Studies On The Cloning, Characterization And Expression Of Endochitinase Gene From Trichoderma Viride In Escherichia Coli

The Chitinase from Trichoderma viride is a kind of endochitinase which can hydrolysis the internalβ-1,4 glucosidic bonds of chitin to produce chito-oligosaccharides.More than ten billion tons of chitin is produced annually in nature,and it is one of the second most abundant compounds after cellulose. Currently,many researches about chitinase acquired some achievements,but not realized the industrilization.Separating new chitinase genes and constructing the engineering strains that can efficiently express the chitinase are significant to realize the industrialization of chitinase.In this paper,endochitinase gene DNA and cDNA sequences from Trichoderma viride was amplified by the polymerase chain reaction(PCR) and reverse transcript polymerase chain reaction(RT-PCR) technology and sequence analysis was made.The prokaryotic expression vector containing the mature endochitinase was constructed and transformed into Escherichia coli BL21(DE3). The expressed product was analyzed by SDS-PAGE and the recombinant endochitinase activities were determined by DNS method.The biochemical characterization of the recombinant endochitinase and the optimization of the culture conditions of the recombinant strains were also studied.They are as follows:1.Based on the analysis on the homology of cloned endochitinase genes,a set of the degenerated primers were designed.A special DNA fragment about 1467bp was separated from Trichoderma viride by the PCR technology and a special eDNA fragment about 1276bp was also separated from Trichoderma viride by the RT-PCR technology.Alignment of DNA and cDNA sequences showed that there introns existed in DNA sequence whose length was 52bp,69bp and 64bp, respectively.2.Through analyzing sequence homology of Trichoderma viride endochitinase cDNA sequence and other reported endochitinase sequences,the results showed that the cloned fragment's homology was up to 100%with the endochitinase gene sequence from Trichoderma harzianum X79381.1 and Trichoderma viride AF188924,above 95%with Trichoderma sp.w512 DQ462415.1,Trichoderma virideEF635427.1,AF208842.1,Trichoderma aureoviride AY850032.1 and Trichoderma hamatum U88560.1.3.Endochitinase gene from Trichoderma viride encoded an acidic endochitinase with pI 4.95.The predicted amino acid number is 424 and has a calculated molecular mass 46kDa.Chitinase family 18 active site is located in endochitinase of Trichoderma viride and the consensus pattern is "FDGIDVDWE". The 3D-structure model of endochitinase from Trichoderma viride was predicted by homology modeling.4.Based on the sequence analysis,two set of the degenerated primers were designed and subsequently endochitinase gene containing signal peptide(ECH) and no signal peptide(MECH) were cloned.Utilizing the primitive vector pET-28a to construct prokaryotic expression vector pET28a-ECH and pET28a-MECH. Sequence analysis indicated that the cloned ECH and MECH genes were inserted the correct reading frame of expression vector,respectively.5.The recombinant strains containing the expression vectors pET28a-ECH and pET28a-MECH were induced to express,respectively.SDS-PAGE analysis indicated that the expressed protein molecular weight was 42kDa.The results of the parameter optimization,including the induced time,IPTG concentration and inoculum,showed that the best induced time of the expression vector pET28a-ECH was 3 hour,the best induced time of pET28a-MECH was 3-4 hours; the best induced IPTG concentration of expression vector pET28a-ECH was 1.0mmol/L.The preliminary activity determination results showed that the activity of the strain containing the vector pET28a-MECH was higher than that of the strain containing pET28a-ECH.6.The biochemical properties of the recombinants endochitinase were studied and the results showed that the optimal reaction temperature for the enzyme was 35℃and the optimal reaction pH was 7.0.Thermal stability of the recombinant endochitinase was not good and easily deactivate.Adding low concentration (1.0mM) metal ions such as Mg²⁺,Ba²⁺,Cu²⁺,Fe²⁺,Mn²⁺,Na²⁺ could enhance the enzyme activity,and adding metal ions such as Ca²⁺,Zn²⁺,Co²⁺ could inhibit the enzyme activity.In addition,the colloidal chitin concentration also had heavy effect on chitinase activity.7.The studies on the recombinant E.coli cultural conditions indicated that adding 1.0mmol/L IPTG after induced for one hour was beneficial to the enhancement of the recombinant endochitinase.The highest endochitianse activituy was achieved when the inoculum and the medium volumn were 5%and 100mL/500mL,respectively.