Information on EC 3.2.1.4 - cellulase - BRENDA Enzyme Database

-

beta-1,4-glucanase

-

Cellulase

-

EfPh

-

EGPh

-

679714, 690256, 721354, 724605, 725685

endocellulase

-

endoglucanase

-

PH1171

Starch and sucrose metabolism

9.5 cellulase

-

Abscission cellulase

-

alkali cellulase

-

Alkaline cellulase

-

avicelase

-

beta-1,4-endoglucan hydrolase

-

beta-1,4-glucanase

-

Carboxymethyl cellulase

-

Carboxymethyl-cellulase

-

carboxymethylcellulase

-

CEL1

-

cellobiohydrolase

-

celluase A

-

celludextrinase

-

Cellulase

-

cellulase A 3

-

Cellulase E1

-

Cellulase E2

-

Cellulase E4

-

Cellulase E5

-

Cellulase SS

-

Cellulase V1

-

cellulosin AP

-

CMCase

-

CX-cellulase

-

EG1

-

EG2

-

EG3

-

EGA

-

EGB

-

EGC

-

EGCCA

-

EGCCC

-

EGCCD

-

EGCCF

-

EGCCG

-

EGD

-

EGE

-

EGF

-

EGH

-

EGI

-

EGIV

-

EGM

-

EGSS

-

EGX

-

EGY

-

EGZ

-

endo-1,4-beta-D-glucanase

-

endo-1,4-beta-glucanase

-

endo-1,4-beta-glucanase E1

-

endo-1,4-beta-glucanase V1

-

endocellulase E1

-

endoglucanase

-

endoglucanase D

-

FI-CMCASE

-

pancellase SS

-

Thermoactive cellulase

-

hydrolysis of O-glycosyl bond

-

BRENDA

cellulose degradation

KEGG

-

4-beta-D-glucan 4-glucanohydrolase

Will also hydrolyse 1,4-linkages in beta-D-glucans also containing 1,3-linkages.

9012-54-8

-

4-nitrophenyl cellobioside + H2O

4-nitrophenol + cellobiose

avicel + H2O

?

-

678809, 723993

-

?

avicel + H2O

cellobiose + ?

carboxymethyl cellulose + H2O

?

4 entries

cellulose + H2O

?

-

721354, 724605

-

?

cellulose + H2O

cellobiose + ?

the substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the beta-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. Analysis of the enzyme-substrate structure suggests that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 is released from the active site

-

?

cellulose + H2O

cellooligosaccharide

-

-

?

crystalline cellulase + H2O

?

-

-

?

crystalline cellulose

?

-

664873

-

?

crystalline cellulose + H2O

?

-

-

?

lichenan + H2O

?

-

-

?

p-nitrophenyl cellobiose + H2O

?

-

-

?

p-nitrophenyl cellobiose + H2O

p-nitrophenol + cellobiose

additional information

?

-

cellulose + H2O

cellooligosaccharide

-

-

?

Zn2+

zinc ions tightly bound between the two catalytic glutamate residues, which present an obstacle for the entrance of ligand in the active site

cellobiose

the non-complete saccharification of cellulose by thr enzyme seems to be due to product-feedback inhibition by cellobiose

0.37

4-nitrophenyl cellobioside

pH 6.0, 70°C

0.35 - 6.69

p-nitrophenyl cellobiose

23 entries

additional information

-

9.55

4-nitrophenyl cellobioside

pH 6.0, 70°C

0.003 - 0.91

p-nitrophenyl cellobiose

23 entries

additional information

-

1.51

4-nitrophenyl cellobioside

pH 6.0, 70°C

additional information

-

9.08

cellobiose

pH 5.5, 50°C

5

assay at

5 - 7

wild-type and mutant enzymes C106A/C159A/C372A/C412A, C106A/C159A and C372A/C412A

5.5

5.5 - 6

optimal pH for wild-type enzyme and mutant enzymes E201Q, E342Q, and D385N

5.6

assay at

7

mutant enzyme H297N

8.5

mutant enzyme Y299F

100

optimal temperature is above 100°C

50

assay at

80

assay at

85

90

mutant enzyme C106A/C159A/C372A/C412A

97

-

5.6

calculated from sequence, wild-type enzyme (389 amino acids) is expressed with both N (28 residues)- and C (42 residues)-terminal truncations (full length EGPh contains 458 amino acids) with a signal peptide sequence of 28 residues and a C-terminal region involved in anchoring

-

664873, 678809, 679714, 681348, 690256, 717220, 721354, 723993, 724605, 724939, 725685, 730141, 744265, 746401

