Wang Shihui, Jeyaseelan Jenasia, Liu Yun, Qin Wensheng
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, P7B 5E1, Canada.
Appl Biochem Biotechnol. 2016 Sep;180(1):136-51. doi: 10.1007/s12010-016-2089-5. Epub 2016 Apr 26.
The costs of amylase represent ca. 24 % of the expenditures in the starch industry and an increase in amylase production and/or activity will greatly cut down on production costs. In the present study, we obtained a high amylase-producing strain of bacteria, WangLB, and identified it as a member of the Bacillus genus based on 16S rDNA analysis. The fermentation conditions for amylase production in the strain were optimized, and the maximum amylase activity we obtained was 26,670 ± 1390 U/mL, under the optimized conditions of 48-h incubation in liquid starch medium, 35 °C, pH 10, 1 % v/v inoculum concentration, 20 g/L starch concentration, and 0.1 % w/v peptone. The influences of 16 small organic inducers on amylase production were tested, and the results showed that 20 mmol/L alanine greatly enhanced amylase production to 290 % of the baseline level. We also conducted an amylase enzymology analysis. The molecular weight of the amylase was 55 kD, determined by SDS-PAGE. The optimum temperature and pH for the amylase were 55 °C and pH 9, respectively. The enzyme also showed high activity over a wide range of temperatures (50-85 °C) and pH values (3-10), and the activity of the amylase was Ca(2+) independent. The kinetic parameters K m and V max were 0.37 ± 0.02 mg/mL and 233 U/mg, respectively. Finally, the amylase was applied to the hydrolysis of five different brands of starch. It was found that the hydrolyzability of the substrate by amylase increased along with starch solubility.
淀粉酶的成本约占淀粉工业支出的24%,淀粉酶产量和/或活性的提高将大大降低生产成本。在本研究中,我们获得了一株高产淀粉酶的细菌菌株WangLB,并通过16S rDNA分析将其鉴定为芽孢杆菌属的一员。对该菌株产淀粉酶的发酵条件进行了优化,在液体淀粉培养基中于35℃、pH 10、接种浓度1% v/v、淀粉浓度20 g/L和蛋白胨0.1% w/v的优化条件下培养48小时,我们获得的最大淀粉酶活性为26,670±1390 U/mL。测试了16种小有机诱导剂对淀粉酶产生的影响,结果表明20 mmol/L丙氨酸可将淀粉酶产量大幅提高至基线水平的290%。我们还进行了淀粉酶的酶学分析。通过SDS-PAGE测定,淀粉酶的分子量为55 kD。淀粉酶的最适温度和pH分别为55℃和pH 9。该酶在较宽的温度范围(50 - 85℃)和pH值范围(3 - 10)内也表现出高活性,并且淀粉酶的活性不依赖于Ca(2+)。动力学参数Km和Vmax分别为0.37±0.02 mg/mL和233 U/mg。最后,将淀粉酶应用于五种不同品牌淀粉的水解。发现淀粉酶对底物的水解能力随淀粉溶解度的增加而提高。
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