Expression Of Two Lipase Gene In Pichia Pastoris And The Assess In Deinking Of Old Newspaper

Lipase is an important industrial enzyme which have a wide range of applications in food,chemical, pharmaceutical and pulp and paper-making industries. In recent years, withincreasing emphasis on environmental protection and shortage of resources, the application ofbiological enzyme in the paper industry have received wide spread attention. The applicationsof lipase in the paper industry is mainly concentrated in the waste paper deinking and resincontrol. Studies have shown that using lipase to deinking of waste paper, not only canimprove the brightness of the waste paper, but also significantly reduce the use of chemicalagents such as caustic soda, sodium silicate and hydrogen peroxide, and no damage to thepulp fibers, greatly reducing the pressure of the sewage treatment, The comprehensive cost issignificantly reduced. However, the commercial enzymes currently on the market specificallyfor deinking is shortage, and the effect is not satisfactory, which hampered the lipase widelyused in the paper industry.In this study, Cloning and expression of lipase for the purpose of adapting to therequirements of the deinking process, two alkaline resistance lipase gene have been clonedand expressed in the pichia pastoris. Study its enzymatic properties, evaluated the Deinkingcapacity of the recombine lipases,we found that these two lipases worked well in deinking ofold news paper, specific contents are as follows:1. Cloned the gene from the plasmid pMD18-BSL which preservate by our lab, thenamplificated the gene and cloned into the carrier pPICZαA, constructed a recombinantplasmid pPICZaA-BSL, the recombinant plasmid was linearized by the restrictionendonuclease pmeI, then transformed into Pichia pastoris X-33using the LiCl method. Therecombinant yeast X-33/pPICZαA-BSL could secrete alkaline lipase, and the maximumactivity of the culture supernatant was4.78U/ml when the recombinant strain wascultured in a rotary shaker. The properties of the recombinant lipase was be researched, theresults showed that the optimal temperature and pH of this lipase were40-60℃and9.0,and the lipase was extreme alkaline-resistant. 2. a lipse gene from Acinetobacter radioresistens was optimized by the softwareOptimumGene,we synthesized the gene and cloned into carrier pPICZαA, constructed arecombinant plasmid pPICZaA-ARL, the recombinant plasmid was linearized by therestriction endonuclease pmeI, then transformed into Pichia pastoris X-33using the LiClmethod. The recombinant yeast X-33/pPICZαA-ARL could secrete alkaline lipase, andthe maximum activity of the culture supernatant was60U/ml when the recombinantstrain was cultured in a rotary shaker. the optimal temperature and pH of this lipase were50℃and9.0.3. Deinking with these two recombined lipases and compared the effect with twocommercial deinking enzymes and chemical method. We found that the two recombinedlipases are better than commercial deinking enzymes. And the Physical properties of theenzymatic deinking pulp are better than the chemical deinking pulp, and the pollutioncharacteristics of the enzymatic deinking waste water was better than the chemicaldeinking waste water.