Literature summary for 3.2.1.4 extracted from

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 (2012), Appl. Environ. Microbiol., 78, 3059-3067.

Search for more literature for 3.2.1.4

Application

Application	Comment	Organism
synthesis	when the enzyme is used in combination withbeta-glucosidase, cellulose is completely hydrolyzed to glucose at high temperature, suggesting great potential for EGPh in bioethanol industrial applications	Pyrococcus horikoshii

Cloned(Commentary)

Cloned (Comment)	Organism
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	Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
additional information	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	Pyrococcus horikoshii

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	KM-values of N-terminal and C-terminal deletion mutants	Pyrococcus horikoshii
0.37	-	4-nitrophenyl cellobioside	pH 6.0, 70°C	Pyrococcus horikoshii

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
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	Pyrococcus horikoshii

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	O58925	-	-
Pyrococcus horikoshii DSM 12428	O58925	-	-

Purification (Commentary)

Purification (Comment)	Organism
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	Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
4-nitrophenyl cellobioside + H2O	-	Pyrococcus horikoshii	4-nitrophenol + cellobiose	-	?
4-nitrophenyl cellobioside + H2O	-	Pyrococcus horikoshii DSM 12428	4-nitrophenol + cellobiose	-	?
cellulose + H2O	-	Pyrococcus horikoshii	?	-	?
cellulose + H2O	-	Pyrococcus horikoshii DSM 12428	?	-	?

Subunits

Subunits	Comment	Organism
?	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	Pyrococcus horikoshii

Synonyms

Synonyms	Comment	Organism
EGPh	-	Pyrococcus horikoshii

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	the N-terminalbeta-sheet is critical for enzyme thermostability	Pyrococcus horikoshii

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
additional information	-	additional information	kcat-values of N-terminal and C-terminal deletion mutants	Pyrococcus horikoshii
9.55	-	4-nitrophenyl cellobioside	pH 6.0, 70°C	Pyrococcus horikoshii

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
4	9	wild-type and deletion mutants retain more than 70% of its activity	Pyrococcus horikoshii

pI Value

Organism	Comment	pI Value Maximum	pI Value
Pyrococcus horikoshii	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	-	5.6

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
additional information	-	additional information	kcat/KM-values of N-terminal and C-terminal deletion mutants	Pyrococcus horikoshii
1.51	-	4-nitrophenyl cellobioside	pH 6.0, 70°C	Pyrococcus horikoshii

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Release 2023.1 (January 2023)