一种来源于喜热梭孢壳的耐热型β-甘露聚糖酶  

Characterization of a hyperthermostableβ-mannanase from Thermothelomyces thermophilus

作　　者：施鑫磊 方韵颖 尉俏女 王佳堃[2] 斯越秀 陈忠法[1] 钱国英[1] 尹尚军[1] 王谦 Xinlei Shi;Yunying Fang;QiaonüWei;Jiakun Wang;Yuexiu Si;Zhongfa Chen;Guoying Qian;Shangjun Yin;Qian Wang(College of Biological&Environmental Sciences,Zhejiang Wanli University,Ningbo 315100,Zhejiang Province,China;College of Animal Sciences,Zhejiang University,Hangzhou 310058,Zhejiang Province,China;School of Basic Medical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang Province,China)

机构地区：[1]浙江万里学院生物与环境学院,浙江宁波315100 [2]浙江大学动物科学学院,浙江杭州310058 [3]浙江中医药大学基础医学院,浙江杭州310053

出　　处：《微生物学报》2021年第9期2815-2828,共14页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31902201);浙江省教育厅一般科研项目(Y201840329);浙江省“生物工程”一流学科开放基金(KF2020005)。

摘　　要：【目的】本文旨在挖掘满足工业加工所需的新型耐热β-甘露聚糖酶。【方法】从丝状真菌喜热梭孢壳中克隆甘露聚糖酶基因Mtman1,利用毕赤酵母表达系统进行异源表达,研究重组酶的酶学性质和热处理过程中蛋白构象变化情况。【结果】序列分析显示,Mtman1基因编码409个氨基酸,包含一段20个氨基酸的信号肽序列和一个GH5家族结构域;经毕赤酵母表达的重组MtMAN1蛋白约为60 kDa,存在N-糖基化修饰,该酶的最适反应pH和温度分别为6.0和70℃,催化刺槐豆胶底物的K_(m)和V_(max)分别为4.28±0.73 mg/mL和203.9±14.61μmol/(s·mg);MtMAN1在60℃下十分稳定,在80℃、90℃和100℃下处理10 min后,分别能维持(68.23±7.47)%、(56.01±5.69)%和(14.91±2.92)%的残余活性,且二级结构和最大发射波长基本没有发生变化,提示其构象维持稳定;此外,该酶对较低浓度的Fe^(2+)(<0.1 mmol/L)、Cu^(2+)(<0.1 mmol/L)、Ca^(2+)(<0.5 mmol/L)、Mg^(2+)(<0.1 mmol/L)或Zn^(2+)(<0.1 mmol/L)耐受能力较强。【结论】MtMAN1是一种耐热的β-甘露聚糖酶,在饲料制粒等需要高温处理的工业加工工艺中具有较好的应用前景。[Objective]This study aims to obtain a novel thermostable mannanase to satisfy industrial application.[Methods]Sequence analysis revealed that the Mtman1 encodes for 409 amino acids(AAs),including a signal peptide and a glycoside hydrolase(GH)5 catalytic domain.Recombinant MtMAN1 heterologously expressed in Pichia pastoris showed an optimal pH and temperature of 6.0 and 70℃,respectively.The K_(m) and V_(max) values of MtMAN1 were 4.28±0.73 mg/mL and 203.9±14.61μmol/s·mg against locust bean gum,respectively.[Results]The MtMAN1 was stable under 60℃incubation for 1 h.After treatment at 80℃,90℃and 100℃for 10 min,the enzyme maintained(68.23±7.47)%,(56.01±5.69)%and(14.91±2.92)%residual activities,respectively.Interestingly,its secondary structures and maximum wavelength remained as unchanged,suggesting relatively stable conformational structure.Additionally,the MtMAN1 was considerably resilient to Fe^(2+)(<0.1 mmol/L),Cu^(2+)(<0.1 mmol/L),Ca^(2+)(<0.5 mmol/L),Mg^(2+)(<0.1 mmol/L)or Zn^(2+)(<0.1 mmol/L).[Conclusion]MtMAN1 is a hyperthermostableβ-mannanase and could be a candidate for handling industry tanks at high temperatures,such as feed pelleting.

关 键 词：喜热梭孢壳 甘露聚糖酶 热稳定性 聚沉 二级结构 

分 类 号：S816.7[农业科学—饲料科学]