Purifying And Cloning Of Chitinase From Trichoderma Koningii Used For Biological Control Of Wood Blue-Stain

In the present study,purifying,character analyzing and molecular cloning of chitinase from Trichoderma koningii were carried out with T. koningii(6111-ET)as testing fungus.The composing sequence and biological features of cloned chitinase genes were analyzed in order to provide the necessary data for further study on sapstain control mechanisms and biological agent production and application in bio-control of wood sapstain.T.koningii was cultured in PDA broth at 28℃on a rotary shaker at 200r/min for 10 days.The extracellular chitinases was purified to SDS-PAGE homogeneous by ammonium sulfate fraction,Q-HP chromatography,Phenyl-Sepharose chromatogram,CM-Sepharose chromatogram and Phenyi-Sepharose chromatogram from T.koningii.The molecular weights of the chitinases were about 36kD.The basic properties of chitnases were tested in this study,and the results were as the followings:The optimal pH range and temperature of the enzyme hydrolysis of chitin are pH 3～6 and at 40℃respectively.The enzyme activities kept unchanged after 80 min at 30℃and 60℃.When temperature is over 60℃, the chitinase acticities would begin to decline.This enzyme has certain resistibility on high temperature,with temperature increasing,the chitinases remained 60%of its activity.Different metal ions should different effects on the chitinase acticities.Ca²⁺,Fe²⁺significantly increased activities of the chitinases,whereas K⁺,Na⁺,Cu²⁺caused serious depression.In the present study,chromosomal DNA of T.koningii was extracted by the improved CTAB method and one pair of primers was designed according to the reported chitinase DNA homology sequence of T. koningii.The chitinase gene from T.koningii was cloned in E.coli JM109 with pMD-18T cloning vector by polymerase chain reaction(PCR) amplification method.The positive clone was identified by PCR and used for sequencing.Compared with the Trichoderma chitinase which has been reported,the results shown that similarity values are 90%upwards.The chitinase gene from T.koningii was cloned,sequenced and analyzed to provide the basis for selection of individual fungus of T.koningii with high chitinas yield and for chitinase prodution in commercial scale.