Screening, Properties And Application Of Acid Pullulanase

With the development of science and biotechnology,microbial starch hydrolyzing enzymes have attracted researchers'considerable interests because of their potential application in food,brewing,fodder,detergent and so on.Pullulanase is a very important starch debranching enzyme.In this study,we cloned successfully the pullulanase gene of Bacillus megaterium Y103 isolated by our laboratory previous research,optimizing the level of expression and researching the characteristics and application of pullulanase.Then a wild strain of production pullulanase was isolated and identified from soil,the production,purification and characterization of pullulanase from the Streptomyces venezuelae C105 was investigated.The mainly conclusions are as follows:1.Cloning and expression of the pullulanase gene of Bacillus megaterium,purification,properties and application of the recombinant pullulanaseThe pullulanase gene of Bacillus megaterium Y103 which was screened and identified previously in our laboratory was cloned and expressed in Escherichia coli.The specific primers were designed by using the pullulanase gene in Bacillus megaterium Y103 as template,and the target gene was amplified by PCR.The recombinant plasmid was successfully constructed with Escherichia coli pET27b as the vector plasmid,then successively constructed in Escherichia coli JM109 and BL21?DE3?successively.The expression levels of pullulanase gene was optimized by single factor experiments.The enzyme activity of recombinant pullulanase reached 6.38 U?mL^-1,which was 5.5 times that of 1.16 U?mL^-1 before optimization?P<0.05?.The recombinant pullulanase crude enzyme solution was obtained by ultrasonication,further purified by Ni-IDA affinity chromatography to obtain electrophoretic purity.The recovery was 54.0%,the specific activity of pullulanase was 43.2 U?mg-1,and the molecular weight was 110.8 KDa by SDS-PAGE.The optimum pH and temperature were 6.0,55°C,and the stable pH and temperature was 6.0-8.0 and below 45°C,respectively.The metal Ca²⁺and urea have a great role in promoting pullulanase activity,reaching 289.2%,221.8%,respectively.The pullulanase can hydrolyze pullulan,soluble starch,amylopectin,and also has a certain hydrolysis effect on amylose,the products of hydrolyzed pullulan are mainly maltotriose and oligomeric oligosaccharides,indicating that it is a type?pullulanase.The enzymatic properties make it have great application value in bread baking.Our study also illustrated that the bread volume is increased by 36.7%after adding the pullulanase,the hardness is smaller?reduced 53.7%?,the elasticity is larger?incerased17.5%?,the shelf life is extended,and the overall acceptability of the bread is greatly increased.2.Isolation and identification of Streptomyces venezuelae C105,optimization,purification and characterization of the wild pullulanase.The strain was identified as Streptomyces venezuelae C105 by morphological observation,physiological and biochemical identification and 16S rDNA molecular biological test from the Dongchuan red land,Yunnan,China.The ability of Streptomyces venezuelae C105 to produce pullulanase was optimized by single factor experiments.The optimized medium was obtained:glutinous rice starch 2%,peptone 3%,KH₂PO₄ 0.05%,MgSO₄·7H₂O 0.01%,MnSO₄·7H₂O 0.01%.The initial pH of the culture medium,fermentation temperature and fermentation temperature were identified as 6.0,28°C,72 h,respectively.The enzyme activity was 0.97 U?mL^-1,6.06 times compared before?P<0.05?.The fermentation supernatant was obtained by centrifugation,precipitated by ammonium sulfate,purified by anion exchange chromatography to obtain electrophoretic purity.The recovery was 38.6%,the specific activity of pullulanase was 3.60 U mg^-1,and the molecular weight was 25.7 KDa by SDS-PAGE,the low molecular mass was rarely reported.The optimum pH and temperature were 5.0,45°C,and the stable pH was 5.0-8.0 and40°C,respectively.Ca²⁺,Mn²⁺,urea,Triton X-100 and mercaptoethanol have a significant effect on its enzyme activity.The production of hydrolyzing pullulan by the pullulanase is substantially all maltotriose,indicating that it should be a type I pullulanase,it posed great application value in the production of resistant starch,high glucose syrup,high fructose syrup and ultra-high maltose syrup.