Abstract
A novel β-1,3–1,4-glucanase gene was identified in Bacillus sp. SJ-10 (KCCM 90078) isolated from jeotgal, a traditional Korean fermented fish. We analysed the β-1,3–1,4-glucanase gene sequence and examined the recombinant enzyme. The open reading frame of the gene encoded 244 amino acids. The sequence was not identical to any β-glucanases deposited in GenBank. The gene was cloned into pET22b(+) and expressed in Escherichia coli BL21. Purification of recombinant β-1,3–1,4-glucanase was conducted by affinity chromatography using a Ni-NTA column. Enzyme specificity of β-1,3–1,4-glucanase was confirmed based on substrate specificity. The optimal temperature and pH of the purified enzyme towards barley β-glucan were 50 °C and pH 6, respectively. More than 80 % of activity was retained at temperatures of 30–70 °C and pH values of 4–9, which differed from all other bacterial β-1,3–1,4-glucanases. The degradation products of barley β-glucan by β-1,3–1,4-glucanase were analysed using thin-layer chromatography, and ultimately glucose was produced by treatment with cellobiase.
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Introduction
β-1,3–1,4-Glucan (β-glucan) is found in the cell wall of Poaceae such as avena, barley, maize, oat, and rice. A total of 70 % of the barley cell wall consists of β-glucan [1]. The plant primary structure is based on the structure and characteristics of hemicelluloses such as mannans, xylans, and β-glucan. Among them, β-1,3–1,4-glucan is the third major hemicellulose [2]. Lichenan (lichenin, moss starch) from lichen (moss) is a representative β-glucan. In addition, β-glucan has been found in algae, bryophytes, fern, and fungi [3–6].
β-1,3–1,4-Glucanase (EC 3.2.1.73) is an endo-β-glucanase that cleaves the β-(1 → 4) in the presence of a β-(1 → 3) linkage in mixed-linkage β-1,3–1,4-glucans. The enzyme produces 3-O-cellobiosyl-d-glucose (G3) and 3-O-cellotriosyl-d-glucose (G4) from β-glucan. The β-1,3–1,4-glucanase is used commonly in the brewing industry to improve the efficiency of wort filtration and production of beer from barley glucan [7]. Instead of malt enzyme, thermostable β-1,3–1,4-glucanase has been used in the kilning and mashing process due to its heat stability [8]. Also, it is used in the barley-based diets of broiler chicken and piglet to reduce enteritis and increase the digestion–absorption rate [9–11]. Furthermore, β-glucan digested products (oligosaccharides) have been investigated extensively. Jaskari et al. [12] reported that oligosaccharides improve the growth of probiotic bacteria, and Kim et al. [13] found that they have anti-hypercholesterolemic effects. Recently, β-1,3–1,4-glucanase has commonly been used for the production of ethanol from lichenan and barley [14–16]. The addition of the β-1,3–1,4-glucanase reduces the viscosity and increases the mixing of yeast and nutrients.
Bacillus species produce diverse extracellular polysaccharide-degrading and industrially important enzymes [13]. β-1,3–1,4-Glucanase was cloned from Bacillus spp., such as B. halodurans, B. subtilis, B. brevis, B. licheniformis, and B. amyloliquefaciens, and its activity was determined [17–24]. However, most reported enzymes were active at narrow temperature and pH ranges. Therefore, we aimed to identify a β-1,3–1,4-glucanase with high activity under broad conditions. In previous study, we isolated Bacillus sp. SJ-10 producing halotolerant extracellular protease from joetgal, a traditional Korea fermented fish, preserved with salt [25]. We considered that this strain produces various tolerant enzymes due to which it can grow at high temperature of 55 °C and high concentrations of 0–14 % NaCl.
In this study, a novel β-1,3–1,4-glucanase gene from Bacillus sp. SJ-10 was cloned and expressed, and the biochemical properties of the enzyme were evaluated.
