<Emphasis Type="Italic">Neurospora crassa</Emphasis> FKS Protein Binds to the (1,3)β-Glucan Synthase Substrate,UDP-Glucose

The essential fungal cell-wall polymer (1,3)β-glucan is synthesized by the enzyme (1,3)β-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)β-glucan synthase. We have purified (1,3)β-glucan synthase from Neurospora crassa ∼400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[β-³²P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H⁺-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)β-glucan synthase. Received: 31 May 2002 / Accepted: 27 July 2002     

Structural Characterization of <Emphasis Type="Italic">β</Emphasis>-glucans of <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis> in Different Stages of Fruiting Body Maturity and their Use in Nutraceutical Products

β-Glucans of Agaricus brasiliensis fruiting bodies in different stages of maturity were isolated and characterized by FTIR and NMR. These fractions had greater amount of (1→6)-β-glucan and the (1→3)-β-glucan increased with fruiting bodies maturation. Yields of β-glucans increased from 42 mg β-glucans g⁻¹ fruiting bodies (dry wt) in immature stage to 43 mg g⁻¹ in mature stage with immature spores, and decreased to 40 mg g⁻¹ in mature stage with spore maturation. Mature fruiting bodies, which included these glucans, have potential therapeutical benefits for use in nutraceutical products.     

Molecular cloning and sequence analysis of the β-1,3-glucan synthase catalytic subunit gene from a medicinal fungus, Cordyceps militaris

The entomopathogenic fungus Cordyceps militaris belongs to vegetable wasps and plant worms and is used as herbal medicine, but β-1,3-glucan biosynthesis has been poorly studied in C. militaris. The fungal FKS1 gene encodes an integral membrane protein that is the catalytic subunit of β-1,3-glucan synthase. Here, we isolated cDNA clones encoding a full-length open reading frame of C. militaris FKS1. Cordyceps militaris Fks1 protein is a 1981 amino acid protein that shows significant similarity with other fungal Fks proteins. This study is the first report of molecular cloning of the β-1,3-glucan synthase catalytic subunit gene from vegetable wasps and plant worms.     

Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium

When Phanerochaete chrysosporium was grown with laminarin (a β-1,3/1,6-glucan) as the sole carbon source, a β-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear β-1,3-glucan, branched β-1,3/1,6-glucan, and β-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (β-d-Glcp-(1–>6)-β-d-Glcp-(1–>3)-β-d-Glcp-(1–>3)-d-Glc) and 4-O-glucosyl-laminaribiose (β-d-Glcp-(1–>4)-β-d-Glcp-(1–>3)-d-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes β-d-Glcp-(1–>3)-d-Glcp at subsites −2 and −1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a β-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched β-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular β-1,3-glucanases.     

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several β-glucan hydrolases including an exo-1,3-β-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different β-glucans, including laminaran, a 1,3-β-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K _m 1.66 mg mL⁻¹; V _max 7.69 IU mL⁻¹) and laminari-oligosaccharides and did not yield activity against 1,4-β-glucans, 1,3;1,4-β-glucans or 4-nitrophenyl- and methylumbelliferyl-β-d-glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-β-glucanase was inhibited by glucono-δ-lactone (K _i 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as β-glucosidase. ‘De novo’ sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-β-glucanase is yet another novel enzyme present in the β-glucanolytic enzyme system of T. emersonii.     

Gene Cloning and Heterologous Expression of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete Chrysosporium

The basidiomycete Phanerochaete chrysosporium produces several β-1,3-glucanases when grown on laminarin, a β-1,3/1,6-glucan, as the sole carbon source. To characterize one of the major unknown β-1, 3-glucanases with a molecular mass of 83 kDa, identification, cloning, and heterologous over-expression were carried out using the total genomic information of P. chrysosporium. The cDNA encoding this enzyme included an ORF of 2337 bp and the deduced amino acid sequence contains a predicted signal peptide of 26 amino acids and the mature protein of 752 amino acids. The amino acid sequence showed a significant similarity with glycoside hydrolase family 55 enzymes from filamentous fungi and was named Lam55A. Since the recombinant Lam55A expressed in the methylotrophic yeast Pichia pastoris degraded branched β-1,3/1,6-glucan as well as linear β-1,3-glucan, the kinetic features of the enzyme were compared with those of other β-1,3-glucanases.     

