【作者】 杨梦华；

【导师】 李颖；

【作者基本信息】 中国农业大学 ， 微生物学， 2005， 硕士

【摘要】 本研究以海栖热袍菌（Thermotoga maritima）MSB8菌株基因组DNA为模板,通过PCR获得木聚糖酶（XylanaseB）基因mxynB₆₄和mxynB₅₇₇。经与Karen E.Nelson等人在GertBank中注册的木聚糖酶基因xynB（AF100063）进行同源性比较得知,mxynB₆₄的第64位碱基由腺嘌呤（A）转变为鸟嘌呤（G）,因此,该基因产物的第22位氨基酸由原来的天冬酰胺（Asn）转变为天冬氨酸（Asp）;而mxynB₅₇₇的第577位碱基由胸腺嘧啶（T）转变为胞嘧啶（c）,该基因产物的第193位氨基酸也由原来的酪氨酸（Tyr）转变为组氨酸（His）。将木聚糖酶基因mxynB₆₄克隆至表达载体pET-28a（+）,转化大肠杆菌。该基因可在大肠杆菌细胞内高效表达。其表达产物具有酶活性高、耐90℃高温、纯化方便等特点,采用RBB-木聚糖法测定酶活为121.6U/mg蛋白,但其表达产物不能分泌至细胞外。 分别将木聚糖酶基因mxynB₆₄和mxynB₅₇₇克隆至表达载体pPIC9K,通过转化整合至巴斯德毕赤酵母（Pichiapastoris）GS115菌株基因组中。摇瓶培养发现,重组木聚糖酶mXynB₆₄和mXynB₅₇₇分泌表达量较高,在甲醇诱导培养108小时后,胞外木聚糖酶酶活分别可达到0.6U/ml和0.5U/ml,并具有良好的酶学性质。两种重组木聚糖酶的最适反应温度分别为90℃和80℃,并在pH 5.0-10.8范围内稳定,推测该酶的第193位氨基酸残基仅与酶的耐热性有关,并不影响其pH稳定性。在5升自动发酵罐上对毕赤酵母重组菌株GS115-9K-mxynB₆₄Ⅱ进行高密度甲醇诱导产酶培养,经108小时培养后其细胞密度可达OD₆₀₀=144,酶活水平达到6U/ml,高于摇瓶水平10倍,适于工业化应用。 本实验通过PCR分别获得了多形汉逊酵母（Hansenula polymorpha）中的3个启动子序列,并将酿酒酵母α因子分泌信号肽序列和整合外源基因(mxynB₆₄)的rDNA序列组合,成功构建四种可在大肠杆菌和汉逊酵母中穿梭表达的载体,包括含有诱导型启动子的pMOXp-mxynB₆₄（10.6Kb）,pFMDP-mxynB₆₄（9.7Kb）,pAOX1p-mrynB₆₄（9.8Kb）和含有组成型启动子的pPMA1p-mxynB₆₄（11.1Kb）。将它们分别转化并整合至多形汉逊酵母A16菌株基因组中,摇瓶培养发现,重组耐高温木聚糖酶的分泌表达量比毕赤酵母略低,在摇瓶培养诱导108小时后,多形汉逊酵母重组菌株中,具有诱导型启动子的菌株A16-pMOXp-mxynB₆₄胞外木聚糖酶活力最高,达0.25U/ml,但比毕赤酵母菌株GS115-9K-mxynB₆₄Ⅱ的分泌水平低一倍。在7.5升自动发酵罐上的高密度培养结果证明,组成型表达的汉逊酵母菌株A16-pPMA1p-mxynB₆₄具有不需要甲醇诱导、生长迅速的特点,具有工业化应用的潜力。更多还原

【Abstract】 In this study, Two mutated xylanase genes were obtained from PCR by using the genomic DNA of Thermotoga maritima MSB8 as template, termed as mxynB（64） and mxynB（577）, which represented that the bases of the 64^th and 577^th sites of the gene xynB muftated from A to G and from T to C and the amino acids mutated correspondingly from Asn²² to Asp and from Tyr¹⁹³ to His respectively, by comparing with the xylanase gene published in GenBank with the registration number of AE001693. With the assay of the advanced structure of xylanaseB by bioinformatics, vaxynB（64） was cloned into E. coli expression vector pET-28a （+） , and was successfully expressed in E. coli BL21 as solublef protein product. The recombinant mXynB_（64） in E. coli showed great enzyme activity which reach to 121.6U/ml （tested by RBB-xylans methods）, and it showed high activity in 90℃. The thermostability of this protein offered it to be purified easily. But the recombinant mXynB_（64） expressed in E. coli could not be secreted out of the cell which would be an obstacle for it to be applied in practice.Both of the two xylanase genes, vaxynB_<sub>（64） and mxynB_（577） were inserted into the shuttle and integrated expression vector pPIC9k respectively and expressed by Pichia pastoris as an active extracellular product efficiently. The two recombinant xylanases expressed in P. pastoris still show extreme thermostability and pH stability. mXynB_（64） and mXynB_（577） were optimally active at 90℃ and 80℃ respectively and both quite stable over the pH range of pH 5.0-10.8 at 70℃, from which that the mutated amino acid in mXynB_（577） only affected the thermostability of this enzyme but none on its pH properties could be suggested. In the conditions of high cell density cultivation, P. pastoris recombinant strain GS115-9K- mxynB_（64） II could secreted the recombinant protein of mXynB_（64） efficiently, which indicated that it was of great use in a variety of industrial and agricultural applications.Another methylotrophic yeast, Hansenula polymorpha, expression system was also established in this study. Three promoter sequences were obtained by PCR, and integrated into the four H. polymorpha shuttle and integrated expression vectors with the xylanase expression cassette, among which pMOX_p-mxynB_（64） （10.6Kb） , pFMD_p-mxynB_（64） （9.7Kb） , pAOX1_p-mxynB_（64） （9.8Kb） were of inducible promoters, while pPMA1_p-mxynB_（64） （11.1Kb） , constitutive promoter.. The four H. polymorpha expression vectors were used to heterologously express mxynB₍（64） gene in H. polymorpha A16. After cultivation in shake flash for 108 hours, the H. polymorpha recombinant strain of A16-pMOX_p-mxynB_（64） showed the highest intercellular xylanase activity of 0.25U/ml. High cell fermentation of H. polymorpha recombinant strain, A16- pPMA1_p-mxynB_（64）, showedthat the relatively simple fermentation process would make better sense in future industrial application.更多还原