Molecular Modification Of Caulobacter Crescentus Sucrose Hydrolase And Its Application In The Preparation Of Turanose

Turanose is naturally present in honey and is an isomer of sucrose,with half the sweetness of sucrose.Compared with sucrose,turanose has the advantages of low calorie and no caries,and is expected to replace sucrose as a new functional sweetener.At present,cyclodextrin glucosyltransferase（CGTase） and amylosucrase（ASase） are mainly used to prepare turanose.However,the use of CGTase to prepare turanose is expensive,and the malto-oligosaccharides and trehalulose,which are the by-products in the preparation of turanose by ASase,are not conducive to the separation and purification of turanose,and both enzymes are not conducive to the large-scale industrial production of turanose.In order to solve the above problems,this research took the Caulobacter crescentus sucrose hydrolase mutant S271A（CcSHS271A）with transglycosidation ability obtained in the early stage of the laboratory as the research object,and its molecular modification and enzyme conversion process optimization were carried out to improve the conversion rate and purity.The obtained CcSHS271A mutant I382Q（CcSHS271A/I382Q） was recombined and expressed in the food-safe bacteria Bacillus subtilis,and its expression level was improved through promoter screening and fermentation optimization.The main findings are as follows:（1）The enzymatic properties of CcSHS271A and the enzymatic conversion for the preparation of turanose were investigated.The sucrose hydrolysis mutant CcSHS271A derived from C.crescentus was analyzed for enzymatic properties.The optimum temperature and pH of CcSHS271A were 45℃and 8.0,respectively.The half-life at 30℃was 312 h,and the thermal stability was good.Then,CcSHS271A was used to optimize the preparation of turanose.The results showed that under the conditions of pH 8.0 and 30℃,when 2 mol·L^-1 sucrose was used as the substrate,60 U·g^-1 sucrose CcSHS271A was added,the maximum yield of turanose was 63.3%.However,the yield of trehalulose in the product produced by CcSHS271A was9.1%,which would affect the subsequent separation of turanose.（2）The effect of mutation of CcSHS271A receptor site on the yield and purity of turanose was explored.CcSHS271A was molecularly modified to obtain mutant CcSHS271A/I382Q,which had a high conversion rate of turanose and a low conversion rate of trehalulose in the product,which was 2.3%.The enzymatic properties of CcSHS271A/I382Q were explored.The optimum temperature and pH were 45℃and 8.0,respectively.The half-life of this enzyme at 30℃was 384 h,and the half-life was increased by 72 h compared with CcSHS271A.The process of CcSHS271A/I382Q for the preparation of turanose was optimized,and the results showed that under the condition of pH 5.0 and 30℃,when 2 mol·L^-1 sucrose was used as the substrate,the highest yield of turanose prepared by adding 60 U·g^-1 sucrose CcSHS271A/I382Q was 70.3%.At the same time,trehalulose was not detected by the HPLC of the product,which was beneficial to the separation and purification of turanose.（3）The intracellular expression of CcSHS271A/I382Q in B.subtilis was investigated,and its intracellular expression level in B.subtilis was improved through promoter screening and fermentation optimization.A recombinant B.subtilis strain containing CcSHS271A/I382Q was constructed.By optimizing the promoter,the obtained promoter P_ahpF increased the shake flask fermentation enzyme activity of the recombinant strain to 2.7 U·m L^-1,which was 4.5 times higher than before optimization.By optimizing the carbon source and nitrogen source components of the recombinant strain shake flask fermentation medium,the best carbon source was 20 g·L^-1 glycerol,and the best nitrogen source was 20 g·L^-1 soy peptone and 15 g·L^-1 bone peptone,and the shake flask fermentation enzyme activity of the recombinant enzyme was increased to 3.8 U·m L^-1,which was 1.4 times higher than before optimization.Finally,after 74hours of fermentation in a 3-L fermenter,the enzyme activity of the recombinant bacteria reached the highest 41.0 U·m L^-1,which was 10 times the enzyme activity of shake flask fermentation.In addition,the yield obtained when using B.subtilis recombinant bacteria 3-L fermenter fermentation enzyme solution to prepare turanose was 70.8%,which was basically consistent with its recombinant expression in Escherichia coli to produce turanose.