重组胶原酶Bacillus cereus ColM13的酶学及结构特性分析
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摘要
以前期成功构建出降解胶原蛋白的工程菌pET30a-ColM13/BL21为出发菌,对工程菌表达纯化出的重组胶原酶(ColM13)的酶学性质进行分析。通过研究温度、pH、抑制剂对其的影响,得出ColM13的最适反应温度为50℃,最适反应pH为8.0;在20~55℃和pH6.0~9.0范围内有良好的稳定性;金属蛋白酶抑制剂乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)、乙二醇双(2-氨基乙基醚)四乙酸(ethylenebis(oxyethylenenitrilo tetraacetic acid,EGTA)对ColM13有显著的抑制作用。采用紫外光谱(ultraviolet and visible spectrum,UV)、差示量热扫描(differential scanning calorimeter,DSC)、荧光光谱(FS)、傅里叶红外光谱(FT-IR)对骨胶原蛋白及其酶解产物进行结构特性分析,分析可知酶解作用使骨胶原蛋白的三股螺旋结构遭到破坏,释放出大量的游离氨基酸。酶解后胶原蛋白的肽链上-NH_2~+-相互排斥减弱,疏水氨基酸残基作用增强,具有更高的热稳定性。与B.cereus MBL13-U分离纯化出的胶原酶(BSC)相比,ColM13处理的胶原蛋白的降解效果更明显。
In this study,the enzymatic properties of the recombinant collagenase( ColM13) from engineering bacteria pET30 a-ColM13/BL21 were analyzed after expression and purification. By studying the effects of temperature,pH and inhibitor,the optimal reaction temperature and pH for the ColM13 enzyme activity were determined as 50 ℃ and 8. 0 respectively. The ColM13 enzyme was stable when the temperature was 20-55 ℃. The ColM13 enzyme activity was stable between pH 6. 0-9. 0. Metalloproteinase inhibitors EDTA and EGTA had a significant inhibitory effect on ColM13. The structural properties of bone collagen and its hydrolysate were characterized by ultraviolet( UV),differential scanning calorimetry( DSC),fluorescence and fourier transform infrared spectroscopy( FT-IR).The results showed that the enzymatic hydrolysis damaged the triple helical structure of collagen protein and released a large amount of free amino acids. The enzymolysis reduced the-NH~(2 +)-mutual exclusion on the collagen peptide chain and enhanced the hydrophobic amino acid residues. The collagen hydrolysate had higher thermal stability. Compared with the collagenase( BSC) isolated from B. cereus MBL13-U,the degradation of collagen treated by ColM13 was more significant.
In this study,the enzymatic properties of the recombinant collagenase( ColM13) from engineering bacteria pET30 a-ColM13/BL21 were analyzed after expression and purification. By studying the effects of temperature,pH and inhibitor,the optimal reaction temperature and pH for the ColM13 enzyme activity were determined as 50 ℃ and 8. 0 respectively. The ColM13 enzyme was stable when the temperature was 20-55 ℃. The ColM13 enzyme activity was stable between pH 6. 0-9. 0. Metalloproteinase inhibitors EDTA and EGTA had a significant inhibitory effect on ColM13. The structural properties of bone collagen and its hydrolysate were characterized by ultraviolet( UV),differential scanning calorimetry( DSC),fluorescence and fourier transform infrared spectroscopy( FT-IR).The results showed that the enzymatic hydrolysis damaged the triple helical structure of collagen protein and released a large amount of free amino acids. The enzymolysis reduced the-NH~(2 +)-mutual exclusion on the collagen peptide chain and enhanced the hydrophobic amino acid residues. The collagen hydrolysate had higher thermal stability. Compared with the collagenase( BSC) isolated from B. cereus MBL13-U,the degradation of collagen treated by ColM13 was more significant.
引文
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[22]张晓洁,张宇昊,马良,等.超声辅助提取兔皮胶原蛋白及其理化特性[J].食品与机械,2017,33(1):167-171.
[23]GHRIBI A M,GAFSI I M,SILA A,et al.Effects of enzymatic hydrolysis on conformational and functional properties of chickpea protein isolate[J].Food Chemistry,2015,187(11):322-330.
[24]吴雷,郑娟,刘文涛,等.鸡关节软骨Ⅱ型胶原蛋白结构及性能表征[J].食品与发酵工业,2016,42(11):86-90.
[25]魏晓芳,刘会洲.热变性对蛋白质结构及泡沫行为的影响[J].过程工程学报,2000,21(4):379-383.
[26]杨伟,袁芳,高彦祥.鱼胶原蛋白肽与表没食子儿茶素没食子酸酯相互作用的研究[J].光谱学与光谱分析,2015,35(1):184-188.
[27]肖岚,李诚,付刚,等.5种蛋白酶对猪皮胶原蛋白水解效果的比较研究[J].食品研究与开发,2015,36(17):10-14.
[28]琚海燕,刘新华,但卫华,等.牛跟腱Ⅰ型胶原纤维的微观结构与理化性能分析[J].功能材料,2015(15):15 031-15 034.
[29]钟朝辉,李春美,顾海峰,等.温度对鱼鳞胶原蛋白二级结构的影响[J].光谱学与光谱分析,2007,27(10):1 970-1 976.
