Expression Of A Bacterial Alpha-amylase And A Fungal Glucoamylase In Transgenic Rice Seeds

Alpha-amylase and glucoamylase are widely used in feed,food processing and ethanol production.The traditional method of amylase production is based on fermentation of amylase-producing microbes.Although the cost of the production by microbes is decreasing with the continuous improvement on the microbes,a more cost effective method of amylase production is desirable for further reducing the cost for the rapidly expanding ethanol production industry.This study is to explore the feasiblity to develop transgenic rice for mass production of alpha-amylase and glucoamylase in rice seeds.An alpha-amylase gene from Bacillus stearothermophilus(gb:A24549)under the control of the promoter of a major rice seed storage protein Gtlwas transformed into rice.The glucoamylase was found to have been expressed specifically in rice seeds and the expression of alpha-amylase had some influence to the qualities of rice grain.The transgenic line with the highest alpha-amylase activity reached up to 15,000 units per gram of seeds(one unit was defined as the amount of enzyme that produces 1μmol of reducing ends in 1 min at 70℃).The enzyme produced in the seeds displayed an optimum pH of 5.0-5.5 and optimum temperature of 60-70℃.And it still had some residue activities after cultured at 90℃for 10 min.Without extraction or purification,the powder of transgenic rice seeds was able to liquify 100 times of its weight of corn powder in 30 min.Thus the transgenic rice could be used for industrial starch liquefaction.The powder of transgenic rice seeds could also liquify raw com powder and raw rice powder.A glucoamylase gene from Aspergillus awamori(gb:P69327)under the control of the same major rice seed storage protein promoter as above was also transformed into rice.The glucoamylase was found to have been expressed specifically in rice seeds,and the expression of glucoamylase had some influence to qualities of rice grain.The activity in a selected transgenic line was estimated to be at about 530 units per gram of seeds(one unit was defined as the amount of enzyme that produces 1μmol of reducing ends in 1 min at 60℃using soluble starch as substrate).The enzyme produced in the seeds had an optimum pH of 4.0～5.0 and optimum temperature of 50～60℃.After incubation at 55℃for 16h,one part of this transgenic glucoamylase rice seed powder successfully converted 25 parts of corn starch liquefied by an alpha-amylase also produced by a transgenic rice into glucose.Without extraction or purification,the combinantion of the alpha-amylase and the glucoamylase from transgenic rice successfully converted corn starch into glucose without addition of any external enzymes.It implied that the transgenic enzymes from rice only could be sufficient for industrial starch hydrolysis.