谷氨酰胺转氨酶热稳定剂优化
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摘要
谷氨酰胺转氨酶(Transglutaminase,TGase)能够催化蛋白质分子发生交联,被广泛应用于食品、医药和纺织等领域。为提高其热稳定性,研究了糖、盐及醇类等稳定剂对TGase在55℃下的半衰期[t_(1/2)(55℃)]的影响。结果表明,山梨醇、小麦蛋白、Na Cl和葡萄糖能延长TGase的t_(1/2)(55℃)。在此基础上进行正交实验,获得最佳的稳定剂配方:山梨醇50 g/L、小麦蛋白50 g/L、Na Cl 50 g/L、葡萄糖50 g/L。在该复合稳定剂保护下,TGase的t_(1/2)(55℃)和最适反应温度较对照(无稳定剂)分别提高59.73倍和10℃;室温(25℃)储存80 d的残余酶活率为73.84%,较对照提高62.79%。此研究结果将促进TGase的生产改进和应用拓展。
Transglutaminase( EC 2. 3. 2. 13,TGase) can catalyze the cross-linking of protein molecules,and it has been widely used in the fields of food,biology,medicine,textile and so on. In order to improve the thermal stability,the effects of carbohydrates,salts and alcohols on the half-life of TGase at 55 ℃(( t_(1/2)( 55 ℃)) were studied. The results showed that the addition of sorbitol,wheat protein,Na Cl and glucose could effectively prolong the half-life of TGase. On this basis,orthogonal experiments were carried out to obtain the best compound stabilizer:50 g/L sorbitol,50 g/L wheat protein,50 g/L Na Cl,and 50 g/L glucose. Under the protection of the compound stabilizer,the t_(1/2)( 55 ℃) value and the optimum reaction temperature of TGase were increased by 59. 73-fold and 10 ℃respectively compared with the control( no stabilizer),and the residual enzyme activity rate after storage at room temperature( 25 ℃) for 80 days was 73. 84%,62. 79% higher than the control. The results of this study will facilitate the improvement of TGase production and application development.
Transglutaminase( EC 2. 3. 2. 13,TGase) can catalyze the cross-linking of protein molecules,and it has been widely used in the fields of food,biology,medicine,textile and so on. In order to improve the thermal stability,the effects of carbohydrates,salts and alcohols on the half-life of TGase at 55 ℃(( t_(1/2)( 55 ℃)) were studied. The results showed that the addition of sorbitol,wheat protein,Na Cl and glucose could effectively prolong the half-life of TGase. On this basis,orthogonal experiments were carried out to obtain the best compound stabilizer:50 g/L sorbitol,50 g/L wheat protein,50 g/L Na Cl,and 50 g/L glucose. Under the protection of the compound stabilizer,the t_(1/2)( 55 ℃) value and the optimum reaction temperature of TGase were increased by 59. 73-fold and 10 ℃respectively compared with the control( no stabilizer),and the residual enzyme activity rate after storage at room temperature( 25 ℃) for 80 days was 73. 84%,62. 79% higher than the control. The results of this study will facilitate the improvement of TGase production and application development.
引文
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[24]彭灿,常忠义,王疆元,等.谷朊粉水解物对微生物谷氨酰胺转氨酶稳定性的影响[J].西北农林科技大学学报(自然科学版),2007,35(8):171-175.
[25]KUMAR V,SHARMA V K,KALONIA D S.Effect of polyols on polyethylene glycol(PEG)-induced precipitation of proteins:Impact on solubility,stability and conformation[J].International Journal of Pharmaceutics,2009,366(1-2):38-43.
[26]王希成.生物化学[M].第3版.北京:清华大学出版社,2010:10-16.
[2]叶双双,周丽,周哲敏.基于保护剂筛选及优化策略提高苯丙氨酸羟化酶热稳定性[J].食品与发酵工业,2016,42(6):56-61.
[3]童理明.分子改造提高谷氨酰胺转胺酶热稳定性[D].无锡:江南大学,2016:3-7.
[4]刘中美,坤杜,周哲敏.人工设计二硫键增强谷氨酰胺转胺酶热稳定性[J].食品与生物技术学报,2015,34(10):1 057-1 061.