Materials and methods
Bacterial strains, plasmid, media, and growth conditions
Bacillus sp. SJ-10 (KCCM 90078, JCM 15709) was grown in HM (5 % NaCl, 0.5 % yeast extract, 0.5 % proteose peptone, and 0.1 % glucose) medium at 37 °C overnight. Escherichia coli DH5α and E. coli BL21 were grown in Luria broth (LB) at 37 °C for overexpression. The expression vector used was pET22b(+). The positive transformants were grown in LB supplemented with ampicillin (100 μg/ml) at 37 °C. All strains were mixed with glycerol or DMSO and kept at −70 °C until use.
Sequencing of β-1,3–1,4-glucanase from Bacillus sp. SJ-10
Polymerase chain reaction (PCR) was performed using the Bacillus sp. SJ-10 chromosomal DNA, universal primers and a PCR kit (EX Taq Kit, Takara, Japan). The universal primers (Bacillus-bg1314-UP, 5′-YCTTATCGTATGAAACGAGTG-3′; Bacillus-bg1314-RP, 5′-TTTTTTTGTATARCGYACCCA-3′) were derived from GenBank according to conserved bg1314 gene sequences from Bacillus sp. The initial PCR parameters for 30 cycles were: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s. PCR was then performed for 35 amplification cycles at 94 °C for 45 s, 55 °C for 60 s, and 72 °C for 60 s. Next, the PCR product was reacted with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequencing was performed on an Applied Biosystems model 3730XL (Applied Biosystems, USA). The open reading frame (ORF) was defined using the DNA-Walking Speed-UP Premix Kit (Seegene, Seoul, Korea). The GenBank accession number for the bg1314 gene sequence of Bacillus sp. SJ-10 is JQ782413.
Synthesis of the gene and construction of the expression vector
BSJ-bg1314-UP (5′-GGCCGAATTCGATGTCTTATCGTATGAAACG-3′) and BSJ-bg1314-RP (5′-GGCCCTCGAGTTTTTTTGTATAGCGCACC-3′) primers were used for PCR to construct the expression vector. PCR was performed for 30 amplification cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. The PCR products and pET22b(+) were treated with EcoRI and XhoI restriction enzymes (Roche, USA). The bg1314 gene from Bacillus sp. SJ-10 and plasmid pET22b(+) were ligated using the T4 ligase kit (Takara, Japan) to construct the recombinant plasmid, pETbg1314. pETbg1314 was transformed into E. coli DH5α and finally retransformed into E. coli BL21 (E. coli pETbg1314, KCCM 11264P).
Overexpression and purification of β-1,3–1,4-glucanase
Escherichia coli pETbg1314 was grown until it reached an OD600 of 0.4. IPTG (1 mM) was added to induce expression of the recombinant protein at 37 °C for 4 h. Cells were harvested by centrifugation (4,000 rpm, 4 °C, 10 min) and resuspended in 50 mM Tris–HCl (pH 8.0). After sonication, the homogenates were centrifuged (12,000 rpm, 4 °C, 10 min). Pellets were resuspended in 20 mM Tris–HCl (pH 8.0) containing 6 M urea and dialysed into 20 mM Tris–HCl (pH 8.0) for application to the column. Recombinant protein was purified using the Ni-NTA column system. The presence of purified recombinant β-1,3–1,4-glucanase was confirmed by SDS-PAGE. Protein concentrations were determined using the Bradford assay, with bovine serum albumin as the standard.
β-1,3–1,4-Glucanase activity assay
Activity of recombinant β-1,3–1,4-glucanase was assessed using the method of Okeke et al. [27], with some modifications. Briefly, substrates were dissolved in 100 mM sodium phosphate (pH 6). The enzyme and substrate solution were mixed in a 1:1 ratio and reacted at 50 °C for 30 min. DNS (1 ml) was then added to 100 μl of enzyme reaction mixture and boiled for 10 min. The absorbance was measured at 570 nm. One unit (U) of enzyme activity was defined as the amount of protein that produced 1 μmol of d-glucose per minute.
