微孢根霉华根霉变种CICIM F0088生淀粉酶系的研究
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摘要
本研究从各类样品中分离得到365株具有淀粉水解能力的丝状真菌并保藏于江南大学中国高校工业微生物资源与信息中心,建立了一个丝状真菌资源库。从该资源库中筛选出一株具有较强生淀粉水解能力的混合淀粉酶系产生菌株CICIM F0088。结合形态学观察以及ITS序列同源性分析,确认该菌株为微孢根霉的华根霉变种(Rhizopus microsporus var. chinensis)。研究发现CICIM F0088菌株在固态和液态发酵时所产生的淀粉酶量和淀粉酶系的组成均存在差异。
本文首次对Rhizopus microsporus var. chinensis在固态和液态两种发酵方式下所产生的淀粉酶系进行系统的研究和比较。经过硫酸铵沉淀、双水相分离、离子交换层析、制备电泳和凝胶过滤层析等五种纯化方法,从CICIM F0088菌株的固态发酵产物中分离得到三个电泳纯的酶蛋白GA A,GA B和AA,酶蛋白的分子量依次为53、52和55 kDa;采用硫酸铵沉淀、离子交换层析和制备电泳等三种纯化方法,从液态发酵产物中分离得到两个电泳纯的酶蛋白GA C和GA D,酶蛋白分子量分别为53和52 kDa。
对从CICIM F0088菌株分离纯化得到的5个淀粉酶的酶学性质进行了研究,发现固态发酵时产生的葡萄糖淀粉酶GA A和GA B的温度耐受范围、pH耐受范围以及生淀粉水解能力均优于液态发酵产生的GA C和GA D;上述四个葡萄糖淀粉酶都具有一定的生淀粉水解能力,而固态发酵时产生的α-淀粉酶AA不具有该能力。
结合Rhizopus属来源的葡萄糖淀粉酶氨基酸保守序列及Rhizopus microsporus密码子偏好性等信息设计简并引物,利用PCR技术扩增得到CICIM F0088菌株葡萄糖淀粉酶编码基因的部分DNA序列,再通过反向PCR获得已知序列的侧翼序列,最终获得完整的编码基因gluE。以此为基础,通过RT-PCR的方法扩增出了该基因完整的cDNA序列。通过比较gluE基因的DNA和cDNA序列发现,gluE基因全长2763 bp,内部存在四个内含子;该基因的cDNA全长1818 bp,其中信号肽编码区81 bp,成熟肽编码区共编码579个氨基酸残基。将成熟肽的氨基酸序列与GenBank中其他微生物来源的葡萄糖淀粉酶一级结构进行同源性比对分析发现,该酶与所有已知葡萄糖淀粉酶的最高同源性为80%,是一种新的葡萄糖淀粉酶。
将葡萄糖淀粉酶基因gluE的cDNA序列连入pET-28a(+)表达载体,构建了重组质粒pET-28a-gluE,在Escherichia coli BL21 (DE3)中成功实现了异源活性表达。利用His-tag亲和层析分离纯化了表达后的重组酶蛋白GA E,对重组酶的酶学性质进行了研究,发现其具有水解生淀粉的能力。
A newly isolated active producer of raw-starch-digesting amyloytic enzymes, CICIM F0088, was isolated from the strains library including 365 fungi able to digest starch, and was identified by morphological characteristics and molecular phylogenetic analyses. This fungus was kept in the Culture and Information Center of Industrial Microorganism of China University (http://cicim-cu.jiangnan.edu.cn) at JiangNan University as Rhizopus microsporus var. chinensis CICIM F0088. The amount of enzymes production and their compositions were different between solid-state fermentation (SSF) and submerged fermentation (SmF).