Pyrococcus horikoshii DSM 12428

UniProt

-

721354, 723993, 725685

Pyrococcus horikoshii OT-3

UniProt

-

724939, 744265

UniProt

membrane

-

16020

metabolism

key enzyme involved in the degradation of beta-glucan cellulose biomass acting through hydrolysis of the unbranched beta-1,4-linked glucose homopolymer

746401

O58925 pBLAST

O58925_PYRHO

Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)

458

0

51930

TrEMBL

Show Sequence

Mitochondrion (Reliability: 2)

Pyrococcus horikoshii OT3

7 entries

40000

43000

43970

theoretically, EGPhDELTAC5, truncated form of the enzyme

45561

x * 45561, wild-type enzyme (389 amino acids) expressed with both N(28 residues)- and C(42 residues)-terminal truncations (full length EGPh contains 458 amino acids) with a signal peptide sequence of 28 residues and a C-terminal region involved in anchoring

?

hanging-drop vapor-diffusion method

746401

hanging-drop vapor-diffusion method. The crystals are obtained over a period of about 3 days at 22°C

hanging-drop vapour diffusion method. The crystal of the enzymeligand complex is prepared from the truncated protein lacking five amino acid residues from both the N- and C-terminal ends. Crystal structures of mutants enzymes (E201A, E342A and Y299F) in the complex with either the substrate or product ligands

purified enzyme mutants in complex with either the substrate or product ligands, hanging drop vapour diffusion method, 20 mg/ml protein in 50 mM Tris/HCl, pH 8.0, mixing of 0.0015 ml of both protein and reservoir solution, the latter contaning 1.5 M ammonium phosphate, and 0.1 M MES, pH 6.5, 22°C, 3 days, X-ray diffraction structure determination and analysis at 1.65-2.01 A resolution

X-ray diffraction analysis of crystals of the wild-type full-length EGPh is unsuccessful, sitting-drop vapour-diffusion method

C106A/C159A

kcat/KM for p-nitrophenyl cellobiose is 1.3fold higher than wild-type value. Activity towards carboxymethyl cellulose is increased by 1.7fold

C106A/C159A/C372A/C412A

kcat/KM for p-nitrophenyl cellobiose is 1.4fold higher than wild-type value. Activity towards carboxymethyl cellulose is increased by 2.1fold

C106S

melting temperature of the mutant enzyme is 2°C lower than the wild-type enzyme

C159A

kcat/KM for p-nitrophenyl cellobiose is 1.8fold lower than wild-type value

C372/AC412A

kcat/KM for p-nitrophenyl cellobiose is 2.9fold higher than wild-type value. Activity towards carboxymethyl cellulose is increased by 1.6fold

D385N

activity towards carboxymethyl cellulose is 29.9% of wild-type activity

DELTAQ1-G5

activity towards carboxymethyl cellulose is 135.6% of wild-type activity. kcat/Km for p-nitrophenyl cellobiose is 2.3fold higher than wild-type value. Thermostability is not significantly influenced

E163A

kcat/KM for p-nitrophenyl cellobiose is 6fold lower than wild-type value

E201A

E201Q

activity towards carboxymethyl cellulose is 1.12% of wild-type activity. kcat/Km for p-nitrophenyl cellobiose is 43fold lower than wild-type value

E342A

E342Q

activity towards carboxymethyl cellulose is 0.01% of wild-type activity

G158A

kcat/KM for p-nitrophenyl cellobiose is 2fold lower than wild-type value

H155A

kcat/KM for p-nitrophenyl cellobiose is 140fold lower than wild-type value

H161A

kcat/KM for p-nitrophenyl cellobiose is neatrly identical to wild-type value

H297A

activity towards carboxymethyl cellulose is 0.08% of wild-type activity

H297N

activity towards carboxymethyl cellulose is 1.31% of wild-type activity. pH-optimum is 7.0, compared to 5.5-6 for wild-type enzyme

