Cloning, expression and characterization of novel hyaluronan lyases Vhylzx1 and Vhylzx2 from Vibrio sp. ZG1

IF 2.4 3区化学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Carbohydrate Research Pub Date : 2024-07-22 DOI:10.1016/j.carres.2024.109221

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Abstract

Hyaluronidases are a class of enzymes that can degrade hyaluronic acid and have a wide range of applications in the medical field. In this study, the marine bacterium Vibrio sp. ZG1, which can degrade HA, was isolated, leading to the discovery of two novel hyaluronan lyases, Vhylzx1 and Vhylzx2, through genome sequencing and bioinformatic analysis. These lyases belong to the polysaccharide lyase-8 family. Vhylzx1 and Vhylzx2 specifically degrade HA, with highest activity at 35 °C, pH 5.7 and 50 °C, pH 7.1. Vhylzx1 and Vhylzx2 are endo-type enzymes that can fully degrade HA into unsaturated disaccharides. Sequence homology assessment and site-directed mutagenesis revealed that the catalytic residues of Vhylzx1 are Asn²³¹, His²⁸¹, and Tyr²⁹⁰, and that the catalytic residues of Vhylzx2 are Asn²²⁷, His²⁷⁷, and Tyr²⁸⁶. Moreover, this study used consensus sequences to enhance the specific activity of Vhylzx2 mutants. Notably, the mutants V564I, N742D, L619F, and D658G increases the specific activity by 2.4, 2.2, 1.3, and 1.2-fold. These characteristics are useful for further basic research and applications, and have a promising application in the preparation of biologically active hyaluronic acid oligosaccharides.

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来自弧菌 ZG1 的新型透明质酸裂解酶 Vhylzx1 和 Vhylzx2 的克隆、表达和表征ZG1.

透明质酸酶是一类能降解透明质酸的酶，在医学领域有着广泛的应用。在这项研究中，分离出了能降解 HA 的海洋弧菌 ZG1。通过基因组测序和生物信息学分析，发现了两种新型透明质酸裂解酶 Vhylzx1 和 Vhylzx2。这些裂解酶属于多糖裂解酶-8 家族。Vhylzx1 和 Vhylzx2 专门降解 HA，在 35 °C、pH 值为 5.7 和 50 °C、pH 值为 7.1 时活性最高。Vhylzx1 和 Vhylzx2 是内切型酶，可将 HA 完全降解为不饱和二糖。序列同源性评估和定点突变显示，Vhylzx1 的催化残基为 Asn231、His281 和 Tyr290，Vhylzx2 的催化残基为 Asn227、His277 和 Tyr286。此外，这项研究还利用共识序列来提高 Vhylzx2 突变体的特异性活性。值得注意的是，V564I、N742D、L619F 和 D658G 突变体的比活性分别提高了 2.4、2.2、1.3 和 1.2 倍。这些特性有助于进一步的基础研究和应用，在制备具有生物活性的透明质酸寡糖方面具有广阔的应用前景。

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来源期刊

Carbohydrate Research 化学-生化与分子生物学

CiteScore

5.00

自引率

3.20%

发文量

183

审稿时长

3.6 weeks

期刊介绍： Carbohydrate Research publishes reports of original research in the following areas of carbohydrate science: action of enzymes, analytical chemistry, biochemistry (biosynthesis, degradation, structural and functional biochemistry, conformation, molecular recognition, enzyme mechanisms, carbohydrate-processing enzymes, including glycosidases and glycosyltransferases), chemical synthesis, isolation of natural products, physicochemical studies, reactions and their mechanisms, the study of structures and stereochemistry, and technological aspects. Papers on polysaccharides should have a "molecular" component; that is a paper on new or modified polysaccharides should include structural information and characterization in addition to the usual studies of rheological properties and the like. A paper on a new, naturally occurring polysaccharide should include structural information, defining monosaccharide components and linkage sequence. Papers devoted wholly or partly to X-ray crystallographic studies, or to computational aspects (molecular mechanics or molecular orbital calculations, simulations via molecular dynamics), will be considered if they meet certain criteria. For computational papers the requirements are that the methods used be specified in sufficient detail to permit replication of the results, and that the conclusions be shown to have relevance to experimental observations - the authors'' own data or data from the literature. Specific directions for the presentation of X-ray data are given below under Results and "discussion".

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