| Trehalose is a non-reducing disaccharide that is prevalent in nature.It is non-toxic,stable and widely used.Because trehalose has a protective effect on biological substances in unfavorable environment,trehalose is used in food,cosmetics,medicine and biological sciences,and has wide application prospect.In the method for preparing trehalose,the double enzyme method of maltosyl trehalose synthase(MTSase)and maltosyl trehalose hydrolase(MTHase)has a high application value due to starch as the starting substrate of reaction.For the first time,the treY gene encoding the MTSase and the treZ gene encoding the MTHase were codon-optimized and introduced into Escherichia coli(E.coli).The recombinant expression MTSase and MTHase were studied,and the fermentation conditions of the recombinant bacteria were optimized.The two enzymes of this source were co-expressed in E.coli for the first time,and the fermentation conditions of the recombinant bacteria were optimized.The main findings of the thesis are as follows:(1)The target gene was ligated into the E.coli BL21(DE3)by ligating the expression vector pET-24a(+)with the treY and treZ genes of Arthrobacter ramosus S34,which was optimized by artificial synthesis.E.coli BL21(DE3)/pET-24a-treY and E.coli BL21(DE3)/pET-24a-treZ were constructed respectively.The activity of MTSase in the supernatant of the recombinant strain E.coli BL21(DE3)/pET-24a-treY was determined to be 194.4 U?mL-1,and the enzyme activity of MTHase in the supernatant of recombinant strain E.coli BL21(DE3)/pET-24a-treZ was 528.0 U?m L-1,which reported 17.2 times of the activity of MTSase in the strain,MTHase enzyme activity of 3.0 times.(2)The recombinant strains E.coli BL21(DE3)/pET-24a-treY and E.coli BL21(DE3)/pET-24a-treZ were cultivated in 3L fermentors,and induced temperature and Lactose induced concentration was optimized.The results showed that the optimum conditions for the recombinant strain E.coli BL21(DE3)/pET-24a-treY were the induction temperature of 32? and the lactose flow rate was 0.2 g?L-1?h-1.The OD600 value of the cells was 154,the activity of MTSase was 1608.3 U?mL-1,which was 8.3 times higher than that of shake flask fermentation.The optimum conditions for the recombinant strain E.coli BL21(DE3)/pET-24a-treZ were induction temperature of 27?,lactose flow rate was 0.4 g?L-1?h-1,the OD600 value of the cell was 149.4,the activity of MTSase was 8766.2 U?mL-1,which was 16.6 times of that of shake flask fermentation.(3)The enzymatic properties of MTSase and MTHase were studied.The optimum temperature of MTSase was found to be 45 ?,the optimum pH was 7.0,the optimum temperature of MTHase was 55?,and the optimum pH was 6.0.The effects of reaction temperature,initial pH,substrate DE value,enzyme concentration and substrate concentration on the yield of trehalose were studied.The optimum conditions of the process were as follows: reaction temperature 45?,initial pH 5.5,substrate DE value 8.5,enzyme dosage MTSase minimum dosage 15.75 U?g-1 starch,MTHase minimum dosage 7.5 U?g-1 starch.The yield of trehalose was 67.7%.(4)The treY and treZ genes of Arthrobacter ramosus S34 were co-expressed in E.coli with pETDuet-1 as vector,and the co-expression strain E.coli BL21(DE3)was constructed by adjusting the order of treY and treZ in the vector.E.coli BL21(DE3)/PETDuet-1-treYtreZ and E.coli BL21(DE3)/pETDuet-1-treZtreY were constructed respectively.The two strains were fermented in shake flask,then the results showed that the ratio of MTSase and MTHase activity of recombinant strain E.coli BL21(DE3)/PETDuet-1-treYtreZ was 1: 1.4,and the recombinant strain E.coli BL21(DE3)/pETDuet-1-treZtreY had a ratio of MTSase to MTHase activity of 1: 2.4.Since the ratio of MTSase and MTHase was 1: 0.5 in the process of formation of trehalose by MTSase and MTHase,the MTHase activity of the two recombinant co-expressing engineering bacteria was excessive.The selection of the strain was determined by the enzyme activity of MTSase.While the MTSase activity of recombinant E.coli BL21(DE3)/PETDuet-1-treYtreZ was 158.16 U?mL-1,which was 30% higher than that of the recombinant strain E.coli BL21(DE3)/pETDuet-1-treZtreY,the activity of MTSase was 119.76 U?mL-1.Thus,E.coli BL21(DE3)/PETDuet-1-treYtreZ was selected as co-expressing strain.(5)The recombinant strain E.coli BL21(DE3)/pETDuet-1-treYtreZ was optimized on the 3L fermentor.The most suitable fermentation conditions were as follows: induction temperature 27?,lactose flow rate 0.4 g?L-1?h-1,the OD600 value of the cell was 151.9,the activity of MTSase was 1827.4 U·mL-1,the activity of MTHase was 2944.9 U?m L-1,in which the enzyme activity was 113.6% higher than that of recombinant E.coli BL21(DE3)/pET-24a-treY.The effects of different enzyme contents on the yield of trehalose were studied.The results showed that when the activity of MTSase was greater than or equal to 15.75 U?g-1,the trehalose conversion rate can be maintained at 67%.This is similar to the conversion of trehalose obtained by trehalose conversion with two single expression enzyme solutions. |
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