Characterization of a New 1,3-1,4-β-Glucanase Gene from Bacillus tequilensis CGX5-1

1,3-1,4-β-Glucanase received great interest due to its application in brewing and feed industries. Application of 1,3-1,4-β-glucanase in brewing industry helps make up for the defect that plant-derived β-glucanases are heat-sensitive. A new strain, CGX5-1, exhibited remarkable 1,3-1,4-β-glucanase, was isolated from Asian giant hornet nest and identified Bacillus tequilensis. Moreover, a new 1,3-1,4-β-glucanase gene from B. tequilensis was cloned and measured to be 720 bp encoding 239 amino acids, with a predicted molecular weight of 26.9 kDa. After expressed in Escherichia coli BL21, active recombinant enzyme of 24 kDa was detected in the supernatant of cell culture, with the activity of 2,978.2 U/mL. The new enzyme was stable in the pH 5.0–7.5 with the highest activity measured at pH 6.0. Moreover, it is thermostable within 45 to 60 °C. The property of the new recombinant enzyme makes this enzyme a broad prospect in brewing industry. Moreover, this is the first report on 1,3-1,4-β-glucanase produced by B. tequilensis.     

Production, Purification, Immobilization, and Characterization of a Thermostable β-Galactosidase from Aspergillus alliaceus

A fungal strain isolated from rotten banana and identified as Aspergillus alliaceus was found capable of producing thermostable extracellular ??-galactosidase enzyme. Optimum cultural conditions for ??-galactosidase production by A. alliaceus were as follows: pH?4.5; temperature, 30?°C; inoculum age, 25?h; and fermentation time, 144?h. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 45?°C, 20?min, and 7.2, respectively, for crude and partially purified enzyme. For immobilized enzyme?Csubstrate reaction, these three variable, temperature, time, and pH were optimized at 50?°C, 40?min, and 7.2, respectively. Glucose was found to inhibit the enzyme activity. The K _m values of partially purified and immobilized enzymes were 170 and 210?mM, respectively. Immobilized enzyme retained 43?% of the ??-galactosidase activity of partially purified enzyme. There was no significant loss of activity on storage of immobilized beads at 4?°C for 28?days. Immobilized enzyme retained 90?% of the initial activity after being used four times.     

Isolation, Purification, and Characterization of Two Thermostable Endo-1,4-��-d-glucanase Forms from Opuntia vulgaris

Four endoglucanase temperature isoforms (T (30), T (50), T (70), and T (90)) were identified and purified from the cladodes of the xerophytic plant Opuntia vulgaris. These isoforms exhibited optimum catalytic activity at 30 °C, 50 °C, 70 °C, and 90 °C and yielded an apparent molecular mass of 150, 20, 74, and 45 kDa, respectively, on gel filtration chromatography. These isoforms were purified 24-, 25-, 29-, and 27-fold with a yield of 15%, 12%, 17%, and 19% and having a specific activity of 120, 125, 144, and 136 U/mg, respectively. The thermostable T (70) and T (90) isoforms exhibited optimum activity at pH 4.5 and 7 and also yielded a molecular weight of 66 and 36 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The T (70) had a K (m) of 43 mM and a V (max) of 12.5 μmol min(-1) μg(-1) of protein, and the T (90) isoform had a K (m) of 40 mM, with an apparent V (max) of 10 μmol min(-1) μg(-1) of protein. Western blot, immunodiffusion, and in vitro inhibition assays established the reactivity of the T (90) isoform with polyclonal anti-T (90) antibody raised in rabbit. Cross-reactivity of this antibody with the T (70) endoglucanase isoform was also noted.     

Isolation, Purification, and Certain Properties of β-1,3-Glucanase from the Fungus Verticillium dahliae

Two homogeneous molecular forms of -1,3-glucanase are isolated and purified from the culture fluid ofVerticillium dahliaeKleb. Isolation and purification methods including ion exchange on DEAE-TSK 650M with subsequent gel filtration on TSK HW 50, preparative electrophoresis, and adsorption chromatography on HA-Ultrogel were compared. Certain kinetic properties of the molecular forms of -1,3-glucanase for laminarin were studied. The effect of amino acids on the activity of both forms of -1,3-glucanase was investigated     

