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Literature summary for 3.2.1.4 extracted from

  • Nath, P.; Dhillon, A.; Kumar, K.; Sharma, K.; Jamaldheen, S.B.; Moholkar, V.S.; Goyal, A.
    Development of bi-functional chimeric enzyme (CtGH1-L1-CtGH5-F194A) from endoglucanase (CtGH5) mutant F194A and ?-1,4-glucosidase (CtGH1) from Clostridium thermocellum with enhanced activity and structural integrity (2019), Biores. Technol., 282, 494-501 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of soluble enzyme chimeric mutant CtGH1-L1-CtGH5-F194A in Escherichia coli strain BL21 Acetivibrio thermocellus

Protein Variants

Protein Variants Comment Organism
F194A site-directed mutagenesis, the mutant shows 2fold increased activity compared to wild-type enzyme Acetivibrio thermocellus
additional information enzyme mutant CtGH5-F194A is fused with a beta-1,4-glucosidase, CtGH1 from Clostridium thermocellum to develop a chimeric enzyme. Improved structural integrity, thermostability and enhanced bifunctional enzyme activities of the chimeric mutant compared to the single point mutant Acetivibrio thermocellus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics, overview Acetivibrio thermocellus

Organism

Organism UniProt Comment Textmining
Acetivibrio thermocellus
-
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
40
-
enzyme mutant F194A, pH 5.5, 50°C Acetivibrio thermocellus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
beta-glucan + H2O
-
Acetivibrio thermocellus ?
-
?
carboxymethyl cellulose + H2O
-
Acetivibrio thermocellus cellobiose + cellooligosaccharides
-
?
lichenan + H2O
-
Acetivibrio thermocellus ?
-
?
additional information final hydrolysed product produced from carboxymethyl cellulose by chimera 1 is glucose confirming both beta-1,4-endoglucanase and beta-1,4-glucosidase activities, while the products of CtGH5-F194A point mutant are cellobiose and cello-oligosaccharides. Product indentification by thin layer chromatography. Enzymatic hydrolysis of 1% w/v Sorghum stalk pretreated by 1% NaOH by mutant CtGH5-F194A with or without wild-type enzyme CtGH1, which exhibits beta-glucosidase activity. No activity of the wild-type enzyme and mutant F194A with 4-nitrophenyl beta-D-glucoside, which is a substrate of the beta-glucosidase activity of chimeric mutant CtGH1-CtGH5-F194A Acetivibrio thermocellus ?
-
?

Synonyms

Synonyms Comment Organism
beta-1,4-endoglucanase
-
Acetivibrio thermocellus
CtGH5
-
Acetivibrio thermocellus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
mutant CtGH5 F194A Acetivibrio thermocellus
60
-
wild-type CtGH5 and chimeric mutant CtGH1-CtGH5 F194A Acetivibrio thermocellus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
68
-
melting temperature of mutant enzyme CtGH5-F194A Acetivibrio thermocellus
79
-
melting temperature of wild-type enzyme CtGH5 Acetivibrio thermocellus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.006
-
carboxymethyl cellulose pH 4.2, 60°C, recombinant wild-type enzyme CtGH5 Acetivibrio thermocellus
0.026
-
carboxymethyl cellulose pH 4.2, 60°C, recombinant mutant CtGH5-F194A Acetivibrio thermocellus
0.045
-
beta-Glucan pH 4.2, 60°C, recombinant mutant CtGH1-CtGH5-F194A Acetivibrio thermocellus
0.053
-
lichenan pH 4.2, 60°C, recombinant chimeric mutant CtGH1-CtGH5-F194A Acetivibrio thermocellus
0.09
-
beta-Glucan pH 4.2, 60°C, recombinant wild-type enzyme CtGH5 Acetivibrio thermocellus
0.1
-
lichenan pH 4.2, 60°C, recombinant wild-type enzyme CtGH5 Acetivibrio thermocellus
0.115
-
carboxymethyl cellulose pH 4.2, 60°C, recombinant mutant CtGH1-CtGH5-F194A Acetivibrio thermocellus
0.135
-
beta-Glucan pH 4.2, 60°C, recombinant mutant CtGH5-F194A Acetivibrio thermocellus
0.145
-
lichenan pH 4.2, 60°C, recombinant mutant CtGH5-F194A Acetivibrio thermocellus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2
-
wild-type CtGH5 Acetivibrio thermocellus
5
-
chimeric mutant CtGH1-CtGH5 F194A Acetivibrio thermocellus
5.5
-
mutant CtGH5 F194A Acetivibrio thermocellus

General Information

General Information Comment Organism
evolution the enzyme belongs to the glycoside hydrolase family 5,GH5 Acetivibrio thermocellus