通过生物信息学分析发现了米曲霉(Aspergillus oryzae)中属于氨肽酶M18家族的新成员天冬酰胺氨肽酶(AAP)基因,经反转录克隆获得该基因,实现了其在毕赤酵母GS115中的表达,同时研究了重组AAP的酶学性质及其在大豆蛋白水解中的应用。结果表明,重组AAP的分子质量约54.0 kDa,在较广的pH范围内有活性(pH 6.0~9.5),最适反应温度和pH分别为50 ℃和7.0,Zn2+和Ca2+对该酶起促进作用,EDTA能够显著抑制该酶的酶活。以Asp-pNA为底物时, Km为0.42 mmol/L,Vmax为21.57 μmol/(mL·min),该酶具有水解N末端为天冬氨酸和谷氨酸的底物特异性。重组AAP可显著降低大豆多肽的苦味,提高水解度,具有在食品中的应用潜力。
A novel gene AAP that encodes aspartyl aminopeptidase from the peptidase family M18 in Aspergillus oryzae was discovered by bioinformatics analysis. The gene AAP was cloned and overexpressed in Pichia pastoris GS115. Enzymatic properties of recombinant AAP and its application in the hydrolysis of soybean protein were investigated. The results showed that the recombinant AAP had a molecular weight of about 54 kDa. It was active over a broad pH range (6.0-9.5). Its optimal reaction temperature and pH were 50 ℃ and 7.0, respectively. Moreover, Zn2+ and Ca2+ promoted the activity of recombinant AAP, while EDTA significantly inhibited its activity. Using Asp-pNA as a substrate, the Km and Vmax of the enzyme were 0.42 mmol/L and 21.57 μmol/(mL·min), respectively. Recombinant AAP exhibited a substrate specificity of hydrolyzing N-terminal aspartyl and glutamyl residues. In addition, recombinant AAP significantly decreased the bitterness of soybean pepides and improved its hydrolysis degree, which indicated its potential applications in food industries.
[1] 马俊阳,诸葛斌,方慧英,等. 米曲霉蛋白酶的分离纯化及酶学性质研究[J]. 微生物学通报,2014(1): 83-89.
[2] 孙倩. 三种不同来源(植物、细菌和真菌)蛋白酶的纯化、性质及应用研究[D]. 北京:中国农业大学,2016.
[3] 程晓芳,袁丹丹,张余慧,等. 蛋白酶在食品工业中的应用研究进展[J]. 食品研究与开发,2018, 39(7): 221-224.
[4] 张明赞,王京. 微生物蛋白酶的研究进展[J]. 安徽农学通报, 2018(8): 15-16.
[5] KANG C, YU X W, XU Y. Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization [J]. Journal of Industrial Microbiology & Biotechnology, 2015, 42(2): 263-272.
[6] CERQUEIRA G C, ARNAUD M B, INGLIS D O, et al. The Aspergillus genome database: multispecies curation and incorporation of RNA-Seq data to improve structural gene annotations[J]. Nucleic Acids Research, 2014, 42(Database issue): D705-710.
[7] MACHIDA M, ASAI K, SANO M, et al. Genome sequencing and analysis of Aspergillus oryzae[J]. Nature, 2005, 438(7 071): 1 157-1 161.
[8] 郭兴峰,魏芳,周祥山,等. 苦味肽的形成机理及脱苦技术研究进展[J]. 食品研究与开发, 2017, 38(21): 207-211.
[9] 付静. 食品外肽酶的研究进展[J]. 食品科学, 2013, 34(7): 349-354.
[10] MIKA N, ZORN H, RUHL M. Prolyl-specific peptidases for applications in food protein hydrolysis[J]. Applied Microbiology and Biotechnology, 2015, 99(19): 7 837-7 846.
[11] 黄伟谦. 酱油曲霉亮氨酸氨肽酶sLAP1在毕赤酵母中表达及其酶学性质研究[D]. 广州:华南理工大学, 2014.
[12] 雷芬芬. 海洋来源Bacillus licheniformis SWJS33所产氨肽酶的研究及应用[D]. 广州:华南理工大学, 2017.
[13] 刘伯业. 小麦蛋白低苦味肽的制备及其脱苦机理研究[D]. 无锡:江南大学, 2017.
[14] AASLYNG M D, MARTENS M, POLL L, et al. Chemical and sensory characterization of hydrolyzed vegetable protein, a savory flavoring[J]. Journal of Agricultural and Food Chemistry, 1998, 46(2): 481-489.
[15] LEI F, ZHAO Q, LIN L, et al. Evaluation of the hydrolysis specificity of an aminopeptidase from Bacillus licheniformis SWJS33 using synthetic peptides and soybean protein isolate[J]. Journal of Agricultural and Food Chemistry, 2017, 65(1): 167-173.
[16] 应欣,张连慧,陈卫华. 蛋白水解物苦味形成、评价及功能活性的研究进展[J]. 中国粮油学报, 2017, 32(12): 141-146.
[17] LIU B Y, ZHU K X, PENG W, et al. Effect of sequential hydrolysis with endo- and exo-peptidase on bitterness properties of wheat gluten hydrolysates[J]. RSC Advances, 2016, 6(33): 27 659-27 668.
[18] CHAIKUAO A, PILKA E S, DE RISO A, et al. Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family [J]. BMC Structural Biology, 2012, 12(1): 12-14.
[19] 荆谷,冯静,孔健,等. 微生物金属蛋白酶的研究进展[J]. 中国生物工程杂志, 2002(1): 61-63,56.
