Cloning and Expression of a Gene for an 87-kDa β-1,3-Glucanase of Bacillus circulans IAM1165 in Escherichia coli K-12

Partially Sau3AI-digested fragments of chromosomal DNA from Bacillus circulans IAM1165, a high producer of β-1,3-glucanases able to lyse fungal cell walls, were inserted into a BamHI site of the plasmid vector pHSG399. A gene for the glucanase was cloned in Escherichia coli K-12 by the shotgun method. An 8-kb inserted DNA directed synthesis of an 87-kDa endo-β-1,3-n-glucanase in E. coli. The β-glucanase gene was in a 2.6-kb EcoRI-SmaI segment within the insert DNA. The enzyme activity was found mainly in the periplasmic fraction of E. coli carrying the gene.     

The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity

The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called “pythiosis”. Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.     

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp²⁰² and Glu³⁶¹ were catalytic residues, and Trp²⁷⁰, Tyr⁴⁶¹, and Asn⁴⁶² were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn⁴⁶² and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.     

Structural and Biochemical Analyses of Glycoside Hydrolase Family 26 β-Mannanase from a Symbiotic Protist of the Termite Reticulitermes speratus

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of β-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites −5 and −2, respectively, in addition to accommodation of both glucose and mannose at subsites −3 and −4. RsMan26C has a long open cleft with a hydrophobic platform of Trp⁹⁴ at subsite −5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met⁸⁵ specifically interacts with the equatorial O-2 of glucose at subsite −3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.     

Cloning and Expression of a β-Galactosidase Gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta^®. Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Cloning and Expression of the Exo-β-D-1,3-glucanase Gene (exgS) from Aspergillus saitoi

A gene of exo-1,3-β-D-glucanase (exgS) was cloned from a koji mold, Aspergillus saitoi, genomic DNA using PCR. The exgS has an ORF comprising 2832 bp, which contains one intron of 45 bp, and encodes 945 amino acids. The deduced amino acid sequences showed that the ExgS has a non-homologous linker region consisting of 180 amino acids, which encompassed highly conserved regions observed in Exg homologues from filamentous fungi. A recombinant protein (ExgS) has been recovered from the cultural filtrate of an Aspergillus oryzae strain that carried an expression vector containing full length of the exgS. The N-terminal amino acid sequences of the recombinant exo-1,3-β-D-glucanase (ExgS) were identical to that of native ExgS from A. saitoi.     

Cloning and Expression of an α-1,3-Glucanase Gene from Bacillus circulans KA-304: The Enzyme Participates in Protoplast Formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of α-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity.

The gene of α-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of α-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of α-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (α-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.

The recombinant α-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant α-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-β-D-thiogalactopyranoside. The recombinant α-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

An Electrochemical Assay of β-1,3-Glucanase Gene from Transgenic Capsicum Using Asymmetric PCR

5′-Thiol-derivatized specific DNA probes were added to the single primer polymerase chain reaction (asymmetric PCR) solution. In the PCR process, the DNA probes extended in the presence of target; the extended probes were then immobilized on a glassy carbon electrode (GCE) via gold nanoparticles. Finally, methylene blue and the extended probes were combined and the electrochemical signals were measured. This signal was higher than that of the GCE modified only by the original probe. When there was no target in PCR solution, the probe did not extend and the signal did not increase. The specific sequences of the β-1,3-glucanase gene were detected successfully from three targets with different length: oligonucleotide, molecule clone vector DNA, and total genome DNA of transgenic capsicum. The detection limits of 2.6 × 10^?13, 7.8 × 10^?13, and 9.1 × 10^?13 moll^?1 for oligonucleotide, molecule clone vector DNA, and total transgenic capsicum genome DNA were estimated.     

The Tetramer Structure of the Glycoside Hydrolase Family 27 α-Galactosidase I from Umbelopsis vinacea

The crystal structure of Umbelopsis vinacea α-galactosidase I, which belongs to glycoside hydrolase family 27, was determined at 2.0 Å resolution. The monomer structure was well conserved with those of glycoside hydrolase family 27 enzymes. The biological tetramer structure of this enzyme was constructed by the crystallographic 4-fold symmetry, and tetramerization appeared to be caused by three inserted peptides that were involved in the tetramer interface. The quaternary structure indicated that the substrate specificity of this enzyme might be related to the tetramer formation. Three N-glycosylated sugar chains were observed, and their structures were found to be of the high-mannose type.     