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

Purification of β-1,3-1,4-glucanase from the cell-free culture fluid of Bacillus subtilis GN156 by affinity chromatography of epoxy-activated sepharose 6B and ultrafiltration technique resulted in homogeneous J1 and partially purified pJ2 enzymes. The molecular weight and pI of J1 were 25 kDa and 3.5, respectively, while those for J2 were 90 kDa and 3.6, respectively. Both β-1,3-1,4-glucanase J1 and pJ2 had optimum pH values of 6–6.5 and an optimum temperature of 60°C. Both enzymes were not inhibited by Li²⁺ but were inhibited significantly by Ca²⁺, Cu²⁺, Mn²⁺ and Zn²⁺. However, J1 was slightly inhibited by Fe²⁺, while pJ2 was inhibited by Mg²⁺ as well. They were highly specific to only barley β-glucan. K _m and V _max values of J1 were 1.53 mg/ml and 8,511 μU/ml.min, respectively, while those for pJ2 were 4.36 mg/ml and 7,397 μU/ml.min, respectively. Degradation of barley β-1, 3-1,4-glucan resulted in four different oligosaccharides with 1,3 linkages triose, tetrose, pentose and a high molecular weight (HMW) with 1,3 linkage estimated from their mobilities. The quantitative degradation by the crude enzyme after of incubation yielded in descending order: triose, pentose and tetrose, while that of J1 in descending order yielded: pentose, triose and tetrose. The pJ2 showed low activity yielding a degradation pattern in descending order: pentose, triose, tetraose and a HMW polysaccharide.     

Cloning, Expression, and Characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris

The β-mannanase gene (1,029 nucleotide) from Bacillus subtilis MAFIC-S11, encoding a polypeptide of 342 amino acids, was cloned and expressed in Pichia pastoris. To increase its expression, the β-mannanase gene was optimized for codon usage (mannS) and fused downstream to a sequence-encoding modified α-factor signal peptide. The expression level was improved by 2-fold. This recombinant enzyme (mannS) showed its highest activity of 24,600 U/mL after 144-h fermentation. The optimal temperature and pH of mannS were 50 °C and 6.0, respectively, and its specific activity was 3,706 U/mg. The kinetic parameters V _max and K _m were determined as 20,000 U/mg and 8 mg/mL, respectively, representing the highest ever expression level of β-mannanase reported in P. pastoris. In addition, the enzyme exhibited much higher binding activity to chitin, chitosan, Avicel, and mannan. The superior catalytic properties of mannS suggested great potential as an effective additive in animal feed industry.     

Expression and Characterization of a GH39 β-Xylosidase II from Caulobacter crescentus

In the present work, the gene xynB2, encoding a ??-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5?%. CcXynB2 appeared as a single band of 60?kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 ??-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55?°C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5?C7.5 for 24?h and thermotolerance up to 50?°C. The K _M and V _Max values were 9.3?±?0.45?mM and 402?±?19???mol?min^?1 for ??-nitrophenyl-??-d-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family ??-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.     

Production, Purification, and Characterization of a β-Glucosidase of Penicillium funiculosum NCL1

Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of ??-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular ??-glucosidase production was studied in submerged fermentation. At 30?°C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of ??-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major ??-glucosidase (Bgl3A) with the apparent molecular weight of ~120?kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60?°C, and exhibited a half-life of 1?h at 60?°C. K _m of Bgl3A was found to be 0.057?mM with p-nitrophenyl ??-d-glucoside and V _max was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K _i of 1.5?mM. Bgl3A readily hydrolyzed glucosides with ??-linkage. Bgl3A activity was enhanced (156%) by Zn²⁺ and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.     

Prokaryotic Expression, Purification and Characterization of Aspergillus sulphureus β-Mannanase and Site-Directed Mutagenesis of the Catalytic Residues

Wild type (WT) DNA sequence, which encoded a mature β-mannanase of Aspergillus sulphureus, composed of 1,152 nucleotides (nt), was amplified from pUCm-T-mann by polymerase chain reaction (PCR). Based on this DNA fragment, mutants designated as E²⁰⁶G and E³¹⁴G were constructed by overextension PCR (OE-PCR). Glutamic acids of the 206th and 314th sites in the amino acid sequence of β-mannanase were separately replaced by glycine in these two mutants. The WT and mutant genes were ligated into prokaryotic vector pET-28a (+) and transformed into the Escherichia coli BL21 strain, respectively. The recombinant enzyme proteins were expressed by IPTG induction and detected by Western blot. The recombinant proteins purified with Ni-NTA column were dialyzed to correctly refold. The WT recombinant β-mannanase showed optimal activity at 50 °C and pH 2.4. The kinetic parameters of K _m and V _max for purified β-mannanase were 1.38 mg/ml and 72.99 U/mg, respectively. However, the mutant proteins did not show any activity. It was demonstrated that E²⁰⁶ and E³¹⁴ were the catalytic residues of β-mannanase.     