[5]张春红,高慧楠,常南,等.以壳聚糖为载体固定化谷氨酰胺转氨酶的研究[J].食品科技,2009,34(12):33-35.
[6]刘松,堵国成,陈坚.化学修饰提高谷氨酰胺转胺酶活性与热稳定性[J].食品与生物技术学报,2015,34(11):1 135-1 140.
[7]SEKIGUCHI S,HASHIDA Y,YASUKAWA K,et al.Stabilization of bovine intestine alkaline phosphatase by sugars[J].Bioscience Biotechnology and Biochemistry,2012,76(1):95-100.
[8]BELLUZO S,BOERIS V,FARRUGGIA B,et al.Influence of stabilizers cosolutes on catalase conformation[J].International Journal of Biological Macromolecules,2011,49(5):936-941.
[9]常忠义,荣绍丰,高红亮,等.采用TG和DSC方法研究糖类对谷氨酰胺转胺酶热稳定性的影响[J].食品科学,2007,28(2):202-205.
[10]SZABO A,KOTORMAN M,LACZKO I,et al.Influence of carbohydrates on stability of papain in aqueous tetrahydrofuran mixture[J].Journal of Chemical Technology and Biotechnology,2009,84(1):133-138.
[11]PAZHANG M,MEHRNEJAD F,PAZHANG Y,et al.Effect of sorbitol and glycerol on the stability of trypsin and difference between their stabilization effects in the various solvents[J].Biotechnology and Applied Biochemistry,2016,63(2):206-213.
[12]KUMAR V,CHARI R,SHARMA V K,et al.Modulation of the thermodynamic stability of proteins by polyols:Significance of polyol hydrophobicity and impact on the chemical potential of water[J].International Journal of Pharmaceutics,2011,413(1-2):19-28.
[13]PETERSEN S B,JONSON V,FOJAN P,et al.Sorbitol prevents the self-aggregation of unfolded lysozyme leading to an up to 13 degrees C stabilisation of the folded form[J].Journal of Biotechnology,2004,114(3):269-278.
[14]朱怀梅,常忠义,高红亮,等.盐离子对微生物谷氨酰胺转胺酶热稳定性的影响[J].云南大学学报(自然科学版),2008,30(S1):426-429.
[15]GROSSOWICZ N,WAINFAN E,BOREK E,et al.The enzymatic formation of hydroxamic acids from glutamine and asparagine[J].Journal of Biological Chemistry,1950,187(1):111-125.
[16]O'FAGAIN C.Enzyme stabilization-recent experimental progress[J].Enzyme and Microbial Technology,2003,33(2-3):137-149.
[17]彭灿.微生物谷氨酰胺转胺酶稳定性的研究[D].上海:华东师范大学,2007:46-52.
[18]TIMASHEFF S N.Protein hydration,thermodynamic binding,and preferential hydration[J].Biochemistry,2002,41(46):13 473-13 482.
[19]FERNANDEZ L,GOMEZ L,RAMIREZ H L,et al.Thermal stabilization of trypsin with glycol chitosan[J].Journal of Molecular Catalysis B-Enzymatic,2005,34(1-6):14-17.
[20]COMBES D,MONSAN P.Effect of polyhydric alcohols on invertase stabilization[J].Annals of the New York Academy of Sciences,1984,434:61-63.
[21]毛新焕,李响,王姗姗,等.辣根过氧化物酶的热稳定剂筛选[J].生物工程学报,2009,25(3):388-391.
[22]荣绍丰,高红亮,常忠义,等.一些蛋白质和多肽对谷氨酰胺转胺酶热稳定性的影响[J].食品工业科技,2006,27(12):95-97.
[23]王璋,许时婴,汤坚.食品化学[M].北京:中国轻工业出版社,1999:48-69.
[24]彭灿,常忠义,王疆元,等.谷朊粉水解物对微生物谷氨酰胺转氨酶稳定性的影响[J].西北农林科技大学学报(自然科学版),2007,35(8):171-175.
[25]KUMAR V,SHARMA V K,KALONIA D S.Effect of polyols on polyethylene glycol(PEG)-induced precipitation of proteins:Impact on solubility,stability and conformation[J].International Journal of Pharmaceutics,2009,366(1-2):38-43.
[26]王希成.生物化学[M].第3版.北京:清华大学出版社,2010:10-16.