Cell wall α-1,3-glucans from a biocontrol isolate of Rhizoctonia: immunocytolocalization and relationship with α-glucanase activity from potato sprouts

The interface between plants and pathogens plays an important role in their interaction. Studies of fungal cell walls are scarce and previous results show the existence of α-1,3-glucans in addition to ß-glucans. In addition, α-1,3-glucans are not present in plant cell walls, and α-glucanase activity in plants has not been described before. In a previous work, we purified and characterized an α-1,3-glucan from a binucleated, non-pathogenic Rhizoctonia isolate, which induces plant defence responses. Therefore, in order to study the architecture of the fungal cell wall, and the accessibility and localization of the α-glucan elicitor, we prepared an antibody against the α-1,3-glucan and analysed its localization by TEM. Immunolocalization showed the presence of the α-1,3-glucan in the intercellular spaces and along the cell walls, mainly on the inner layers. This result, and the presence of the α-1,3-glucan in the liquid culture medium in which binucleated non-pathogenic Rhizoctonia was grown, confirmed that the α-glucan had been secreted. The α-1,3-glucan was also immunocytolocalized on potato sprouts tissue elicited with the glucan; gold particles were observed in vacuoles and close to the plasmalemma. In addition, α-glucanase activity in potato sprouts was detected using cell wall glucans from the pathogenic isolate R. solani AG-3 as substrates; whereas, when cell wall glucans from non-pathogenic isolates were used, no α-glucanase activity was detected. Our results suggest that the presence of α-1,3-glucans could be associated with the formation and integrity of the cell wall and also with plant–fungi interactions. This is the first report to describe α-glucanolytic activity in plants.     

Codon optimization of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> β-1,3-1,4-glucanase gene and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant β-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding β-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, β-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l⁻¹. The β-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized β-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45°C, respectively.     

Structural characterisation and biological activities of a unique type β-<Emphasis Type="SmallCaps">d</Emphasis>-glucan obtained from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A β-d-glucan obtained from Aureobasidium pullulans (AP-FBG) exhibits various biological activities: it exhibits antitumour and antiosteoporotic effects and prevents food allergies. An unambiguous structural characterisation of AP-FBG is still awaited. The biological effects of β-d-glucan are known to depend on its primary structures, conformation, and molecular weight. Here, we elucidate the primary structure of AP-FBG by NMR spectroscopy, and evaluate its biological activities. Its structure was shown to comprise a mixture of a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every two residues (major structure) and a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every three residues (minor structure). Furthermore, this β-d-glucan exhibited immunostimulatory effects such as the accumulation of immune cells and priming effects against enterobacterium. To our knowledge, 1-3-β-glucans like AP-FBG with such a high number of 1-6-β-glucopyranosyl side branching have a unique structure; nevertheless, many 1-3-β-glucans were isolated from various sources, e.g. fungi, bacteria, and plants.     

Characterization of endo-1,3–1,4-β-glucanases in GH family 12 from Magnaporthe oryzae

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze β-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3–1,4-β-glucans, such as barley β-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3–1,4-β-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3–1,4-β-glucan during infection and spore formation.     

Cloning and functional characterization of a complex endo-β-1,3-glucanase from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp.

A β-1,3-glucanase gene, encoding a protein of 1,793 amino acids, was cloned from a strain of Paenibacillus sp. in this study. This large protein, designated as LamA, consists of many putative functional units, which include, from N to C terminus, a leader peptide, three repeats of the S-layer homologous module, a catalytic module of glycoside hydrolase family 16, four repeats of the carbohydrate-binding module of family CBM_4_9, and an analogue of coagulation factor Fa5/8C. Several truncated proteins, composed of the catalytic module with various organizations of the appended modules, were successfully expressed and characterized in this study. Data indicated that the catalytic module specifically hydrolyze β-1,3- and β-1,3–1,4-glucans. Also, laminaritriose was the major product upon endolytic hydrolysis of laminarin. The CBM repeats and Fa5/8C analogue substantially enhanced the hydrolyzing activity of the catalytic module, particularly toward insoluble complex substrates, suggesting their modulating functions in the enzymatic activity of LamA. Carbohydrate-binding assay confirmed the binding capabilities of the CBM repeats and Fa5/8C analogue to β-1,3-, β-1,3–1,4-, and even β-1,4-glucans. These appended modules also enhanced the inhibition effect of the catalytic module on the growth of Candida albicans and Rhizoctonia solani.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> juice on cell wall polysaccharides of three <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from different origins

In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the β(1,3)-glucanase lytic activity and the β-glucan/mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake flasks with 30 g l⁻¹ sugar concentration of A. tequilana juice and with the control YPD using 30 g l⁻¹ of glucose. The three yeasts strains showed different levels of β-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast.     

