| As a natural oligosaccharide,trehalose has excellent non-specific protective effect on organisms,and can be used as a component of energy supply in organisms.It is slightly sweet,antioxidant and moisturizing.Therefore,trehalose is widely used in food,medicine and cosmetics.Trehalose is mainly obtained from starch by maltooligosyl trehalose synthase?MTSase,EC5.4.99.15?and maltooligosyl trehalose halohydrolase?MTHase,EC3.2.1.141?.However,most of the expression hosts of these two enzymes was E.coli.endotoxins secreted by these two enzymes are not conducive to the preparation of food enzymes.B.subtilis does not secrete endotoxins,and moderate fermentation period and convenient cultivation are one of the best choices for expression hosts of food-grade enzymes.B.subtilis WS11/pHY300PLK-Y and B.subtilis WS11/pHY300PLK-Z expressing Athrobacter ramosus MTSase and MTSase were constructed earlier in our lab.In this study,fermentation optimization was carried out at shaking flask and 3-L levels,and enzymatic properties of the two recombinases were studied respectively.The recombinant bacteriaB.subtilis WB600/pHY300PLK-Z and B.subtilis RIK1285/pHY300PLK-Z were constructed,and the latter was optimized by shaking flask and 3-L tank fermentation.Then,the technological process of trehalose preparation was optimized,and the pilot test was carried out.Finally,the conditions of trehalose separation and extraction were preliminarily explored.The main results are as follows:?1?The recombinant bacteria B.subtilis WS11/pHY300PLK-Y was fermented in shaking flask,and the enzymatic properties of MTSase were investigated.The results showed that the optimum pH was 6.0,and the optimum temperature was 45?.Under the conditions of pH 6.0,the half-life of 45?was 76 h,similar to the enzyme expressed in E.coli.The fermentation conditions of the recombinant strain producing MTSase were explored in a shake flask.The results showed that the optimum carbon and nitrogen sources were glucose 5 g·L-1,industrial peptone 10 g·L-1,and corn syrup 25 g·L-1,the enzyme activity reached 63.2 U·mL-1 under such culture conditions.On the basis of shaking flask fermentation optimization,the fermentation conditions of recombinant bacteria in 3-L tank were optimized.The results showed that when glucose was used as carbon source,industrial peptone and corn syrup as nitrogen source,C/N1:1 and pH 7.0,the activity of MTSase recombinant bacteria reached 1139.2 U·mL-1,which was 18 times than that of shaking flask fermentation.?2?The recombinant bacteria B.subtilis WS11/pHY300PLK-Z was fermented in shaking flask,and the enzymatic properties of MTHase were investigated.The results showed that the optimum pH was 5.5,and the optimum temperature was 55?.Under the conditions of pH 5.5,the half-life of 45?was 4 h,similar to the enzyme expressed in E.coli.The fermentation conditions of the recombinant strain producing MTSase were explored in a shake flask.The results showed that the optimum carbon and nitrogen sources were glycerol 5 g·L-1,industrial peptone 25 g·L-1,and soybean peptone 10 g·L-1,xylose induction concentration 5 g·L-1,the recombinase enzyme activity reached 74.7 U·mL-1 under such culture conditions.On the basis of shaking flask fermentation optimization,the fermentation conditions of recombinant bacteria in 3-L tank were optimiz that is,the feed C/N ratio was 2:1,and the xylose flow acceleration rate was 0.5 g·L-1·h-1.The enzyme activity eventually reached 294.2 U·mL-1,which was 3.9times than that before shake flask optimization.The enzyme activity decreased after 40 h of fermentation,which resulted in instability of fermentation enlargement.?3?To investigate the effect of different B.subtilis host bacteria on MTHase production.B.subtilis RIK1285/pHY300PLK-Z and B.subtilis WB600/pHY300PLK-Z were constructed,in which the recombinant B.subtilis RIK1285/pHY300PLK-Z shake flask fermentation activity was higher,the enzyme activity was 54.7 U·mL-1.Fermentation optimization of the higher was carried out.The results showed the optimum carbon and nitrogen sources were 5 g·L-1glycerol,soybean peptone 25 g·L-1 and corn syrup 5 g·L-1,the enzyme activity reached 74.7 U·mL-1under such culture conditions.Based on shaking flask fermentation medium,the fermentation conditions of recombinant bacteria were optimized at 3-L tank.On the basis of shaking flask fermentation optimization,the feed C/N of recombinant bacteria in 3-L tank was studied.The final result showed that the feed C/N ratio was 1:1.The enzyme activity eventually reached816.2 U·mL-1,which was 2.8 times than that in B.subtilis WS11.?4?200 g·L-1 substrate with DE value of 17-20,200 g·L-11 was used as substrate to catalyze the conversion of 52.6%at 45??pH of 6.0 in 40 h.The system contains some by-products such as maltose and maltotriose.Cyclodextrin Glycosyltransferase??/?-CGTase?with disproportionation activity can prolong the degree of polymerization to improve the utilization of substrates.The catalytic period was shortened to 30 h and the conversion rate was increased to 68.6%when?/?-CGTase from Bacillus stearothermophilus was mixed.On the basis of this optimization,a pilot test in a 10-t tank was carried out.The conversion rate of trehalose was65.4%.Then trehalose was separated and extracted.The optimum conditions of decolorization,separation and concentration crystallization of activated carbon were determined.Final yield of trehalose was 80.1%. |
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