I157A

kcat/KM for p-nitrophenyl cellobiose is nearly identical to wild-type value

I162A

kcat/KM for p-nitrophenyl cellobiose is 140fold lower than wild-type value

N200A

activity towards carboxymethyl cellulose is 5.43% of wild-type activity

P164A

kcat/KM for p-nitrophenyl cellobiose is 6fold lower than wild-type value

P74C

melting temperature of the mutant enzyme is 2°C lower than the wild-type enzyme

P74C/C106S

melting temperature of the mutant enzyme is 2°C lower than the wild-type enzyme

Q306A

25% of the activity with avicel as compared to wild-type enzyme

R102A

activity towards carboxymethyl cellulose is 0.67% of wild-type activity

R156A

kcat/KM for p-nitrophenyl cellobiose is 10fold lower than wild-type value

T160A

kcat/KM for p-nitrophenyl cellobiose is nearly identical to wild-type value

W377A

W82A

75% of the activity with avicel as compared to wild-type enzyme

Y299A

activity towards carboxymethyl cellulose is 0.21% of wild-type activity

Y299F

additional information

7 entries

sequential deletion analyses from both N and C termini, removing 10 amino acids at a time, are carried out to determine whether a shorter enzyme with improved characteristics could becreated. Among the three C-terminal deletions, only the C10 mutant, which misses the last 10 amino acids, maintained activity. In contrast, all N-terminal deletion mutants (N10, N20, and N30) retains activity except N40. Detailed analysis of the aligned sequences reveals that the highly conserved sequence begins at L35, after the 34 N-terminal residues of EGPh. Therefore, a mutant with a deletion betweenN30 and N40 (N34) is prepared and expressed. This mutant retains enzymatic activity like N30, suggesting that the critical residues for enzyme activity start from L35. Each of the active N-terminal deletions is combined with the C10 deletion to establish the minimal sequence required for activity. When enzyme function is tested in the presence of CMC, only the N10C10 mutant exhibits activity, suggesting that the loss of activity may be due to the loss of thermostability. To test this, enzymatic activity assays are performed at 60°C. At this lower temperature, the N20C10, N30C10, and N34C10 mutants all exhibit carboxymethyl cellulase (CMCase) activity, but the N40, C20, and C30 mutants do not. Therefore, the shortest EGPh sequence maintaining hydrolytic activity isN34C10, representing an 11% reduction in amino acid residues. In addition to the decreased optimal temperature of C mutants,N and C combination mutants (except N10C10) are active at 60°C but not at 80°C. At 80°C, the wild type and the N10 mutant are stable, whereas N20, N30, and N34 show gradually decreasing activity. A longer deletion leads to a more severe decrease in activity, and theN34 mutant exhibits the shortest t1/2 of 8 h. Both C10 andN10C10 lose more than 50% activity in less than 2 h at 80°C, suggesting that the decreased activity of C-terminal deletion mutants is due to decreased thermostability

-

sequential deletion analyses from both N and C termini, removing 10 amino acids at a time, are carried out to determine whether a shorter enzyme with improved characteristics could becreated. Among the three C-terminal deletions, only the C10 mutant, which misses the last 10 amino acids, maintained activity. In contrast, all N-terminal deletion mutants (N10, N20, and N30) retains activity except N40. Detailed analysis of the aligned sequences reveals that the highly conserved sequence begins at L35, after the 34 N-terminal residues of EGPh. Therefore, a mutant with a deletion betweenN30 and N40 (N34) is prepared and expressed. This mutant retains enzymatic activity like N30, suggesting that the critical residues for enzyme activity start from L35. Each of the active N-terminal deletions is combined with the C10 deletion to establish the minimal sequence required for activity. When enzyme function is tested in the presence of CMC, only the N10C10 mutant exhibits activity, suggesting that the loss of activity may be due to the loss of thermostability. To test this, enzymatic activity assays are performed at 60°C. At this lower temperature, the N20C10, N30C10, and N34C10 mutants all exhibit carboxymethyl cellulase (CMCase) activity, but the N40, C20, and C30 mutants do not. Therefore, the shortest EGPh sequence maintaining hydrolytic activity isN34C10, representing an 11% reduction in amino acid residues. In addition to the decreased optimal temperature of C mutants,N and C combination mutants (except N10C10) are active at 60°C but not at 80°C. At 80°C, the wild type and the N10 mutant are stable, whereas N20, N30, and N34 show gradually decreasing activity. A longer deletion leads to a more severe decrease in activity, and theN34 mutant exhibits the shortest t1/2 of 8 h. Both C10 andN10C10 lose more than 50% activity in less than 2 h at 80°C, suggesting that the decreased activity of C-terminal deletion mutants is due to decreased thermostability

4 - 9

wild-type and deletion mutants retain more than 70% of its activity

721354

100

Tm-value for wild-type enzyme

101

melting-temperature of the mutant enzymes P74C/C106S, P74C and C106S

103

wild type enzyme is thermally unfolded at 103.4°C

95 - 96

Tm-value for mutant enzymes C106A/C159A/C372A/C412A, C106A/C159A and C372A/C412A

97

3 h, 20% loss of activity

additional information

-

deletion of the C-terminal region facilitates expression of soluble protein and has no effect on enzyme activity. Therefore the wild-type enzyme (389 amino acids) is expressed with both N (28 residues)- and C (42 residues)-terminal truncations

by removing some of the C-terminal sequence of the ORF of the endoglucanase (PH1171), two types of recombinant proteins are expressed from one ORF, using Escherichia coli

664873

expressed in Escherichia coli

expression in Escherichia coli strain BL21(DE3)

expression of mutant enzymes in Escherichia coli

expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)

heterologous expression in Talaromyces cellulolyticus under the control of a glucoamylase promoter of Talaromyces cellulolyticus. The characteristics of the enzyme are almost same as those prepared by Escherichia coli

high level expression in tobacco chloroplast.

