Heterologous expression, characterization, and application of recombinant thermostable α-amylase from Geobacillus sp. DS3 for porous starch production

IF 2.3 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
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引用次数: 0

Abstract

Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 °C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30–90 °C) and pH range (6–8), with optimal activity at 70 °C and pH 6 with great stability at 60 °C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca2+. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.

多孔淀粉生产中 Geobacillus sp. DS3 的异源表达、表征和重组恒温 α 淀粉酶的应用
从滇西溪当火山口分离出的新型地衣芽孢杆菌(Geobacillus sp.)DS3具有工业应用前景广阔的特性,尤其是在生产恒温α-淀粉酶方面。该研究利用重组技术在大肠杆菌 BL21(DE3)中表达恒温α-淀粉酶,以克服高温生产的挑战。该研究旨在表达、纯化、表征这种新型酶,并探索其潜在应用。该酶成功地在大肠杆菌 BL21(DE3) 中进行了表达,在 18 °C、0.5 mM IPTG 诱导下持续 20 小时。用 Ni-NTA 柱纯化后,比初始粗酶的纯度提高了 69.23%,比活度提高了 3.6 倍。该酶的分子量为 ±70 kDa(±58 kDa 酶+11 kDa SUMO 蛋白)。它在很宽的温度范围(30-90 °C)和 pH 值范围(6-8)内都表现出活性,在 70 °C 和 pH 值为 6 时活性最佳,在 60 °C 时非常稳定。动力学分析表明,在不添加辅助因子的情况下,以糊精为首选底物,其Km和Vmax值分别为324.03 mg/ml和36.5 U/mg。作为一种金属酶,它在 Ca2+ 存在下表现出最佳活性。该酶被用于多孔淀粉的生产,并成功地固定在壳聚糖上,表现出更好的热稳定性。在第四次重复使用后,固定化酶的活性比初始固定化酶保持了 62%。
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来源期刊
Biochemistry and Biophysics Reports
Biochemistry and Biophysics Reports Biochemistry, Genetics and Molecular Biology-Biophysics
CiteScore
4.60
自引率
0.00%
发文量
191
审稿时长
59 days
期刊介绍: Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.
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