论文总字数：20804字

摘 要

本课题前期通过甲壳素吸附解析的方法，从几丁质高效降解菌Chitinolyticbacter meiyuanensis SYBC-H1中挖掘到一个新型双功能几丁质酶CmChi1，其同时具有外切几丁质酶活性和N-乙酰氨基葡萄糖苷酶活性，可以实现单酶法将几丁质转化为单一的N-乙酰氨基葡萄糖。经氨基酸序列分析发现，几丁质酶CmChi1含有一个信号肽序列(signal peptide，SP)、两个几丁质结合域(CBM-A 和CBM-B)和一个GH18家族催化结构域，是一个单催化域双功能的几丁质酶。为进一步研究其双功能催化活性是否由同一个活性中心完成，猜测CmChi1酶活性中心谷氨酸残基Glu406为活性位点。为研究该酶的催化机制，本实验将该酶的活性位点的酸性氨基酸Glu406突变为丙氨酸Ala和谷氨酰胺Gln，构建CmChi1活性突变体(E406A和E406Q)，并进行异源表达。通过镍柱纯化得到纯酶，以胶体几丁质和几丁二糖为底物对野生型酶和突变酶进行酶活测定。结果发现突变体酶完全丧失了双功能催化活性。由此证明了CmChi1的外切几丁质酶活性和N-乙酰氨基葡萄糖苷酶活性都源于同一催化活性中心，也证明了CmChi1的确是一个单催化结构域双功能催化活性的新型几丁质酶。

关键词：双功能几丁质水解酶 CmChi1 活性中心 定点突变

Site-directed mutagenesis of the active site of the bifunctional chitinase CmChi1 in Chitinolyticbacter meiyuanensis

Abstract

A novel chitinase CmChi1 was purified from the culture supernatant of Chitinolyticbacter meiyuanensis SYBC-H1 by colloidal chitin affinity. It has exo-chitinase activity and N-acetylglucosaminidase activity. It can achieve single enzymatic conversion of chitin into single N-acetyl glucosamine. Amino acid sequence analysis found that Chitinase CmChi1 contains a signal peptide (SP), two chitin binding domains (CBM-A and CBM-B) and a GH18 family catalytic domain. It is a single catalytic domain bifunctional chitinase. To further investigate whether its bifunctional catalytic activity is accomplished by the same active center, we speculate that the glutamic acid residue Glu406 in the active site of CmChi1 is the active site. To study the catalytic mechanism of this enzyme, we mutated the acidic amino acid Glu406 of the active site of the enzyme into alanine Ala and glutamine Gln, constructed CmChi1 activity mutants (E406A and E406Q). And heterologous expression. We purified by nickel column, measured the enzymatic activity of wild-type enzymes and mutant enzymes using colloidal chitin and chitosan as substrates. As a result, it was found that the mutant enzyme completely lost the bifunctional catalytic activity. It was proved that both the exochitinase activity and the N-acetylglucosaminidase activity of CmChi1 originate from the same catalytic activity center. It also demonstrated that CmChi1 is indeed a novel chitinase with bifunctional catalytic activity of a single catalytic domain.

Keywords: Bifunctional chitinase;CmChi1;Active site;Site-directed mutagenesis

目 录

摘要 Ⅰ

Abstract Ⅱ

第一章 文献综述 1

1.1生物酶转化法生产N-乙酰氨基葡萄糖是目前几丁质研究和开发利用的热点之一 1

1.2双功能几丁质水解酶的研究现状 2

1.3目前关于单催化域双功能几丁质水解酶的催化机制尚不明晰 3

1.4问题提出以及研究意义 3

第二章 Chitinolyticbacter meiyuanensis双功能几丁质酶CmChi1活性中心的定点突变 4

2.1材料与方法 4

2.1.1实验试剂与仪器 4

2.1.2菌体总DNA的提取 5

2.1.3小量凝胶DNA回收 6

2.1.4质粒的提取 6

2.1.5大肠杆菌普通感受态细胞的制备 6

2.1.6转化感受细胞 6

2.1.7 CmChi1酶的定点突变、克隆及表达载体构建 7

2.1.8野生型、突变型重组酶酶活性测定 11

2.2结果与分析 17

2.2.1 CmChi1全长基因和活性中心定点突变基因扩增 17

2.2.2表达载体pET-29a( )-CmChi1的构建 18

2.2.3野生型、突变型重组酶的诱导表达 19

2.2.4野生型、突变型重组酶的纯化 19

2.2.5野生型、突变型重组酶的活性 19

2.3总结与展望 16

主要参考文献 18

致谢 22

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