The substrates used were β-d-glucan (from barley, Sigma G6513), 1,3–1,4-β-glucan (from oat), β-1,3-glucan (from Euglena gracilis, Sigma 89862), laminarin (Sigma L9634), 1,3–1,6-β-glucan (from yeast), curdlan (Sigma C7821), and cellulose (Sigma C6288). β-d-Glucan was used to evaluate other chemical properties (optimal pH and temperature). To examine the effects of temperature, an enzyme assay was performed after incubating at 10–80 °C (10 °C intervals). Thermostability was evaluated by incubating at 50–80 °C (10 °C intervals) for 60 min (10 min interval sampling), followed by an enzymatic reaction at 50 °C for 30 min. To determine the optimal pH and stability, the substrates were dissolved in each of the following four buffers: 100 mM potassium chloride–HCl (pH 2), 100 mM sodium acetate (pH 3–6), sodium phosphate (pH 7–8), and glycine–NaOH (pH 9–11).
Determination of enzyme degradation products by thin-layer chromatography
Thin-layer chromatography (TLC), following Apiraksakorn et al. [19], was used to verify the enzymatic reaction products. The enzyme reaction mixture (0.5 μl) was spotted on silica gel 60 TLC plastic sheets (Merck). The developing solvent was composed of n-butanol–isopropanol–ethanol–H2O (2:3:3:2, v/v). The spots were visualised by spraying with 10 % sulphuric acid and then baking at 110 °C for 15–20 min. Standard materials were glucose, cellobiose, cellotriose, and cellotetraose.
Results and discussion
Analysis of the nucleotide and amino acid sequences of bg1314
β-1,3–1,4-Glucanase (bg1314) from Bacillus sp. SJ-10 was composed of 732 nucleic acids and encoded a 244-amino acid protein with a calculated molecular weight of 26.8 kDa. The conserved amino acid motif ‘EIDIEF’, which is located in the active site of glycosyl hydrolase 16 (GH16) family proteins, contains two glutamic acid residues (E134, E138) required for hydrolytic activity. This motif was present in the bg1314 gene [7, 28–32]. The bg1314 nucleotide sequence of Bacillus sp. SJ-10 had 95.9 % homology with B. amyloliquefaciens FZB42 (CP000560.1), 94.8 % with B. atrophaeus 1942 (CP002207.1), 93.3 % with Bacillus sp. 289 (FJ031233.1), 93.1 % with B. subtilis subsp. subtilis 168 (Z46862.1), 92.6 % with B. subtilis subsp. spizizenii TU-B-10 (CP002905.1), and 90 % with B. licheniformis XRK4 (GQ901889.1). The amino acid sequence of β-1,3–1,4-glucanase also has homologies of 96.7, 93.8, 94.3, 91.7, 93.4, and 90.9 % with B. amyloliquefaciens FZB42 (ABS75948.1), B. atrophaeus 1942 (ADP32279.1), Bacillus sp. 289 (ACH85242.1), B. subtilis subsp. subtilis 168 (CAA86922.1), B. subtilis subsp. spizizenii TU-B-10 (AEP86905.1), and B. licheniformis XRK4 (ACX42225.1), respectively.
Purification of β-1,3–1,4-glucanase
The recombinant plasmid pETbg1314 for overexpression of β-1,3–1,4-glucanase was transformed into E. coli BL21(DE3). Protein expression after addition of IPTG was confirmed using SDS-PAGE. β-1,3–1,4-Glucanase (30 kDa), expressed from the pET22b(+) vector, contained an additional His-tag sequence at the C-terminus and was expressed in inclusion bodies. His-tag recombinant β-1,3–1,4-glucanase was effectively purified as a clear single band by Ni-NTA His bind resin (Novagen) (Fig. 1).
