The Study On Characteristic And Production Of A Novel Raw Starch Amylase From Marine Yeast Aureobasidium Pullulans

Marine yeasts are a kind of micobial community living in marine environments which plays a important role in marine eco-system. Marine yeast strains can produce many enzymes such as protease,amylase,lipase,phytase,inulinase,cellulase,killer toxin and so on. It is an interesting work for us to investigate the application of these bioresources.In this study, marine yeast strain N13d, producing a high amount of extracellular amylase was isolated from the deep sea sediments of Pacific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identification methods.This is the first report that marine Aureobasidium pullulans produced extracellular amylase. Based on optimal medium and flask cultivation conditions for amylase production by the marine yeast N13d we carried out amylase liquid fermentation experiment by 2L fermentor.Under the optimal aeration rate 6 Lmin-1 and agitation speed 250 rpm the amylase specific activity in crude enzyme preparation reach 58.5U/mg protein. Hydrolysis of raw potato starch was carried out at 60℃.After 6 h of incubation, 59.5% of the raw potato starch with 36% concentration was hydrolyzed. The end product was identified to glucose by TLC and HPLC.In order to explore the potential application of this amylase we purified this enzyme and carried out a comprehensive study about the properties of enzyme. The results showed that this amylase possessing a molecular weight of 98kDa is a kind of glucoamylase. And this enzyme comprised two subunits whose molecular weight are 33kDa and 65kDa respectively. With a good thermal stability and pH stability the residual activity of amylase still kept over 75% after the treatment at pH from 4.0 to 8.0 and 55℃for 3 h and the residual activity of amylase still kept 50% of the control after treatment at 60℃for 3 h. A speciality of this amylase is that it could hydrolyzeα-1,6glycoside linkages efficiencily. This character is scarce for glucoamylases from fungi. This amylase is a kind of raw starch amylase,which can hydrolyze raw potato starch,raw sweet potato starch and raw corn starch, especially raw potato starch. With a concentration of 20U/ml the enzyme could hydrolyze the most of raw potato starch at a concentration of 36%(w/v) in the suspension. After 3h incubation at 60℃, 58% of the raw potato starch was transform into reducing sugar. The capability of hydrolyzing raw potato starch is the strongest comparing the previous reports about raw starch amylase.Based on the above results we conclude that the amylase produced by N13d is a novel raw starch amylase with great potential applications.In order to reduce the cost of hydrolysis the crude enzyme is necessary. Crude enzyme with high concentration of amylase is needed for hydrolysis of large amount of raw potato starch. So we carried out solid state fermentation of marine yeast Aureobasidium pullulans N13d in order to get crude enzyme with a high concentration of amylase. Wheat bran and rice bran the low cost agricultural residues were used in fermentation. After optimization of process parameters, we get crude amylase extraction with 120.5U/mg specific activity successfully. The specific activity of the solid state fermentation extraction was two times than the crude enzyme preparation from liquid fermentation.In our study we get an amylase producing marine yeast N13d which can produce a large amount of novel raw starch amylase. This is the first report that marine yeast Aureobasidium pullulans produce raw starch amylase. The results about purified amylase properties clearly demonstrate that raw starch amylase produced the marine yeast was different from that produced by other yeasts and fungi. One of two specialties of this amylase is that the ability of hydrolyzingα-1,6glycoside linkages efficiencily. Another speciality is the strong ability of hydrolyzing high concentration raw potato starch. By solid state fermentation we get crude enzyme extraction with high concentration raw starch amylase. It may have great potential use in digestion of raw potato starch in food and fermentation industries. The results also suggest that marine yeasts have the potential for the production of novel enzymes which would not be observed from terrestrial yeasts and fungi.These novel enzymes may become enzyme source used in industrial production.