Purification and Characterization of β-Glucosidase from a Newly Isolated Strain Tolypocladium cylindrosporum Syzx4

A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer(ITS) rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27...     

Purification and Characterisation of a β-1,4-Xylanase from Remersonia thermophila CBS 540.69 and Its Application in Bread Making

This work reports the first investigation of Remersonia thermophila hemicellulosic hydrolytic enzyme production, with subsequent purification of an extracellular endo-β-1,4-xylanase (RtXyl) and its application in bread making. The research describes RtXyl purification from sorghum-induced submerged liquid cultures of this moderately thermophilic, aerobic, ascomycete fungus. The purified enzyme is a single subunit protein with a molecular mass of 42 kDa and exhibits glycosyl hydrolase family-10-like activity over a broad pH and temperature range. Optimal activity was measured at pH 6.0 and 65 °C respectively, which is suitable for bread making applications. Substrate specificity studies revealed that RtXyl is purely xylanolytic with no side-activities against other plant polysaccharides. The RtXyl catalytic efficiency (K _cat/K _m) was highest with oats spelt xylan (810.90 mg mL^?1 s^?1), wheat arabinoxylan (809.52 mg mL^?1 s^?1) and beechwood xylan (417.40 mg mL^?1 s^?1) with less efficiency towards insoluble oats spelt xylan (236.40 mg mL^?1 s^?1). Hydrolysis products analysed by thin layer chromatography yielded a range of xylosaccharides, predominantly xylotriose and xylobiose. RtXyl application in a basic wheat bread recipe at low dosages (0.297 XU/g) showed its suitability to increase loaf volume by 8.0 % compared with the control bread. RtXyl increased loaf softness by 19.6 % while reducing bread staling by 20.4 % up to 4 days of storage.     

Purification and Characterization of a Glycoside Hydrolase Family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

The gene encoding a glycoside hydrolase family 43 β-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis–Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-β-d-xylopyranose (4NPX) and p-nitrophenyl-α-l-arabinofuranose (4NPA), and it was found that the ratio k _cat/K _m 4NPA/k _cat/K _m 4NPX was ～7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k _cat/K _m muA/k _cat/K _m muX was ～5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K _i of 6.8?±?0.62 mM and xylose K _i of 76?±?8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 °C, with a t _1/2 of 35 min at 57.5 °C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.     

Purification and Characterization of a Low Molecular Weight of β-Mannanase from Penicillium occitanis Pol6

The highest β-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified β-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 °C and pH 4.0. It was stable for 30 min at 70 °C and has a broad pH stability (2.0–12.0). ManIII showed K _m, V _max, and K _cat values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s⁻¹ with locust bean gum as substrate, respectively. It was inhibited by mannose with a K _I of 0.610⁻³ mg/ml. ManIII was activated by CuSO₄ and CaCl₂ (2.5 mM). However, in presence of 2.5 mM Co²⁺, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.     

Expression,Purification, and Characterization of Endo-β-N-Acetylglucosaminidase H Using Baculovirus-Mediated Silkworm Protein Expression System

Endo-β-N-acetylglucosaminidase (Endo H) from Streptomyces plicatus hydrolyzes the core di-GlcNAc units of asparagine-linked oligosaccharides and is regarded as an important tool for glycobiology research. In the present study, we established a large-scale system to produce secreted Endo H using a silkworm-baculovirus expression system (silkworm-BES). The recombinant Endo H purified from silkworm hemolymph had activity comparable to that from recombinant Escherichia coli. As well as its well-characterized substrate RNase B, the Endo H from silkworm-BES was able to deglycosylate the high-mannose glycoproteins from silkworm hemolymph. Interestingly, the secretion amount of recombinant Endo H was significantly varied among the different silkworm strains, which could provide valuable information for larger-scale protein productions from silkworm-BES.     