For the first time, comparative research of amyloytic enzymes produced in SSF and SmF was carried out in fungus Rhizopus microsporus. Two glucaomylases named as GA A and GA B, and oneα-amylase named as AA from SSF were purified to homogeneity through ammonium sulfate precipitation, aqueous two-phase systems, Toyopearl DEAE-650M chromatography, prep cell and Toyopearl HW-55F gel filtration chromatography. The mocular weights of three proteins were 53, 52, and 55 kDa, respectively. In addition, two amyloytic enzymes from SmF were also purified to homogeneity by the procedures of ammonium sulfate precipitation, Toyopearl DEAE-650M chromatography, prep cell, separately named as GA C and GA D. The mocular weights of two proteins were 53, and 52 kDa, respectively.
Glucoamylases produced from SSF were much more thermostable and pH tolerate than enzymes produced from SmF. Raw-starch-digesting abilities were found in all the four glucoamylases except theα-amylase.
Based on the analysis of conserved amino acid sequences from Rhizopus sp., and the codon preference of Rhizopus microsporus, DNA sequence of gluE was amplified, and then the cDNA sequence of gluE was amplified by RT-PCR. Results showed a 2763 bp gluE gene including 4 introns was cloned. The deduced unprocessed glucoamylase from CICIM F0088 was 579 amino acids long and shows 80% identity to the Rhizopus glucoamylase. It was the first time of glucoamylase gene cloning from Rhizopus microsporus.
GA E cDNA fragment encoding mature peptide was inserted into the plasmid pET-28a(+) and expressed in E. coli BL21. The purified recombinant enzyme displayed raw starch digesting ability.
本文首次对Rhizopus microsporus var. chinensis在固态和液态两种发酵方式下所产生的淀粉酶系进行系统的研究和比较。经过硫酸铵沉淀、双水相分离、离子交换层析、制备电泳和凝胶过滤层析等五种纯化方法,从CICIM F0088菌株的固态发酵产物中分离得到三个电泳纯的酶蛋白GA A,GA B和AA,酶蛋白的分子量依次为53、52和55 kDa;采用硫酸铵沉淀、离子交换层析和制备电泳等三种纯化方法,从液态发酵产物中分离得到两个电泳纯的酶蛋白GA C和GA D,酶蛋白分子量分别为53和52 kDa。
对从CICIM F0088菌株分离纯化得到的5个淀粉酶的酶学性质进行了研究,发现固态发酵时产生的葡萄糖淀粉酶GA A和GA B的温度耐受范围、pH耐受范围以及生淀粉水解能力均优于液态发酵产生的GA C和GA D;上述四个葡萄糖淀粉酶都具有一定的生淀粉水解能力,而固态发酵时产生的α-淀粉酶AA不具有该能力。
结合Rhizopus属来源的葡萄糖淀粉酶氨基酸保守序列及Rhizopus microsporus密码子偏好性等信息设计简并引物,利用PCR技术扩增得到CICIM F0088菌株葡萄糖淀粉酶编码基因的部分DNA序列,再通过反向PCR获得已知序列的侧翼序列,最终获得完整的编码基因gluE。以此为基础,通过RT-PCR的方法扩增出了该基因完整的cDNA序列。通过比较gluE基因的DNA和cDNA序列发现,gluE基因全长2763 bp,内部存在四个内含子;该基因的cDNA全长1818 bp,其中信号肽编码区81 bp,成熟肽编码区共编码579个氨基酸残基。将成熟肽的氨基酸序列与GenBank中其他微生物来源的葡萄糖淀粉酶一级结构进行同源性比对分析发现,该酶与所有已知葡萄糖淀粉酶的最高同源性为80%,是一种新的葡萄糖淀粉酶。
将葡萄糖淀粉酶基因gluE的cDNA序列连入pET-28a(+)表达载体,构建了重组质粒pET-28a-gluE,在Escherichia coli BL21 (DE3)中成功实现了异源活性表达。利用His-tag亲和层析分离纯化了表达后的重组酶蛋白GA E,对重组酶的酶学性质进行了研究,发现其具有水解生淀粉的能力。
A newly isolated active producer of raw-starch-digesting amyloytic enzymes, CICIM F0088, was isolated from the strains library including 365 fungi able to digest starch, and was identified by morphological characteristics and molecular phylogenetic analyses. This fungus was kept in the Culture and Information Center of Industrial Microorganism of China University (http://cicim-cu.jiangnan.edu.cn) at JiangNan University as Rhizopus microsporus var. chinensis CICIM F0088. The amount of enzymes production and their compositions were different between solid-state fermentation (SSF) and submerged fermentation (SmF).
