β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30

Baicalin (baicalein 7-O-β-d-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-d-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.     

Identification and expression analysis of a new glycoside hydrolase family 55 exo-β-1,3-glucanase-encoding gene in Volvariella volvacea suggests a role in fruiting body development

The edible straw mushroom Volvariella volvacea is an important crop in South East Asia and is predominantly harvested in the egg stage. Rapid stipe elongation and cap expansion result in a swift transition from the egg to elongation and maturation stage, which are subjected to fast senescence and deterioration. In other mushrooms, β-1,3-glucanases have been associated with degradation (softening) of the cell wall during stipe elongation and senescence. We present a new glycoside hydrolase family 55 (GH55) exo-β-1,3-glucanase gene, exg2, and highly conserved deduced EXG2 protein. The 3D model and presumed catalytic residues of V. volvacea EXG2 are identical to Lentinula edodes EXG2 and Phanerochaete chrysosporium Lam55A, supporting similar enzymatic functions. In addition to previous association to stipe elongation and senescence, our data clearly indicates a role for cap (pileus) expansion. Digital gene expression, quantitative PCR and isobaric tags for relative and absolute quantification analysis showed low exg2 and EXG2 levels in primordia, button, egg and elongation stages and significantly increased levels in the maturation stage. Subsequent relative quantitative PCR analysis designated expression of exg2 to the stipe in the elongation stage and to the pileus and stipe in the maturation stage. EXG2 cell wall softening activity, close correlation of exg2 expression with the principal expanding mushroom tissues and a strong conservation of expression patterns and protein sequences in other mushrooms, make V. volvacea exg2 an important candidate for future studies on mechanisms of fruiting body expansion and senescence causing commodity value loss.     

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.     

Characterization of a glycoside hydrolase family 42 ��-galactosidase from Deinococcus geothermalis

A putative recombinant β-galactosidase from Deinococcus geothermalis was purified as a single 79 kDa band of 42 U activity/mg using His-Trap affinity chromatography. The molecular mass of the native enzyme was a 158 kDa dimer. The catalytic residues E151 and E325 of β-galactosidase from D. geothermalis were conserved in all aligned GH family 42 β-galactosidases, indicating that this enzyme is also a GH family 42 β-galactosidase. Maximal activity of the enzyme was at pH 6.5 and 60°C. It has a unique hydrolytic activity for p-nitrophenyl(pNP)-β-D-galactopyranoside (k (cat)/K (m) = 69 s(-1) mM(-1)), pNP-β-D-fucopyranoside (13), oNP-β-D-galactopyranoside (9.5), oNP-β-D-fucopyranoside (2.6), lactose (0.97), and pNP-α-L-arabinopyranoside (0.78), whereas no activity, or less than 2% of the pNP-β-D-galactopyranoside activity, for other pNP- and oNP-glycosides.     

Gene cloning and expression of a new acidic family 7 endo-β-1,3-1,4-glucanase from the acidophilic fungus Bispora sp. MEY-1

Most reported microbial β-1,3-1,4-glucanases belong to the glycoside hydrolase family 16. Here, we report a new acidic family 7 endo-β-1,3-1,4-glucanase (Bgl7A) from the acidophilic fungus Bispora sp. MEY-1. The cDNA of Bgl7A was isolated and over-expressed in Pichia pastoris, with a yield of about 1,000 U ml^–1 in a 3.7-l fermentor. The purified recombinant Bgl7A had three activity peaks at pH 1.5, 3.5, and 5.0 (maximum), respectively, and a temperature optimum at 60°C. The enzyme was stable at pH 1.0–8.0 and highly resistant to both pepsin and trypsin. Belonging to the group of non-specific endoglucanase, Bgl7A can hydrolyze not only β-glucan and cellulose but also laminarin and oat spelt xylan. The specific activity of Bgl7A against barley β-glucan and lichenan (4,040 and 2,740 U mg^–1) was higher than toward carboxymethyl cellulose sodium (395 U mg^–1), which was different from other family 7 endo-β-glucanases.     

Biochemical characterization of thermostable β-1,4-mannanase belonging to the glycoside hydrolase family 134 from Aspergillus oryzae

Applied Microbiology and Biotechnology - A β-1,4-mannanase, termed AoMan134A, that belongs to the GH 134 family was identified in the filamentous fungus Aspergillus oryzae. Recombinant...     

