Research On The Breeding Of β-glucosidase Producing Strains And The Enzymatic Properties

P-glucosidase, an enzyme with capacity of hydrolysing glycosidic linkages between aryl group/hydrocarbon and glycosyl to glucoxe, appears an extensive application in the field of bio-energy, food industry, health care industry, agriculture, medicine and ect.At present, the sweeping and efficient industrialization of β-glucosidase was confined by its low enzymatic expression level. Genetic engineering and multiple mutation techniques have been used to improve the yield of β-glucosidase. However, optimization for the fermentation condition is also a method to rapidly improve the yield of P-glucosidase. Meanwhile, the scale up cultivation in fermentor and the purification of P-glucosidase from fermentation broth are both the urgent problems to be resolved.In this paper, the recombinant β-glucosidase-producing strain Pichia was use as the original strain to breed a high yield of β-glucosidase mutant FH6 with atmospheric pressure at room temperature plasma (ARTP) mutagenesis and UV mutagenesis techniques. Also the enzyme-yielding conditions, high density fermentation conditions, purification methods of β-glucosidase and its enzymatic properties were studied. The main results are as follows:(1)The atmospheric and room temperature plasma (ARTP) and ultraviolet mutagenesis techniques was used to mutagenize β-glucosidase-producing strains from Pichia pastoris. After enzyme color circle method and 48 microplates for high-throughput screening rescreening means, finally got a β-glucosidase producing strain FH6, the activity of the starting strain and strain in shake flasks microplate increased by 84.34% and 85.66%, and genetic performance is more stable;optimized Pichia yield β-glucosidase in 48 microplates culture conditions:liquid volume 400μL, add methanol opportunity 24h, the amount of methanol was added 2.5%, temperature 28 ℃, rotation speed 200r/min; studies have shown that mutant in diameter than the size of the color circle, there is a good correlation between the activity of 48-well plates microplates, flasks activity, esculin using chromogenic medium for the mutant bacteria were screened,48 microplate and microplate reader measured enzyme activity in the culture of combining high-throughput screening can be used as a novel and effective method of screening for β-glucosidase producing strains.(2)Statistic method was used to optimize the fermentation of β-glucosidase produced by Pichia pastoris.Glycerol methanol and initial pH were confirmed as the critical factors impacting the enzyme activity according to the results of single factor experiment. Subsequently, using response surface methodology, the optimum condition for P-glucosidase yielding was obtained as following:19.4mL/L of glycerol,15g/L of ammonium sulfate,2% of methanol with initial pH value of 6.12 and 20mL of liquid volume. The β-glucosidase activity was up to 31.87U/mL, which was increased by 162.74% than that of the original.(3)High-density fermentation method, by mutation breeding resulting recombinant Pichia FH6 amplifying cultured in 3L fermentor. Glycerol fermentation process fed mode, methanol feeding and pH control mode is optimized,improved high-density culture of recombinant yeast Pichia FH6 method:constant speed stream glycerol, after the increase the initial methanol concentration of dissolved oxygen in series flow in methanol, pH value segment control (pH value in the control of cell growth stage 5.0, the pH-induced enzyme production phase control in 6.0), P-glucosidase activity reached 195.74 U/mL,specific activity in shake flasks 31.87 U/mL improved 5.14 times.(4)With 80% ammonium sulfate precipitation and Sephadex G-100 gel filtration chromatography, etc. The recombinant Pichia FH6 high density fermentation produced P-glucosidase was isolated and purified, P-glucosidase specific activity increased from 121.91 U/mg to 1392.21 U/mg, purification of 11.42 times, ultimate recovery of P-glucosidase was 16.21%.Identified by SDS-PAGE electrophoresis,p-glucosidase purified purity has reached electrophoretic homogeneity,molecular weight of the P-glucosidase is about 66kDa.(5)Characterization of P-glucosidase recombinant Pichia FH6 produced high density fermentation were studied. The optimum temperature of the β-glucosidase is 50℃,it has good thermal stability at temperatures below 40℃;the enzyme optimum pH is 5.0, suitable for storage at neutral pH 6.0 to 8.0; metal ions Co2+, Cu2+, Ag+, Ba2+, and Hg2+ the β-glucosidase have a strong degree of inhibition,wherein Hg2+ inhibition of the most significant, The Ca2+ and Fe2+could activate the enzyme action; mercaptoethanol, SDS, DMSO, EDTA, sodium azide five kinds of β-glucosidase inhibitors have a certain extent;in the reciprocal substrate concentration as abscissa, the reciprocal of the reaction rate for the vertical axis, as Lineweaver-Burk double reciprocal plot, michaelis constant Km is 1.3319mmol/L, Vmax value of 0.1779μmol·mL-1·min-1.