Molecular Cloning and Characterization of a Novel Exo-β-1,3-Galactanase from Penicillium oxalicum sp. 68

Abstract

β-D-(1→4)-galactan, whereas AG-II is β-D-(1→3) and/or (1→6)-galactan. Large quantities of AGs are present in Larix trees [1][2][3], and have been shown to possess diverse biological properties, including immunological activity [4], antitumor [5], and antiviral effects [6]. AGs from Larix laricina have been reported to play a unique role in reducing the incidence of the common cold [7]. In addition, AGs have been approved by the US Food and Drug Administration (FDA) for use as dietary fiber. AG-mediated biological activity is generally associated with its monosaccharide composition, type of glycosidic linkage, molecular weight, as well as the number and type of substituents and branched chains. Therefore, analysis of the complex fine structure and function is a significant problem in glycobiology.Glycoside hydrolase is an important enzyme that is widely used in the study of polysaccharides [8]. Up to now, about 171 glycoside hydrolase families have been reported in the CAZy database [9]. β-Galactanases are a broad group of enzymes that hydrolyze glycosidic bonds in plant-derived galactans, and have been widely used in the analysis of polysaccharide structures, construction of medicinal plant fingerprints, preparation of galactooligosaccharides, and food quality improvement [10][11][12]. This group of enzymes can be divided into β-1,3-galactanase, β-1,4galactanase and β-1,6-galactanase according to the structure of their substrates [13]. At present, there are many studies on the preparation and functional analysis of β-1,4-galactanase, whereas investigations with β-1,3-and β-1,6-galactanases are relatively rare [14,15]. Therefore, it has become crucial to the field to prepare and characterize β-1,3-and β-1,6-galactanases. Arabinogalactans have diverse biological properties and can be used as pharmaceutical agents.Most arabinogalactans are composed of β-(1 → 3)-galactan, so it is particularly important to identify β-1,3-galactanases that can selectively degrade them. In this study, a novel exo-β-1,3-galactanase, named PoGal3, was screened from Penicillium oxalicum sp. 68, and hetero-expressed in P. pastoris GS115 as a soluble protein. PoGal3 belongs to glycoside hydrolase family 43 (GH43) and has a 1,356bp gene length that encodes 451 amino acids residues. To study the enzymatic properties and substrate selectivity of PoGal3, β-1,3-galactan (AG-P-I) from larch wood arabinogalactan (LWAG) was prepared and characterized by HPLC and NMR. Using AG-P-I as substrate, purified PoGal3 exhibited an optimal pH of 5.0 and temperature of 40°C. We also discovered that Zn 2+ had the strongest promoting effect on enzyme activity, increasing it by 28.6%. Substrate specificity suggests that PoGal3 functions as an exo-β-1,3-galactanase, with its greatest catalytic activity observed on AG-P-I. Hydrolytic products of AG-P-I are mainly composed of galactose and β-1,6-galactobiose. In addition, PoGal3 can catalyze hydrolysis of LWAG to produce galacto-oligomers. PoGal3 is the first enzyme identified as an exo-β-1,3-galactanase that can b...

β‐Galactosidase: Insights into source variability, genetic engineering, immobilisation and diverse applications in food, industry and medicine

“…Furthermore, a structural characteristic is the low content of helical elements, as its structure features a short helix located in the Nterminal region adjacent to the first loop, in close proximity to the negative subsites (Viborg et al 2019). Since galactose in nature is usually linked to other molecules through b-1,3, b-1,4 or b-1,6 glycosidic bonds, the b-Galactosidases can also be classified as b-1,3-Galactosidase, b-1,4-Galactosidase and b-1,6-Galactosidase based on their recognition of glycosidic bonds (Zhou et al 2022a). Most b-1,3-Galactosidases and b-1,6-Galactosidases are belonged to the GH-35 and GH-42 families.…”

“…Since galactose in nature is usually linked to other molecules through β‐1,3, β‐1,4 or β‐1,6 glycosidic bonds, the β‐Galactosidases can also be classified as β‐1,3‐Galactosidase, β‐1,4‐Galactosidase and β‐1,6‐Galactosidase based on their recognition of glycosidic bonds (Zhou et al . 2022a). Most β‐1,3‐Galactosidases and β‐1,6‐Galactosidases are belonged to the GH‐35 and GH‐42 families.…”