Secreted Expression And Application Of Thermo-Stable And Acid-Stable α-Amylase From Thermococcus Siciuli HJ21

Thermo-stable α-amylase is adopted to hydrolyze starch, and the application of theenzyme plays a key role in the entire liquefaction process. Extreme thermophile bacteriumwhich can grow in the range of80-110℃, is an important source of thermo-stable α-amylase.Since most of archaea are anaerobic, they are difficult to cultivate, and the yield of enzyme islow. Therefore, cloning the thermo-stable α-amylase gene and transforming it into the hostcells which can grow in the room temperature is a main measure to develop and apply newthermo-stable α-amylase. In this study, the α-amylase gene was cloned from the genomicDNA isolated from Thermococcus siculi HJ21. The mature α-amylase was successfullyexpressed in Yarrowia lipolytica Po1h. The characterizations of recombinant α-amylase werestudied. The fermentation conditions for the recombinant α-amylase production withrecombinant Yarrowia lipolytica were studied. Some factors which could influence enzymatichydrolysis were studied. The results were as follows:(1) The gene encoding a thermo-stable and acid-stable α-amylase was amplified by PCRusing Thermococcus siculi HJ21genomic DNA as template. Then the gene was cloned intothe vector of pINA1317. The recombinant plasmid was linearized and transformed intoYarrowia lipolytica Po1h. The recombinant α-amylase was expressed and secreted out of thecells. A strain called G7-5that can secrete higher α-amylase activity was got.(2) The fermentation supernatant of G7-5was concentrated by ultrafiltration andanalysed by SDS-PAGE. A40kDa protein was found and the activity staining showed that theprotein is the cloned α-amylase. But the apparent molecular weight of the protein is nearly9kDa smaller than the theoretical molecular weight. The recombinant α-amylase exhibitedmaximal activity at90℃and at pH5.0. The enzyme is so thermostable that after disposed at100℃for5hours about60%of activity was retained. The thermal stability of the enzyme isnot dependent on Ca2+.(3) A orthogonal test was designed to explore the effects of4main cultivation conditionssuch as carbon amount, KH2PO4amount, MgSO4amount and pH on the expressed activity ofthe thermo-stable and acid-stable α-amylase based on single factors research during flaskcultivation. The fermentation conditions of thermal-stable and acid-stable α-amylase weregiven as follows:4%（w/v）molasses,0.080%（w/v） KH2PO4,0.0198%（w/v） MgSO4andpH6.5. The activity of thermal-stable and acid-stable α-amylase was13.06U/mL on theoptimal fermentation conditions.(4) A kind of thermo-stable and acid-stable α-amylase was adopted to hydrolyze cornstarch. Some factors which could influence enzymatic hydrolysis were studied. Thehydrolysis pH was4.5, which is almost the same as the nature pH of the starch and theoptimum pH of glucoamylase.