黑曲霉酸性蛋白酶EXPA的克隆表达与酶学性质解析
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摘要
酸性蛋白酶在食品、酿造、饲料和皮革等行业具有重要的应用价值。然而,现有的酸性蛋白酶在50℃以上或pH> 3. 0时不稳定,限制了其应用范围。该研究通过分子克隆技术将黑曲霉CICIM F0510的酸性蛋白酶基因exp A在毕赤酵母中进行了克隆表达,构建获得了重组菌GS115 (p PIC-expA)。摇瓶发酵条件下,重组酶EXPA的酶活为257 380 RFU/h。生物信息学分析的结果显示,该酶属于天冬氨酸蛋白酶A1A家族。酶学性质的研究表明,该重组酶的最适反应温度和pH分别为50℃和3. 0;分别在40～50℃或pH 2. 5～3. 5孵育1 h后,仍能保留80%左右的活力。Zn~(2+)、Ca~(2+)、Fe~(2+)和Mn~(2+)对其活性有一定的促进作用;而Cu~(2+)、Co~(2+)、Fe~(3+)、EDTA和SDS则对其活性有显著的抑制作用。此外,重组酶EXPA对大豆分离蛋白、水溶性玉米蛋白和小麦水解蛋白均具有较好的水解作用。较好的耐热性和pH稳定性为EXPA在食品、饲料等领域的应用奠定了基础。
Acid protease has wide applications in foods,brewing,feeds,and leather industries. However,current acid proteases are unstable when the temperature or pH values are higher than 50 ℃ or 3. 0,respectively,which restricts their applications. Thus,acid protease encoding gene expA from Aspergillus niger CICIM F0510 was successfully cloned and expressed in Pichia pastoris,and the recombinant strain GS115(p PIC-expA) was generated in this study. In shaking flask experiments,the activity of recombinant enzyme EXPA was 257 380 RFU/h. Bioinformatics analysis showed that this enzyme belonged to family A1 A of aspartic proteases. The optimal temperature and pH of EXPA were 50 ℃ and 3. 0,respectively. After incubating at 40-50 ℃ or pH 2. 5-3. 5 for 1 h,the recombinant enzyme still retained more than 80% of its maximum activity. Zn~(2+),Ca~(2+),Fe~(2+),and Mn~(2+)enhanced EXPA activity,whereas Cu~(2+),Co~(2+),Fe~(3+),EDTA,and SDS had significant inhibitory effects on EXPA activity. Besides,EXPA also hydrolyzed soybean protein isolates,water-soluble corn protein,and wheat protein. Good thermostability and pH stability of EXPA provide a solid foundation for its applications in food and feed industries.
Acid protease has wide applications in foods,brewing,feeds,and leather industries. However,current acid proteases are unstable when the temperature or pH values are higher than 50 ℃ or 3. 0,respectively,which restricts their applications. Thus,acid protease encoding gene expA from Aspergillus niger CICIM F0510 was successfully cloned and expressed in Pichia pastoris,and the recombinant strain GS115(p PIC-expA) was generated in this study. In shaking flask experiments,the activity of recombinant enzyme EXPA was 257 380 RFU/h. Bioinformatics analysis showed that this enzyme belonged to family A1 A of aspartic proteases. The optimal temperature and pH of EXPA were 50 ℃ and 3. 0,respectively. After incubating at 40-50 ℃ or pH 2. 5-3. 5 for 1 h,the recombinant enzyme still retained more than 80% of its maximum activity. Zn~(2+),Ca~(2+),Fe~(2+),and Mn~(2+)enhanced EXPA activity,whereas Cu~(2+),Co~(2+),Fe~(3+),EDTA,and SDS had significant inhibitory effects on EXPA activity. Besides,EXPA also hydrolyzed soybean protein isolates,water-soluble corn protein,and wheat protein. Good thermostability and pH stability of EXPA provide a solid foundation for its applications in food and feed industries.
引文
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[21] LI J,CHI Z,WANG X. Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli[J]. Microbiological Research,2010,165(3):173-182.
[22]马纳纳,徐冬冬,黄伟谦,等.一种新颖的毕赤酵母酸性蛋白酶基因的克隆和异源表达以及酶学性质研究[J].广东农业科学,2015,42(12):141-146.
[23]王晓亮,周樱,张庆丽.酶制剂在猪生产中的应用[J].饲料工业,2013,34(6):23-25.
