【作者】 连文浩；

【导师】 林娟；

【作者基本信息】 福州大学 ， 微生物学， 2015， 硕士

【摘要】 几丁质(chitin)又名甲壳素,是乙酰-D-氨基葡萄糖(NAG)单糖脱水缩合通过β-1,4糖苷键聚合而成的大分子多糖。几丁质酶可将大分子的几丁质催化水解成小分子的几丁寡糖或乙酰-D-氨基葡萄糖。通过酶法降解贝壳类废弃物生产几丁寡糖能够实现废弃物的高价值利用,增加经济效益。本文从15个环境样品中筛选得到40株产几丁质酶微生物,获得了 25条几丁质酶基因片段,克隆其中两条基因全长,并对这两条基因进行表达和性质研究,研究结果如下:1、从一些特殊环境(如广西北海红树林、大布苏盐碱湖等)采集样品,从中筛选得到40株能够分解几丁质产生透明圈的菌株。经分子生物学鉴定,筛选到的产酶微生物主要有弧菌属(Vibriosp.),产微球茎菌属(Microbulb ifersp.),气单胞菌属(Aeromonas sp.)等。对几丁质酶粗酶性质研究表明,其最适反应温度普遍在40℃～60℃之间,最适pH在5.0～6.0之间。2、选择酶活力较高或酶学性质特异的菌株,设计简并引物,扩增几丁质酶基因片段,通过测序分析,共得到25条基因片段。序列分析表明这些基因片段与已知序列的一致性在67%～100%。其中,chiG31-2基因的一致性只有67%,属于比较新颖的基因。3、利用TAIL-PCR方法从菌株Vibriosp.GR52中克隆得到两个几丁质酶全长基因chiGR52-1和chiGR52-2。序列分析表明这两个基因全长分别为2553 bp与2385 bp,各编码850与794个氨基酸。Blastp分析表明这两个几丁质酶蛋白均与Vibrio fluvialis来源的几丁质酶一致性最高,分别为80%和78%,属于比较新颖的酶蛋白。4、几丁质酶基因chiGR52-1和chiGR52-2分别在大肠杆菌中实现了异源表达,并利用镍柱对重组蛋白进行纯化。重组蛋白rChiGR52-1最适反应pH和温度分别为6.0和50℃,在45℃以下及pH5.0～10.0稳定性良好;以胶体几丁质为底物时Vmax为4.829μmol/(mg·min),Km为1.302 mg/mL。rChiGR52-2最适反应pH和温度分别为6.0和50℃,以胶体几丁质为底物时Vmax为0.9606μmol/(mg·min),Km为2.777 mg/mL。本课题通过从不同环境中筛选产几丁质酶微生物,并进行基因克隆表达以及酶学性质的研究,旨在为寻找新的几丁质酶基因和几丁质酶的开发应用奠定基础。更多还原

【Abstract】 Chitin is a biotic macromolecular polymer of a N-acetyl-D-glucosamine(NAG)linked with β-1,4 glycosidic bonds.Chitinase catalyzes the hydrolysis of chitin into chitooligosaccharides and N-acetyl-D-glucosamine.By enzymatic degradation of shellfish waste to produce chitooligosaccharides,it can achieve high-value waste utilization and increase economic efficiency.In this study,40 microorganisms of chitinase-producing were isolated from 15 different samples.26 chitinase gene fragments were gained.Two chitinase genes were cloned,expressed and characterized.The details of results were listed as follows:(1)We collected samples from some special circumstances(such as Beihai mangroves,Dabusu salt lake etc.),and isolated 40 strains which had chitinase-produc ing capabilities.By 16s rDNA sequence analysis,isolated microorganisms of chitinase-producing were mainly belong to the genus such as Vibrio,Microbulbifer,Aeromonas.The characterization of chitinase crude enzyme showed that the optimum reaction temperature was generally between 40℃～60℃,the optimum pH was generally between 5.0～6.0.(2)Selected strains with higher enzyme activity or specific enzymatic properties,designed degenerate primers,used PCR to aplificate chitinase gene and then by sequencing analysis,25 gene fragments were obtained.Sequence analysis showed that these genes had identities from 67%to 100%with known sequences.Among them,the highest identities of chiG31-2 genes was only 67%,it was a relatively novel gene.(3)By TAIL-PCR,we cloned two full-length chitinase genes(chiGR52-1 and chiGR52-2)from Vibrio sp.GR52.Sequence analysis showed that two genes respectively have a total length of 2553 bp and 2385 bp,each of them encoded 850 amino acids and 794 amino acids.Blastp analysis showed that the two proteins were chitinase and the highest identities in known proteins were 80%and 78%,which sources from Vibrio fluvialis.They were relatively novel enzyme proteins.(4)Chitinase genes(chiGR52-land chiGR52-2)were expressed in Escherichia coli BL21(DE3),the recombinant proteins were purified by N i-affinity chromatography.The optima reaction pH and temperature of recombinant protein rChiGR52-1 was 6.0 and 50℃.And the enzyme was stable under 45℃ and pH5.0～10.0.By used colloidal chitin as substrate,the Vmax and Km was 4.829 μmol/(mg·min)and 1.302 mg/mL.For rChiGR52-2,the optima reaction pH and temperature was 6.0 and 50℃.By used colloidal chitin was as substrate,the Vmax and Km was 0.9606μmol/(mg-min)and 2.777 mg/mL.In this study,we isolated chitinase-producing microorganisms from various environments,and then cloned,expressed chitinase gene,studied the enzyme properties.These results may pave the way for mining new chitinase gene and the application of chitinase in industry in the future.更多还原