Phylogenetic Analysis of the Plant Endo-β-1,4-Glucanase Gene Family

Phylogenetic analysis of the endo--1,4-glucanase gene family of Arabidopsis and other plants revealed a clear distinction in three subfamilies (, , and ). The - and -subfamily contains proteins believed to be involved in a number of physiological roles such as elongation, ripening, and abscission. The -subfamily is composed of proteins that are predicted to have a membrane-spanning domain and to be localized at the plasma membrane. Some of these proteins have been linked to cellulose biosynthesis by serving to hydrolyze a lipid-linked intermediate that acts as a primer for the elongation of -glucan chains during cellulose synthesis at the plasma membrane. Similar glucanases are important in cellulose biosynthesis in bacteria. Searches in the genomes of unrelated organisms that make cellulose, such as Ciona intestinalis and Dictyostelium discoideum, revealed the presence of membrane-linked endo--1,4-glucanases and it is suggested that these might also have a role in cellulose synthesis.     

A Novel β-N-Acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: Gene Cloning,Expression, and Assignment to Family 3 of the Glycosyl Hydrolases

A β-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an M_r of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned β-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical β-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable β-glucosidase activity, revealed homology with microbial β-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of β-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60°C and pH 5.0, and the apparent K_m and V_max values for p-nitrophenyl-β-N-acetylglucosamine were 425.7 μM and 24.8 μmol min⁻¹ mg of protein⁻¹, respectively.Streptomycetes are gram-positive, mycelial soil bacteria with a high G+C content. In addition to having the ability to synthesize a wide variety of antibiotics and chemotherapeutic agents, they produce extracellular hydrolytic enzymes to obtain nutrients and energy by solubilizing polymeric compounds in soil. These enzymes include proteases, nucleases, lipases, and a variety of enzymes that hydrolyze different types of polysaccharides such as cellulose, chitin, and xylan (13). This last class of enzymes has received considerable attention not only from the standpoint of the utilization of renewable resources but also from that of basic research. Among actinomycetes, Streptomyces spp. make up one group regarded as particularly efficient in the breakdown of chitin (10). Following cellulose, chitin is the second most abundant polymer (β-1,4-linked polymer of N-acetylglucosamine) in nature. Efficient degradation of chitin by microorganisms is achieved by the concerted action of chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) (1, 19, 20).We have been studying the chitinolytic system of Streptomyces thermoviolaceus OPC-520 to clarify the roles of individual enzymes involved in chitin degradation, the relationship between structure and function, and the regulation of gene expression. When S. thermoviolaceus OPC-520 is cultivated in the presence of chitin, this strain secretes three different chitinases and only one β-N-acetylglucosaminidase and the production is repressed by glucose (unpublished data). Previously, we purified and characterized a major chitinase (Chi40) produced by the strain, which shows a high optimum temperature (70 to 80°C), high optimum pH (pH 8.0 to 10.0), and heat stability (22), and recently reported the cloning and expression of the Chi40 gene (23).While a number of chitinase genes have been isolated from a wide variety of organisms, including bacteria, fungi, insects, plants, and animals, examples of cloning of the β-N-acetylglucosaminidase gene involved in a chitinolytic system are few. To understand the role of β-N-acetylglucosaminidase in chitin degradation by strain OPC-520, its relationship to similar proteins isolated from other sources, and the regulatory system involved in the induction of the enzyme, we have isolated and expressed the gene encoding β-N-acetylglucosaminidase. Here we report the molecular cloning and biochemical characterization of a β-N-acetylglucosaminidase, designated NagA, from S. thermoviolaceus OPC-520. This novel enzyme, which is clearly different from the N-acetylglucosaminidases so far reported, is assigned to family 3 of the glycosyl hydrolases on the basis of sequence comparison. This is the first report of a β-N-acetylglucosaminidase gene isolated from the genus Streptomyces.     

