纤维单胞菌Cellulomonas sp.中性β-半乳糖苷酶新基因的克隆、表达及酶学性质分析
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摘要
β-半乳糖苷酶通常被称为乳糖酶,它广泛存在于植物、细菌、真菌、放线菌以及动物肠道中。该酶能将乳糖水解为半乳糖和葡萄糖,并具有半乳糖苷的转移作用,主要用来治疗乳糖不耐受症,处理加工牛乳、乳清,生产低乳糖牛奶和乳制品。乳糖酶的研究具有重要的实践价值和理论意义。本论文的目的是从特殊环境分离产乳糖酶的菌株,克隆得到具有特殊性质的乳糖酶基因,为进一步的工业化生产提供材料。
本研究从来源于火焰山的土壤中筛选获得一株产乳糖酶的菌株Dm,通过对菌株生理生化特性的研究及16SrDNA的分子鉴定,初步认为该菌株属于纤维单胞菌属(Cellulomonas sp.)。
利用PCR(简并PCR,热不对称交错PCR(TAIL-PCR)和反向PCR)的方法,克隆得到乳糖酶基因,命名为galc。galc全长2076bp,(G+C)%含量72.9%,编码692个氨基酸,无信号肽序列。经过序列分析和结构预测,推测galc为属于糖基水解酶第42家族的乳糖酶基因,编码的蛋白GALC含有三个结构域。通过序列比对分析,galc与已报道的乳糖酶的序列相似性都在为40～60%之间,其中与来源于Arthrobacter sp. FB24相似性最高,为59%。可以初步确定galc为新的乳糖酶基因。本研究为首次在Cellulomonas sp.中克隆获得乳糖酶基因。
将galc分别在大肠杆菌Escherichia coli BL21(DE3)和Pichia pastoris GS115中进行了表达,并对原核表达的乳糖酶进行纯化和酶学性质分析。成熟蛋白的分子量为76kD,最适pH为6.4,最适温度47℃,比活为188.30 u/mg,动力学分析表明其Km为19.33mmol/L,Vmax为416.67nmol/min。
β-D-galactosidase (β-D-galactoside galactohydrolase, EC3.2.1.23) hydrolyzes the dlsaccharide lactose to glucose and galactose, also referred to as lactase. It is widely distributedin various microorganisms such as bacteria, fungi, and also in various Plants and animals’intestine. Lactase can be used for the treatment of‘lactose intolerance’, and the Production of low-lactose milk, whey and other dairy Products. Lactase study has important theoretical and practical value. In this study, novel genes encoding lactase with advantage properties were cloned and expressed from microorganism strains isolated from special enviroments, provide material for the further industrial production.
We have isolated a strain Dm from soil of The Flaming Mountain. The phylogenetic analysis of the 16s ribosomal DNA sequence from this strain resulted in that it can be classed in the genus cellulomonas sp. .
A PCR approach including degenerate PCR, thermal asymmetric interlaced (TAIL) PCR and IPCR we used to clone lactase gene galc. Sequences analysis reveals that the lactase gene with the length of 2076bp encoding a polypeptide of 692 amino acid residues. (G+C) % is 72.9%, and no signal peptide. Structure prediction indicated that galc belonged to the glycosyl hydrolase 42 family, and its protein GALC, have three domains. Use BLAST, the deduced amino acid sequence of this lactase showed 40～60% identities to the previously reported genes,and the highest is to Arthrobacter sp. FB24, is 59%. Suggesting the novelty of this lactase, and it is first reported from Cellulomonas sp.
The lactase gene galc was expressed in Escherichia coli BL21 (DE3) and Pichia pastoris GS115 and the properties of prokaryotic expression lactase was purified and characterized. The purfied recombinant lactase had a molecular mass of 76kD. The optimum pH and temperature were 6.4 and 47℃, respectively. At 47℃and pH6.4, the Km was 19.33mmol/L and the Vmax was 416.67nmol/min. Specific activity of galc was about 188.2954 u/mg.
本研究从来源于火焰山的土壤中筛选获得一株产乳糖酶的菌株Dm,通过对菌株生理生化特性的研究及16SrDNA的分子鉴定,初步认为该菌株属于纤维单胞菌属(Cellulomonas sp.)。
利用PCR(简并PCR,热不对称交错PCR(TAIL-PCR)和反向PCR)的方法,克隆得到乳糖酶基因,命名为galc。galc全长2076bp,(G+C)%含量72.9%,编码692个氨基酸,无信号肽序列。经过序列分析和结构预测,推测galc为属于糖基水解酶第42家族的乳糖酶基因,编码的蛋白GALC含有三个结构域。通过序列比对分析,galc与已报道的乳糖酶的序列相似性都在为40～60%之间,其中与来源于Arthrobacter sp. FB24相似性最高,为59%。可以初步确定galc为新的乳糖酶基因。本研究为首次在Cellulomonas sp.中克隆获得乳糖酶基因。
将galc分别在大肠杆菌Escherichia coli BL21(DE3)和Pichia pastoris GS115中进行了表达,并对原核表达的乳糖酶进行纯化和酶学性质分析。成熟蛋白的分子量为76kD,最适pH为6.4,最适温度47℃,比活为188.30 u/mg,动力学分析表明其Km为19.33mmol/L,Vmax为416.67nmol/min。
β-D-galactosidase (β-D-galactoside galactohydrolase, EC3.2.1.23) hydrolyzes the dlsaccharide lactose to glucose and galactose, also referred to as lactase. It is widely distributedin various microorganisms such as bacteria, fungi, and also in various Plants and animals’intestine. Lactase can be used for the treatment of‘lactose intolerance’, and the Production of low-lactose milk, whey and other dairy Products. Lactase study has important theoretical and practical value. In this study, novel genes encoding lactase with advantage properties were cloned and expressed from microorganism strains isolated from special enviroments, provide material for the further industrial production.
We have isolated a strain Dm from soil of The Flaming Mountain. The phylogenetic analysis of the 16s ribosomal DNA sequence from this strain resulted in that it can be classed in the genus cellulomonas sp. .
A PCR approach including degenerate PCR, thermal asymmetric interlaced (TAIL) PCR and IPCR we used to clone lactase gene galc. Sequences analysis reveals that the lactase gene with the length of 2076bp encoding a polypeptide of 692 amino acid residues. (G+C) % is 72.9%, and no signal peptide. Structure prediction indicated that galc belonged to the glycosyl hydrolase 42 family, and its protein GALC, have three domains. Use BLAST, the deduced amino acid sequence of this lactase showed 40～60% identities to the previously reported genes,and the highest is to Arthrobacter sp. FB24, is 59%. Suggesting the novelty of this lactase, and it is first reported from Cellulomonas sp.
The lactase gene galc was expressed in Escherichia coli BL21 (DE3) and Pichia pastoris GS115 and the properties of prokaryotic expression lactase was purified and characterized. The purfied recombinant lactase had a molecular mass of 76kD. The optimum pH and temperature were 6.4 and 47℃, respectively. At 47℃and pH6.4, the Km was 19.33mmol/L and the Vmax was 416.67nmol/min. Specific activity of galc was about 188.2954 u/mg.
引文
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