Abstract

This study presents the exploration of laccase-like enzyme produced by thermophilic bacterial strains present in the Tattapani hotspring located in Chhattisgarh, India. Two bacterial isolates namely TPNR1 and TPNR6 were found to be positive for laccase-like enzyme production on screening with guaiacol as a substrate. The biochemical and 16S rRNA gene sequence analysis indicated that the isolates have similarity with Bacillus licheniformis strains and, thus, named as B. licheniformis TPNR1 and B. licheniformis TPNR6. The activity of crude enzymes was estimated using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as a substrate and measured as 3.1 U/mL and 7.1 U/mL for TPNR1 and TPNR6, respectively. The SDS-PAGE of purified enzymes showed that the enzymes are monomers with molecular weight of 44 (TPNR1) and 38 (TPNR6) kDa. The native-PAGE technique followed by activity staining using ABTS confirms the presence of purified active enzyme with laccase-like activity in both cases. Kinetic studies displayed that catalytic efficiency of the enzymes was higher for the substrate ABTS than 2,6-dimethoxyphenol for both enzymes. The maximum enzyme activity was observed at 50°C for both enzymes while the optimal pH for TPNR1 and TPNR6 was 5.0 and 6.0, respectively. The TPNR1 exhibited half-life of 4 h at 70°C and was stable at 60°C for 180 min with a residual activity of 79%. Similarly, the TPNR6 possessed half-life of 3.1 h at 70°C and retained 88% of its activity at 60°C for 180 min. In addition, the catalytic efficiency of the enzymes was tested by decolourization of toxic dyes which showed that the both enzymes are highly potential to degrade them.

中文翻译：

---

地衣芽孢杆菌菌株的胞外热类似漆酶的酶：生产，纯化和表征。

摘要

这项研究提出了对位于印度恰蒂斯加尔邦的塔塔帕尼温泉中嗜热细菌菌株产生的漆酶样酶的探索。在以愈创木酚为底物筛选时，发现两种细菌分离株，即TPNR1和TPNR6，对漆酶样酶的生产呈阳性。生化和16S rRNA基因序列分析表明，分离株与地衣芽孢杆菌菌株相似，因此被命名为地衣芽孢杆菌TPNR1和地衣芽孢杆菌。TPNR6。以2,2'-叠氮基双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）为底物评估粗酶的活性，TPNR1和TPNR6分别为3.1 U / mL和7.1 U / mL。纯化酶的SDS-PAGE结果表明，该酶为分子量为44（TPNR1）和38（TPNR6）kDa的单体，其native-PAGE技术和ABTS活性染色证实了漆酶样漆酶的存在。动力学研究表明，底物ABTS的酶催化效率均高于2,6-二甲氧基苯酚，两种酶均在50°C时观察到最大酶活性，而TPNR1和TPNR1的最佳pH值则观察到。 TPNR6分别为5.0和6.0。TPNR1在70°C下显示4小时的半衰期，并在60°C下稳定180分钟，残留活性为79％。同样，TPNR6在70°C下的半衰期为3.1小时，在60°C下的180min保留其88％的活性。另外，通过有毒染料的脱色测试了酶的催化效率，这表明两种酶都具有很高的降解它们的潜力。