Recombinant expression and characterization of an α-galactosidase from Thermoclostridium stercorarium subsp. thermolacticum DSM 2910 and its application in the hydrolysis of raffinose

IF 2.4 3区化学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Carbohydrate Research Pub Date : 2025-01-29 DOI:10.1016/j.carres.2025.109410

Lin Ge , Ruobing Jia , Yingying Liu , Lingling Zhan

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Abstract

Soybean contains anti-nutritional factor raffinose oligosaccharide (RFOs), which can cause flatulence, gastrointestinal dysfunction and low feed utilization rate. However, α-galactosidase can hydrolyze raffinose oligosaccharides (RFOs). Therefore, in this study, a novel α-galactosidase from Thermoclostridium stercorarium subsp. thermolacticum DSM 2910 (TstGal) which can hydrolyze raffinose was cloned, expressed, purified, and characterized. The gene fragment size is 2208 bp, and the enzyme TstGal consists of 736 amino acids. Under the optimum culture conditions, the maximum enzyme activity of the target protein TstGal was 23.8 U/mL. The enzyme was purified 11.3-fold by Ni-NTA agarose resin with an overall recovery of 51.4 % and specific activity of 9.0 U/mg, and its relative molecular weight was about 85 kDa. The optimal temperature of TstGal was 70 °C, and it exhibited excellent thermal stability at 60 °C. Furthermore, The TstGal had the highest activity at pH 6.5 and good pH stability at pH 5.0–7.5. Besides, the enzyme has a good sugar tolerance to galactose and sucrose. In addition, K⁺ and Fe²⁺ could significantly enhance the enzyme activity at a concentration of 5 mM. The values of K_M, V_max, k_cat, and k_cat/K_M for pNPGal were found to be 0.507 mM, 13.979 U/mg, 19.735 s⁻¹, and 38.895 s⁻¹mM⁻¹, respectively. Under the optimum conditions, the maximum hydrolysis rate of raffinose by TstGal reached 99.8 %, which shows that the enzyme had potential application value in food and feed industry.

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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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阿拉丁

p-Nitrophenyl α-d-galactopyranoside (pNPGal)