Inhibition of Neurospora crassa Growth by a Glucan Synthase-1 Antisense Construct

We have used the filamentous fungus, Neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)β-glucan synthase [E.C. 2.4.1.34; UDP glucose: 1,3-β-D-glucan 3-β-D-glucosyltransferase], leads to a corresponding decrease in growth of the organism. Previously, our laboratory isolated a gene (glucan synthase-1, gs-1) that is required for (1,3)β-glucan synthase activity. Wild-type cells were transformed with DNA vectors encoding various RNAs complementary to the gs-1 messenger RNA (antisense RNA) cloned downstream from an inducible promoter (quinic acid-2 [qa-2p]). Stable transformants, expressing a partially inverted antisense message of gs-1 (pMYX107), exhibited dramatic reduction in growth compared with empty vector controls. Hyphal measurements of these transformants grown on race tubes indicated that all of the transformants showed various degrees of inhibition. Microscopic observations of transformants revealed shorter hyphal lengths when grown under conditions expressing antisense. Further characterization revealed that the specific activities of (1,3)β-glucan synthase were decreased by as much as 63% relative to empty vector controls. Together, these observations suggest that antisense against (1,3)β-glucan synthase led to a reduction in enzyme levels that resulted in altered cell-wall morphology and inhibition of growth. It is possible that antisense oligonucleotides against gs-1 may be useful antifungal agents. Received: 20 September 1996 / Accepted: 1 November 1996     

Characterization and function of wall-bound exo-β-glucanases of Lilium longiflorum pollen tubes

Two exo-β-glucanases (LP-ExoI, 83 kDa and LP-ExoII, 71 kDa) were extracted and partially purified from the cell wall of Lilium longiflorum pollen tubes. Both LP-ExoI and LP-ExoII hydrolyzed laminarin (1,3-β-glucan). These enzymes also exhibited some activity toward 1,3:1,4-β-glucans of Hordeum vulgare and Cetraria islandica and the 1,6-β-glucan of Umbilicaria papullosa. The pH for optimum activity for both exo-β-glucanases was 5.5. Methylation analysis of the reaction products revealed that purified LP-ExoI decreased both 1,3- and 1,4-glucosyl linkages in hemicellulosic polysaccharides isolated from the cell wall of lily pollen tubes. D-gluconolactone and nojirimycin, inhibitors of glucosidase, inhibited activities of both exo-β-glucanases, as well as growth of the lily pollen tubes. These results disclosed that the wall-bound exo-β-glucanases play an important role in the regulation of lily pollen tube growth. Received: 3 January 2000 / Revision accepted: 8 March 2000     

Critical roles of Asp270 and Trp273 in the α-repeat of the carbohydrate-binding module of endo-1,3-β-glucanase for laminarin-binding avidity

A carbohydrate-binding module from family 13 (CBM13), appended to the catalytic domain of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, was overexpressed in E. coli, and its interactions with β-glucans, laminarin and laminarioligosaccharides, were analyzed using surface plasmon resonance biosensor and isothermal titration calorimetry. The association constants for laminarin and laminarioligosaccharides were determined to be approximately 10⁶ M⁻¹ and 10⁴ M⁻¹, respectively, indicating that 2 or 3 binding sites in the α-, β-, and γ-repeats of CBM13 are involved in laminarin binding in a cooperative manner. The binding avidity is approximately 2-orders higher than the monovalent binding affinity. Mutational analysis of the conserved Asp residues in the respective repeats showed that the α-repeat primarily contributes to β-glucan binding. A Trp residue is predicted to be exposed to the solvent only in the α-repeat and would contribute to β-glucan binding. The α-repeat bound β-glucan with an affinity of approximately 10⁴ M⁻¹, and the other repeats additionally bound laminarin, resulting in the increased binding avidity. This binding is unique compared to the recognition mode of another CBM13 from Streptomyces lividans xylanase.     

