| Transglutaminase(TGase,EC2.3.2.13)has been widely used in the field of food,because it can catalyze the cross-linking reaction between proteins or polypeptide molecules.In recent years,TGase has been used in biotechnology and medicine fields,it is utilized for a large number of applications to specifically conjugate proteins or peptides to DNA,chemical material and pharmaceutical protein,and it also has been used as the medical hemostatic material.The commericial TGase is mainly microbial transglutaminase(MTGase),which derived from Streptomyces mobaraensis.But the quality of current MTGase can not meet the requirements in various fields,especially in the field of biotechnology and medicine fields,because its purity is low and the stability of the product is uneven.So the most important thing is to solve the problem of improving purity and stability of commericial MTGase.In order to solve the above problems,MTGase derived from Streptomyces mobaraensis was used in this study.Firstly,established the the test and screening methods of MTGase stability;then the MTGase stability effect factors of structure and purity were studied;and it had been found that the stability of MTGase was related to the ratio of MTG I1 and MTG 12;finally,purified and stable MTGase was obtained by fine purification,so the effects of impurities was eliminated.The main conclusions were as follows:1.The test and screening methods of MTGase stability were established.Electrophoresis method:MTGase was dissolved in buffer A and buffer B,and then detected by SDS-PAGE or Native-PAGE.The ratio of A1/A2 or A1/A3 was positively correlated with the MTGase stability(Al:the SDS-PAGE band area of MTGase dissolved in buffer A;A2:the SDS-PAGE band area of MTGase dissolved in buffer B;A3:the Native-PAGE band area of MTGase dissolved in buffer A).37? preservation method:Liquid MTGase was stored at 37? for 1-5 days,and the stability of MTGase can be accurately detected.Combining with two methods,when the ratio of A1/A2 or A1/A3 was higher than 0.7,the enzyme activity preservation rate was higher than 80%under 37?after 5 days;when the ratio of Al/A2 or Al/A3 was lower than 0.2,the enzyme activity preservation rate was less than 60%under 37? after 5 days.Therefore,these two methods could accurately screen out the stable MTGase.2.Different structures of MTGase were found and separated,which were the MTGase isomers(MTG I1 and MTG I2).And the relationship between isomers ratio and stability of MTGase has been established.MTGase isomers were separated by pH-mediated cation exchange chromatography,the separation method was stable and repeatable.The method was:MTGase isomers were separated by Hitrap SP FF chromatographic column(1 mL),the starting buffer was pH 5.5 20 mM phosphate buffer,elution buffer was pH 9.0 20 mM phosphate buffer with 0-100%in 30 CV,the flow rate was 1 mL/min.The yield of MTG I1 and MTG 12 was ?80%,the yield of MTG I1 was 60-65%,the yield of MTG 12 was 15-20%.MTG I1 was eluted between pH 5.5-6.5,MTG 12 was eluted between pH 6.6-7.5.The Rs of MTG II and MTG 12 was about 1.2.The ratio of MTG I2/MTGI1 was negatively correlated with the stability of MTGase.3.The study of the properties of MTGase.The molecular weight of MTG I1 and MTG 12 were 37.816 KDa,four N-terminal sequences were Asp-Ser-Asp-Glu.The isoelectric point of MTG I1 and MTG 12 were 7.94 and 8.03,respectively.The catalytic activity of MTG I1 was significantly higher than MTG 12 at 30-70?,and the catalytic activity of MTG 12 was higher than MTG I1 below 30?;MTG I1 temperature stability was significantly higher than MTG 12 at 40-60?.The optimum pH of MTG I1 was 6-8,and the optimum pH of MTG 12 was 5-8,the optimum pH range of MTG 12 was wider than MTG I1;at 37?,the pH stability of MTG I1 was pH5-9,the pH stability of MTG 12 was pH5-8,at 4?,the pH stability of MTG I1 was pH4-10,the pH stability of MTG 12 was pH5-10,the pH stability range of MTG I1 was wider than MTG 12;and at 37?,the pH stability of MTG I1 was significantly higher than MTG 12 when pH>6 and pH<5,and at 4?,the pH stability of MTG I1 was significantly higher than MTG 12 when 7<pH<10 and pH<5;so the temperature tolerance and the acid-base tolerance of MTG I1 were significantly higher than MTG 12.The Vmax of MTG I1 and MTG 12 were 0.84 U/mL*min and 0.56 U/mL*min,respectively;the Km of MTG I1 and MTG 12 were 19.08 mM/mL and 10.49 mM/mL,respectively;the affinity of MTG 12 with substrates was higher than MTG I1.The ratio and content of MTG I1 and MTG 12 were constant value,but MTG I1 had dynamic conversion phenomenon,when MTG 12 was separated from MTGase,MTG I1 would continue to transform to MTG 12,but MTG I2 was unable to transform to MTG I1.Therefore,the higher the proportion of MTG I1,the higher the stability of MTGase.4.The fine purification of MTGase.The method was:MTGase was purified by Hitrap Q FF chromatographic column(1 mL),the starting buffer was pH 7.0 10 mM phosphate buffer,elution buffer was pH 7.0 10 mM phosphate buffer +1 M NaCl with 0-20%in 25 CV,the flow rate was 1 mL/min.Loading quantity of sample was 5-10 mg,the yield of MTGase was about 65%,specific activity of MTGase was 23.62 U/mg,the purification method was stable and repeatable.After desalination and freeze drying,the yield of MTGase was about 47%,specific activity of MTGase was 25.88 U/mg.When the volume of anion exchange column was increased to 200 mL,the yield of MTGase was about 77%,specific activity of MTGase was 27.89 U/mg.The stability of purified MTGase was improved,and the electrophoretic pure was above 95%.Therefore,purified and stable MTGase cound be obtain by the methods.In summary,in this study,the rapid test methods of MTGase stability were established,which could quickly and effectively screen out the high stability of MTGase.And the relationship between MTGase stability and its structure was obtained by studying the stability of MTGase in different structures.Finally,In order to improve the purity and stability of MTGase,the purified and stable MTGase was obtained by fine purification.Therefore,it could be better used in various fields. |
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