| Summary: | 碩士 === 靜宜大學 === 食品營養研究所 === 94 === The aims of this study were to overexpress the xylanase gene of Trichoderma reesei in Escherichia coli and determine the characteristics of recombinant enzyme. Recombinant xylanase gene was constructed by ligating the cDNA of xylanase, obtained from reverse transcription -polymerase chain reaction, with the expression vector pET-43.1b(+). Several expression hosts, including E. coli AD494(DE3), E. coli DH5α, E. coli DH10B, E. coli JM105, E. coli BL21(DE3) and E. coli AD494(DE3)pLys, were used to express the recombinant xylanase II. DNA sequencing data showed that the cloned xylanase I and xylanase II contained 534 and 570 bp, respectively, and had the homology of 100 and 99% with xylanase I and xylanase II reported by Trrnen et al, respectively. Compared to the reported xylanase II, the cloned xylanase II had two different nucleotides. One of the different nucleotide made no change on the gene codon of praline. The other changed the gene codon of alanine to the gene codon of valine. Fortunately, the two different gene bases did not locate at the gene sequences for the catalytic residues of the enzyme (Glu 86 and Glu 177). Xylanase I expressed as inclusion bodies in all expression hosts and none of them presented as active enzyme. For the expression of recombinant xylanase II, all of the expression hosts expressed the enzyme in the form of inclusion body, except the AD494(DE3)pLys. Denature-Ni-NTA purification and three-phase partitioning were attempted to soluble the insoluble form of recombinant xylanase II produced AD494(DE3)pLys; however, none of these methods worked successfully. Molecular weight of the soluble xylanase II was approximately 21 kDa and the specific activity and pI were 188.8 U/mg and 6.37, respectively. Optimal reaction pH and temperature for the recombinant xylanase II were 5.0 and 50℃, respectively. The recombinant xylanase II was stable at pH range of 5.0-8.0 and maintained 70% residual activity after incubating at 30℃ for 1 hr; however, the enzyme activity decreased dramatically when pH was below 4.0. For thermal stability of enzyme activity, about 70% of the original activity was left even at 50℃for 1 hr. Kinetic parameters Km and Vmax of the recombinant xylanase II were 12.63 mg/ml and 0.552 umole/mg/min, respectively. The activation energy of the recombinant xylanase II was 1.013 Kcal/mole. Birchwood xylan and beechwood xylan could be hydrolyzed easily by the recombinant xylanase II. Xylanase activity was apparent inhibited by 8 mM mercuric ion, but was apparent enchanced by 8 mM EDTA. Since the catalytic residue of recombinant xylanase II was glutamine, the enzyme activity was expected to be inhibited by Woodward’s reagent. Surprised, the activity was increased. We suggested the Woodward’s reagent modified the protein structure outside the catalytic center, and resulted in the increase of enzyme activity.
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