For the first time, comparative research of amyloytic enzymes produced in SSF and SmF was carried out in fungus Rhizopus microsporus. Two glucaomylases named as GA A and GA B, and oneα-amylase named as AA from SSF were purified to homogeneity through ammonium sulfate precipitation, aqueous two-phase systems, Toyopearl DEAE-650M chromatography, prep cell and Toyopearl HW-55F gel filtration chromatography. The mocular weights of three proteins were 53, 52, and 55 kDa, respectively. In addition, two amyloytic enzymes from SmF were also purified to homogeneity by the procedures of ammonium sulfate precipitation, Toyopearl DEAE-650M chromatography, prep cell, separately named as GA C and GA D. The mocular weights of two proteins were 53, and 52 kDa, respectively.
Glucoamylases produced from SSF were much more thermostable and pH tolerate than enzymes produced from SmF. Raw-starch-digesting abilities were found in all the four glucoamylases except theα-amylase.
Based on the analysis of conserved amino acid sequences from Rhizopus sp., and the codon preference of Rhizopus microsporus, DNA sequence of gluE was amplified, and then the cDNA sequence of gluE was amplified by RT-PCR. Results showed a 2763 bp gluE gene including 4 introns was cloned. The deduced unprocessed glucoamylase from CICIM F0088 was 579 amino acids long and shows 80% identity to the Rhizopus glucoamylase. It was the first time of glucoamylase gene cloning from Rhizopus microsporus.
GA E cDNA fragment encoding mature peptide was inserted into the plasmid pET-28a(+) and expressed in E. coli BL21. The purified recombinant enzyme displayed raw starch digesting ability.
引文
本章部分内容已发表于《工业微生物》, 2010, 40(1):68-72;
本章部分内容已被《食品工业科技》所接受.
本章部分内容已发表于《食品与发酵工业》, 2010, 36(1):1-4;
本章部分内容已被《应用与环境生物学报》所接受.
本章部分内容已发表于《Journal of Microbiology and Biotechnology》, 2010, 20(2):383-390.
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81. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the folin-phenol reagent [J]. J Biol Chem, 1951, 193: 265-275.
82.李彧娜,石贵阳,王武等.利用Native制备电泳分离来源于Rhizopus microsporus var. chinensis的葡萄糖淀粉酶同工酶[J].食品与发酵工业, 2010, 36(1): 53-56.
83. Clark A H. Direct analysis of experimental tie line data (two polymer–one solvent systems) using Flory–Huggins theory [J]. Carbohyd Polym, 2000, 42(4): 337-351.
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88. Nguyen CH, Tsurumizu R, Sato T, et al. Taka-amylase A in the conidia of Aspergillus oryzae RIB40 [J]. Biosci Biotechnol Biochem, 2005, 69:2035-2041.
89. Hamilton L M, Kelly C T, Fogarty W M. Production and properties of the raw starch-digestingα-amylase of Bacillus sp. IMD 435 [J]. Process Biochem, 1999, 35: 27-31.
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95. Sauer JC, T Frandsen. Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger [J]. Biochemistry, 2001, 40(31): 9336-9346.
96. Svensson B, Larscn K,et al. The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger [J]. I. & Bocl, E, 1983, 48: 529-544.
97.李育阳.基因表达技术[M].科学出版社, 2001.
98. Martinez A, Krappskreg P M, Olafsdottir S, et al. Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases [J]. J Biotechnol, 1995, 306: 589-597.
99. Luli G W, Strolh W R. Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations [J]. Appl Environ Microbiol, 1990, 56: 1004-1011.
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