Kinetics of Phase Separation of Oat β-Glucan/Whey Protein Isolate Binary Mixtures

The kinetics of phase separation and microstructure of oat β-glucan/whey protein binary mixtures varying in concentration (4–16% w/v protein, 0.3–1.2% w/v β-glucan) and β-glucan molecular weight (1.3 × 10⁶, 640 × 10³, 180 × 10³, and 120 × 10³ g/mol) was investigated by turbidimetry and fluorescent microscopy. The phase separation of the mixed systems was followed at pH 7.0 and at room temperature under quiescent conditions. Application of first principles revealed that phase separation of the systems follows first-order kinetics. Acceleration of the phase-separation process was observed with increase of β-glucan concentration for the three lowest-MW samples but the highest molecular weight (1.3 × 10⁶ g/mol) exhibited the opposite trend. Changes in the polysaccharide molecular weight resulted in considerable differences in β-glucan aggregate morphology in the mixed systems. The change in the continuity of the mixed system from polysaccharide-, to bi-, to protein-continuous was confirmed for a wide range of mixed systems differing in biopolymer concentration, and β-glucan molecular weight.     

Characterization of the catalytic domains of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> endoglucanase I,II, and III,expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A) from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins. Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and 15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β-d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β-d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β-d-glucan, xyloglucan, xylan, and mannan.     

Arabinoxylan and (1→3),(1→4)-β-glucan deposition in cell walls during wheat endosperm development

Arabinoxylans (AX) and (1→3),(1→4)-β-glucans are major components of wheat endosperm cell walls. Their chemical heterogeneity has been described but little is known about the sequence of their deposition in cell walls during endosperm development. The time course and pattern of deposition of the (1→3) and (1→3),(1→4)-β-glucans and AX in the endosperm cell walls of wheat (Triticum aestivum L. cv. Recital) during grain development was studied using specific antibodies. At approximately 45°D (degree-days) after anthesis the developing walls contained (1→3)-β-glucans but not (1→3),(1→4)-β-glucans. In contrast, (1→3),(1→4)-β-glucans occurred widely in the walls of maternal tissues. At the end of the cellularization stage (72°D), (1→3)-β-glucan epitopes disappeared and (1→3),(1→4)-β-glucans were found equally distributed in all thin walls of wheat endosperm. The AX were detected at the beginning of differentiation (245°D) in wheat endosperm, but were missing in previous stages. However, epitopes related to AX were present in nucellar epidermis and cross cells surrounding endosperm at all stages but not detected in the maternal outer tissues. As soon as the differentiation was apparent, the cell walls exhibited a strong heterogeneity in the distribution of polysaccharides within the endosperm.     

Ability of rumen protozoa <Emphasis Type="Italic">Diploplastron affine</Emphasis> to utilize β-glucans

The ability of the rumen ciliates to utilize β-glucans other than cellulose and xylan is currently being recognized. The objective of the present study was to characterize the ability of the ciliate Diploplastron affine to digest some pachyman, laminarin, pustulan, curdlan and lichean. The protozoa were isolated from the rumen of sheep and either grown in vitro or inoculated into the rumen of ciliate-free sheep and maintained in natural conditions. In vitro culture studies showed that the enrichment of culture medium with the examined saccharides results in an increase in the number of ciliates in comparison to the control cultures. The increase was over 36 and 15 % when the growth medium was supplemented with pachyman (1,3-β-glucan) and pustulan (1,6-β-glucan), respectively. A positive correlation was also found between the population density of ciliates and the dose of saccharide supplemented to the growth medium. Enzyme studies were performed using the crude enzyme preparation obtained from ciliates treated with antibiotics. The ability of ciliates to digest the examined β-glucans was tested by the quantification of reducing sugars released from the mentioned substrates during the incubation with crude enzyme preparation. The results showed that D. affine ciliates were able to digest both of them. The mean degradation rate varied between 6.7 and 28.2 μmol/L glucose per mg protein per h for pustulan and lichean, respectively, whereas the digestion velocity was the highest at 5.0–5.5 pH and 45–50°C.     

A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography

A new screening method for β-(1,3–1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.