雅致放射毛霉的分离鉴定及其羧肽酶Y的基因克隆与表达
发布时间：2018-07-31 20:49
【摘要】：腐乳白点是毛霉蛋白酶水解腐乳大豆蛋白形成过饱和游离氨基酸的结晶,主要成份为酪氨酸(L-Tyr)。腐乳中游离酪氨酸主要由毛霉内切蛋白酶和羧肽酶共同水解大豆蛋白产生,其中羧肽酶在这一过程中起主要作用。因此,羧肽酶对腐乳白点的形成有重要作用,但目前没有毛霉中羧肽酶基因序列的报道,无法对其进行深入研究。本论文从腐乳中鉴定出一株生产菌株雅致放射毛霉(Actinomucor elegans PEP001),并从雅致放射毛霉中调取羧肽酶Y(carboxypeptidase Y,CPY)的基因,同时研究了重组羧肽酶Y酶原(proCPY)在大肠杆菌(Escherichia coli Rosetta)和毕赤酵母(Pichia pastoris GS115)中的表达,并对重组酶进行了纯化、体外激活及酶学性质研究。主要研究结果包括:(1)鉴定了腐乳白点主要成份为氨基酸(占白点干重的92.6%),其中酪氨酸占氨基酸总量90%以上;检测腐乳后发酵过程中发酵前期与后期发酵液中游离氨基酸变化,发现后发酵后期发酵液中酪氨酸含量较后发酵前期增加了近9倍,说明酪氨酸大量积累是在腐乳后发酵过程中受蛋白酶,特别是外切蛋白酶作用下产生的。(2)从腐乳中分离、筛选出多种微生物并鉴定了一株腐乳发酵菌株,命名为雅致放射毛霉PEP001(A.elegans PEP001),并从毛霉发酵液中检测到可释放多肽C末端酪氨酸的羧肽酶活性,推测其可能为CPY;以亲缘性较近来源的真菌中CPY氨基酸保守序列设计简并引物调取部分目的基因序列,利用cDNA末端快速扩增技术(RACE)获得基因全长信息:基因全长1557个碱基,编码全长为518个氨基酸;通过软件分析预测:其由23个氨基酸的信号肽、67个氨基酸的前导肽与428个氨基酸的成熟酶构成,酶活性中心位点为S228、D434与H495。(3)proCPY在大肠杆菌中以包涵体形式表达;在毕赤酵母中成功地分泌表达。毕赤酵母表达的proCPY纯化后,通过胰蛋白酶体外激活获得成熟酶,酶活为157.2 U·mg-1。酶学性质分析表明,其最适反应pH为6.0,最适反应温度为45℃,在40℃下有较高稳定性,在60℃下很快失活,在pH 4.0-7.0下都有较高的稳定性。
[Abstract]:The white spot of sufu is the crystallization of supersaturated free amino acid formed by the hydrolysis of sufu soybean protein by Mucor protease. The main component is tyrosine (L-Tyr). The free tyrosine in sufu is mainly produced by the hydrolysis of soybean protein by Mucor endopepsin and carboxypeptidase, in which carboxypeptidase plays a major role. Therefore, carboxypeptidase plays an important role in the formation of white spots in sufu, but there is no report on the sequence of carboxypeptidase gene in Mucor. In this paper, a producing strain (Actinomucor elegans PEP001) was identified from sufu, and the gene of carboxypeptidase (Y (carboxypeptidase YPY) was obtained from Rhizopus rhinorrhoeae. The expression of recombinant carboxypeptidase Y (proCPY) in Escherichia coli (Escherichia coli Rosetta) and Pichia pastoris (Pichia pastoris GS115 was studied. The recombinant enzyme was purified and activated in vitro. The main results are as follows: (1) the main components of white spots of sufu were identified as amino acids (accounting for 92.6% of the dry weight of white spots), in which tyrosine accounted for more than 90% of the total amino acids. It was found that the content of tyrosine in the fermentation broth at the later stage of post-fermentation was increased by nearly 9 times compared with that in the early stage of post-fermentation, which indicated that the accumulation of tyrosine was produced by protease, especially the exo-protease, in the process of post-fermented fermentation. (2) Separation of tyrosine from sufu. A variety of microbes were screened and a suckling fermentation strain named PEP001 (A.elegans PEP001) was identified. The carboxypeptidase activity of C terminal tyrosine was detected from the fermentation broth of Mucor. The conserved sequence of CPY amino acids in fungi with close genetic origin was used to design degenerate primers to extract some of the target gene sequences, and the full length of the gene was obtained by cDNA terminal rapid amplification technique (RACE): the gene was 1557 bases in length. It is predicted by software analysis that it consists of 23 amino acid signal peptides, 67 amino acid precursor peptides and 428 amino acid mature enzymes. The enzyme activity sites were S228D434 and H4955.3.The proCPY was expressed as inclusion body in Escherichia coli and secreted successfully in Pichia pastoris. After purification of proCPY expressed by Pichia pastoris, the mature enzyme was obtained by activation of trypsin in vitro. The enzyme activity was 157.2 U mg-1. The analysis of enzymatic properties showed that the optimum reaction pH was 6.0, the optimum reaction temperature was 45 鈩，
本文编号：2156786
[Abstract]:The white spot of sufu is the crystallization of supersaturated free amino acid formed by the hydrolysis of sufu soybean protein by Mucor protease. The main component is tyrosine (L-Tyr). The free tyrosine in sufu is mainly produced by the hydrolysis of soybean protein by Mucor endopepsin and carboxypeptidase, in which carboxypeptidase plays a major role. Therefore, carboxypeptidase plays an important role in the formation of white spots in sufu, but there is no report on the sequence of carboxypeptidase gene in Mucor. In this paper, a producing strain (Actinomucor elegans PEP001) was identified from sufu, and the gene of carboxypeptidase (Y (carboxypeptidase YPY) was obtained from Rhizopus rhinorrhoeae. The expression of recombinant carboxypeptidase Y (proCPY) in Escherichia coli (Escherichia coli Rosetta) and Pichia pastoris (Pichia pastoris GS115 was studied. The recombinant enzyme was purified and activated in vitro. The main results are as follows: (1) the main components of white spots of sufu were identified as amino acids (accounting for 92.6% of the dry weight of white spots), in which tyrosine accounted for more than 90% of the total amino acids. It was found that the content of tyrosine in the fermentation broth at the later stage of post-fermentation was increased by nearly 9 times compared with that in the early stage of post-fermentation, which indicated that the accumulation of tyrosine was produced by protease, especially the exo-protease, in the process of post-fermented fermentation. (2) Separation of tyrosine from sufu. A variety of microbes were screened and a suckling fermentation strain named PEP001 (A.elegans PEP001) was identified. The carboxypeptidase activity of C terminal tyrosine was detected from the fermentation broth of Mucor. The conserved sequence of CPY amino acids in fungi with close genetic origin was used to design degenerate primers to extract some of the target gene sequences, and the full length of the gene was obtained by cDNA terminal rapid amplification technique (RACE): the gene was 1557 bases in length. It is predicted by software analysis that it consists of 23 amino acid signal peptides, 67 amino acid precursor peptides and 428 amino acid mature enzymes. The enzyme activity sites were S228D434 and H4955.3.The proCPY was expressed as inclusion body in Escherichia coli and secreted successfully in Pichia pastoris. After purification of proCPY expressed by Pichia pastoris, the mature enzyme was obtained by activation of trypsin in vitro. The enzyme activity was 157.2 U mg-1. The analysis of enzymatic properties showed that the optimum reaction pH was 6.0, the optimum reaction temperature was 45 鈩，
本文编号：2156786
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