An Aspergillus nidulans endo-β-1,3-glucanase exhibited specific catalytic features and was used to prepare 3-O-β-cellobiosyl-d-glucose and 3-O-β-gentiobiosyl-d-glucose with high antioxidant activity from barley β-glucan and laminarin, respectively

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Highlights

  • A distinctive β-glucanase for preparation of glucooligosaccharide from β-glucan

  • Superdex™ 30 open column can efficiently purification of glucooligosaccharides.

  • Antioxidant activity increases with increase of DP of glucooligosaccharides.

  • β-1,4/1,6-Linkage at non-reducing end enhance antioxidant activity of oligosaccharide.

  • β-1,4-Linkage at reducing end decreases antioxidant activity of glucooligosaccharide.

Abstract

An endo-β-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of β-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-β-gentiobiosyl-d-glucose and hydrolyzed barley β-glucan to mainly produce 3-O-β-cellobiosyl-d-glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-β-cellobiosyl-d-glucose and 3-O-β-gentiobiosyl-d-glucose, respectively, from AnENG16A-hydrolysate of barley β-glucan and E. bicyclis laminarin. Further study reveals that 3-O-β-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley β-glucan and 3-O-β-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-β-cellobiosyl-d-glucose and 3-O-β-gentiobiosyl-d-glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that β-1,4-linkage or -1,6-linkage at non-reducing end of β-glucotrioses had enhancing effect on antioxidant activity compared to β-1,3-linkage. Furthermore, 3-O-β-cellobiosyl-d-glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley β-glucan, indicating that β-1,4-linkage at reducing end of β-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to β-1,3-linkage.

Introduction

Carbohydrates are classified according to their degree of polymerization (DP) to form monosaccharides, oligosaccharides or polysaccharides [1], [2]. Oligosaccharides are polymers of monosaccharides with a low degree of polymerization that contain two to ten monosaccharide residues and are usually produced by chemical or enzymatic synthesis from monosaccharides or monosaccharide derivatives or by the hydrolysis of polysaccharides [2], [3], [4]. Many oligosaccharides, such as fructooligosaccharides, galactooligosaccharides, chitooligosaccharides, xylooligosaccharides and mannan oligosaccharides, have been found to have beneficial effects on human health, including prebiotic, immunomodulatory, antioxidant, antitumor and cardioprotective effects [3], [4], [5], [6], [7]. Therefore, some oligosaccharides have been employed as food ingredients, medicinal ingredients, dietary supplements, and animal feed ingredients [3], [8]. The bioactivities of oligosaccharides are closely correlated with their molecular structures, such as their monosaccharide composition, position of glycosidic linkages, degree of polymerization, anomeric configuration, etc. [9], [10], [11]. A basic understanding of the structure and biological activities of oligosaccharides is essential for their successful application in functional foods and other multifunctional applications [5], [12].

Recently, β-glucan oligosaccharides have attracted attention because they are beneficial for the maintenance of human health [13], [14], [15]. The mixed linkage β-oligosaccharides mixture from enzyme-hydrolyzed barley β-glucan was reported to show DPPH radical-scavenging activity [16] and cholesterol-lowering effects in diabetic rats [17] and to promote of growth of probiotic lactic acid bacteria [18]. The laminarin oligosaccharides mixture from enzyme-hydrolysate of laminarin, curdlan or laminarioligosaccharide was found to possess enhanced antioxidant activity [19] and immunostimulatory activity [20] compared to their original materials. However, these enzyme hydrolysates of β-glucan or its oligosaccharides were composed of complicated oligosaccharide mixtures. Therefore, the antioxidant activity of the hydrolysate was determined using heterogeneous and/or relatively poorly characterized oligomer mixtures, and it was difficult to determine which molecule was critical for the antioxidant activity observed. It is clearly essential that well-characterized and highly purified oligosaccharides be used in both biological activity and structural studies to understand the antioxidant activity of barley β-glucan or laminarin oligosaccharides in depth. In this study, we report that an endo-β-1,3(4)-glucanase, AnENG16A, from Aspergillus nidulans exhibits distinctive catalytic features against both barley β-glucan and Eisenia bicyclis laminarin, and that it can be used to hydrolyze E. bicyclis laminarin and barley β-glucan to produce mainly glucooligosaccharide triose with small amounts of other oligosaccharides. We successfully isolated and purified a 3-O-β-cellobiosyl-d-glucose from the AnENG16A-hydrolysate of barley β-glucan and a 3-O-β-gentiobiosyl-d-glucose from the AnENG16A-hydrolysate of E. bicyclis laminarin, respectively, and proved that 3-O-β-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley β-glucan and 3-O-β-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin.

