• Journal of Life Science
• /
• v.26 no.10
• /
• pp.1113-1120
• /
• 2016

Two bacterial strains producing extracellular man nanase were isolated from doenjang, a traditionally fermented soybean paste in Korea. The isolates, WL-6 and WL-11, were identified as Bacillus subtiis on the basis of their 16S rRNA gene sequences, morphological, and biochemical properties. Two genes encoding the mannanase of both B. subtilis WL-6 and B. subtilis WL-11 were each cloned into Escherichia coli, and their nucleotide sequences were determined. Both mannanase genes consisted of 1,086 nucleotides, encoding polypeptides of 362 amino acid residues. The deduced amino acid sequences of the two WL-6 and WL-11 mannanases, designated Man6 and Man11, respectively, differed from each other by eight amino acid residues, and they were highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The 26 amino acid stretch in the N-terminus of Man6 and Man11 was a predicted signal peptide. Both Man6 and Man11 were localized at the level of 94–95% in an intracellular fraction of recombinant E. coli cells. The enzymes hydrolyzed both locust bean gum and mannooligosaccharides, including mannotriose, mannotetraose, mannopentaose, and mannohexaose, forming mannobiose and mannotriose as predominant products. The optimal reaction conditions were 55°C and pH 6.0 for Man6, and 60°C and pH 5.5 for Man11. Man11 was more stable than Man6 at high temperatures.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.8
• /
• pp.1064-1071
• /
• 2022

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℃. 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.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.11
• /
• pp.1295-1300
• /
• 2009

A 2,172-bp full-length gene (aga-F78), encoding a protease-resistant $\alpha$-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an $\alpha$-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one-step purification with a Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and nondenaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was a trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at $50^{\circ}C$ and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose, and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of $415.58\;{\mu}g/g$ soybean meal. When combined with trypsin, the enzyme retained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.