Aspergillus Niger β-MANNANASE Gene Cloning And Expression Of Prokaryotic And Eukaryotic

β-mannanase (endo-1 ,4-β-D-mannanase; mannohydrolase; EC3.2.1.78) are a kind of class able to hydrolyze oligosaccharides, mannose polysaccharides (including mannan, galactomannan, glucomannan, etc.) of 1,4-β-D-mannosidase endo bond hydrolase and a semi-cellulose enzymes. The application prospect ofβ-mannanase is very extensive study of its high commercial value and scientific significance. Nowβ-mannanase has been widely used in the food, pharmaceutical, paper making, textile printing and dyeing, oil exploration and other aspects of biological research techniques. Industrial production of Aspergillus niger is the main species mannanase. However, the current production strain exists a large number of disadvantages , such as a number of spores during fermentation, short fermentation period , low enzyme production. In this case, the application of construction of genetic engineering engineering bacteria mannanase, simplified fermentation conditions, increased enzyme production, have important research value.In this research, mannanase gene producing strain of Aspergillus niger as a gene donor, cloned mannanase gene (MAN), were seperately constructed the E. coli expression vector, Pichia pastoris expression vector and the Agrobacterium-mediated gene MAN homologous recombination vector, respectively, in the meantime, were transferred to E. coli BL21, Pichia pastoris GS115 and glucoamylase producing strain of Aspergillus niger for laying the foundation for highly expressed genes.The main results are as follows:It appliedβ-mannanase producing strain of Aspergillus niger as the material and obtained by RT-PCR amplification to gainβ-mannanase gene MAN of the cDNA fragments. Though PCR amplification ofβ-mannanase gene gained MAN of the DNA fragments, the material that is Aspergillus niger glucoamylase production obtained by PCR amplification GlA gene 5 'and 3' homologous arm of the DNA fragments. As a result, the sequence is correct.1. MAN gene expression in E. coliIt appliedβ-mannanase producing strain of Aspergillus niger as the material and obtained by RT-PCR amplification to gainβ-mannanase gene MAN of the cDNA fragments.MAN gene of E. coli was constructed to express vector pET-MAN. After restriction endonuclease analysis is correct, the vector was introduced into E. coli BL21. Recombinant strain were induced by IPTG. Though SDS-PAGE, the expressed products were fusion protein by 62 KD band, indicating that the Aspergillus nigerβ-mannanase successful expression in E. coli. The determination of DNS method used different temperatures and different pH to dentify mannan fusion protein activity. It shows that recombinant mannanase optimum temperature was 55℃, the optimum pH of 5.5. In this condition, mannanase activity was 48 IU / mL. 2. MAN gene expression in Pichia pastorisThe cDNA fragment of the MAN as a template amplified by PCR to remove its signal peptide sequence fragment. MAN gene was constructed yeast expression vector pGAPHα-MAN. After restriction endonuclease analysis is correct, the vector was linearized and transformed into Pichia pastoris by electroporation GS115. Identification of transformants obtained by recombinant PCR. In the proper fermentation conditions.3. MAN gene expression in Aspergillus nigerThough PCR amplification ofβ-mannanase gene gained MAN of the DNA fragments, the material that is Aspergillus niger glucoamylase production obtained by PCR amplification Gla gene 5 'and 3' homologous arm of the DNA fragments.By overlap extension PCR method to MAN, Gla5 , Gla3 extension of the three genes overlap with the GMG. It construct the Gene vector pSZH-MAN.pSZH-MAN plasmid was transferred into Agrobacterium tumefaciens EHA105 by the freeze-thaw. The Aspergillus niger glucoamylase production co-culture, screening transformants highly expressed mannanase engineering strain. Recombinant strain induced, the expressed products were SDS-PAGE, and the determination by DNS method mannan of the protein activity.