Molecular Modification To Increase The Enzyme Activity Of Mtsase From Sulfolobus Acidocaldarius

Trehalose is a highly safe non-reducing disaccharide,which has excellent non-specific protective effects on organisms or biological macromolecules;it can also be used as a structural component of organisms to participate in the composition of some microbial cell walls;it also can be used as a carbon source and energy substance.Therefore,trehalose is widely used in medicine,food,cosmetics and agriculture.Maltooligosyl trehalose synthase?MTSase,EC 5.4.99.15?derived from S.acidocaldarius ATCC 33909 is one of the key enzymes for producing trehalose from starch,but the activity of the enzyme is low.It's disadvantages are not conducive to industrial applications.Therefore,a high-throughput screening method suitable for the screening of MTSase mutant libraries was constructed in this study.On this basis,directed evolution was used to increase the enzyme activity of MTSase from S.acidocaldarius ATCC 33909.The main results were listed as follows:?1?The influence of different substrate concentrations on high-throughput screening method was explored.A high-throughput screening system for MTSase with 0.2%?w/v?maltodextrin solution as a substrate was established.An error-prone PCR system for the construction of MTSase mutant libraries was explored.It was found that when the final concentrations of Mn²⁺and Mg²⁺in the PCR system were 0.4 mmol?L^-11 and 5.0 mmol?L^-1respectively,the mutation frequency of the mutation library was most suitable.According to the well-established error-prone PCR system,using MTSase gene treY as a template for error-prone PCR,a recombinant plasmid pET-24a?+?-treY was constructed and introduced into E.coli BL21?DE3?to construct an MTSase mutant library.?2?The first screening of MTSase mutant library based on screening system,F-7,E-3and D-6 with improved enzyme activity were obtained,and the shake flask fermentation enzyme activity was 50.64.U?mL^-1,40.51 U?mL^-11 and 41.22 U?mL^-1,which are 1.90,1.52 and1.54 times of the wild-type MTSase,respectively.The wild-type MTSase and mutants were isolated and purified,and their characterization was studied.The results showed that the optimal pH values for the different mutants and the wild-type were both 6.0.The stability of the different mutants was similar to wild type under the conditions of pH 5.5-9.0.The optimum temperature of mutants and wild-type MTSase is between 60-70?and the half-life at 60?is all above 14 d.The kinetic constants(K_m and k_cat)of the different mutants differed from the wild-type MTSase and the catalytic efficiency of the substrate was improved.?3?The mutant F-7 gene was used as a template to conduct the second round of directed evolution,an enzyme activity-enhancing mutant D-4 was obtained,and the shake flask fermentation enzyme activity was 63.96 U?m L^-1,which was 2.40 times that of the wild type.The mutant was isolated and purified,and characterization was studied.The results showed that the optimal pH value of 4 was 6.0.The stability of D-4 was similar to the wild type under the conditions of pH 5.5-9.0.The optimum temperature of D-4 was 60?and the half-life at60?was above 14 d.The catalytic efficiency of D-4 was 2.33 times that of the wild type.?4?Based on the mutant gene sequence analysis and site-directed mutagenesis technique,it was found that Phenylalanine at position 284 and threonine at position 439 were the most significant sites for the improvement of enzyme activity.When these two sites were changed to valine and alanine,respectively,the enzyme activity of the mutant was 1.45 and 1.50 times that of the wild type,respectively.Analysis of the mutation sites by molecular modeling suggested that the increase in enzyme activity may be due to the weakening of the hydrophobic interaction and the polar interaction makes the loop of the catalytic residue Asp443 more flexible,which facilitates the binding of the residue to the substrate.?5?The difference in enzyme production between the mutant D-4 and the wild-type MTSase in 3.6 L fermenter was investigated.The results showed that incubate at 37?until the OD₆₀₀00 reaches 40,adjust the temperature to 25°C,lactose was induced at a constant rate of0.1 g?L^-1?h^-1.At 18 h,the highest activity of the mutant was 624.7 U?mL^-1,while the wild-type MTase was 316.7 U?mL^-1.At the same time,the differences in the production of trehalose by using maltodextrin as a substrate were explored.The results showed that the trehalose conversion rate of both was 72.8%when the same amount of enzyme was added.The use of mutant D-4 would benefit for the cost of trehalose production.