分子改造提高谷氨酰胺转氨酶的催化活性
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摘要
谷氨酰胺转氨酶(EC2. 3. 2. 13,Transglutaminase,TGase)是一种重要的食品酶。为提高其催化活性,通过Discovery Studio 2017预测了Streptomyces mobaraense TGase中影响其与底物α-N-CBZ-GLN-GLY结合自由能的氨基酸位点,构建得到结合自由能下降的TGase突变体:Y24W、E300W和Y302R。与野生TGase相比,E300W的比酶活提高了31%; Km、kcat和kcat/Km值分别提高了10%、42%和29%,说明E300W比酶活的提高主要是由于酶转换数的增加。Y24W、E300W和Y302R的热稳定性均有不同程度下降。作用力分析发现,Y24W、E300W和Y302R主链-主链氢键分别减少1、2和4个。上述结果表明,基于蛋白质结合自由能分析的策略能迅速鉴定影响TGase催化活性关键氨基酸,进一步突变能有效提高其催化活性。
Transglutaminase( EC2. 3. 2. 13,TGase) is an important food enzyme. In order to enhance its catalytic activity,the amino acid sites that could affect the binding energy of S. mobaraense TGase and its substrate α-NCBZ-GLN-GLY were predicted through Discovery Studio 2017. Then mutants( Y24 W,E300 W and Y302 R) with reduced binding energy were constructed. Compared with wild type of TGase,the specific activity of E300 W was increased by 31%. Km,kcatand kcat/Kmvaule of E300 W were increased by 10%,42% and 29% respectively. The results showed that the increase of specific enzyme activity was mainly due to the increase of enzymatic conversion number. While the thermal stability of all the mutants decreased in varying degrees. And the structure analysis indicated that the main-chain hydrogen bonds of Y24 W,E300 W and Y302 R decreased by 1,2 and 4,respectively,compared with original TGase. These results suggested that the strategy based on the analysis of binding free energy could rapidly identify the key amino acids that affect the catalytic activity of TGase,and further mutation might effectively improve its catalytic activity.
Transglutaminase( EC2. 3. 2. 13,TGase) is an important food enzyme. In order to enhance its catalytic activity,the amino acid sites that could affect the binding energy of S. mobaraense TGase and its substrate α-NCBZ-GLN-GLY were predicted through Discovery Studio 2017. Then mutants( Y24 W,E300 W and Y302 R) with reduced binding energy were constructed. Compared with wild type of TGase,the specific activity of E300 W was increased by 31%. Km,kcatand kcat/Kmvaule of E300 W were increased by 10%,42% and 29% respectively. The results showed that the increase of specific enzyme activity was mainly due to the increase of enzymatic conversion number. While the thermal stability of all the mutants decreased in varying degrees. And the structure analysis indicated that the main-chain hydrogen bonds of Y24 W,E300 W and Y302 R decreased by 1,2 and 4,respectively,compared with original TGase. These results suggested that the strategy based on the analysis of binding free energy could rapidly identify the key amino acids that affect the catalytic activity of TGase,and further mutation might effectively improve its catalytic activity.
引文
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[21] TAGAMI U,SHIMBA N,NAKAMURA M,et al. Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis[J]. Protein Engineering Design and Selection,2009,22(12):747-752.
[2] KIELISZEK M,MISIEWICZ A. Microbial transglutaminase and its application in the food industry. A review[J]. Folia Microbiologica,2014,59(3):241-250.
[3] SANTHI D,KALAIKANNAN A,MALAIRAJ P,et al.Application of microbial transglutaminase in meat foods:A review[J]. Critical Reviews in Food Science and Nutrition,2017,57(10):2 071-2 076.
[4] TATSUKAWA H,FURUTANI Y,HITOMI K,et al.Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death[J].Cell Death and Disease,2016,7(6):e2 244.
[5] MARINIELLO L,PORTA R,SORRENTINO A,et al.Transglutaminase-mediated macromolecular assembly:production of conjugates for food and pharmaceutical applications[J]. Amino Acids,2014,46(3):767-776.
[6] ECKERT R L,KAARTINEN M T,NURMINSKAYA M,et al. Transglutaminase regulation of cell function[J].Physiological Reviews,2014,94(2):383-417.
[7] KUO T F,TATSUKAWA H,KOJIMA S. New insights into the functions and localization of nuclear transglutaminase2[J]. Febs Journal,2011,278(24):4 756-4 767.
[8] FU Jia-jia,SU Jing,WANG Ping,et al. Enzymatic processing of protein-based fibers[J]. Applied Microbiologyand Biotechnology,2015,99(24):1 0387-1 097.
[9] GRIFFIN M,CASADIO R,BERGAMINI C M. Transglutaminases:Nature's biological glues[J]. Biochemical Journal,2002,368(Pt 2):377-396.
[10] BOMMARIUS A S,BLUM J K,ABRAHAMSON M J.Status of protein engineering for biocatalysts:how to design an industrially useful biocatalyst[J]. Current Opinion in Chemical Biology,2011,15(2):194-200.
[11] YOKOYAMA K,UTSUMI H,NAKAMURA T,et al.Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis[J]. Applied Microbiology and Biotechnology,2010,87(6):2 087-2 096.
[12] ZHANG Jun-hui, JIANG Yu-yan, LIN Ying, et al.Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids[J]. PLo S One,2013,8(7):e67 892.
[13] LIPPOW S M,WITTRUP K D,TIDOR B. Computational design of antibody-affinity improvement beyond in vivo maturation[J]. Nature Biotechnology,2007,25(10):1 171-1 176.
[14] LIU Song,WAN Dan,WANG Miao,et al. Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization[J]. BMC Biotechnology,2015,15(1):75.
[15] XUAN Jian-wu,FOURNIER P,GAILLARDIN C. Cloning of the LYS5 gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration[J]. Current Genetics,1988,14(1):15-21.
[16]刘松. Streptomyces hygroscopicus谷氨酰胺转胺酶的异源表达研究[D].无锡:江南大学,2011.
[17] KASHIWAGI T,YOKOYAMA K,ISHIKAWA K,et al.Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense[J]. The Journal of Biological Chemistry,2002,277(46):44 252-44 260.
[18] LIU Song,ZHANG Dong-xu,WANG Miao,et al. The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli[J]. FEMS Microbiology Letters,2011,324(2):98-105.
[19] CHEONG D E,KO K C,HAN Y,et al. Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5'coding region[J]. Biotechnology and Bioengineering,2015,112(4):822-826.
[20] XIE Yuan,AN Jiao,YANG Guang-yu,et al. Enhanced enzyme kinetic stability by increasing rigidity within the active site[J]. Journal of Biological Chemistry,2014,289(11):7 994-8 006.
[21] TAGAMI U,SHIMBA N,NAKAMURA M,et al. Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis[J]. Protein Engineering Design and Selection,2009,22(12):747-752.