Cloning And Expression Of An α-amylase Gene From The Potato And A γ-amylase Gene From The Aspergillus Niger In Pichia Pastoris GS115

The traditional way of producing Ethanol was through high-temperature boiling fermentation.Considering the disadvantages of traditional alcohol fermentation industry such as high consuming of energy, lacking of origins, high pollution, low alcohol level, etc., we chose potato stark as raw material for fermentation in this work, and it met the national industry policy of energy saving and exhaust reduction. We cloned potato originatedα-amylase gene and acid and high temperature proof Aspergillus nigerγ-amylase gene by gene manipulation technology. After construction in vitro, it was transformed into yeast, and a transgenic engineering Pichia pastoris which could degrade potato stark. By inoculating dry yeast under aerobic fermentation, Ethanol was attained.The main research results are as following:1,α-amylase gene (amyA) was attained through RT-PCR, total RNA of the potato cv.Shepody as the template, primers designed and synthesized according to the sequence of a-amylase gene conservative region. It was cloned to pET32a by the method of blunt end clone technology and sequenced. The sequencing result showed that the whole length of the gene was 1224 bp, and it coded 406 amino acids. Bioinformatics analysis showed that 54-1224 bp coded mature peptide gene. Also we designed primers to clone and attained mature peptide gene (Namy), subcloned it to pPIC9k, constructed recombined expression plasmid pkamyA, transformed Pichia pastoris GS115, and then attained high expressed amylase active strain GSamy05 by Trypan blue-BMMY medium screening. The strain was cultivated, and inductively expressed in 0.5%(v/v) Methanol for 72 hours. The expressed supernatant was analyzed by SDS-PAGE, and specific protein stripe expressed by GSamyA was found, about 42kDa. Study of enzymology properties of recombined raw enzyme showed that the most proper reaction pH value was 6.0, temperature 45℃, low heat stability, low acid and base tolerance, and no dependence on metal ions.2,γ-amylase gene (amyC) was attained through RT-PCR, total RNA of Aspergillus niger as the template, primers designed and synthesized according to the sequence of y-amylase gene conservative region. It was cloned to pET32a by the method of blunt end clone technology and sequenced. The sequencing result showed that the whole length of the gene was 1996 bp, and it coded 604 amino acids. Also we subcloned it to pPIC9k, constructed recombined expression plasmid pkamyC, transformed Pichia pastoris GS115, and then attained high expressed amylase active strain GSamyC3 by Trypan blue-BMMY medium screening. The strain was cultivated, and inductively expressed in 0.5%(v/v) Methanol for 72 hours. The expressed supernatant was analyzed by SDS-PAGE, and specific protein stripe expressed by GSamyC3 was found, about 62kDa. Study of enzymology properties of recombined raw enzyme showed that the most proper reaction pH value was 4.6, temperature 60℃, good heat stability, good acid and base tolerance, and no dependence on metal ions.3,We produced alcohol through aerobic fermentation, which took potato stark as raw material, inoculated transgenicα-amylase andγ-amylase engineering yeast separately in the amount of 1%, inoculated transgenicγ-amylase yeast again after expressing induced by methanol for 36 hours, and added 5% active dry yeast. We studied the influence on alcohol yield and hydrolysis rate with different time, fermenting temperature and inoculating amount and concluded that the most appropriate condition for this fermentation technology is 5% inoculating amount, 30℃and 72 hours. Alcohol yield is 9.6%,7.77%,9.75%.