Improvement in the Temperature Properties of Xylanase by Site-Saturated Mutagenesis

doi:10.3969/j.issn.1673-1689.2019.07.009

Home > Archive>Volume 38, Issue 7, 2019 >65-70. DOI:10.3969/j.issn.1673-1689.2019.07.009

Improvement in the Temperature Properties of Xylanase by Site-Saturated Mutagenesis
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                        10.3969/j.issn.1673-1689.2019.07.009
                    
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To improve the temperature properties of AoXyn11A，a glycoside hydrolase family 11 mesophilic xylanase from Aspergillus oryzae CICC40186，the amino acid residue of Gly²¹that possessing the highest B-factor value was replaced randomly based on the homology modeling of AoXyn11A and molecular dynamics（MD） simulation for its three-dimensional structure. Using the recombinant plasmid pET-28a-Aoxyn11A as a template，the codon of Gly²¹in the xylanase-encoding gene（Aoxyn11A） was site-saturated mutagenesis with the two-stage whole-plasmid PCR technique. And then，the mutational transformant library was constructed by transforming the mutants of pET-28a-Aoxyn11A into E. coli BL21. In reference to the thermostability of enzymes，optimal mutational transformant（E. coli/Aoxyn11A^G21I） was screened from library. The DNA sequencing results showed that E. coli/Aoxyn11A^G21I expressed a mutant enzyme（AoXyn11A^G21I） with the amino acid residue of Gly²¹ changed by Ile²¹. The analytical results indicated that the optimal temperature（T^opt） of AoXyn11A^G21Iwas 65 ℃，which was 10 ℃ higher than that of AoXyn11A. AoXyn11A^G21Iwas thermostable at or below 55 ℃，being 7 ℃ higher than that of AoXyn11A. In addition，the pH properties of AoXyn11AG21I did not obviously change compared with AoXyn11A.

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WU Qin, ZANG Jia, HU Die, WANG Rui, WU Minchen. Improvement in the Temperature Properties of Xylanase by Site-Saturated Mutagenesis[J]. Journal of Food Science and Biotechnology,2019,38(7):65-70.

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