Functional characterization and effect of polysaccharides on the activity of GH16 β-agarase from Gelidibacter salicanalis PAMC21136

IF 3.4 3区化学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Carbohydrate Research Pub Date : 2026-01-01 Epub Date: 2025-10-28 DOI:10.1016/j.carres.2025.109719

Lakshan Paudel , Bashu Dev Pardhe , Sushma Gupta , So-Ra Han , Jun Hyuck Lee , Tae-Jin Oh

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Abstract

β-agarase is the key enzyme for the primary degradation of agar, a major component of red algae. This study demonstrates cloning, overexpression and characterization of a recombinant GH16 β-agarase (WP_199597959) from Antarctic bacterium Gelidibacter salicanalis PAMC21136. Biochemical properties demonstrated that the maximum activity was observed at pH and temperature of 7.0 and 50°C, respectively using agarose as a substrate. The agarolytic activity of WP_199597959 on agarose was found to be endo-type with production of neoagarobiose (NA2) as a major product. Mn²⁺ metal ion significantly enhanced the activity, doubling the reaction rate as compared with other metal ions. Neoagarooligosaccharides (NAOSs) like neoagarotetraose (NA4), and neoagarohexaose (NA6) hydrolyzed into NA2. In-silico analysis demonstrated that the key catalytic residues E184 and E189 are involved in retaining hydrolytic mechanisms. In-vitro, interaction with negatively charged carboxymethyl cellulose (CMC) enhanced the enzyme activity by 15% while positively charged chitosan (CS) decreased the enzyme activity by 12%, highlighting the effect of biopolymers on enzyme activity within the microenvironment interaction. These findings may provide insight into biochemical properties and application of WP_199597959 for agarose degradation.

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水杨胶杆菌PAMC21136中多糖对GH16 β-琼脂酶活性的影响及功能表征

β-琼脂酶是红藻主要成分琼脂初级降解的关键酶。本研究从南极细菌Gelidibacter salicanalis PAMC21136中克隆、过表达重组GH16 β-琼脂酶（WP_199597959）并对其进行了鉴定。以琼脂糖为底物，在pH为7.0、温度为50℃时活性最高。发现WP_199597959对琼脂糖的溶糖活性为内溶型，主要产物为新琼脂糖（NA2）。Mn2+金属离子显著提高了反应活性，反应速率是其他金属离子的两倍。新琼脂低聚糖（NAOSs）如新琼脂四糖（NA4）和新琼脂己糖（NA6）水解成NA2。硅分析表明，关键催化残基E184和E189参与保持水解机制。在体外，与带负电的羧甲基纤维素（CMC）相互作用，酶活性提高了15%，而带正电的壳聚糖（CS）使酶活性降低了12%，这突出了生物聚合物对微环境相互作用中酶活性的影响。这些发现为WP_199597959降解琼脂糖的生化特性及应用提供了新的思路。

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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".