Purification, characterization and specificity of a new GH family 35 galactosidase from Aspergillus awamori

https://doi.org/10.1016/j.ijbiomac.2020.04.013Get rights and content

Highlights

  • A. awamori produces galactosidase with both α and β-galactosidase activities.

  • Purified enzyme (118 kDa) had broad substrate specificity.

  • Optimum activity for α and β-galactosidase between 55–60 °C and pH 5.0–5.5.

  • In-gel digestion and MS/MS analysis revealed probable β-galactosidase A.

Abstract

Galactosidases, ubiquitous in nature, are complex carbohydrate-active enzymes and find extensive applications in food, pharma, and biotechnology industries. The present study deals with the production of galactosidases from fungi by solid-state fermentation. Fifteen fungi were screened and Aspergillus awamori (MTCC 548), exhibited the highest α and β-galactosidase activities of 75.11±0.29 U/g and 155.34±1.26 U/g, respectively. 30 g of wheat bran substituted with 6% defatted soy flour, at 28°C, pH 5.0 for 120 h, was established as the optimum production conditions by one-factor approach. The enzyme was purified to homogeneity with an apparent mass of 118 ± 2 kDa by ammonium sulfate precipitation (50–80%), ion exchange and hydrophobic interaction chromatography. Specific activities for α and β-galactosidase were 22 and 74 U/mg, respectively. Optimum temperature and pH ranges for enzyme activities were 55–60 °C, 5.0–5.5, respectively. The thermal inactivation mid-point was 65 °C. The purified enzyme not only exhibited α and β-galactosidase activities, but also exhibited β-xylosidase and β-glucosidase activities, indicating the enzyme has broad substrate specificity. Sequence analysis by in-gel digestion and tandem mass spectrometry (MS/MS) revealed that the enzyme was a probable β-galactosidase A, belonging to glycoside hydrolase 35 family, and is being reported for the first time.

Introduction

Carbohydrate active enzymes are one of the most diverse and interesting enzymes found in nature. Glycoside hydrolases (GHs; EC 3.2.1.x) are a group of carbohydrate-active enzymes that are currently classified into 116 families (www.cazy.org). They are known to cleave O, N, and S-glycosidic bonds in glycoconjugates, glycosides, and glycans [1]. Alpha-galactosidase (α-gal; EC 3.2.1.22) and beta-galactosidase (β-gal; EC 3.2.1.23) are two such glycoside hydrolases with important applications in the food, feed as well as pharmaceutical industries [2,3]. The enzyme α-gal is known to hydrolyze the α-galactosyl (α 1–6 linkages) terminal moieties of glycoproteins and glycolipids [4], whereas β-gal hydrolyzes the D-galactosyl (β 1–4 linkage) residues from secondary metabolites, polymers or oligosaccharides [5]. The α-gal and β-gal enzymes are produced by microorganisms, plants, and animals [3,6,7]. Among the different microbial sources, fungi are typically preferred due to extracellular secretion into the medium, which then facilitates easier downstream processing, various biochemical properties, and comparatively higher yields.

Although both galactosidases are ubiquitous in nature, their deficiency in mammals, especially in humans, causes many digestive disorders. The inability of humans to digest the milk sugar lactose, due to lack or under-production of β-gal is termed as lactose intolerance [8]. It is known to affect at least 75% of the global population; however, the percentage varies from race to race and region to region [8]. The other related carbohydrate digestive disorders are due to lack of brush border enzymes, such as lactase phlorizin hydrolase and sucrase-isomaltase, commonly known as brush border malease [9]. This may be due to old age, diseased conditions, and mutations (like in celiac and Fabry's disease) [10]. Lack of carbohydrate digestion and absorption causes flatulence, discomfort, nausea, and irritable bowel syndrome [3].

This study is focused on the production of both extracellular α-gal and β-gal, purification, characterization, and understanding its mode of action, to further find objective applications in the food and dairy industries. However, purification, sequence analysis and other investigations revealed that the enzyme was a broad substrate specific glycoside hydrolase. This new enzyme, exhibiting both α-gal and β-gal properties, is being reported for the first time at the experimental or protein level.

Section snippets

Chemicals and media

All media were purchased from Hi-media Laboratory Pvt. Ltd. (Mumbai, India). The reagents for the assay and gel electrophoresis were purchased from Sigma Aldrich (India). The wheat bran was obtained from the automated wheat roller flour mill, International school of milling technology (Buhler, Switzerland) at CSIR-CFTRI, Mysuru, India. The rice bran, green gram husk, chickpea husk, defatted soy flour, and spent coconut meal were obtained from the local markets in Mysuru, India.

Microorganisms and culture conditions

Fifteen fungal

Screening for galactosidases from fungi

There are many biotechnologically relevant fungi, which produce carbohydrate active enzymes, primarily galactosidases. There is an added advantage if these enzymes were produced by solid state fermentation. First, it acts as a natural medium for the growth of fungi, and second, it allows very little scope for enzyme hydrolysis by proteases [21,22]. Therefore, this study began with screening of 15 fungi for galactosidases production by solid state fermentation, galactosidases activity and

Conclusion

In this study, an effort was made to enhance the production of galactosidase by one-factor approach. The enzyme was purified to homogeneity. Sequence analysis revealed enzyme belongs to GH family 35 (probable β-galactosidase A) and is broad substrate specific nature. Further analysis of this enzyme will enable its applications in both food and pharmaceutical industries.

Author statement

C H Vidya have performed SSF experiments and downstream processing and enzyme characterization experiments and written original draft.

C V Chinmayee has performed SSF experiments.

Gnanesh Kumar contributed to the LC-MS/MS analysis and edited manuscript.

Sridevi Annapurna Singh has designed the experiments, checked analysis reviewed and edited the manuscript.

Acknowledgments

The authors would like to thank the Director, CSIR-CFTRI, for providing the facilities. Vidya CH would like to thank DST-INSPIRE for the fellowship granted. Chinmayee CV would like to thank UGC for the fellowship granted.

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