文献类型： 外文期刊

作者： Wang YaPing ¹ ; Rao Ben ² ; Zhang Ling ¹ ; Ma Lixin ¹ ;

作者机构： 1.Hubei Univ, Coll Life Sci, Hubei Key Lab Ind Biotechnol, Hubei Collaborat Innovat Ctr Green Transformat Bi, Wuhan 430062, Hubei Province, Peoples R China

2.Hubei Acad Agr, Natl Biopesticide Engn Res Ctr, Wuhan 430064, Hubei Province, Peoples R China

关键词： Yarrowialipolytica;Mannanse;Multi-copy expression cassette;High-density fermentation;Thermophilic

期刊名称：PROTEIN EXPRESSION AND PURIFICATION （ 影响因子：1.65； 五年影响因子：1.548 ）

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收录情况： SCI

摘要： Two thermophilic beta-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pH similar to 5 and similar to 70 degrees C, and for manB activity were pH similar to 7 and 60 degrees C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications. (C) 2017 Elsevier Inc. All rights reserved.