The filamentous fungi from the Huanghai sea sludge were screened according to their ability to produce cold-active α-amylase. The strain with the highest amylase activity was identified as Penicillium species. The ...The filamentous fungi from the Huanghai sea sludge were screened according to their ability to produce cold-active α-amylase. The strain with the highest amylase activity was identified as Penicillium species. The α-amylase purified by ammonium sulphate precipitation and column chromatography on DEAE-sepharose and sephadex G-100 shows a molecular weight of about 55000 and a pI of 4.38. The enzyme is stable in a pH range of 5.5—8.0 and has a maximum activity at pH 6.0. Compared with the α-amylase from mesophiles and thermophiles, the cold-active enzyme shows a high enzyme activity at lower temperatures and a high sensitivity at temperatures higher than 50 ℃. The optimal temperature is 40 ℃ and the activity decreases dramatically at temperatures above 50 ℃. Ca 2+ shows a significant effect on maintaining the structure and the activity of the enzyme. EDTA and Cu 2+ are its inhibitors. The products from the hydrolysis of soluble starch with the cold-active enzyme are maltose and other oligosaccharides.展开更多
One Gram-positive bacteria,Bacillus sp.strain BG-CSN,was isolated from the muds with hypersaline and alkaline water at the beach of Banger Soda Lake in Tibet,China.After cultivating in liquid medium for 48 hours,an ex...One Gram-positive bacteria,Bacillus sp.strain BG-CSN,was isolated from the muds with hypersaline and alkaline water at the beach of Banger Soda Lake in Tibet,China.After cultivating in liquid medium for 48 hours,an extracellular α-amylase AmyBGC from the strain BG-CSN was purified 23-fold reaching to electrophoretic homogeneity by sequentially ammonium sulfate precipitation,Octyl-Sepharose CL-4B column chromatography,DEAE-Sepharose Fast Flow chromatography,DEAE-Toyopearl 650M chromatography and Sephadex G-100 chromatography.The enzyme had a molecular mass of 87 kD estimated by SDS-PAGE.The enzyme was optimally active at pH 10.5 and 52.5℃ and showed stability at pH range of 5.0 to 11.5 at the temperature below 35℃. The enzyme activity was inhibited by Hg+ and Fe 3+.The activity was not prevented at all by chelating reagents EDTA and SDS at high concentrations.This enzyme efficiently hydrolyzed starch to yield a series of maltooligosaccharides,including maltose after completion of the reaction.These results indicate that AmyBGC is classified as a liquefying endo-1,4-D-α-amylase(EC-3.2.1.1).展开更多
将去信号肽的耐酸性高温α-淀粉酶突变基因amyd克隆到大肠杆菌表达载体pET-30a上，实现重组质粒pET-amyd在大肠杆菌BL21（DE3）中的高效表达。经硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶层析，重组酶AMYD...将去信号肽的耐酸性高温α-淀粉酶突变基因amyd克隆到大肠杆菌表达载体pET-30a上，实现重组质粒pET-amyd在大肠杆菌BL21（DE3）中的高效表达。经硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶层析，重组酶AMYD的比活达到354．6U·mg^-1、纯化倍数为83．83，获得凝胶电泳条带单一的蛋白样品，经SDS-PAGE检测，AMYD酶分子量为63．5kDa。重组酶AMYD的最适温度80℃、最适反应pH值为4．5，在温度低于90℃、反应pH值4．0～6．5的条件下，酶活较稳定。展开更多
文摘The filamentous fungi from the Huanghai sea sludge were screened according to their ability to produce cold-active α-amylase. The strain with the highest amylase activity was identified as Penicillium species. The α-amylase purified by ammonium sulphate precipitation and column chromatography on DEAE-sepharose and sephadex G-100 shows a molecular weight of about 55000 and a pI of 4.38. The enzyme is stable in a pH range of 5.5—8.0 and has a maximum activity at pH 6.0. Compared with the α-amylase from mesophiles and thermophiles, the cold-active enzyme shows a high enzyme activity at lower temperatures and a high sensitivity at temperatures higher than 50 ℃. The optimal temperature is 40 ℃ and the activity decreases dramatically at temperatures above 50 ℃. Ca 2+ shows a significant effect on maintaining the structure and the activity of the enzyme. EDTA and Cu 2+ are its inhibitors. The products from the hydrolysis of soluble starch with the cold-active enzyme are maltose and other oligosaccharides.
文摘One Gram-positive bacteria,Bacillus sp.strain BG-CSN,was isolated from the muds with hypersaline and alkaline water at the beach of Banger Soda Lake in Tibet,China.After cultivating in liquid medium for 48 hours,an extracellular α-amylase AmyBGC from the strain BG-CSN was purified 23-fold reaching to electrophoretic homogeneity by sequentially ammonium sulfate precipitation,Octyl-Sepharose CL-4B column chromatography,DEAE-Sepharose Fast Flow chromatography,DEAE-Toyopearl 650M chromatography and Sephadex G-100 chromatography.The enzyme had a molecular mass of 87 kD estimated by SDS-PAGE.The enzyme was optimally active at pH 10.5 and 52.5℃ and showed stability at pH range of 5.0 to 11.5 at the temperature below 35℃. The enzyme activity was inhibited by Hg+ and Fe 3+.The activity was not prevented at all by chelating reagents EDTA and SDS at high concentrations.This enzyme efficiently hydrolyzed starch to yield a series of maltooligosaccharides,including maltose after completion of the reaction.These results indicate that AmyBGC is classified as a liquefying endo-1,4-D-α-amylase(EC-3.2.1.1).
文摘将去信号肽的耐酸性高温α-淀粉酶突变基因amyd克隆到大肠杆菌表达载体pET-30a上，实现重组质粒pET-amyd在大肠杆菌BL21（DE3）中的高效表达。经硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶层析，重组酶AMYD的比活达到354．6U·mg^-1、纯化倍数为83．83，获得凝胶电泳条带单一的蛋白样品，经SDS-PAGE检测，AMYD酶分子量为63．5kDa。重组酶AMYD的最适温度80℃、最适反应pH值为4．5，在温度低于90℃、反应pH值4．0～6．5的条件下，酶活较稳定。