Section snippets

Chemicals

Laminarin from Laminaria digitata, CMC-Na, and p-nitrophenyl-β-D-galactopyranoside (pNPG) were purchased from Sigma (USA); laminarin from E. bicyclis and Avicel were purchased from TCI (Japan); barley β-glucan, CM-pachyman, xyloglucan and laminarioligosaccharides (n = 2–6) were purchased from Megazyme (Ireland); and pustulan and cellotriose were purchased from Elicityl-OligoTech (France).

Strains

The A. nidulans TN02A7 strain was kindly provided by Dr. Lu at the Nanjing Normal University and Pichia

Cloning, heterologous expression, and purification

An uncharacterized hypothetical protein (GenBank accession: XP_657849.1) from A. nidulans FGSC A4 was revealed to share 45.83% identity with ENG16A from Coprinopsis cinerea [35] and belongs to the glycoside hydrolase 16 (GH16) family, and is named AnENG16A, as determined by protein-protein BLAST analysis. A. nidulans AnENG16A consists of 342 amino acids with a molecular weight of 36,854 Da and a PI of 4.43. According to the analysis of the Signal 5.0 server (//www.cbs.dtu.dk/services/SignalP

Discussion

Barley β-D-glucan is a mixed-linkage β-1,3-1,4-D-glucan that is composed of groups of two or three contiguous β-1,4-glucosidic linkages separated by single β-1,3-glucosidic linkages. Laminarinases or β-1,3(4)-D-glucanases cleave the β-1,4 glucosidic bond adjacent to the reducing end of a β-1,3-linkage in barley β-1,3-1,4-glucan to yield a series of mixed linkage glucooligosaccharides, including laminaribiose, 3-O-β-cellobiosyl-d-glucose and 3-O-β-D-cellotriosyl-d-glucose [31]. Laminarin is a

Conclusion

The enzyme-hydrolysate of barley β-glucan or laminarin has been reported to possess enhanced antioxidant activity compared to their original polysaccharides but it was difficult to determine which molecule was critical for the antioxidant activity observed due to the enzyme-hydrolysate consisting of complicated oligosaccharide mixtures. In this study, the β-1,3(4)-glucanase AnENG16A from A. nidulans was explored to possess distinctive catalytic capacity to hydrolyze E. bicyclis laminarin to

CRediT authorship contribution statement

Cuicui Liu: Design of the study, Experiments, Data analysis, Drafting of the manuscript. Songling Yan: Experiments, Data analysis. Jing Zhao: Experiments. Miao Lin: Experiments. Baiyun Duan: Data analysis. Zhenqing Zhang: MS analysis. Yao Yang: Supervision, Data curation. Zhonghua Liu: Supervision, Data curation. Sheng Yuan: Supervision, Writing – review & editing, Conceptualization, Funding acquisition.

Declaration of competing interest

We declare no competing financial interests.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 31870033), and the Natural Science Foundation of Jiangsu Province (No BK20140918), the Program for Jiangsu Excellent Scientific and Technological Innovation Team (17CXTD00014), and the Priority Academic Development Program of Jiangsu Higher Education Institutions.

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    These authors contributed equally: Cuicui Liu, Songling Yan.

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