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Cloning and characterization of two Chondroitin Sulfate ABC Lyases from Edwardsiella tarda LMG2793
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2021-02-01 , DOI: 10.1016/j.enzmictec.2020.109701
Guanrui Song 1 , Junhao Sun 1 , Mingliu Zhao 1 , Zheng Wang 1 , Qianhong Gong 1 , Wengong Yu 1
Affiliation  

Chondroitinase ABC can be used to prepare chondroitin sulfate (CS) oligosaccharides efficiently and environmentally. It also promotes nerve recovery through enzymatic degradation of glycosaminoglycan chains in damaged nerve tissue. In this study, two new chondroitin sulfate ABC lyases were expressed and characterized from Edwardsiella tarda LMG2793, with molecular weight of 116.8 kDa and 115.9 kDa, respectively. Two lyases ChABC I and ChABC II belonged to the polysaccharide lyase (PL) family 8. ChABC I and ChABC II showed enzyme activity towards chondroitin sulfate A (CS-A), CS-B, CS-C and CS-D, but had no activity towards hyaluronan (HA). The optimal temperature for ChABC I to exhibit the highest activity against CS-A was 40 °C and the optimal pH was 7.0. ChABC II showed the highest activity to CS-A at optimal temperature of 40 °C and pH of 9.0. ChABC I and ChABC II were stable at 37 °C and remained about 90 % of activity after incubation at 37 °C for 3 h. Many metal ions had no effect on the activity of ChABC I and ChABC II. These properties were beneficial to their further basic research and application. ChABC I was an endo-type enzyme while ChABC II was an exo-type enzyme. A group of amino acids were selected for further study by evaluating the sequence homology with other CS degradation lyases. Mutagenesis studies speculated that the catalytic residues in ChABC I were His522, Tyr529 and Arg581. The catalytic residues of ChABC II were His498, Tyr505 and Arg558. This work will contribute to the structural and functional characterization of biomedically relevant CS and promote the application of CS lyase in further basic research and therapeutics.

中文翻译:

迟发爱德华氏菌 LMG2793 中两种硫酸软骨素 ABC 裂解酶的克隆和表征

软骨素酶 ABC 可用于高效、环保地制备硫酸软骨素 (CS) 寡糖。它还通过酶促降解受损神经组织中的糖胺聚糖链来促进神经恢复。在本研究中,两种新的硫酸软骨素 ABC 裂解酶被表达并表征自迟发爱德华氏菌 LMG2793,分子量分别为 116.8 kDa 和 115.9 kDa。两种裂解酶 ChABC I 和 ChABC II 属于多糖裂解酶 (PL) 家族 8。ChABC I 和 ChABC II 对硫酸软骨素 A (CS-A)、CS-B、CS-C 和 CS-D 显示出酶活性,但对乙酰透明质酸 (HA) 没有活性。ChABC I 对 CS-A 表现出最高活性的最佳温度为 40 °C,最佳 pH 值为 7.0。ChABC II 在 40 °C 的最适温度和 9.0 的 pH 值下对 CS-A 表现出最高的活性。ChABC I 和 ChABC II 在 37 °C 下稳定,在 37 °C 下孵育 3 小时后仍保持约 90% 的活性。许多金属离子对 ChABC I 和 ChABC II 的活性没有影响。这些特性有利于其进一步的基础研究和应用。ChABC I 是一种内型酶,而 ChABC II 是一种外型酶。通过评估与其他 CS 降解裂解酶的序列同源性,选择一组氨基酸用于进一步研究。诱变研究推测 ChABC I 中的催化残基是 His522、Tyr529 和 Arg581。ChABC II 的催化残基是 His498、Tyr505 和 Arg558。这项工作将有助于生物医学相关 CS 的结构和功能表征,并促进 CS 裂解酶在进一步的基础研究和治疗中的应用。
更新日期:2021-02-01
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