茂原链霉菌(Streptomyces mobaraenesis)来源的谷氨酰胺转氨酶(transglutaminase,TGase)是一种重要的食品酶,广泛应用于蛋白基食品改性。为强化高温条件下的应用效果,拟通过构建蛋白分子内二硫键提高TGase的热稳定性。首先,基于野生型TGase晶体结构(PDB:1iu4)构建已报道TGase耐热突变体MS(S2P-S23V-Y24 N-S199A-K294L)的模拟结构。其次,通过Disulfide by design 2.0进行二硫键预测,根据二硫键成键自由能选择12种二硫键突变体在大肠杆菌(Escherichia coli)BL21(DE3)中表达。最后,对纯化后突变体的酶学性质进行表征和稳定化机制解析。结果显示,经60 ℃处理20 min,D118C-K121C、P244C-E249C和P22C-Q328C残余酶活力较MS分别提升了77.39%、71.58%和91.06%。与MS相比,MS-P22C-Q328C的t1/2 (60 ℃)和tm分别提升了2.06倍和1.06 ℃,但比酶活力下降13.2%。模拟结构分析表明,新增的二硫键可能降低了MS-P22C-Q328C中2个高柔性 loop的刚性,从而使其热稳定性增强。该研究结果将为高热稳定性TGase的开发提供基础数据。
Streptomyces mobaranensis transglutaminase TGase is an important food enzyme, which is widely used in protein-based food modification. In order to improve the thermostability of TGase, intramolecular disulfide bonds were introduced. Firstly, based on the crystal structure of wild-type TGase (PDB∶1iu4), the simulated structure of TGase thermostable mutant MS (S2P-S23V-Y24N-S199A-K294L) was constructed. Secondly, the disulfide bonds were predicted by Disulfide Design 2.0. According to the free energy of disulfide bonds formation, 12 disulfide bonds mutant were selected and expressed in Escherichia coli BL21(DE3). Finally, the enzymatic properties of the purified mutants were characterized, and the stabilization mechanism was analyzed. The results showed that the residual enzyme activities of D118C-K121C, P244C-E249C and P22C-Q328C were 77.39%, 71.58% and 91.06% higher than MS after treated at 60 ℃ for 20 min, respectively. Compared to MS, the t1/2 (60 ℃) and tm of MS-P22C-Q328C increased by 2.06 times and 1.06 ℃, respectively, but its specific activity was decreased by 13.2%. The simulated structure analysis showed that the new disulfide bonds might by reducing the rigidity of the two highly flexible loops in MS-P22C-Q328C to enhance the thermostability of TGase.
[1] KASHIWAGI T,YOKOYAMA K I,ISHIKAWA K,et al.Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense[J].Journal of Biological Chemistry,2002,277(46):44 252-44 260.
[2] ANDO H,ADACHI M,UMEDA K,et al.Purification and characteristics of a novel transglutaminase derived from microorganisms[J].Agricultural and Biological Chemistry,1989,53(10):2 613-2 617.
[3] STROP P.Versatility of microbial transglutaminase[J].Bioconjugate Chemistry,2014,25(5):855-862.
[4] LESIOW T,RENTFROW G K,XIONG Y L.Polyphosphate and myofibrillar protein extract promote transglutaminase-mediated enhancements of rheological and textural properties of PSE pork meat batters[J].Meat Science,2017,128:40-46.
[5] SERNA N,LAURA S G,UNZUETA U,et al.Protein-based therapeutic killing for cancer therapies[J].Trends in Biotechnology,2018,36(3):318-335.
[6] MARX C K,HERTEL T C,PIETZSCH M.Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants[J].Journal of Biotechnology,2008,136(3-4):156-162.
[7] BUETTNER K,HERTEL T C,PIETZSCH M.Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis[J].Amino Acids,2012,42(2-3):987-996.
[8] LIU Y H,HUANG L,SHAN M Y,et al.Enhancing the activity and thermostability of Streptomyces mobaraensis transglutaminase by directed evolution and molecular dynamics simulation[J].Biochemical Engineering Journal,2019,151:107 333.
[9] MU D D,LU J J,SHU C,et al.Improvement of the activity and thermostability of microbial transglutaminase by multiple-site mutagenesis[J].Bioscience,Biotechnology,and Biochemistry,2018,82(1):106-109.
[10] MATSUMURA M,SIGNOR G,MATTHEWS B W.Substantial increase of protein stability by multiple disulphide bonds[J].Nature,1989,342(6247):291-293.
[11] ZHANG Z J,YANG J,XIE P J,et al.Characterization of a thermostable phytase from Bacillus licheniformis WHU and further stabilization of the enzyme through disulfide bond engineering[J].Enzyme and Microbial Technology,2020,142:109 679.
[12] 朱方剑. 二硫键理性设计提高Thermobifida fusca角质酶热稳定性研究[D].无锡:江南大学,2020.
ZHU F J.Study on rational design of disulfide bonds to improve thermal stability of Thermobifida fusca cutinase[D].Wuxi:Jiangnan University,2020.
[13] 王睿, 喻晓蔚,徐岩.理性设计二硫键提高华根霉脂肪酶热稳定性[J].微生物学通报,2018,45(11):2 311-2 319.
WANG R,YU X W,XU Y.Rational design of disulfide bond in Rhizopus chinensis lipase to improve thermostability[J].Microbiology China,2018,45(11):2 311-2 319.
[14] YANG J Y,ZHANG Y.I-TASSER server:New development for protein structure and function predictions[J].Nucleic Acids Research,2015,43(W1):W174-W181.
[15] CRAIG D B,DOMBKOWSKI A A.Disulfide by Design 2.0:a web-based tool for disulfide engineering in proteins[J].BMC Bioinformatics,2013,14:1 471-2 105.
[16] 杜坤, 周丽,堵国成,等.内含肽介导谷氨酰胺转胺酶酶原的活化[J].食品科学,2013,34(9):90-94.
DU K,ZHOU L,DU G C,et al.Intein-mediated activation of transglutaminase from Streptomyces hygroscopicus[J].Food Science,2013,34(9):90-94.
[17] FOLK J E,COLE P W.Transglutaminase:Mechanistic features of the active site as determined by kinetic and inhibitor studies[J].Biochimica et Biophysica Acta (BBA)-Enzymology and Biological Oxidation,1966,122(2):244-264.
[18] MARK J A,TEEMU M,ROLAND S,et al.GROMACS:High performance molecular simulations through multi-level parallelism from laptops to supercomputers[J].SoftwareX,2015,1-2:19-25.
[19] PEZESHGI M H,MOFRAD M R,SANATI N A.H.Protein thermostability engineering[J].RSC Advances,2016,6(116):115 252-115 270.
[20] 郭超, 王志彦,甘一如,等.理性设计改造牛肠激酶的热稳定性[J].中国生物工程杂志,2016,36(8):46-54.
GUO C,WANG Z Y,GAN Y R,et al.Engineering thermostability of bovine enterokinase by rational design method[J].China Biotechnology,2016,36(8):46-54.
[21] MASAZUMI M,WAYNE J B,MICHAEL L,et al.Stabilization of phage T4 lysozyme by engineered disulfide bonds[J].Proceedings of the National Academy of Sciences of the United States of America,1989,86(17):6 562-6 566.
