Homologous Expression Of Tannase Gene In The Aspergillus Niger

Tannase is a specific hydrolytic enzyme. It can hydrolyze ester bon, condensing phenol carboxylic key of gallate tannins, generate gallic acid and other compounds. It can also hydrolyze the precipitate in the cold tea or drink(milk tea produced in the cold tea or beverage). Tannase has a great application prospect in the food, feed, cosmetics, pharmaceuticals, leather and other aspects. It has aroused people’s attention,at present, the production of enzymes tannins mainly relys on fermentation of Aspergillus niger the productivity is low. The enzyme activity is mostly at200U/g,but the price is as high as200yuan/kg. Hence, it is in urgent need to build safe and efficient engineering bacteria to increase the producing capacity of tannase.This research based on food-grade Aspergillus niger expression system, which is established by our laboratory and had independent property right. We amplified the coding region of the tannase gene from the genome of Aspergillus niger, and constructed the express vector:pSZH-tan, The tannase gene was transferred into Aspergillus niger by Agrobacterium-mediated method and integrated it to the saccharifying enzyme gene loci which is high expression.Then we could achieve homologous high expression of tannase gene in Aspergillus niger, it laid the foundation of cultivating engineering bacteria of tannase. The main results are as follows:1Obtaining of Aspergillus niger transformants from tannase geneThe positive rate of tan gene conversion was50%by PCR.We identified four positive strains by PCR using homologous recombination primers. All of four positive strains could be amplified purpose bands at about1701bp. They were all homologous recombinants and the homologous recombination rate was100%. We obtained four homologous recombinants with recombinant screening and PCR primers. And then we analyzed the expression product of the transformant with SDS-PAGE and detected its enzyme activity.2Expression of tannase gene in the Aspergillus nigerThe tannase activity of fermentation supernatant of recombinant was up to41.12U/mL in the shaking flask conditions, and it was13.80times of starting strain(2.98U/mL). We took fermentation supernatant for SDS-PAGE and find that there was an apparent protein band at about76KDa. Analysed with ALPHAIMAGER SYSTEMS software.The tannase expression of recombinant strains was322μg/mL～581μg/mL. It described that tannin gene was highly expressed in the Aspergillus niger.