724605

shorter version mutants exhibiting activity at 80°C are expressed in Escherichia coli with a C-terminal His6 tag to facilitate purification using immobilized-metal affinity chromatography. Both the wild type and the deletion mutants are highly expressed in Escherichia coli after IPTG induction

truncated mutants are constructed by deleting five residues from C-termini of endoglucanase catalytic domain. The truncated gene is inserted into the expression vector pET11a (Novagen, Madison, WI). The constructed plasmids are introduced into Escherichia coli strain BL21(DE3)

industry

synthesis

Kashima, Y.; Mori, K.; Fukada, H.; Ishikawa, K.

Analysis of the function of a hyperthermophilic endoglucanase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose

Extremophiles

9

37-43

2005

Pyrococcus horikoshii (O58925)

15375673

Kim, H.W.; Takagi, Y.; Hagihara, Y.; Ishikawa, K.

Analysis of the putative substrate binding region of hyperthermophilic endoglucanase from Pyrococcus horikoshii

Biosci. Biotechnol. Biochem.

71

2585-2587

2007

Pyrococcus horikoshii (O58925)

17928686

Kang, H.J.; Uegaki, K.; Fukada, H.; Ishikawa, K.

Improvement of the enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshii

Extremophiles

11

251-256

2007

Pyrococcus horikoshii (O58925), Pyrococcus horikoshii

17072684

Kang, H.J.; Ishikawa, K.

Analysis of active center in hyperthermophilic cellulase from Pyrococcus horikoshii

J. Microbiol. Biotechnol.

17

1249-1253

2007

Pyrococcus horikoshii (O58925), Pyrococcus horikoshii

18051592

Kim, H.W.; Mino, K.; Ishikawa, K.

Crystallization and preliminary X-ray analysis of endoglucanase from Pyrococcus horikoshii

Acta Crystallogr. Sect. F

64

1169-1171

2008

Pyrococcus horikoshii (O58925)

19052378

Kim, H.W.; Ishikawa, K.

Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots

Biochem. J.

437

223-230

2011

Pyrococcus horikoshii (O58925), Pyrococcus horikoshii

21557724

Yang, T.C.; Legault, S.; Kayiranga, E.A.; Kumaran, J.; Ishikawa, K.; Sung, W.L.

The N-terminal beta-sheet of the hyperthermophilic endoglucanase from Pyrococcus horikoshii is critical for thermostability

Appl. Environ. Microbiol.

78

3059-3067

2012

Pyrococcus horikoshii (O58925), Pyrococcus horikoshii, Pyrococcus horikoshii DSM 12428 (O58925)

22344652

Ando, S.; Ishida, H.; Kosugi, Y.; Ishikawa, K.

Hyperthermostable endoglucanase from Pyrococcus horikoshii

Appl. Environ. Microbiol.

68

430-433

2002

Pyrococcus horikoshii (O58925), Pyrococcus horikoshii DSM 12428 (O58925)

11772658

724605

Nakahira, Y.; Ishikawa, K.; Tanaka, K.; Tozawa, Y.; Shiina, T.

Overproduction of hyperthermostable beta-1,4-endoglucanase from the archaeon Pyrococcus horikoshii by tobacco chloroplast engineering

Biosci. Biotechnol. Biochem.

77

2140-2143

2013

Pyrococcus horikoshii (O58925)

24096651

Kim, H.W.; Ishikawa, K.

The role of disulfide bond in hyperthermophilic endocellulase

Extremophiles

17

593-599

2013

Pyrococcus horikoshii (O58925), Pyrococcus horikoshii, Pyrococcus horikoshii OT-3 (O58925)

23624891

Kim, H.W.; Ishikawa, K.

Complete saccharification of cellulose at high temperature using endocellulase and beta-glucosidase from Pyrococcus sp.

J. Microbiol. Biotechnol.

20

889-892

2010

Pyrococcus horikoshii (O58925), Pyrococcus horikoshii, Pyrococcus horikoshii DSM 12428 (O58925)

20519912

Kishishita, S.; Fujii, T.; Ishikawa, K.

Heterologous expression of hyperthermophilic cellulases of archaea Pyrococcus sp. by fungus Talaromyces cellulolyticus

J. Ind. Microbiol. Biotechnol.

42

137-141

2015

Pyrococcus furiosus (E7FHY8), Pyrococcus furiosus, Pyrococcus horikoshii (O58925), Pyrococcus horikoshii

25387612