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Cloning and Expression of Polygalacturonase Gene from Aspergillus Niger EIM-6

Author: QiangHuiNi
Tutor: HuangJianZhong；TianBaoYu
School: Fujian Normal University
Course: Microbiology
Keywords: Aspergillus niger Polygalacturonase gene Bioinformatics analysis Heterologous expression Co-fermentation
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 28
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Abstract


Polygalacturonase of the pectinase family is one of the most important components in degrading pectin and widely used in many fields including industry, agriculture, etc. In this study, the polygalacturonase gene was cloned from a pectin-producing Aspergilhis niger EIM-6 strain and expressed in Pichia pastoris GS115. Based on the recomminants, we launched the efficiency study of polygalacturonase to the whole enzymes. The polygalacturonase gene was first cloned based on the extracted Aspergillus niger EIM-6 genomic DNA as templet. After on-line Blast comparison, results show that pga gene was 1163 bp in size interrupted by one intron sited in 200-250bp and encoded a polygalacturonase of 370 amino acids with approximate molecular mass of 38 kDa. Furthermore, the primary structure, the hydrophobic interactions, and the spatial structure and other aspects were analysized and predicted. The studies showed polygalacturonase had high similarity to those reported A. niger polygalacturonases.The cDNA sequence of Polygalacturonase gene was amplified by using overlap-PCR and the coding region of pga was.obtained. The polygalacturonase cDNA was subcloned into pET30a vector and the recombinant plasmid was transformed into E. coli BL21. The polygalacturonase was overexpressed in E. coli strain BL21 after induction by IPTG, but the polygalacturonase accumulated in the cells in an insoluble form as inclusion bodies. So. the encoding gene of polygalacturonase was inserted into the plasmid pPIC3.5k and was successfully expressed in Pichia pastoris GS115 under the control of methanol. The culturing of the rcombinant Pichia pastoris strain yielded an additional protein band with an approximate molecular mass of 38 kDa in SDS-PAGE. The maximum activity of recombinant polygalacturonase was 560U/mL in the culture medium.The optimum pH and temperature is 4.0 and 50℃respectively, which are the same as that from wild strain.Based on the recombinant Pichia pastoris strain, the improvement efficiency of the polygalacturonase to the whole pecinase enzymes was studied. In the research of co-fermentation using with polygalacturonase and pectin lyase engineering strains, we found that when the initial inoculum between the two were equal, the color of the hydrolysis circles on the pectin plate was significantly deeper than that from individual recombinant. And the enzymatic properties of polygalacturonase and pectin lyase have not been changed. The whole results showed that the effect from co-fermentation on pectin-degrading was greater than that from the individual component.

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