β‐glucuronidase of family‐2 glycosyl hydrolase: A missing member in plants

Glycosyl hydrolases hydrolyze the glycosidic bond in carbohydrates or between a carbohydrate and a non‐carbohydrate moiety. β‐glucuronidase (GUS) is classified under two glycosyl hydrolase families (2 and 79) and the family‐2 β‐glucuronidase is reported in a wide range of organisms, but not in plants. The family‐79 endo-β-glucuronidase (heparanase) is reported in microorganisms, vertebrates and plants. The E. coli family‐2 β‐glucuronidase (uidA) had been successfully devised as a reporter gene in plant transformation on the basis that plants do not have homologous GUS activity. On the contrary, histochemical staining with X‐Gluc was reported in wild type (non-transgenic) plants. Data shows that, family‐2 β‐glucuronidase homologous sequence is not found in plants. Further, β‐glucuronidases of family‐2 and 79 lack appreciable sequence similarity. However, the catalytic site residues, glutamic acid and tyrosine of the family‐2 β‐glucuronidase are found to be conserved in family‐79 β‐glucuronidase of plants. This led to propose that the GUS staining reported in wild type plants is largely because of the broad substrate specificity of family‐79 β-glucuronidase on X‐Gluc and not due to the family‐2 β‐glucuronidase, as the latter has been found to be missing in plants.     

A novel nucleoside hydrolase from Lactobacillus buchneri LBK78 catalyzing hydrolysis of 2′-O-methylribonucleosides

2′-O-Methylribonucleosides (2′-OMe-NRs) are promising raw materials for nucleic acid drugs because of their high thermal stability and nuclease tolerance. In the course of microbial screening for metabolic activity toward 2′-OMe-NRs, Lactobacillus buchneri LBK78 was found to decompose 2′-O-methyluridine (2′-OMe-UR). The enzyme responsible was partially purified from L. buchneri LBK78 cells by a four-step purification procedure, and identified as a novel nucleoside hydrolase. This enzyme, LbNH, belongs to the nucleoside hydrolase superfamily, and formed a homotetrameric structure composed of subunits with a molecular mass around 34 kDa. LbNH hydrolyzed 2′-OMe-UR to 2′-O-methylribose and uracil, and the kinetic constants were K_m of 0.040 mM, k_cat of 0.49 s^?1, and k_cat/K_m of 12 mM^?1 s^?1. In a substrate specificity analysis, LbNH preferred ribonucleosides and 2′-OMe-NRs as its hydrolytic substrates, but reacted weakly with 2′-deoxyribonucleosides. In a phylogenetic analysis, LbNH showed a close relationship with purine-specific nucleoside hydrolases from trypanosomes.     

A Shinella β-N-acetylglucosaminidase of glycoside hydrolase family 20 displays novel biochemical and molecular characteristics

β-N-Acetylglucosaminidases (GlcNAcases) are important for many biological functions and industrial applications. In this study, a glycoside hydrolase family 20 GlcNAcase from Shinella sp. JB10 was expressed in Escherichia coli BL21 (DE3). Compared to many GlcNAcases, the purified recombinant enzyme (rJB10Nag) exhibited a higher specificity activity (538.8 µmol min⁻¹ mg⁻¹) or V _max (1030.0 ± 82.1 µmol min⁻¹ mg⁻¹) toward p-nitrophenyl β-N-acetylglucosaminide and N,N′-diacetylchitobiose (specificity activity of 35.4 µmol min⁻¹ mg⁻¹) and a higher N-acetylglucosaminide tolerance (approximately 50% activity in 70.0 mM N-acetylglucosaminide). The degree of synergy on enzymatic degradation of chitin by a commercial chitinase and rJB10Nag was as high as 2.35. The enzyme was tolerant to most salts, especially 3.0–15.0% (w/v) NaCl and KCl. These biochemical characteristics make the JB10 GlcNAcase a candidate for use in many potential applications, including processing marine materials and the bioconversion of chitin waste. Furthermore, the enzyme has the highest proportions of alanine (16.5%), glycine (10.5%), and random coils (48.8%) with the lowest proportion of α-helices (24.9%) among experimentally characterized GH 20 GlcNAcases from other organisms.

