Mohamed Sara, Bouacem Khelifa, Mechri Sondes, Addou Nariman Ammara, Laribi-Habchi Hassiba, Fardeau Marie-Laure, Jaouadi Bassem, Bouanane-Darenfed Amel, Hacène Hocine
Laboratory of Cellular and Molecular Biology, Microbiology Team, Faculty of Biological Sciences (FSB), University of Sciences and Technology of Houari Boumediene (USTHB), PO Box 32, El Alia, Bab Ezzouar, 16111, Algiers, Algeria.
Laboratory of Cellular and Molecular Biology, Microbiology Team, Faculty of Biological Sciences (FSB), University of Sciences and Technology of Houari Boumediene (USTHB), PO Box 32, El Alia, Bab Ezzouar, 16111, Algiers, Algeria; Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax, 3018, Tunisia.
Carbohydr Res. 2019 Feb 1;473:46-56. doi: 10.1016/j.carres.2018.12.017. Epub 2018 Dec 29.
An extracellular acido-thermostable endochitinase (called ChiA-Mt45) from thermohalophilic Melghiribacillus thermohalophilus strain Nari2A gen. nov. sp. nov., was purified and biochemically characterized. The maximum chitinase activity recorded after 48-h of incubation at 55 °C was 9000 U/mL. Pure enzyme was obtained after heat treatment (20 min at 90 °C) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on MALDI-TOF/MS analysis, the purified enzyme is a monomer with a molecular mass of 45201.10 Da. The 27 residue NH-terminal sequence of the enzyme showed high homology with Bacillus GH-18 chitinases family. The optimum pH and temperature values for chitinase activity were pH 3.5 and 90 °C, respectively. In addition, the enzyme was halotolerant and can be classified as an extremozyme. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). Its K and k values were 0.253 mg colloidal chitin/mL and 47000 s, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Hh59 from Hydrogenophilus hirchii KB-DZ44 and chitodextrinase from Streptomyces griseus, and N-acetyl-β-glucosaminidase from Trichoderma viride. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Additionally, thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Mt45 acted as an endosplitting enzyme. Overall, the chitinase ChiA-Mt45 may have great potential for the enzymatic degradation of chitin.
从嗜热嗜盐菌Melghiribacillus thermohalophilus菌株Nari2A gen. nov. sp. nov.中纯化并对一种细胞外酸热稳定内切几丁质酶（称为ChiA-Mt45）进行了生化特性分析。在55°C孵育48小时后记录的最大几丁质酶活性为9000 U/mL。经过热处理（90°C 20分钟），然后在快速蛋白质液相色谱（FPLC）和高效液相色谱（HPLC）上进行连续柱色谱后获得了纯酶。基于基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）分析，纯化后的酶是一种分子量为45201.10 Da的单体。该酶的27个残基N端序列与芽孢杆菌GH-18几丁质酶家族具有高度同源性。几丁质酶活性的最佳pH值和温度值分别为pH 3.5和90°C。此外，该酶具有耐盐性，可归类为极端酶。纯酶被对氯汞苯甲酸（p-CMB）和N-乙基马来酰亚胺（NEM）完全抑制。其Km和kcat值分别为0.253 mg胶体几丁质/mL和47000 s-1。有趣的是，其催化效率高于来自 Hirschii嗜氢菌KB-DZ44的几丁质酶ChiA-Hh59、灰色链霉菌的壳糊精酶以及绿色木霉的N-乙酰-β-葡萄糖苷酶。所研究的几丁质酶对胶体几丁质、几丁质天青、糖基几丁质表现出高活性，而不水解壳二糖和直链淀粉。此外，几丁质寡糖的薄层色谱（TLC）分析表明ChiA-Mt45是一种内切酶。总体而言，几丁质酶ChiA-Mt45在几丁质的酶促降解方面可能具有巨大潜力。