Substrate specificity
Various substrates were used to evaluate the substrate specificity of β-1,3–1,4-glucanase. β-1,3–1,4-Glucanase had activity towards β-glucan (60.8 U/mg) from barley and β-glucan (2.8 U/mg) from oat (Table 1). In contrast, β-1,3–1,4-glucanase did not digest β-1,3–1,6-glucan, curdlan, cellulose or dextran. Also, activities against β-1,3-glucan and laminarin were only 1.4 and 0.4 U/mg, respectively. Teng et al. [22] reported that substrate specificity depends on the number of β-1,3 and β-1,4 glycosidic linkages, the branching mode and their distribution in the substrates. β-Glucan from barley and oat is composed of β-1,3 and β-1,4 linkages; and the ratio of β-1,3:β-1,4 linkage is 1:1.9–2.8 in barley and 1:1.5–2.3 in oat [33]. β-1,3-Glucan and curdlan comprised glucose β-1,3 linkages while laminarin and β-glucan from yeast consist of β-1,3 and β-1,6 linkages. Cellulose is a polysaccharide composed of β-1,4 linkages. The four endo-type enzymes that break down β-glucan from barley and oat are β-1,4-d-glucan-4-glucanohydrolase (cellulase, EC 3.2.1.4), β-1,3-d-glucan-3-glucanohydrolase (β-1,3-glucanase, EC 3.2.1.39), β-1,3–1,4-d-4-glucanohydrolase (β-1,3–1,4-glucanase, lichenase, EC 3.2.1.73), and β-1,3(4)-glucanase (laminarinase, EC 3.2.1.6) [17]. Of these, β-1,3–1,4-glucanase can hydrolyse only the β-1,4-glycosidic bond adjacent to β-1,3-glycosidic bond in the mixed glycosidic linkages of β-glucan, but cannot hydrolyse the β-1,4-glycosidic bond in cellulose [7]. Therefore, recombinant Bacillus sp. SJ-10 is likely a β-1,3–1,4-glucanase. These results also suggest that the activity of β-1,3–1,4-glucanase is proportional to the number of β-1,4-linkage bonds [22]. On the other hand, its hydrolysis activity against barley β-glucan is higher than that against oat β-glucan, despite having the same linkage bond, because of the different proportions of β-glycosidic bonds and the use of low yield β-glucan (8.8 %) from oat as a substrate.
Enzyme assay and stability test
Purified β-1,3–1,4-glucanase (1 μg) was reacted with 0.5 % barley β-glucan under various conditions to determine the optimal temperature and pH. In Fig. 2, the optimal temperature and pH were 50 °C and pH 6, respectively. Moreover, >80 % of enzymatic activity was retained at 30–70 °C and pH 4–9.
The temperature stability test showed that β-1,3–1,4-glucanase was highly stable between 10 and 50 °C, retaining more than 95 % activity. The enzyme activity decreased by 70 and 10 % after a 30-min incubation at 60 and 70 °C, respectively (Fig. 2a). The stability was also evaluated at various pH (3–11) values, and the residual activity was measured under optimal conditions, namely, 50 °C and pH 6. The enzyme was highly stable at pH 3–11 (Fig. 2b).
We next compared the activity of the β-1,3–1,4-glucanase of Bacillus sp. SJ-10 to those of other bacteria (Table 2). The optimal conditions for the β-1,3–1,4-glucanase from Bacillus spp. were 50–60 °C and pH 6, excluding B. brevis and Bacillus strain N137.
Recombinant β-1,3–1,4-glucanase from Bacillus sp. SJ-10 showed 82 and 84 % activity at pH 4 and 9, while the activity of enzymes from other Bacillus spp. was less than 50 % under these conditions. B. brevis, Bacillus strain N137 and Rhizopus microspores enzymes had activities of 100 % (pH 9), 85 % (pH 9) and 98 % (pH 4), respectively. Unlike the β-1,3–1,4-glucanase from Bacillus sp. SJ-10, these enzymes showed high activity at narrow pH ranges. Also, Bacillus sp. SJ-10 β-1,3–1,4-glucanase activity was 75, 88, 90 and 80 % at 20, 40, 60 and 80 °C, respectively, whereas the enzymes from other Bacillus spp. and Rhizopus had activities of less than 40 % at 20 and 80 °C (Table 2). Therefore, the β-1,3–1,4-glucanase of Bacillus sp. SJ-10 is a novel enzyme with high activity at broad temperature and pH ranges.