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.     

Cloning, expression, and characterization of a glycoside hydrolase family 50 β-agarase from a marine Agarivorans isolate

The gene for a thermostable β-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to β-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant β-agarase was purified to homogeneity. It had maximal activity at 40°C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl₂. The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.     

Evolution of the βGRP/GNBP/β-1,3-glucanase family of insects

The βGRP/GNBP/β-1,3-glucanase protein family of insects includes several proteins involved in innate immune recognition, such as the β-glucan recognition proteins of Lepidoptera and the Gram-negative bacteria-binding proteins of Drosophila. A phylogenetic analysis supported the existence of two distinct subfamilies, designated the pattern recognition receptor (PRR) and glucanase subfamilies, which originated by gene duplication prior to the origin of the Holometabola. In the C-terminal region (CTR) shared by both subfamilies, the PRR subfamily has evolved significantly more rapidly at the amino acid sequence level than has the glucanase subfamily, implying a relative lack of constraint on the amino acid sequence of this region in the PRR subfamily. PRR subfamily members also include an N-terminal region (NTR), involved in carbohydrate recognition, which is not shared by glucanase subfamily members. In comparisons between paralogous PRR subfamily members, there were no conserved amino acid residues in the NTR. However, when pairs of putatively orthologous PRR subfamily members were compared, the NTR was most often as conserved as the CTR or more so. This pattern suggests that the NTR may be important in functions specific to the different paralogs, while amino acid sequence changes in the NTR may have been important in functional differentiation among paralogs, specifically with regard to the types of carbohydrates that they recognize.     

Distribution and Properties of Endo-β-1,4-glucanase from a Lower Termite,Coptotermes formosanus (Shiraki)

A multi-enzyme distribution of endo-β-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract.

EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and K _m were 48 kDa, 6.0, 50°C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-β-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-β-1,4-glucanases of family 9.     

Purification and Some Properties of an Endo-β,1,6-glucanase from Neurospora crassa

An endo-β-1,6-glucanase (E.C. 3.2.1.75) was purified from the culture filtrate of Neurospora crassa IFO-6O68 by chromatographies on CM-cellulofine, Con-A Sepharose 4B, and Sepharose Cl-6B followed by preparative affinity gel electrophoresis. The purified enzyme had an apparent molecular weight of 47,000. The pH and temperature optima for the activity were 5.0 and 50°C. The enzyme acted on β-1,6-glucan (Pustulan) and yielded a series of gentio-oligosaccharides with endo- type action, and finally, glucose and gentiobiose were produced. The enzyme was also able to act on N. crassa cell wall β-glucan, and a small amount of hydrolysis fragments were liberated without apparent change of the cell wall glucan molecules.     

Specificity of Endo-β-1,3-glucanase G A from Cellulomonas cellulans towards Structurally Diversified Acceptor Molecules in Transglycosylation Rreaction

The synthetic properties of homogeneous endo- &#103 -1,3-glucanase G A from Cellulomonas cellulans were studied. Thirty-one synthetic and natural &#102 - and &#103 -glycosides were examined as acceptor molecules in transglycosylation reactions conducted in an aqueous-organic solvent environment with &#103 -1,3-glucan as a polymeric donor. Seventeen acceptors underwent significant glycosylation. The best acceptors for glucanase G A were &#102 - or &#103 -glycosides that, in the favoured chair conformation of the pyranose ring, had all the equatorial substituents. Glycosides having an axial 4-hydroxyl or 2-hydroxyl group in the pyranose ring represent a poor class of acceptors.     

Amino Acid Sequence and Stereoselective Hydrolytic Reaction of an Endo-1,4-β-glucanase from a Bacillus Strain

Bacillus sp. KSM-522 produces three different extracellular endo-l,4-β-glucanases [EGs; Okoshi et al., Agric. Biol. Chem., 54, 83–89 (1990)]. Here, we report the molecular cloning and sequencing of the gene for the fourth EG (EG-IV) of the organism and the mechanism of its hydrolytic reaction. The structural gene contained an open reading frame of 1911 bp, corresponding to 636 amino acids, the amino acid sequence of which was very close to that of an EG of Clostridium cellulovorans, belonging to the cellulase family E2. The molecular mass of the extracellular mature enzyme (Ser²⁶ through Lys⁶³⁶) was calculated to be 69,076 Da, a value close to the 69.2 kDa measured for the recombinant EG-IV expressed in Bacillus subtilis. The optimum pH and temperature for activity of the recombinant enzyme were pH 8.0 and 50°C, respectively. By ¹H-NMR spectroscopy, we demonstrated that the hydrolysis of p-nitrophenyl β-d-cellotrioside by EG-IV proceeded with inversion of the anomeric configuration.     

