Heterologous Expression And Immobilization Of MTSase And MTHase In Bacillus Subtilis

Trehalose has good chemical stability and unique biological properties,and is widely used in food processing and protection of biological macromolecules.At present,the production of trehalose is dominated by a two-enzyme method,which uses maltodextrin as a substrate,and uses maltooligosyl trehalose synthase(MTSase,EC5.4.99.15)and maltooligosyl trehalose trehalohydrolase(MTHase,EC 3.2.1.141)synergistically catalyzes the production of trehalose.Although trehalose has achieved industrial production,the main production bacteria are Escherichia coli,which contains endotoxins and is not conducive to the preparation of high-quality trehalose with low endotoxins.In this study,the safety-grade strain Bacillus subtilis without endotoxin was used as the host bacteria,and the MTSase and MTHase derived from Sulfolobus acidocaldarius ATCC 33909 were expressed heterologously.This study will explore the enzymatic properties of MTSase and MTHase,the optimization of the fermentation medium of MTSase and MTHase recombinant bacteria,and the optimization of trehalose conversion conditions.Due to the stable enzymatic properties of MTSase and MTHase,in order to improve the utilization rate of the enzyme,reduce the production cost,and facilitate the separation of trehalose,a one-step reaction can be used to achieve the purification and immobilization of Spy Tag-MTSase and Spy Tag-MTHase recombinase.Spy Catcher immobilized carrier,through optimization of immobilization conditions,realizes the multiple cycles of Spy Tag-MTSase and Spy Tag-MTHase recombinase.The main work of this paper is as follows:(1)MTSase and MTHase derived from S.acidocaldarius ATCC 33909 were heterologously expressed in B.subtilis WB800N/?amy E::Spc respectively.It was determined that the optimum temperature of MTSase was 65 ?,the optimum p H was5.5,and the specific enzyme activity under optimum conditions was 106.7 U/mg;the optimum temperature of MTHase was 75 ?,the optimum p H was 6.0,and the ratio under optimum conditions The enzyme activity is 243.9 U/mg.(2)Through optimization of the fermentation medium of recombinant bacteria,it was determined that 12 g/L sucrose was the carbon source,18 g/L Angel yeast extract powder FM888 was the nitrogen source,the phosphate concentration was 1 mmol/L,and glucose was added to maintain reducing sugar.At a concentration of 5 g/L,the enzyme activities of MTSase and MTHase increased to 6652.68 U/g and 5543.23 U/g,respectively.(3)Through optimization of trehalose conversion conditions,it was determined that the DE value of maltodextrin was 8.8,the addition amount of Promozyme D2 was5.0 U/g,the conversion temperature was 60 ?,the p H was maintained at 6.0,the addition amount of MTSase was 95.0 U/g,and the addition of MTHase The amount is35.0 U/g,the amount of CGTase added is 1.0 U/g,200 g/L maltodextrin is used as the substrate,and the conversion time is 48 h.After saccharification with glucoamylase,the conversion rate of trehalose is increased to 74.7%.(4)Using microcrystalline cellulose as the matrix to prepare cellulose microspheres;according to the principle that the carbohydrate binding domain(CBM)can adsorb cellulose,fix the Spy Catcher and CBM fusion protein Spy Catcher-CBM on the cellulose microspheres to prepare Obtain the Spy Catcher immobilized vector;according to the principle that Spy Catcher and Spy Tag can spontaneously form isopeptide bonds,the Spy Tag-MTSase and Spy Tag-MTHase recombinases were purified and immobilized respectively.(5)Through optimization of immobilization conditions,it was determined that the immobilization temperature was 25 ?,p H 7.4,the molar ratio of the target enzyme to the Spy Catcher immobilized carrier was 1:1.4,and the immobilization time was 20 min,the immobilization rate of the target enzyme was 76.6%.Based on the optimization of the immobilization conditions,the Spy Tag-MTSase and Spy Tag-MTHase in the crude enzyme solution were purified and immobilized respectively,and the immobilization rates were 68.9% and 68.0%,respectively.(6)Based on the optimization of trehalose conversion conditions,add 95.0 U/g of Spy Tag-MTSase immobilized enzyme and 35.0 U/g of Spy Tag-MTHase immobilized enzyme for trehalose conversion,and the immobilized enzyme is recycled3 times,trehalose The conversion rate is higher than 50%.