To measure the thermostability of β-1,3–1,4-glucanase, the enzyme was incubated at 50–80 °C for 1 h. The residual enzyme activity was more than 95 % at 50 °C for 30 min, and 75, 10 and 10 % at 60, 70 and 80 °C, respectively. Also, the enzyme showed residual activities of 95 and 40 % after incubation at 50 and 60 °C (Fig. 3). The high thermostability of F. succinogenes β-1,3–1,4-glucanase was caused by five key amino acids: Gly63, Trp54, Trp141, Trp148 and Trp203 [34, 35]. Bacillus sp. SJ-10 β-1,3–1,4-glucanase also had these five amino acids: Gly141, Trp122, Trp180, Trp187 and Trp237. Moreover, Teng et al. [22] reported that five key amino acids, Gly113, Trp104, Trp152, Trp159 and Trp209, were found in B. licheniformis β-1,3–1,4-glucanase, which had 50 % residual activity after 10 min at 70 °C [22].
Thin-layer chromatography analysis
To examine the products of β-glucan degradation by β-1,3–1,4-glucanase, the hydrolysate was collected after 1-, 6-, 12- and 24-h reactions. The flow rate (Rf) of hydrolysate from β-glucan differed from that of maltotriose and maltotetraose as standard sugar, mainly because G3 and G4 were produced after 1, 6, 12 and 24 h. This demonstrates that β-1,3–1,4-glucanase is an endo-type enzyme (Fig. 4a). Generally, the primary products of β-glucan degradation by β-1,3–1,4-glucanase are triose (G3), tetraose (G4) and a small quantity of cellodextrin-like oligosaccharides (5–10 %) from the polymer regions that contain more than three continuative 4-O-linked glucose residues. The rates of G3 and G4 production from barley β-glucan are 52–69 and 25–33 %, respectively [33]. In Fig. 4a, the hydrolysate appeared mainly as two spots (G3, G4). The upper spot likely represents G3 and has a greater intensity than the G4 spot.
Kim et al. found that G3 and G4 have cholesterol-lowering activities. They found that total cholesterol is effectively decreased after injection of G3 and G4 for 6 weeks in diabetic rats [13]. Thus, we predict that G3 and G4 oligosaccharides can be used as bio-health products. The biological effects of hydrolysed oligosaccharides from various polysaccharides have been investigated extensively, but the effects of oligosaccharides from β-glucan remain unclear. Further studies are required to determine the biological effects of β-glucan hydrolysate.
Next, G3 and G4 were incubated with cellobiase from Aspergillus niger. Almost all of the oligosaccharides were degraded into glucose (Fig. 4b). Thus, we confirmed that glucose was the ultimate product of β-glucan degradation by β-1,3–1,4-glucanase and cellobiase. Recently, Menon et al. reported the application of lichenase to produce ethanol from lichenan. They found that a high yield of ethanol was produced from complete hydrolysate using the synergistic interaction of lichenase and β-glucosidase. More specifically, lichenase from Thermomonospora sp. effectively increased glucose production [14]. Therefore, β-1,3–1,4-glucanase may be applicable to ethanol production in alcohol fermentation using the synergistic interaction with cellobiase.
In this study, a novel β-1,3–1,4-glucanase with a wide pH and temperature range was identified from Bacillus sp. SJ-10, itself isolated from jeotgal, and the enzyme was expressed and purified using an E. coli overexpression system. Purified β-1,3–1,4-glucanase may be used for production of oligosaccharides and bio-ethanol.