Cloning and Characterization of the nagA Gene that Encodes β-N-Acetylglucosaminidase from Aspergillus nidulans and Its Expression in Aspergillus oryzae

We isolated a β-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed β-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of β-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of β-N-acetylglucosaminidase in a wheat bran solid culture.     

Salecan Enhances the Activities of β-1,3-Glucanase and Decreases the Biomass of Soil-Borne Fungi

Salecan, a linear extracellular polysaccharide consisting of β-1,3-D-glucan, has potential applications in the food, pharmaceutical and cosmetic industries. The objective of this study was to evaluate the effects of salecan on soil microbial communities in a vegetable patch. Compositional shifts in the genetic structure of indigenous soil bacterial and fungal communities were monitored using culture-dependent dilution plating, culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR. After 60 days, soil microorganism counts showed no significant variation in bacterial density and a marked decrease in the numbers of fungi. The DGGE profiles revealed that salecan changed the composition of the microbial community in soil by increasing the amount of Bacillus strains and decreasing the amount of Fusarium strains. Quantitative PCR confirmed that the populations of the soil-borne fungi Fusarium oxysporum and Trichoderma spp. were decreased approximately 6- and 2-fold, respectively, in soil containing salecan. This decrease in the amount of fungi can be explained by salecan inducing an increase in the activities of β-1,3-glucanase in the soil. These results suggest the promising application of salecan for biological control of pathogens of soil-borne fungi.     

News & Notes: Production, Purification, and Properties of an Endo-1,3-β-Glucanase from the Basidiomycete Agaricus bisporus

Agaricus bisporus H 25 produced extracellular endo-1,3-β-glucanase when grown in a static culture at 25°C in a minimal synthetic medium supplemented with A. bisporus cell walls plus fructose. Endo-1,3-β-glucanase was purified 17.85-fold from 20-day-old culture filtrates by precipitation at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and preparative PAGE followed by electroelution. The purified enzyme yielded a single band in both native and SDS-polyacrylamide gels with a molecular mass of 32 kDa (SDS-PAGE) and 33.7 kDa (MALDI-MS), showing an isoelectric point of 3.7. The enzyme was active against β-1,3- linkages and, to a lesser extent, against β-1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase against laminarin and pustulan were observed between pH 4 and 5.5, and between 40° and 50°C for laminarin, and between 30° and 50°C for pustulan. The optimum pH and temperature were 4.5 and 45°C for both substrates. Received: 17 June 1998 / Accepted: 24 September 1998     

Cloning,Sequencing, and Expression of the Gene Encoding an Intracellular β-D-Xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular β-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50°C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with β-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with β-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-β-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50°C.     

Cloning, Molecular Characterization, and mRNA Expression of the Thermostable Family 3 β-Glucosidase from the Rare Fungus Stachybotrys microspora

The filamentous fungus Stachybotrys microspora possess a rich β-glucosidase system composed of five β-glucosidases. Three of them were already purified to homogeneity and characterized. In order to isolate the β-glucosidase genes from S. microspora and study their regulation, a PCR strategy using consensus primers was used as a first step. This approach enabled the isolation of three different fragments of family 3 β-glucosidase gene. A representative genomic library was constructed and probed with one amplified fragment gene belonging to family 3 of β-glucosidase. After two rounds of hybridization, seven clones were obtained and the analysis of DNA plasmids leads to the isolation of one clone (CF3) with the largest insert of 7 kb. The regulatory region shows multiple TC-rich elements characteristic of constitutive promoter, explaining the expression of this gene under glucose condition, as shown by zymogram and RT-PCR analysis. The tertiary structure of the deduced amino acid sequence of Smbgl3 was predicted and has shown three conserved domains: an (α/β)₈ triose phosphate isomerase (TIM) barrel, (α/β)₅ sandwich, and fibronectin type III domain involved in protein thermostability. Zymogram analysis highlighted such thermostable character of this novel β-glucosidase.     

Cloning and Characterization of the Gene Encoding Endo-β-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-β-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-β-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-β-1,3-glucanases, but GluA was partially similar to two fungal exo-β-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two β-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-β-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.