[2] SUNDARARAJAN S,KANNAN C N,CHITTIBABU S.Alkaline protease from Bacillus cereus VITSN04:Potential application as a dehairing agent[J]. Journal of Bioscience&Bioengineering,2011,111(2):128-133.
[3] OH S,CHANG W,SUH J. An aspartic protease analogue:intermolecular catalysis of peptide hydrolysis by carboxyl groups[J]. Bioorganic&Medicinal Chemistry Letters,2001,11(11):1 469-1 472.
[4] V M-GONZL,L V-T,MA A-R,et al. Secreted fungal aspartic proteases:A review[J]. Revista Iberoamericana De Micologia,2016,33(2):76-82.
[5]张美香,刘娜,蔡静平,等.微生物源酸性蛋白酶的研究进展[J].河南工业大学学报(自然科学版),2009,30(6):88-92.
[6] SOUZA P M,WERNECK G,ALIAKBARIAN B,et al.Production,purification and characterization of an aspartic protease from Aspergillus foetidus[J]. Food and Chemical Toxicology,2017,109:1 103-1 110.
[7] MURTHY P S,KUSUMOTO K-I. Acid protease production by Aspergillus oryzae on potato pulp powder with emphasis on glycine releasing activity:A benefit to the food industry[J]. Food and Bioproducts Processing,2015,96:180-188.
[8] WANG Y C,XUE W,SIMS A H,et al. Isolation of four pepsin-like protease genes from Aspergillus niger and analysis of the effect of disruptions on heterologous laccase expression[J]. Fungal Genetics and Biology,2008,45(1):17-27.
[9] LU J F,INOUE H,KIMURA T,et al. Molecular cloning of a c DNA for proctase B from Aspergillus niger var. macrosporus and sequence comparison with other Aspergillopepsins I[J]. Bioscience,Biotechnology and Biochemistry,1995,59(5):954-955.
[10] YIN L J,HSU T H,JIANG S T. Characterization of acidic protease from Aspergillus niger BCRC 32720[J]. Journal of Agricultural&Food Chemistry,2013,61(3):662-666.
[11] JARAI G,VAN DEN HOMBERGH H,BUXTON F P.Cloning and characterization of the pep E gene of Aspergillus niger encoding a new aspartic protease and regulation of pep E and pep C[J]. Gene,1994,145(2):171-178.
[12]董自星,肖华,王静培,等.黑曲霉果糖基水解酶的重组表达及酶学特征分析[J].食品工业科技,2017,38(10):178-184.
[13]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994:413-450.
[14]奥斯伯F,金斯顿R,塞德曼J,等.精编分子生物学实验指南[M].北京:科学出版社,2008:42-50.
[15] JONES L J,UPSON R H,HAUGLAND R P,et al.Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement[J]. Analytical Biochemistry,1997,251(2):144-152.
[16] SCHADE S Z. Fluorescence polarization assays of enzymes and substrates therefore:United States,US005804395A[P]. 1998-09-08.
[17] TAMURA K,PETERSON D,PETERSON N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution,2011,28(10):2 731-2 739.
[18] REVUELTA M V,VAN KAN J A,KAY J,et al. Extensive expansion of A1 family aspartic proteinases in fungi revealed by evolutionary analyses of 107 complete eukaryotic proteomes[J]. Genome Biology and Evolution,2014,6(6):1 480-1 494.
[19] CHEN C C,CHO Y C,LAI C C,et al. Purification and characterization of a new rhizopuspepsin from Rhizopus oryzae NBRC 4749[J]. Journal of Agricultural and Food Chemistry,2009,57(15):6 742-6 747.
[20] TSUJITA Y,ENDO A. Purification and characterization of the two molecular forms of Aspergillus oryzae acid protease[J]. Biochim Biophys Acta,1976,445(1):194-204.
[21] LI J,CHI Z,WANG X. Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli[J]. Microbiological Research,2010,165(3):173-182.
[22]马纳纳,徐冬冬,黄伟谦,等.一种新颖的毕赤酵母酸性蛋白酶基因的克隆和异源表达以及酶学性质研究[J].广东农业科学,2015,42(12):141-146.
[23]王晓亮,周樱,张庆丽.酶制剂在猪生产中的应用[J].饲料工业,2013,34(6):23-25.