Characterization of a Thermostable Endo-β-1,4-D-galactanase from the Hyperthermophile Thermotoga maritima

A putative endo-β-1,4-D-galactanase gene of Thermotoga maritima was cloned and overexpressed in Escherichia coli. The recombinant enzyme hydrolyzed pectic galactans and produced D-galactose, β-1,4-D-galactobiose, β-1,4-D-galactotriose, and β-1,4-D-galactotetraose. The enzyme displayed optimum activity at 90 °C and pH 7.0. It was slowly inactivated above pH 8.0 and below pH 5.0 and stable at temperatures up to 80 °C.     

Cloning and characterization of a novel cellobiase gene, cba3, encoding the first known β-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea

A novel cellobiase gene, designated cba3, was cloned from Cellulomonas biazotea. Although cellobiase genes of C. biazotea were previously cloned, published and/or patented, they encoded β-glucosidases all belonging to glycoside hydrolase family 3 (GH3); the new Cba3 cellobiase was identified to be a glycoside hydrolase family 1 (GH1) member, which represents the first discovered GH1 β-glucosidase of C. biazotea. Escherichia coli transformants expressing recombinant Cba3 were shown to grow readily in minimal media using cellobiose as the sole carbon source, supporting the conclusion that Cba3 is a genuine cellobiase. The full-length cba3 gene was revealed by sequencing to be 1344 bp long. Cba3 deletants lacking either the N-terminal 10 amino acids or the C-terminal 10 residues were found to be biologically inactive, supporting the importance of both ends in catalysis. Like other GH1 β-glucosidases, Cba3 was shown to contain the highly conserved NEP and ENG motifs, which are crucial for enzymatic activity. Despite lacking a classical N-terminal signal peptide, Cba3 was demonstrated to be a secretory protein. The findings that Cba3 is a cellobiase, and that it was expressed well as an extracellular protein in E. coli, support the potential of Cba3 for use with other cellulases in the hydrolysis of cellulosic biomass.     

Over-production of a glycoside hydrolase family 50 β-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product

The gene for β-agarase of an Agarivorans sp. JA-1 was expressed in Bacillus subtilis strain DB104 for efficient and economical mass-production of the enzyme. We isolated 360 mg protein with a specific activity of 201 U/mg from the culture broth. The efficiency of production was approximately 130-fold higher than that in E. coli. The enzyme produced neoagarohexaose, neoagarotetraose and neoagarobiose from agar. Neoagarooligosaccharides produced by the enzyme had a whitening effect and inhibited tyrosinase activity in the murine melanoma cell line, B16F10. Neoagarooligosaccharides were not cytotoxic to B16F10 or normal cells. β-Agarase could therefore be a good whitening, cosmetic additive.     

NMR characterization of a 264-residue hyperthermostable endo-β-1,3-glucanase

Insight into the hyperthermostable endo-β-1,3-glucanase pfLamA from Pyrococcus furiosus is obtained by using NMR spectroscopy. pfLamA functions optimally at 104 °C and recently the X-ray structure of pfLamA has been obtained at 20 °C, a temperature at which the enzyme is inactive. In this study, near-complete (>99%) NMR assignments are presented of chemical shifts of pfLamA in presence and absence of calcium at 62 °C, a temperature at which the enzyme is biologically active. The protein contains calcium and the effects of calcium on the protein are assessed. Calcium binding results in relatively small chemical shift changes in a region distant from the active site of pfLamA and thus causes only minor conformational modifications. Removal of calcium does not significantly alter the denaturation temperature of pfLamA, implying that calcium does not stabilize the enzyme against global unfolding. The data obtained form the basis for elucidation of the molecular origins involved in conformational stability and biological activity of hyperthermophilic endo-β-1,3-glucanases at extreme temperatures.