Isolation Of Acid-resistant α-amylase-producing Strain And Studies On Fermentation Conditions And Acid-resistant α-amylase Properties

With the development of starch processing industry, more and more starch processingtechnologies needα-amylase which could keep stable under low pH conditions. A specialenzyme, acid-resistantα-amylase, can be used in starch processing under low pH. Theapplication of the enzyme can improve and modify the starch processing, reduce the costof the production and help to solve their disposal and pollution problems etc. The objectiveof this study was the selection of suitable acid-resistantα-amylase-producing strain, studieson fermentation conditions and acid-resistantα-amylase properties.The strain ZY8 producing acid-resistantα-amylase was isolated from acidic soilnear a starch processing factory. The strain had high ability to produce acid-resistantα-amylase. According to analysis of the culture characteristics and morphology, the straincould be identified as Aspergillus niger.The liquid state fermentation condition of ZY8 was optimized, and the optimalmedium contained soluble starch 50g/L, ammonium citrate dibasic 20g/L, KH₂PO₄3g/L,CaCl₂0.1g/L, FeSO₄·7H₂O 0.01g/L, MgSO₄·7H₂O 1g/L; The optimal initial medium pH,culture temperature and inoculums size was 4.0, 30℃and 2% respectively. The activityof acid-resistantα-amylase could reach 19.86u/mL after cultured in the optimizedcondition for 72h, which was 1.8 times higher than preoptimized culture condition.The solid state fermentation condition of ZY8 was also optimized. The results were asfollows, the optimal medium contained wheat bran 5g, soluble starch 0.5g, soybean 0.5g,salt solution (KH₂PO₄ 2g/L, NH₄PO₄ 5g/L, NaCl 1g/L, MgSO₄·7H₂O 1g/L) 14mL in250mL flask, The optimal initial medium pH, initial moisture, culture temperature andinoculums size were 4.0, 70%, 35℃, and 5% respectively.The crude acid-resistantα-amylase preparation was obtained by solid statefermentation of ZY8 strain, the extract conditions and crude enzyme produce technologieswere studied. The optimum extract condition was that the fermentation productions weredipped in 3 times volume of water as the fermentation productions at 35℃and pH5.0 for 3hours. The extract solution obtained by filtration was mixed with alcohol and the finalconcentration of alcohol reached to 70%. After centrification, The refined solid was driedat 40℃. under the optimized conditions, the total product yield was abve 70%, and theactivity of acid-resistantα-amylase was 150.57u/mg。The study of the enzyme properties showed that optimal pH value and temperature were 4.0 and 70℃,respectively. The enzyme was stable under pH2.0～5.0, and the residue enzyme activity was 50.2% under 90℃for 30min. The residue activity was 85%～96% after the enzyme was kept at 40℃,pH2～5for 1 h. A relative molecular weight about 65KD appeared in SDS-PAEG, the clear zone on a bluebackground was appeared in nondenaturing-PAGE; The results of thin-layer chromatographydemonstrate that the products from starch catalyzed by the enzyme are glucose,maltose and otheroligosaccharide. This enzyme should be classified as an endoenzyme. The properties of theenzyme imply it has good peospect of use in the future.