嗜热甘露聚糖酶毕赤酵母工程菌的表达及该酶在果汁澄清中的应用  

Expression in Pichia pastoris and Characterization of a Thermophilic Mannanase and Its Application in Juice Clarification

作　　者：陈伟 谷新晰[1] 黄蕾[1] 李晨[1] 田洪涛[1] 卢海强[1] CHEN Wei;GU Xinxi;HUANG Lei;LI Chen;TIAN Hongtao;LU Haiqiang(College of Food Science and Technology,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北农业大学食品科技学院

出　　处：《食品科学》2019年第22期81-87,共7页Food Science

基　　金：国家自然科学基金青年科学基金项目(31501417);河北省食品科学与工程学科“双一流”建设资金项目(2016SPGCA18)

摘　　要：探究嗜热真菌Neosartorya sp.HBFH9中甘露聚糖酶NsMan5B的酶学性质及其在果汁加工中的应用潜力。本研究从菌株HBFH9中获得nsMan5B基因,该基因全长1491 bp(2个内含子),cDNA全长1371 bp,编码456个氨基酸和1个终止密码子。NsMan5B酶分子由1个信号肽序列、碳水化合物结合序列、linker序列和催化区组成。重组NsMan5B的最适反应温度为60℃,最适反应pH 4.0,在pH 2.0~10.0和50℃条件下稳定。NsMan5B成熟蛋白分子质量约为49 kDa,不存在糖基化修饰。金属离子和化学试剂对该酶活性的影响差异较大,其中K^+、Mg^2+和Cu^2+等离子对酶活性促进较明显,分别使酶活力提高了271%、123%和100%。随着醇类分子碳链的延长和浓度的增加,醇类物质对重组酶NsMan5B活性的抑制作用增强,其中在正丁醇体积分数为2.5%条件下,使该酶活性降低了86%。重组NsMan5B酶对柿子汁、苹果汁、桃子汁、葡萄汁和橘子汁澄清实验结果显示,除橘子汁外,重组酶对其他果汁的澄清度都有不同程度的提高,其中柿子汁澄清度提高了31.8%,其他3种果汁的澄清度分别提高7%、4%和4%。综上所述,甘露聚糖酶NsMan5B在果汁加工中具有较好的应用前景。Aβ-mannanase gene,nsMan5B,was cloned from the thermophilic fungus Neosartorya sp.HBFH9 and successfully expressed in Pichia pastoris.Sequence analysis indicated that the complete DNA sequence of nsMan5B was 1491 bp in length(containing 2 Introns).The cDNA sequence of nsMan5B was 1371 bp in length,which could encode a protein of 456 amino acids with a molecular mass of 49 kDa.Structural analysis indicated the presence of a putative signal peptide,a carbohydrate-binding module of family 1(CBM 1),a linker region and a catalytic domain of GH5 in NsMan5B.The recombinant NsMan5B was optimally active at pH 4.0 and 60℃,and had good stability over the wide range of pH 2.0–10.0 and at 50℃or below.The effects of metal ions and chemical reagents on its enzymatic activity were different.The enzymatic activity was increased by 271%,123%and 100%in the presence of K^+,Mg^2+and Cu^2+,respectively.With increasing carbon chain length and concentration of alcohol,the inhibitory effect on NsMan5B activity was markedly enhanced.Its enzymatic activity was reduced by 86%by n-butanol(2.5%,V/V).It was observed that NsMan5B was effective in the clarification of orange juice as well as persimmon,apple,peach and grape juices,whose clarity was increased by 31.8%,7%,4%and 4%,respectively.Consequently,it was concluded that the NsMan5B could be successfully used in the fruit juice industry.

关 键 词：甘露聚糖酶 果汁澄清 异源表达 食品酶 嗜热酶 

分 类 号：Q789[生物学—分子生物学]