| Xylan,a kind of poly five carbon sugar,is the major component of hemicelluloses in plant cell and the most abundant polysaccharide in nature except the content of cellulose. It is composed of beta-D- pyranose xylose unit and linked by 1-4 glycosidic. The complete degradation of the xylan requires a synergistic interaction between various enzymes in the xylanase system, in which xylanase play a critical role, especially beta-D-1,4 endo xylanase. The products of hydrolysis are mainly xylose, xylobiose and a small amount of arabinose.Xylanase widely exist in various kinds of bacteria, fungi and plants and animals. With the utilization of biomass resources and agricultural by-products and the research on physiological function of oligo xylose, the enzyme is widely used as a biomass resource in papermaking, foodstuff, feed, textiles, brewing, energy and other many industries in recent years,. Although there are a lot of wood poly enzyme was cloned and expressed at home and abroad, the application of most xylanase is limited for the reason of their narrow temperature and pH adaptability.With the development of molecular biology, structural biology and the application of protein engineering, the research on the relationship between structure and function of xylanase has been deeply studied. In this paper, we have synthesied the xylanase gene from Aspergillus kawachii and Bacillus sp and expressed both genes in Pichia pastoris GS115 respectively. On this basis, we reformed the xylanase gene from A. kawachii using computer aided design to change its pH adaptability. We also analyzed thermal stability of th he xylanase from the basophilic bacteria Bacillus sp.Main research results are as follows:According to the amin acid sequence of xylanase from Aspergillus niger and Bacillus sp. published in NCBI, the xylanase gene was desined based on the biaos codon of yeast and synthesized by PTDS methods. The total length of Aspergillus kawachii Xylanase Xyn C-C is 636 bp, which encoding a protein with one different site on Thr64 Ser from the Aspergillus niger(accession number AY178045). The GC content in the synthesized gene was adjusted to 52.2%. The whole gene of xylanase from Bacillus sp(accession number is U51675.1) is 606 bp. and the GC content in synthesized gene was adjusted to 48.3%. Both synthesized XynC-C and Bsxyn A genes was inserted into the cloning vector pMD18-T and the correct clone was selected by sequencing. Then both Xylanase genes were cloned into yeast expression vector pYPX88 and transformed to Pichia pastoris G115. Subsequently, the activity of Xylanase was tested to be 82.27U/m L and 82.27U/m L from the the fermentation culture medium for XynC and BsxynA yeast strain, respectively. The specific activity of XynC and Bsxyn A were 600.5 U/mg and 291.87 U/mg, respectively. High expression of xylanase in Pichia pastoris was achieved. The yeast expression strains for Xylanase had good genetic stability. The recombinant xylanase was purified by ammonium sulfate precipitation following Ni2+ chelating affinity chromatography. SDS-PAGE identification results showed that the molecular weight of XynC-C and BsxynA were about 23 k D and 28 kD. The molecular weight of XynC-C was similar to the predictive value, while Bsxyn A was smaller than the predictive value. It indicated that XynC-C had no glycosylation, and BsxynA had the occurrence of glycosylation. The degree of glycosylation for BsxynA was about 4 kD.After purification, the enzymatic properties of recombinant xylanase Xyn C-C and BsxynA were analyzed. The optimal pH of XynC-C and Bsxyn A were 3.8 and 6.6, respectively. The optimum temperature for both enzyme is 45°C. However BsxynA has better temperature stability than XynC-C. The value of Km and Vmax for XynC-C and BsxynA is 10 mg/mL, 1250 μg/mL/min and 8 mg/mL, 83.3 μg/m L/min respectively. The reducing agent DTT and diffent metal ion(Zn2+, K+, Mg2+, Co2+, Ca2+, Al3+, NH4+, Fe3+, Fe2+,, Ni2+, Na+, and Cr3+ can improve the activation of xylanase XynC-C. However, metal ion Cu2+, Mn2+, and surface active agent SDS have inhibitory effects on XynC-C and the inhibition of SDS and Mn2+ on XynC-C is relatively strong. No effect of the stomach and trypsin on the activity of xylanase XynC-C. Ni2+,DTT,Fe2+,Ca2+,Mg2+ and K+ can improve the activation of xylanase BsxynA. Among them, DTT and Fe2+ had stronger activation effect on xylanase. Mn2+, Zn2+, Co2+, SDS, Fe3+, Cu2+, Na+, and Al3+ have inhibitory effect on the activation of BsxynA, in which SDS, Al3+, Fe3+ and Cu2+ have stronger inhibition.Multiple sequence alignment of homologs indicated that XynC-C and BsxynA belong to the GH11. The catalytic site of XynC-C is located in Glu90 and Glu181, the catalytic residue site of Bsxyn A is Glu86 and Glu180.The results of homology modeling, sequence comparison and mutagenesis, showed that in xylanase XynC-C several amino acids on β-sheet of A5()is related to xylanase optimum pH. But the effect of Asp48 on xylanase optimum pH is more significant. In addition, Tyr77, Tyr81, Tyr92, and Trp183 most likely act as substrate binding site.This results have laid an important foundation for the further research on the relathion of structure and function for xylanase. |
PDF Full Text Request