References
Buliga GS, Brant DA, Fincher GB (1986) The sequence statistics and solution confirmation of a barley (1 → 3,1 → 4)- β-d-glucan. Carbohydr Res 157:139–156
Wue X, Fry SC (2012) Evolution of mixed-linkage (1 → 3,1 → 4)-β-d-glucan (MLG) and xyloglucan in Equisetum (horsetails) and other monilophytes. Ann Bot 109:873–886
Ford CW, Percival E (1965) The carbohydrates of Phaeodactylum tricornutum. Part I. Preliminary examination of the organism, and characterisation of low molecular weight material and of a glucan. J Chem Soc 1965:7035–7041
Nevo Z, Sharon N (1969) Cell wall of Peridinium westii: a noncellulosic glucan. Biochim Biophys Acta 173:161–175
Popper ZA, Fry SC (2003) Primary cell wall composition of bryophytes and charophytes. Ann Bot London 91:1–12
Burton RA, Fincher GB (2009) (1,3;1,4)-β-d-Glucans in cell walls of the Poaceae, lower plants, and fungi: a tale of two linkages. Mol Plant 2:873–882
Bielecki S, Galas E (1991) Microbial β-glucanase different from cellulases. Crit Rev Biotechnol 10:275–305
Cantwell BA, McConnell DJ (1983) Molecular cloning and expression of Bacillus subtilis β-glucanase gene in Escherichia coli. Gene 23:211–219
Beckmann L, Simon O, Vahjen W (2006) Isolation and identification of mixed linked β-glucan degrading bacteria in the intestine of broiler chickens and partial characterization of respective 1,3–1,4-beta-glucanase activities. J Basic Microbiol 46:175–185
Li S, Sauer WC, Huang SX, Gabert VI (1996) Effect of beta-glucanase supplementation to hulless barley- or wheat-soybean meal diets on the digestibilities of energy, protein, beta-glucans and amino acid in young pigs. J Anim Sci 74:1649–1656
Planas A (2000) Bacterial 1,3–1,4-β-glucanases: structure, function and protein engineering. Biochim Biophys Acta 1543:361–382
Jaskari J, Kontula P, Sitonen A, Jousimies SH, Mattila ST, Poutanen K (1998) Oat β-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains. Appl Microbiol Biotechnol 49:175–181
Kim KH, Kim YO, Ko BS, Youn HJ, Lee DS (2004) Over-expression of the gene (bglBC1) from Bacillus circulans encoding of oligosaccharides from barley β-glucan. Biotechnol Lett 26:1749–1755
Menon V, Divate R, Rao M (2011) Bioethanol production from renewable polymer lichenan using lichenase from an alkalothermophilic Thermomonospora sp and thermotolerant yeast. Fuel Process Technol 92:401–406
Nghiem NP, Hicks KB, Johnston DB, Senske G, Kurantz M, Li M, Shetty J, Konieczny-Janda G (2010) Production of ethanol from winter barley by the EDGE (enhanced dry grind enzymatic) process. Biotechnol Biofuels 3:8
Nghiem NP, Taylor F, Johnston DB, Shetty JK, Hicks KB (2011) Scale-up of ethanol production from winter barley by the EDGE (enhanced dry grind enzymatic) process in fermentors up to 300 l. Appl Biochem Biotechnol 165:870–882
Akita M, Kayatama K, Hatada Y, Ito S, Horikoshi K (2005) A novel β-glucanase gene from Bacillus halodurans C-125. FEMS Microbiol Lett 248:9–15
Furtado GP, Ribeiro LF, Santos CR, Tonoli CC, Souza AR, Oliveira RR, Murakami MT, Ward RJ (2011) Biochemical and structural characterization of a β-1,3–1,4-glucanase from Bacillus subtilis 168. Process Biochem 46:1202–1206
Apiraksakorn J, Nitisinprasert S, Levin RE (2008) Grass degrading β-1,3–1,4-d-glucanases from Bacillus subtilis GN156: purification and characterization of glucanase J1 and pJ2 possessing extremely acidic pI. Appl Biochem Biotechnol 149:53–66
Qiao J, Dong B, Li Y, Zhang B, Cao Y (2009) Cloning of a β-1,3–1,4-glucanase gene from Bacillus subtilis MA139 and its functional expression in Escherichia coli. Appl Biochem Biotechnol 152:334–342
