luga iandB.We le,Vaccine, 2018, 36, 3622–3628.
21 A. V. Almeida, A. J. Ca alho and A. S. Pe ei a, Coo d. Chem.
Re ., 2021, 448, 214188.
22 O. Pec
´anac, C. Ma in, S. Sa ino, H. J. Rozeboom, M. W.
F aaije and N. Lonc
ˇa , ChemBioChem, 2024, 25, e202400713.
23 E. E en, B. Wang, D. C. Winkle , N. R. Wa s, A. C. S e en
and P. T. Wing ield, S uc u e, 2022, 30,551–563.
24 N. Lonc
ˇa , H. L. an Beek and M. W. F aaije, In . J. Mol. Sci.,
2019, 20, 6148.
25 A. N. Gabash ili, N. S. Chmelyuk, M. V. E emo a, J. A.
Malino skaya, A. S. Semkina and M. A. Abakumo , Bio-
molecules, 2020, 10, 966.
26 C. Engle , M. Youles, R. G ue zne , T. M. Ehne , S. We ne ,
J. D. Jones, N. J. Pa on and S. Ma illonne , ACS Syn h. Biol.,
2014, 3, 839–843.
27 J. Zi ano , T. Nakane, B. O. Fo sbe g, D. Kimanius, W. J.
Hagen, E. Lindahl and S. H. Sche es, eLi e, 2018, 7, e42166.
28 T. Wagne , F. Me ino, M. S ab in, T. Mo iya, C. An oni,
A. Apelbaum, P. Hagel, O. Si sel, T. Raisch, D. P umbaum,
D. Quen in, D. Rode e , S. Tacke, B. Siebolds, E. Schube ,
T. R. Shaikh, P. Lill, C. Ga sogiannis and S. Raunse , Com-
mun. Biol., 2019, 2, 218.
29 J. Jumpe , R. E ans, A. P i zel, T. G een, M. Figu no ,
O. Ronnebe ge , K. Tunyasu unakool, R. Ba es, A. Z
ˇı
´dek,
A. Po apenko, A. B idgland, C. Meye , S. A. A. Kohl, A. J.
Balla d, A. Cowie, B. Rome a-Pa edes, S. Nikolo , R. Jain,
J. Adle , T. Back, S. Pe e sen, D. Reiman, E. Clancy, M. Zielinski,
M. S einegge , M. Pacholska, T. Be ghamme , S. Bodens ein,
D.Sil e ,O.Vinyals,A.W.Senio ,K.Ka ukcuoglu,P.Kohliand
D. Hassabis, Na u e, 2021, 596, 583–589.
30 T. I. C oll, Ac a C ys allog ., Sec . D: S uc . Biol., 2018, 74,
519–530.
31 D. Liebschne , P. V. A onine, M. L. Bake , G. Bunko
´czi,
V. B. Chen, T. I. C oll, B. Hin ze, L.-W. Hung, S. Jain, A. J.
McCoy, N. W. Mo ia y, R. D. Oeffne , B. K. Poon, M. G.
P isan , R. J. Read, J. S. Richa dson, D. C. Richa dson,
M. D. Sammi o, O. V. Sobole , D. H. S ockwell, T. C.
Te willige , A. G. U zhum se , L. L. Videau, C. J. Williams
and P. D. Adams, Ac a C ys allog ., Sec . D: S uc . Biol., 2019,
75, 861–877.
32 C. J. Williams, J. J. Headd, N. W. Mo ia y, M. G. P isan ,
L. L. Videau, L. N. Deis and V. Ve ma, e al.,P o ein Sci.,
2018, 27, 293–315.
33 T. A. Kunkel, P oc. Na l. Acad. Sci. U. S. A., 1985, 82, 488–492.
Ma e ials Ad ances Pape
Open Access A icle. Published on 26 June 2025. Downloaded on 11/23/2025 1:23:21 PM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
