Purification And Properties Of Fructosyltransferase And Comparative Study Of Difference In Enzyme Activity

Fructooligosaccharides（FOS）have been widely used in the food industry because of their physiological functions.Fructooligosaccharides come from a wide range of sources.In modern industrial production of Fructooligosaccharides,the main method is use the strains which can produce Fructosyltransferase though sucrose as the substrate.At present,researches on fructosyltransferases（FTase）were mainly focused on enzyme screening,purification and properties.Compared with mutant strains,wild-type strains have many shortcomings such as low thermal stability and low catalytic efficiency.However,little research has been done on the differences in enzyme characteristics and enzyme-producing mechanisms,which makes this study appear especially important.In the early stage of this laboratory,a strain of Aspergillus oryzae ZT65 producing FTase was selected from the coastal mud in the northwestern sea of Guangxi province,China.In the preliminary work of the laboratory,the Aspergillus oryzae ZT65 was obtained by ultraviolet mutagenesis to obtain a strain with high FTase Aspergillus oryzae S719.In this study,the FTase produced by the original strain Aspergillus oryzae ZT65 and the mutant Aspergillus oryzae S719 were isolated and purified,and their enzymatic properties were studied.The FTase gene,transcriptomics,and fermentation morphology of the two strains were analyzed afterwards.The reasons for the differences between the enzyme and enzyme production mechanisms are as follows:（1）Purified electrophoretic pure FTase from strains Aspergillus oryzae ZT65 and S719 by ammonium sulfate fractionation precipitation,DEAE-sepharose ion exchange chromatography,Mono Q strong anion exchange chromatography and Sepharose CL-6B gel column chromatography.The molecular weight of FTase detected by SDS-PAGE was about 65KD and 95KD.The specific activity of FTase purified by mutant S719 was 4200 U mg-1,the purification rate was 66 times,and the recovery was 26%.The specific activity of FTase purified by original strain ZT65 was 3587 U mg-1,and the purification rate was 54 times,the recovery rate was 16%.（2）Enzymatic properties of FTase1 and FTase2 isolated and purified from Aspergillus oryzae S719 and Aspergillus oryzae ZT65 were studied.Deglycosylation experiments on the purified FTase1 showed that the increased apparent molecular weight of about 30k Da was due to glycosylation.Under the non-denaturing conditions,after the glycosylation treatment,it was found that the N-glycosylation modification had no effect on the activity of FTase1 Significant effect,but may have an important effect on the expression and secretion of FTase1.Studies on the enzymatic properties of the enzyme showed that the optimal temperature and pH of FTase1 purified from mutants were 55°C and 6.0,respectively,and its activity remained stable at a pH range of 4.0to 11.0 and in environments less than room temperature more than 80%.The Km and kcat values of FTase were 310 mmol/L and 2.0×103 min^-1,respectively.5m mol L-1 Mg²⁺and 10m mol L-1 Na⁺promoted FTase1.With 50%（w/v）sucrose as the substrate and FTase as the catalyst,the yield of FOS reached 64%within 4h.The optimal temperature and pH of FTase2 purified from the original strain were 55°C and 5.5.It has high stability in the pH range of 4.0 to 8.0 and in environments less than 40°C.The Km of FTase2 is 381 mmol/L/min,and the catalytic efficiency is low.（3）Under the same fermentation conditions,the maximum enzyme activities of strain ZT65 were 18.1U/ml,while the maximum enzyme activities of strain S719 were 155.4U/ml.By comparing the FTase gene fragments of the original strain Aspergillus oryzae ZT65 and the mutant strain Aspergillus oryzae S719,the results showed that they were completely identical.The nucleotide sequence analysis showed that the gene was 1782bp in length and the encoded protein was composed of 594amino acids.Transcriptomics analysis showed that there were significant differentially expressed up-regulated genes 2488 and down-regulated genes 2636.The expression level of fructosyltransferase gene of Aspergillus oryzae S719 was 2.733 times that of Aspergillus oryzae ZT65.It was verified by RT-q PCR that the expression level of the mutant S719fructosyltransferase gene was 6.75 times,4.37 times,5.51 times of the original bacteria at 12 h,24 h and 36 h,which indicated that the overexpression of the fructosyltransferase gene of strain S719 promotes the increase of FTase1 enzyme activity.Observation of the morphology of the bacteria in the fermentation broth by electron microscopy and electron microscopy showed that the high enzyme activity of the mutant strain Aspergillus oryzae S719 was closely related to the morphology of the fungal colonies with loose and uniform hyphae.These results are important for the biotechnology industry,as they outline ways to increase the efficiency of FOS production.