Discovery Of Novel Cellobiose Phosphorylase And Study On Enzymology Properties

Recently,the cellobiose phosphorylase has been attracted much attention.Cellobiose phosphorylase belonging to glycoside hydrolase 94 family,can specificially degrad beta-1,4-glucosidic bond and catalytic cellobiose to ?-D-glucose and ?-D-glucose-1-phosphate.It is considered that the cellobiose phosphorylase has good prospects in the biofuels production and glycoside synthesis.In our study,we use cellobiose as the sole carbon source,screened out a cellobiose phosphorylase producing strain by measured special activity,and study its conditions for enzyme production.Gene clone the genomic DNA of two thermophilic bacteria strains obtained objective genes.We heterologous expression the gene in the E.coli and analyze the heterologous expression product,the results are as follows:(1)The strain BY-? producing cellobiose phosphorylase is a rod-shaped Gram-negative bacteria,after the sequence analysis of 16 S r RNA can determined that it belongs to Klebsiella.(2)Optimization the fermentation conditions we determine BY-?'s optimal fermentation temperature was 37 ?,fermentation time was 24 h,culture medium volume was 30 m L / 250 m L,shaker culture.(3)According to the gene sequence of Ta CBP and Tp CBP published by database,design primers,PCR amplification get size suitable nucleic acids fragments,double digestion and enzyme-linked,plasmid extraction and sequenced,finally,we get the Ta CBP protein sequence meet to protein database information completely,and the Tp CBP protein with four amino acid mutations.(4)The purpose gene was induced in Escherichia coli,collecting bacteria and ultrasonic disruption,detection by SDS-PAGE and then the target genes was be found successful expression in Escherichia coli and the concentration of the purified protein is 0.0527mg/m L and 0.0496mg/m L,respectively.(5)The enzymatic properties of the purified cellobiose phosphorylase were studied,the enzyme from Thermotoga petrophila has the optimum p H of 7.5,the optimum temperature of 80? and the half inactivation time of 48.29min;from thermosipho africanus TCF52 B has the optimum p H of 8.0,the optimum temperature of 75? and the half inactivation time of 64.66 min.(6)After the enzymatic kinetic analysis,when adjusted the concentrations of D-xylose,the Vmax values of two enzymes were 5.22?mol/ml* min?4.44?mol/ml* min,the Km values were 6.37mg/ml?5.18mg/ml,the kcat value were 30.831/s?34.261/s,respectively.when adjusted the concentrations of G-1-P,the Vmax values of two enzymes were 3.54?mol/ml*min?3.78?mol/ml* min,the Km values were2.73mg/ml?2.68mg/ml,the kcat value were 15.49/s?17.32/s,respectively.