Expression And Optimization Of Lacto-N-biose Phosphorylase

Lacto-N-biose(LNB)is an important component of oligosaccharides in breast milk,it is also an important substrate for life-sustaining activities of bifidobacteria,and plays an important role in promoting the colonization and proliferation of bifidobacteria in infantile intestines.Bifidobacteria can metabolize LNB to obtain the energy required for life activity through lacto-N-biose phosphorylase(LNBP).LNBP(EC 2.4.1.211)is a reversible phosph orylase that catalyzes the production of N-acetylglucosamine(Glc NAc),N-acety lgalacto samine(Gal NAc)and LNB and GNB(galacto-N-biose).?-D-galactose 1-phosphate(Gal 1-P).Wada et al.demonstrated that there is a gene cluster encoding a metabolic LNB in Bifidobacteria,wherein LNB is transported to the cytoplasm by a specific ABC-type transporter,and is metabolized by LNBP and ultimately in the glycolysis and amino sugar metabolic pathways.The lacto-N-biose phosphorylase gene was cloned into Bifidobacterium longum JCM1217,and the recombinant expression plasmid p ET28a-LNBP was constructed by ligation with plasmid p ET28 a.The recombinant plasmid p ET28a-LNBP was first transformed into E.coli BL21 to construct a recombinant plasmid.Bacteria E.coli BL21/p ET28a-LNBP.Next,the recombinant expression plasmid p HT43-LNBP was constructed by ligation with plasmid p HT43,and the recombinant plasmid p HT43-LNBP was transformed into Bacillus subtilis WB800 N to construct the recombinant strain B.subtilis WB800N/p HT43-LNBP.The recombinant lacto-N-biose phosphorylase was expressed in E.coli and Bacillus subtilis by IPTG induction.The expression conditions of recombinant E.coli BL21/p ET28a-LNBP and B.subtilis WB800N/p HT43-LNBP were optimized by single factor test,including initial bacterial density,inducer concentration,induction temperature,induction time and shaking culture speed.Optimization;in addition,the crude enzyme solution of the recombinant lacto-N-biose phosphorylase was purified using a Ni-NTA column,and its enzymatic properties were analyzed,including the optimum temperature and the optimum p H.The results of experiment are as follows:1.The optimal initial bacterial density of recombinant lactobacillus E.coli BL21/p ET28a-LNBP expressing lacto-N-biose phosphorylase was OD600=0.5,the final concentration of IPTG was 0.1 mmol/L,the induction temperature was 30 °C,and the expression time was induced.The optimum speed for 18 h shaking culture was 180 rpm.After the recombinant E.coli BL21a/p ET28a-LNBP was cultured under the above conditions,the total enzyme activity of LNBP obtained was 353.94 U/L,and the specific activity was 15.18U/mg.2.The optimal initial bacterial density OD600=0.5,0.1 mmol/L of the recombinant bacterium B.subtilis WB800N/p HT43-LNBP expressed as the final concentration of IPTG,37 °C is the optimal induction temperature,9h The optimum induction time and 120 rpm were the optimal rotation speed of shaking culture.After the recombinant B.subtilis WB800N/p HT43-LNBP was cultured under the above conditions,the total LNBP activity was778.82 U/L and the specific activity was 31.36 U./mg.3.The recombinant Ni-NTA phosphorylase was purified by Ni-NTA,and the target protein was eluted with an eluent containing 200 mmol/L imidazole,which had a good purification effect.The results of enzymatic analysis showed that the optimal reaction temperature of recombinant lactobiose phosphorylase was 20 °C,and the optimum p H was 7.4.In this paper,the optimal expression of recombinant lacto-N-biose phosphorylase was significantly improved by optimizing the expression conditions of recombinant E.coli BL21/p ET28a-LNBP and recombinant B.subtilis WB800N/p HT43-LNBP.The results showed that B.subtilis significantly increased the production of recombinant lacto-N-biose phosphorylase relative to E.coli.Therefore,the subsequent experiments selected Bacillus subtilis expression system for protein expression,provide technical support and guidance for the industrial production of lacto-N-biose phosphorylase.