Enzymatic characterization of a novel thermostable and alkaline tolerant GH10 xylanase and activity improvement by multiple rational mutagenesis strategies

Autor: Yanhe Ma, Xiaochen Ma, Yanfen Xue, Lai Zhihua, Cheng Zhou
Rok vydání: 2020
Předmět:
Models
Molecular
Protein Denaturation
Hot Temperature
Protein Conformation
Mutant
Mutagenesis (molecular biology technique)
Bacillus
02 engineering and technology
Molecular Dynamics Simulation
medicine.disease_cause
Biochemistry
Substrate Specificity
03 medical and health sciences
Bacterial Proteins
Structural Biology
medicine
Enzyme kinetics
Amino Acid Sequence
Cloning
Molecular
Molecular Biology
Escherichia coli
Conserved Sequence
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
Endo-1
4-beta Xylanases
Base Sequence
Sequence Homology
Amino Acid
Chemistry
Protein Stability
Rational design
General Medicine
Hydrogen-Ion Concentration
021001 nanoscience & nanotechnology
Kinetics
Enzyme
Xylanase
Mutagenesis
Site-Directed
Specific activity
Xylans
0210 nano-technology
Sequence Alignment
Zdroj: International journal of biological macromolecules. 170
ISSN: 1879-0003
Popis: Thermo-alkaline xylanases are widely applied in paper pulping industry. In this study, a novel thermostable and alkaline tolerant GH10 xylanase (Xyn30Y5) gene from alkaliphilic Bacillus sp. 30Y5 was cloned and the surface-layer homology (SLH) domains truncated enzyme (Xyn30Y5-SLH) was expressed in Escherichia coli. The purified Xyn30Y5-SLH was most active at 70 °C and pH 7.0 and showed the highest specific activity of 349.4 U mg−1. It retained more than 90% activity between pH 6.0 to 9.5 and was stable at pH 6.0–10.0. To improve the activity, 47 mutants were designed based on eight rational strategies and 21 mutants showed higher activity. By combinatorial mutagenesis, the best mutant 3B demonstrated specific activity of 1016.8 U mg−1 with a doubled catalytic efficiency (kcat/Km) and RA601/2h value, accompanied by optimal pH shift to 8.0. The molecular dynamics simulation analysis indicated that the increase of flexibility of α5 helix and loop7 located near to the catalytic residues is likely responsible for its activity improvement. And the decrease of flexibility of the most unstable regions is vital for the thermostablity improvement. This work provided not only a novel thermostable and alkaline tolerant xylanase with industrial application potential but also an effective mutagenesis strategy for xylanase activity improvement.
Databáze: OpenAIRE
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