Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability
| Autor: | Aleksandra M. Rozhkova, Arkady P. Sinitsyn, Alexander V. Gusakov, Veronika Yu. Matys, Ivan N. Zorov, Yury A. Denisenko, Dmitry O. Osipov, I. V. Uporov |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Models Molecular Protein Conformation Mutant Biology Biochemistry law.invention 03 medical and health sciences Structural Biology law Enzyme Stability Amino Acid Sequence Site-directed mutagenesis Molecular Biology Thermostability chemistry.chemical_classification Endo-1 4-beta Xylanases Penicillium Temperature Promoter General Medicine 030104 developmental biology Enzyme chemistry Penicillium canescens Mutation Recombinant DNA Xylanase Mutagenesis Site-Directed |
| Zdroj: | International journal of biological macromolecules. 104 |
| ISSN: | 1879-0003 |
| Popis: | In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50–60 °C was 2–2.5–fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1 °C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability. |
| Databáze: | OpenAIRE |
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