College of Animal Science and Technology, Hebei Normal University of Science &Technology, Qinhuangdao 066600, PR China; Key Laboratory of Environmental and Applied Microbiology, Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR China.
Key Laboratory of Environmental and Applied Microbiology, Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Int J Biol Macromol. 2021 Oct 1;188:482-490. doi: 10.1016/j.ijbiomac.2021.07.153. Epub 2021 Jul 29.
A novel chitinase (P1724) was discovered from a Qinghai-Tibetan plateau microbial metagenome. P1724 contains two GH18 family catalytic domains and is phylogenetically distant from any of the chitinases studied. P1724 and its truncated versions, P1724(∆cGH18) and P1724(∆nGH18), were produced in Escherichia coli and characterized. Using colloidal chitin as substrate, the three recombinant proteins showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive, as they remained active at 4 °C; their chitinase activities were decreased with the presence of Cu and EDTA, but increased with Ba and Ca; they all showed both chitobiosidase and endochitinase activities. Compared to P1724(∆nGH18), P1724 and P1724(∆cGH18) shared more similarities in temperature and pH stabilities, NaCl tolerance, and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. k/K value of P1724 was significantly higher than the sum values of P1724(∆cGH18) and P1724(∆nGH18), which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin. This study has not only broadened the understanding of unknown chitinases in nature but also discussed the strategy of adding additional catalytic domains in enzyme engineering.
从青藏高原微生物宏基因组中发现了一种新型几丁质酶（P1724）。P1724 含有两个 GH18 家族催化结构域，与研究过的任何几丁质酶在系统发育上都相距甚远。在大肠杆菌中产生并表征了 P1724 及其截断版本 P1724(∆cGH18)和 P1724(∆nGH18)。使用胶体几丁质作为底物，三种重组蛋白在 40°C、pH5.0-6.0 和 0-0.5M NaCl 下表现出最大水解活性，并且具有冷适应性，因为它们在 4°C 时仍保持活性；它们的几丁质酶活性随着 Cu 和 EDTA 的存在而降低，但随着 Ba 和 Ca 的存在而增加；它们都表现出壳二糖酶和内切几丁质酶活性。与 P1724(∆nGH18)相比，P1724 和 P1724(∆cGH18)在温度和 pH 稳定性、NaCl 耐受性和底物亲和力方面具有更多的相似性，这表明 N 端 GH18 结构域比 C 端 GH18 结构域对 P1724 的生化特性贡献更大。P1724 的 k/K 值明显高于 P1724(∆cGH18)和 P1724(∆nGH18)的总和值，这表明 P1724 的两个 GH18 结构域在降解几丁质时协同作用。这项研究不仅拓宽了对自然界中未知几丁质酶的认识，还讨论了在酶工程中添加额外催化结构域的策略。
