耐热β-甘露聚糖酶基因的克隆与表达及酶学性质

doi:10.3969/j.issn.1673-1689.2014.06.005

首页 > 过刊浏览>2014年第33卷第6期 >590-596. DOI:10.3969/j.issn.1673-1689.2014.06.005

耐热β-甘露聚糖酶基因的克隆与表达及酶学性质
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                        10.3969/j.issn.1673-1689.2014.06.005
                    
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Gene Cloning and Expression of a Thermostable β-Mannanase and Its Enzymatic Properties

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基于生物信息学和基因组学分析的结果，采用RT-PCR技术从宇佐美曲霉（Aspergillus usamii） E001中克隆出一种糖苷水解酶（GH） 26家族β-甘露聚糖酶（AuMan26A）成熟肽编码基因（Auman26A），成功实现其在毕赤酵母（Pichia pastoris） GS115中的异源表达。重组β-甘露聚糖酶（reAuMan26A）的发酵上清液对角豆胶的酶活性为281.9 U/mL，纯化后比酶活为2281.7 U/mg。其最适反应温度Topt为40 ℃；在80 ℃处理60 min后残留酶活性为60%；90 ℃时的半衰期t1/290为10 min，处理60 min后残留酶活性仍为33%；其最适pH为5.5，在pH 5.0~7.0的范围内较稳定；Fe2+和Fe3+对其有明显的抑制作用，其它所测金属离子和EDTA对其没有明显的影响；所测酶的最适底物为角豆胶，其次是魔芋粉和瓜尔豆胶，其对角豆胶的Km和Vmax值分别为15.25 mg/mL和7 841.9 U/mg。reAuMan26A良好的热稳定性使其在食品、饲料和纺织等工业具有广阔的应用前景。

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Based on the bioinformatics and genomics analysis, a glycoside hydrolase(GH) family 26 gene, Auman26A, which encodes β-mannanase mature peptide was cloned from Aspergillus usamii E001 by RT-PCR. Then, it was successfully expressed in Pichia pastoris GS115. The recombinant enzyme(reAuMan26A) activity of the fermentation supernatant towards locust bean gum was 281.9 U/mL. The specific activity of the purified enzyme was 2281.7 U/mg. The optimal temperature of reAuMan26A was 40 ℃. After incubating at 80 ℃ and 90 ℃ for 60 min, the residual activities were 60% and 33%, respectively. The half-life (t1/290) of reAuMan26A at 90 ℃ was 10 min. The optimal pH of reAuMan26A was 5.5, and it was stable over a range of pH 5.0~7.0. Its activity was not significantly affected by metal ions and EDTA, but inhibited by Fe2+ and Fe3+. Its most favorable substrate was locust bean gum, followed by konjac flour and guar gum. The Km and Vmax of reAuMan26A towards locust bean gum were 15.25 mg/mL and 7 841.9 U/mg, respectively. The superior properties of reAuMan26A, especially the excellent thermostability, make it have the broad application prospects in food, paper making and textile industry.

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赵梅,魏喜换,王春娟,董运海,李剑芳,邬敏辰.耐热β-甘露聚糖酶基因的克隆与表达及酶学性质[J].食品与生物技术学报,2014,33(6):590-596.

ZHAO Mei, WEI Xihuan, WANG Chunjuan, DONG Yunhai, LI Jianfang, WU Minchen. Gene Cloning and Expression of a Thermostable β-Mannanase and Its Enzymatic Properties[J]. Journal of Food Science and Biotechnology,2014,33(6):590-596.

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在线发布日期: 2014-10-19
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