Cloning, over-expression and characterization of an alkali-tolerant endo-¦Â-1,4-mannanase from Penicillium freii F63

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• 作者：Yawei Wang¹ ; ² ; Pengjun Shi¹ ; Huiying Luo¹ ; Yingguo Bai¹ ; Huoqing Huang¹ ; Peilong Yang¹ ; Hairong Xiong² ; Bin Yao¹ ; ^{binyao@caas.net.cn}
• 关键词：Penicillium freii F63 ; ¦Â-Mannanase ; Pichia pastoris ; Over-expression ; Alkali-tolerant
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2012
• 出版时间：June, 2012
• 年：2012
• 卷：113
• 期：6
• 页码：710-714
• 全文大小：694 K

文摘

A glycosyl hydrolase family 5 endo-¦Â-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 g l^-1 in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60¡ãC and exhibited good stability over a broad pH range from acidic to alkaline (> 85.0 % activity at pH 4.0-9.0, > 70.0 % activity at pH 10.0 and 43.7 % even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co^2 +, Cu^2 +, Mn^2 + and ¦Â-mercaptoethanol and was strongly inhibited by Hg^2 + and SDS. The specific activity, K_m and V_max values were 47.5 U mg^?#xA0;1, 7.8 mg ml^?#xA0;1 and 70.4 ¦Ìmol min^?#xA0;1 mg^?#xA0;1, respectively, for locust bean gum, and 40.3 U mg^?#xA0;1, 2.3 mg ml^?#xA0;1 and 61.7 ¦Ìmol min^?#xA0;1 mg^?#xA0;1, respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries.