發(fā)布時間：2018-07-31 20:49

【摘要】：腐乳白點是毛霉蛋白酶水解腐乳大豆蛋白形成過飽和游離氨基酸的結(jié)晶,主要成份為酪氨酸(L-Tyr)。腐乳中游離酪氨酸主要由毛霉內(nèi)切蛋白酶和羧肽酶共同水解大豆蛋白產(chǎn)生,其中羧肽酶在這一過程中起主要作用。因此,羧肽酶對腐乳白點的形成有重要作用,但目前沒有毛霉中羧肽酶基因序列的報道,無法對其進行深入研究。本論文從腐乳中鑒定出一株生產(chǎn)菌株雅致放射毛霉(Actinomucor elegans PEP001),并從雅致放射毛霉中調(diào)取羧肽酶Y(carboxypeptidase Y,CPY)的基因,同時研究了重組羧肽酶Y酶原(proCPY)在大腸桿菌(Escherichia coli Rosetta)和畢赤酵母(Pichia pastoris GS115)中的表達,并對重組酶進行了純化、體外激活及酶學(xué)性質(zhì)研究。主要研究結(jié)果包括:(1)鑒定了腐乳白點主要成份為氨基酸(占白點干重的92.6%),其中酪氨酸占氨基酸總量90%以上;檢測腐乳后發(fā)酵過程中發(fā)酵前期與后期發(fā)酵液中游離氨基酸變化,發(fā)現(xiàn)后發(fā)酵后期發(fā)酵液中酪氨酸含量較后發(fā)酵前期增加了近9倍,說明酪氨酸大量積累是在腐乳后發(fā)酵過程中受蛋白酶,特別是外切蛋白酶作用下產(chǎn)生的。(2)從腐乳中分離、篩選出多種微生物并鑒定了一株腐乳發(fā)酵菌株,命名為雅致放射毛霉PEP001(A.elegans PEP001),并從毛霉發(fā)酵液中檢測到可釋放多肽C末端酪氨酸的羧肽酶活性,推測其可能為CPY;以親緣性較近來源的真菌中CPY氨基酸保守序列設(shè)計簡并引物調(diào)取部分目的基因序列,利用cDNA末端快速擴增技術(shù)(RACE)獲得基因全長信息:基因全長1557個堿基,編碼全長為518個氨基酸;通過軟件分析預(yù)測:其由23個氨基酸的信號肽、67個氨基酸的前導(dǎo)肽與428個氨基酸的成熟酶構(gòu)成,酶活性中心位點為S228、D434與H495。(3)proCPY在大腸桿菌中以包涵體形式表達;在畢赤酵母中成功地分泌表達。畢赤酵母表達的proCPY純化后,通過胰蛋白酶體外激活獲得成熟酶,酶活為157.2 U·mg-1。酶學(xué)性質(zhì)分析表明,其最適反應(yīng)pH為6.0,最適反應(yīng)溫度為45℃,在40℃下有較高穩(wěn)定性,在60℃下很快失活,在pH 4.0-7.0下都有較高的穩(wěn)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，

本文編號：2156786