Heterologous expression and biochemical characterization of a thermostable endo-β-1,4-glucanase from Colletotrichum orchidophilum View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2020-08-09

AUTHORS

Hai-Yan Zhou, Jian-Bao Zhou, Xiao-Nan Yi, Yan-Mei Wang, Ya-Ping Xue, De-Shui Chen, Xin-Ping Cheng, Mian Li, Hong-Yan Wang, Kai-Qian Chen, Zhi-Qiang Liu, Yu-Guo Zheng

ABSTRACT

To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55–75 °C and high thermostability (about 85% residual activity) at the temperature of 55 °C after incubation for 3 h. The highest activity of CoCel5A was detected when 100 mM citric acid buffer (pH 4.0–5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100 mM citric acid buffer (pH 3.0–6.0) at 4 °C for 24 h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and β-d-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters Vmax, Km, and Kcat/Km values against CMC were 290.70 U/mg, 2.65 mg/mL, and 75.67 mL/mg/s, respectively; and 228.31 U/mg, 2.06 mg/mL, and 76.45 mL/mg/s against β-d-glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields. More... »

PAGES

67-79

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s00449-020-02420-7

DOI

http://dx.doi.org/10.1007/s00449-020-02420-7

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1130001386

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/32772153


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25 schema:description To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55–75 °C and high thermostability (about 85% residual activity) at the temperature of 55 °C after incubation for 3 h. The highest activity of CoCel5A was detected when 100 mM citric acid buffer (pH 4.0–5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100 mM citric acid buffer (pH 3.0–6.0) at 4 °C for 24 h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and β-d-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters Vmax, Km, and Kcat/Km values against CMC were 290.70 U/mg, 2.65 mg/mL, and 75.67 mL/mg/s, respectively; and 228.31 U/mg, 2.06 mg/mL, and 76.45 mL/mg/s against β-d-glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields.
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32 schema:keywords CMC
33 D-glucan
34 Km
35 Km values
36 Vmax
37 acid buffer
38 activity
39 affinity
40 applications
41 biochemical characterization
42 buffer
43 carboxymethyl
44 catalytic efficiency
45 cellulases
46 characterization
47 citric acid buffer
48 efficiency
49 efficient utilization
50 endo
51 endoglucanase gene
52 environmental fields
53 excellent pH stability
54 expression
55 field
56 genes
57 glucanase
58 good substrate
59 heterologous expression
60 high activity
61 high affinity
62 high thermostability
63 incubation
64 kcat/Km values
65 kinetic parameters Vmax
66 lignocellulose
67 new cellulases
68 optimal activity
69 pH stability
70 parameters Vmax
71 potential applications
72 properties
73 purification
74 salt
75 secretory expression
76 sodium salt
77 stability
78 substrate
79 temperature
80 thermostability
81 thermostable endo
82 utilization
83 values
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