Studies On The Purity And Properties Of The Pectinase And Xylanase From Bacillus Circulars

Pectinase (EC.3.2.1.15) is multiplex enzymes catalysing pectine hydrolysis.There are two kinds according to reaction mechanism,they are depolymerizing Enzyme and Pectinesterase.Peclinase are generally discovered in fruitage and microorganism. It was widely used to defecation procession of juice in food industry, procession being retted of flax in spinning industry and feedingstuff additive in farming.Xylanase is hydrolase acting on xylan.They have the development future in food,feedihgstuff and pharmacy industry.In order to enlarge their utilization of pectinanse and xylanase,it was studied that the purity,structure and properties of the pectinase and xylanase from Bacillus Circulars A₆ in the paper.The main results are as follows:(1)In order to find out the catalysis character of pectinase and xylanse in bacillus circularise A₆ and exhibit their maxium catalysis rate,the optimum condition for determination of pectinase and xylanase activity in bacillus circularise A₆ was studied in aspect of pH,temperature,substance concentration,reaction time,anion concentration.The optimum condition for determination pectinase activity showed as:0.5% pectine solution, pH 10.5,0.2mol/L glycine-sodium hydroxide buffer, at 45℃, reaction for five-minutes; The optimum condition of xylanse activity was: 0.8% xylan solution, pH 7.0,0.2mol/L phosphate buffer,at 55℃, reaction for ten-minutes.(2) After both of pectinase and xylanse were separated and purified by (NH₄)₂SO₄ sedimentation ,the purity of the pectinase was further improved by dialysis,CM-Sephadex C-50 gel anion exchange filtration and Sephadex G-50 gel filtration. The purity of pectinase and xylanase was determined by PAGE.The molecular weight of pectinase and xylanase was determined by SDS-PAGE. The results showed that the purity of the pectinase was improved about 41.2 times, the purity of the xylanse was improved about 18.3 times.The electrophoretic purity of pectinase and xylanase were get. The molecular weight of two subunits in the pectinase is respectively 32 253 and 27 400 ; the molecular weight of two subunits in the xylanse is respectively 86 489 and 57 422 .(3) The enzymology properties of the pectinase and xylanase was studied.Pectinase 1 is depolymerizing Enzyme, the depolymerizing Enzyme and xylanase is stable on 30-50℃; the optimal reaction temperature of depolymerizing Enzyme and xylanase was 45℃; the depolymerizing Enzyme was stable on pH 9-11, The optimal reaction pH of depolymerizing Enzyme was pH 10.5;and the xylanse is stable on pH 6-8, The optimal reaction pH of xylanse was 7.(4)The depolymerizing Enzyme was lightly inhibited by K⁺,Mg²⁺ ,and was seriously inhibited by Fe⁺,Cu²⁺,and a little activated by Ca²⁺;The xylanase was not inhibited by K⁺,Mg²⁺,and was seriously inhibited by Cu²⁺;and lightly activated by Fe⁺,Ca²⁺.(5)The experiment result showed: the depolymerizing Enzyme was used to retting in flax stem and juiceprocession; The xylanase was used to produce xylooligosaccharide with flax stem and rice stem and was usedto paper blanching.