[2]刘丽莉,马美湖,余秀芳,等.胶原蛋白酶产生菌的筛选及酶的分离纯化[J].生物工程学报,2010,26(2):194-200.
[3]GELSE K,POSCHL E,AIGNER T.Collagens-structure,function,and biosynthesis[J].Advanced Drug Delivery Reviews,2003,55(12):1 531-1 546
[4]HORN M M,MARTINS V C A,PLEPIS A M D G.Interaction of anionic collagen with chitosan:effect on thermal and morphological characteristics[J].Carbohydrate Polymers,2009,77(2):239-243.
[5]柳林,韦术敏,程仕伟,等.产胶原酶的琥珀葡萄球菌分离鉴定及其培养优化研究[J].中国酿造,2016,35(1):62-67.
[6]BICSAK T A,HARPER E.Purification of nonspecific protease-free collagenase from Clostridium histolyticum[J].Analytical Biochemistry,1985,145(2):286-291.
[7]OKAMOTO M,YONEJIMA Y,TSUJIMOTO Y,et al.A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp.strain MO-1[J].Applied Microbiology and Biotechnology,2001,57(1):103-108.
[8]LIMA C A,RODRIGUES P M B,PORTO T S,et al.Production of a collagenase from Candida albicans,URM3622[J].Biochemical Engineering Journal,2009,43(3):315-320.
[9]KANG S I,JANG Y B,KONG C J Y.Purification and properties of a collagenolytic protease produced by marine bacterium Vibrio vulnificus CYK279H[J].Biotechnology and Bioprocess Engineering,2005,10(6):593-598.
[10]ZHANG X X,LI Y,WANG S Y,et al.Identification of a collagenase produced by Bacillus cereus R75E isolated from human colostrum[J].Applied Biochemistry and Microbiology,2015,51(5):511-521.
[11]杨光垚.胶原蛋白酶的纯化及其基因的克隆与表达研究[D].成都:四川大学,2004.
[12]赵妍嫣,胡林林,方芳,等.骨胶原蛋白的酶解工艺条件[J].食品科学,2010,31(22):153-155.
[13]ZHANG Yu-hao,MA Liang,CAI Lu-yun,et al.Effect ofcombined ultrasonic and alkali pretreatment on enzymatic preparation of angiotensin converting enzyme(ACE)inhibitory peptides from native collagenous materials[J].Ultrasonics Sonochemistry,2017,36(5):88-94.
[14]Lee J M,Lee J,Nam G H,et al.Heterologous expression and enzymatic characterization ofγ-glutamyltranspeptidase from Bacillus amyloliquefaciens[J].Journal of Microbiology,2017,55(2):147-152.
[15]万骥,王丹,傅婷,等.韭菜β-木糖苷酶的分离纯化与部分性质研究[J].食品科学,2016,37(7):104-109.
[16]李陈.一种新的短小芽孢杆菌胶原蛋白酶的分离、纯化及酶学性质研究[D].成都:四川农业大学,2008.
[17]刘丽莉.Bacillus cereus MBL13-U胶原蛋白酶的分离纯化及其降解动力学分析[J].食品与发酵工业,2017,43(12):13-19.
[18]俞园园,刘学谦.类人胶原蛋白与Ca(Ⅱ)相互作用的荧光光谱研究[J].化学工程,2013,41(9):1-4.
[19]庄志凯.凡纳滨对虾虾头内源性蛋白酶分离纯化与酶学特性研究[D].湛江:广东海洋大学,2011.
[20]温慧芳,陈丽丽,白春清,等.基于不同提取方法的鮰鱼皮胶原蛋白理化性质的比较研究[J].食品科学,2016,37(1):74-81.
[21]LIN Y K,DENG C L.Comparison of physical-chemical properties of type I collagen from different species[J].Food Chemistry,2006,99(2):244-251.
[22]张晓洁,张宇昊,马良,等.超声辅助提取兔皮胶原蛋白及其理化特性[J].食品与机械,2017,33(1):167-171.
[23]GHRIBI A M,GAFSI I M,SILA A,et al.Effects of enzymatic hydrolysis on conformational and functional properties of chickpea protein isolate[J].Food Chemistry,2015,187(11):322-330.
[24]吴雷,郑娟,刘文涛,等.鸡关节软骨Ⅱ型胶原蛋白结构及性能表征[J].食品与发酵工业,2016,42(11):86-90.
[25]魏晓芳,刘会洲.热变性对蛋白质结构及泡沫行为的影响[J].过程工程学报,2000,21(4):379-383.
[26]杨伟,袁芳,高彦祥.鱼胶原蛋白肽与表没食子儿茶素没食子酸酯相互作用的研究[J].光谱学与光谱分析,2015,35(1):184-188.
[27]肖岚,李诚,付刚,等.5种蛋白酶对猪皮胶原蛋白水解效果的比较研究[J].食品研究与开发,2015,36(17):10-14.
[28]琚海燕,刘新华,但卫华,等.牛跟腱Ⅰ型胶原纤维的微观结构与理化性能分析[J].功能材料,2015(15):15 031-15 034.
[29]钟朝辉,李春美,顾海峰,等.温度对鱼鳞胶原蛋白二级结构的影响[J].光谱学与光谱分析,2007,27(10):1 970-1 976.