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

Abstract

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 1,356-bp 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 Zn²⁺ 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 be used in building glycan blocks of crucial glycoconjugates to assess their biological functions.

Keywords: Exo-β-1,3-galactanase; Penicillium oxalicum; glycoside hydrolase family 43; larch wood arabinogalactans.

Fig. 1. Phylogenetic analysis of amino acid sequences of PoGal3 and eight exo-β-1,3-galactanases from different sources.

exo-β-1,3-galactanase Il3Gal (GenBank: BAH29957.1), BLLJ_1840 (GenBank: BAJ67504.1), Fo1,3Gal (GenBank: BAG80558.1), Pc1,3Gal43A (GenBank: BAD98241.1), Ct1,3Gal43A (GenBank: ABN51896.1), Sa1,3Gal43A (GenBank: BAC69820.1) and SGalase2/SGalase1 (GenBank: AFH55135.1 / AFH55134.1).

Fig. 2. SDS-PAGE analysis of recombinant PoGal3.

(A) Expression of recombinant PoGal3 in GS115 after ammonium sulfate precipitation method: lane 1, protein markers; lane 2, PoGal3 after ammonium sulfate precipitation method. (B) Purification of recombinant PoGal3 by Sephacryl HR S-100: lane 1, protein marker; lane 2 to lane 8, elated by Na-acetate buffer (50 mM). (C) Purification of recombinant PoGal3 after two-step chromatography: lane 1, protein markers; lane 2, PoGal3 after two-step chromatography.

Fig. 3

Structural characterization of AG-P-I by HPLC (A) and ¹³C NMR (B).

Fig. 4. The effect of pH on activity (A) and stability (B) of PoGal3 used AG-P-1 as substrate.

The activity of the enzyme before incubation was defined as 100%. Results are presented as means±standard deviations (n = 3).

Fig. 5. The effect of temperature on activity (A) and stability (B) of PoGal3 using AG-P-1 as substrate.

The activity of the enzyme before incubation was set as 100%. Results are presented as the mean ± standard deviation (n = 3).

Fig. 6. HPAEC of degradation products of different polysaccharides hydrolyzed by PoGal3.

Reactions were incubated with recombinant enzyme at 40°C for 12 h. The bottom of each graph was the standard sample of galactose or 1,6- galactoise, and oligosaccharides produced in the reaction were detected by HPAEC at 0 h and 12 h.