Louw ME, Reid SJ, Watson TG (1993) Characterization, cloning and sequencing of a thermostable endo-(1,3–1,4) β-glucanase-encoding gene from an alkalophilic Bacillus brevis. Appl Microbiol Biotechnol 38:507–513
Teng D, Wang JH, Fan Y, Yang YL, Tian ZG, Lou J, Yang GP, Zhang F (2006) Cloning of β-1,3–1,4-d-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21(DE3). Appl Microbiol Biotechnol 72:705–712
Borriss R, Olsen O, Thomsen KK, Wettstein DV (1989) Hybrid Bacillus endo-(1–3,1–4)-β-glucanases: construction of recombinant genes and molecular properties of the gene products. Carlsberg Res Commun 54:41–54
Tabernero C, Coll PM, Fernández-abalos JM, Perez P, Santamariaía RI (1994) Cloning and DNA sequencing of bgaA, a gene encoding an endo- β-1,3–1,4-glucanase, from an alkalophilic Bacillus strain (N137). Appl Environ Microbiol 60:1213–1220
Kim EY, Kim DG, Kim YR, Choi SY, Kong IS (2009) Isolation and identification of halotolerant Bacillus sp. SJ-10 and characterization of its extracellular protease. Korean J Microbiol 45:193–199
Celestino KRS, Cunha RB, Felix CR (2006) Characterization of a β-glucanase produced by Rhizopus microspores var. microspores, and its potential for application in the brewing industry. BMC Biochem 7:23
Okeke BC, Obi SKC (1995) Saccharification of agro-waste materials by fungal cellulases and hemicellulases. Bioresour Technol 51:23–27
Parrish FW, Perlin AS, Reese ET (1960) Selective enzymolysis of poly-β-d glucan, and the structure of the polymers. Can J Chem 38:2094–2104
Wolf M, Geczi A, Simon O, Borriss R (1995) Genes encoding xylan and β-glucan hydrolyzing enzyme in Bacillus subtilis: characterization, mapping and construction of strain deficient in lichenase, cellulose and xylanase. Microbiology 4:281–290
Meldgaard M, Harthill J (1994) Diffferent effect of N-glycosylation on the thermostability of highly homologous bacterial (1,3–1,4)-beta-glucanase secreted from yeast. Microbiology 140:153–157
Hahn M, Olsen O, Politz O, Borriss R, Heinemann U (1995) Crystal structure and site-directed mutagenesis of Bacillus maceransedo-1,3–1,4-glucanase. J Biol Chem 270:3081–3088
Juncosa M, Pons J, Dot T, Querol E, Planas A (1994) Identification of active site carboxylic residues in Bacillus licheniformis 1,3–1,4-beta-d-glucan 4-glucanohydrolase by site-directed mutagenesis. J Biol Chem 269:14530–14535
Lazaridou A, Biliaderis CG (2007) Molecular aspects of cereal β-glucan functionality: physical properties, technological applications and physiological effects. J Cereal Sci 46:101–118
Chen JL, Tsai LC, Wen TN, Tang JB, Yuan HS, Shyur LF (2001) Directed mutagenesis of specific active site residues on Fibrobacter succinogenes 1,3–1,4-β-glucanase significantly affects catalysis and enzyme structural stability. J Biol Chem 276:17895–17901
Cheng HL, Tsai LC, Lin SS, Yuan HS, Yang NS, Lee SH, Shyur LF (2002) Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3–1,4-beta-d-glucanase significantly affects catalytic activities of the enzyme. Biochemisty 41:8759–8766
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This research was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
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Kim, YR., Kim, EY., Lee, J.M. et al. Characterisation of a novel Bacillus sp. SJ-10 β-1,3–1,4-glucanase isolated from jeotgal, a traditional Korean fermented fish. Bioprocess Biosyst Eng 36, 721–727 (2013). https://doi.org/10.1007/s00449-013-0896-4
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DOI: https://doi.org/10.1007/s00449-013-0896-4