Expression,Fermentation Optimization Of Dictyoglomus Thermophilum Cellobiose 2-epimerase In Bacillus Subtilis And Study Of Application

Lactulose is a functional disaccharide which is a yellow clear liquid,and its sweetness is slightly lower than sucrose.Lactulose is widely used in food,medicine,animal fodder and other fields due to its physiological and pharmacological.Biology enzymatic preparation of lactulose has the advantages of mild reaction conditions and low environment pollution,so it is gradually become a research hotspot.Cellobiose 2-epimerase belongs to N-acyl-D-glucosamine 2-epimerase superfamily?AGEs?,and is capable of catalyzing the isomerization of the D-glucose residue of the oligosaccharide reducing end of the?-1,4-glycosidically linked to the D-fructose residue to produce lactulose.In this study,we synthesized the Dictyoglomus thermophilum cellobiose 2-epimerase gene.The gene was expressed in Bacillus subtilis CCTCC M 2016536 and its enzymatic properties were studied.Then shake-flask and 3-L fermentor fermentation was optimized to increase the CEase activity.Finally,the process optimization of lactulose production by recombinant enzyme was studied,which provided the foundation for enzymatic preparation of lactulose.The main results are as follows:?1?The cellobiose 2-epimerase gene ce was ligated to the expression vector pBSMuL3,and transformed vector into B.subtilis CCTCC M 2016536 to obtain recombinant strain B.subtilis/pBSMuL3-ce.The intracellular enzyme activity reached 7.5 U·mL^-1 after 48 hours of fermentation in the shake flask.SDS-PAGE showed that the target band appeared at 43 kDa,indicating that the cellobiose epimerase gene from D.thermophilum was successfully expressed in B.subtilis.Then the enzymatic properties of the recombinant CEase were examined.The results showed that the optimum pH was 8.5,and the optimum temperature was 85?.Under the conditions of pH8.5,the half-life of 85?was 120 min.The stability of the reombinant CEase was excellent.?2?In order to increase the expression level of the recombinant enzyme,the fermentation conditions of the recombinant strain producing CEase were explored in a shake flask.The results showed that the optimum medium was glycerol 5 g·L^-1,soybean meal 35 g·L^-1,1 mM Ba²⁺,K₂HPO₄·3H₂O 16.43 g·L^-1,KH₂PO₄ 2.31 g·L^-1,the recombinase intracellular enzyme activity reached 12.13 U·mL^-1 under such culture conditions.The fermentation conditions of the recombinant bacteria were further explored in 3-L fermentor.The effects of inorganic nitrogen source and organic nitrogen source on fermentation were compared.Then the effects of different carbon sources,constant flow feed and gradient feed,feed C/N ratio,fermentation temperature on growth and enzyme production were studied.The final result showed that 100g·L^-1 glucose,60 g·L^-1 soy peptone,175 g·L^-1 soybean meal powder as feed medium,that is,the feed C/N ratio was 2:1,and the fermentation temperature was 33?,the intracellular enzyme activity eventually reached 70.6 U·mL^-1,which was 9.35 times of shake flask.?3?The effects of different reaction temperature,initial reaction pH,substrate concentration,reaction time and enzyme dosage on the preparation of lactulose by recombinant CEase were studied.The results showed that the optimal enzyme conversion conditions were:reaction temperature 90°C,initial reaction pH8.5,substrate concentration 400 g·L^-1,amounts of CEase30 U·mL^-1,and the highest conversion rate was 60.2%after 4 h reaction,at this time,the content of epilactose in the product also reached a minimum of 7.8%.