Isolation, Purification, and Properties of α-Amylase from Bacillus subtilis-7A

Cultivation ofBacillus subtilis-7A on waste from alcohol production yielded an active extracellular enzyme -amylase with MW 75 kDa. The enzyme was isolated from the culture medium by 60% saturated ammonium sulfate and purified until homogeneous by gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. The optimum temperatures for the complex and purified enzyme are 30 and 50°C, respectively. The optimum activity for both preparations occurred at pH 6.5. The substrate specificity of the isolated preparations was studied.     

Purification, Sequencing, and Biochemical Characterization of a Novel Calcium-Independent α-Amylase AmyTVE from Thermoactinomyces vulgaris

α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS–PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable α-amylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl₂, and CaCl₂, and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl₂, suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function.     

Characterization of β-Galactosidase Isoforms from Bacillus circulans and Their Contribution to GOS Production

A β-galactosidase preparation from Bacillus circulans consists of four isoforms called β-gal-A, β-gal-B, β-gal-C, and β-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms may be relevant for GOS production, which is typically done at high substrate concentrations. At initial lactose concentrations in between 0.44 % and 0.68 % (w/w), β-gal-A showed the least oligosaccharide formation, followed by β-gal-B and β-gal-C; most oligosaccharides were formed by β-gal-D. The differences in behavior were confirmed by studying the thermodynamics of lactose conversion with isothermal titration calorimetry since especially β-gal-A showed a different profile than the other isoforms. Also during the conversion of allolactose and 4-galactosyllactose at 0.44 % and 0.61 % (w/w), respectively, β-gal-A and β-gal-D showed clear differences. In contrast to above findings, the selectivity of the isoforms did hardly differ at an initial lactose concentration of 30 % (w/w), except for a slightly higher production of galactose with β-gal-A. These differences were hypothesized to be related to the different accessibility of the active sites of the isoforms for different-sized reactants. The initial GOS formation rates of the isoforms indicate that β-gal-A and β-gal-B are the best isoforms for GOS production at high lactose concentrations.     

Production and Characterization of β-Glucanase Secreted by the Yeast Kluyveromyces marxianus

An extracellular β-glucanase secreted by Kluyveromyces marxianus was identified for the first time. The optimal conditions for the production of this enzyme were evaluated by response surface methodology. The optimal conditions to produce β-glucanase were a glucose concentration of 4 % (w/v), a pH of 5.5, and an incubation temperature of 35 °C. Response surface methodology was also used to determine the pH and temperature required for the optimal enzymatic activity. The highest enzyme activity was obtained at a pH of 5.5 and a temperature of 55 °C. Furthermore, the enzyme was partially purified and sequenced, and its specificity for different substrates was evaluated. The results suggest that the enzyme is an endo-β-1,3(4)-glucanase. After optimizing the conditions for β-glucanase production, the culture supernatant was found to be effective in digesting the cell wall of the yeast Saccharomyces cerevisiae, showing the great potential of β-glucanase in the biotechnological production of soluble β-glucan.     

Heterologous Expression of Aspergillus niger β-d-Xylosidase (XlnD): Characterization on Lignocellulosic Substrates

The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T _max was determined using differential scanning microcalorimetry (DSC) to be 78.2 °C; the K _m and k _cat were found to be 255 μM and 13.7 s⁻¹, respectively, using pNP-β-d-xylopyranoside as substrate. End-product inhibition by d-xylose was also verified and shown to be competitive; the K _i for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.     

A Novel α-Amylase from Bacillus mojavensis A21: Purification and Biochemical Characterization

α-Amylase from Bacillus mojavensis A21 (BMA.2) was purified to homogeneity by ultrafiltration, Sephadex G-75 gel filtration and Sepharose mono Q anion exchange chromatography, with a 15.3-fold increase in specific activity and 11% recovery. The molecular weight of the BMA.2 enzyme was estimated to be 58 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. The optimum temperature and pH were 80?°C and 6.5, respectively. BMA.2 belonged to the EDTA-sensitive α-amylase, but its activity was not stimulated by the presence of Ca²⁺ ions. The major end-products of starch hydrolysis were maltohexaose, maltopentaose and maltotriose. The N-terminal amino acid sequence of the first ten amino acids of the purified α-amylase was ASVNGTLMQY. Compared to sequences of other amylases, the ten amino acid sequence contains Val at position 3, while amylases from Bacillus licheniformis NH1 and Bacillus sp. SG-1 have Leu and Thr at position 3, respectively.