Study On The Improvement Of Thermal Stability Of Streptomyces Hygroscopicus Transglutaminase

Transglutaminase?EC 2.3.2.13,TGase?catalyzes the acyl transfer reaction to promote the cross-linking of protein or small molecules containing both primary amine and glutamine.Based on the catalytic reaction,TGase shows great potential in food,textile and medicine fields.But the poor thermal stability seriously affects the application of TGase.The stable mutant P132I of Streptomyces hygroscopicus WSH03-13 TGase was used as the research object in this study.We improved the thermal stability of TGase by random mutation,and also investigated the effects of small heat shock proteins?sHSPs?and different stabilizer formulations on the stability of the enzyme.What follows are the main research contents:?1?Increasing the thermal stability of TGase by random mutationThe TGase gene was randomly mutated by using error prone PCR technology,and 8mutants named 2-9C,1-3D,5-2E,1-8H,6-7D,6-5G,6-2E and 3-12G were screened from4500 TGase mutants and purified by nickel column affinity.Compared with P132I,the half-life(t_1/2)of 3-12G at 50 ^oC was 10.04 min,which is 100.80%higher than that of P132I,and other mutants also increased by 20.20%-82.40%.In addition,the specific activity of mutant 3-12G and 6-2E respectively increased by 15.57%and 7.39%compared with P132I.According to the analysis of the intramolecular force of TGase,most of mutants increased their interactions and the mutation site of 3-12G increased an additional hydrogen bond than that of P132I.The increase of the intermolecular force of enzymes may be one of the reasons for the increased thermal stability of mutants.?2?Effects of small heat shock proteins on the thermal stability of TGaseThe sHSPs IbpA and IbpB derived from Escherichia coli JM109 were expressed in E.coli BL21?DE3?and purified by nickel column affinity.The sHSPs and 3-12G were mixed at different ratios to investigate the effect of the former on the thermal stability of the latter.The results showed that the effect of adding IbpA on the stability of TGase was not obvious,and the addition of Ibp B(500?g·m L^-1,final concentration)increased the t_1/2?50??of 3-12G(50?g·mL^-1,final concentration)to 16.33 min,which was improved 62.65%compared with that of the enzyme without stabilizer?control?.And adding mixed sHSPs(500?g·mL^-1,total concentration)can increase the t_1/2?50??to 19.62 min,95.42%higher than that of the control.The analysis of transmission electron microscopy showed that the mixture of sHSPs and 3-12G formed larger particles under heat treatment,while 3-12G could not.The above results indicated that the formation of polymers by sHSPs and 3-12G may be the reason for the increased thermal stability of TGase.Moreover,we constructed fusion proteins by using flexible linker peptides with different lengths to connect TGase and sHSPs,and found that the expressed fusion proteins cannot increase the thermal stability of TGase.?3?Increasing the thermal stability of TGase by conventional stabilizersScreening and combining conventional stabilizers to study the effects of enzyme stabilizers on the thermal stability of TGase.The results showed that 40 g·L^-1 NaCl,50 g·L^-1sorbitol,50 g·L^-1 glucose and 50 g·L^-1 wheat protein can make the corresponding t_1/2?50??of TGase(50?g·mL^-1,final concentration)reach 89.79 min,36.71 min,37.52 min,and106.22 min,which was respectively improved 7.94,2.66,2.74 and 9.58 times than that of the control.Based on these stabilizers,we designed orthogonal tests to obtain the best compound stabilizer:wheat protein 50 g·L^-1,NaCl 50 g·L^-1,glucose 50 g·L^-1 and sorbitol 50 g·L^-1.Under the condition of this stabilizer,the t_1/2?50??of 3-12G reached 331.50 min,which was31.97 times higher than that of the control;After 60-day storage at room temperature,the residual enzyme activity of 3-12G was 66.5%,which was improved 17.07 times than that of the control.