     

Chalcone oxides—potent selective inhibitors of cytosolic epoxide hydrolase

The systematic screening of over 150 compounds for inhibitory activity on mammalian cytosolic epoxide hydrolase led to identification of chalcone oxide (trans-1-benzoyl-2-phenyloxirane) as an optimal inhibitory structure. Important structural features for inhibition include two hydrophobic moieties preferably orientating in a trans manner from an electrophilic center such as an activated olefin or epoxide, with the epoxide giving maximal activity. Synthesis of chalcone oxide derivatives bearing a single p-substituent on either phenyl ring has led to very potent inhibitors of the enzyme, the best being 4-phenylchalcone oxide (50% inhibition at 6.4 × 10^?8m). Multiple factorial analysis on the inhibition data for the two series of chalcone oxides prepared (phenyl or benzoyl substituted) revealed both the essentialness of hydrophobic interactions and the apparent nonequivalence of the two hydrophobic sites involved in the inhibitory process. Steric factors were considerably less crucial while electronic effects were unimportant in the compounds examined. The chalcone oxides were either inactive or only weak inhibitors of the other major epoxide-metabolizing enzymes in mouse liver, cytosolic glutathione S-transferase, and microsomal epoxide hydrolase. The nature of the inhibition of cytosolic epoxide hydrolase by chalcone oxides was further investigated through steady-state kinetic analysis and the use of amino acid modifiers. Chalcone oxides give a slowly reversible mixed-noncompetitive inhibition. They may interact covalently with a cysteine residue possibly essential to the catalytic action of cytosolic epoxide hydrolase, and may indeed be alternative substrates with very low turnover. The cytosolic and microsomal epoxide hydrolases can be clearly distinguished by these inhibitors, further indicating different catalytic mechanisms.     

Molecular identification of β-citrylglutamate hydrolase as glutamate carboxypeptidase 3

β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.     

Enzymatic synthesis of β-xylosyl-oligosaccharides by transxylosylation using two β-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4

Two β-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k_cat/K_m) than BxlB towards para-nitrophenyl β-d-xylopyranoside (pNPX) and β-1,4-xylo-oligosaccharides (degree of polymerisation 2–6). For both enzymes k_cat/K_m decreased with increasing β-1,4-xylo-oligosaccharide chain length. Using pNPX as donor with 9 monosaccharides, 7 disaccharides and two sugar alcohols as acceptors 18 different β-xylosyl-oligosaccharides were synthesised in 2–36% (BxlA) and 6–66% (BxlB) yields by transxylosylation. BxlA utilised the monosaccharides d-mannose, d-lyxose, d-talose, d-xylose, d-arabinose, l-fucose, d-glucose, d-galactose and d-fructose as acceptors, whereas BxlB used the same except for d-lyxose, d-arabinose and l-fucose. BxlB transxylosylated the disaccharides xylobiose, lactulose, sucrose, lactose and turanose in upto 35% yield, while BxlA gave inferior yields on these acceptors. The regioselectivity was acceptor dependent and primarily involved β-1,4 or 1,6 product linkage formation although minor products with different linkages were also obtained. Five of the 18 transxylosylation products obtained from d-lyxose, d-galactose, turanose and sucrose (two products) as acceptors were novel xylosyl-oligosaccharides, β-d-Xylp-(1→4)-d-Lyxp, β-d-Xylp-(1→6)-d-Galp, β-d-Xylp-(1→4)-α-d-Glcp-(1→3)-β-d-Fruf, β-d-Xylp-(1→4)-α-d-Glcp-(1→2)-β-d-Fruf, and β-d-Xylp-(1→6)-β-d-Fruf-(2→1)-α-d-Glcp, as structure-determined by 2D NMR, indicating that GH3 β-xylosidases are able to transxylosylate a larger variety of carbohydrate acceptors than earlier reported. Furthermore, transxylosylation of certain acceptors resulted in mixtures. Some of these products are also novel, but the structures of the individual products could not be determined.     

Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism β-xylosidases

We obtained Cx1 from a commercial supplier, whose catalog listed it as a β-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein sequencing indicates Cx1 is similar to but not identical to β-xylosidases of GH52, including Q09LZ0, that have retaining mechanisms. Q09LZ0 β-xylosidase had been characterized biochemically in kinetic reactions that contained Tris. We overproduced Q09LZ0 and demonstrated that Tris is a competitive inhibitor of the β-xylosidase. Also, the previous work used grossly incorrect extinction coefficients for product 4-nitrophenol. We redetermined kinetic parameters using reactions that omitted Tris and using correct extinction coefficients for 4-nitrophenol. Cx1 and Q09LZ0 β-xylosidases were thus shown to possess similar kinetic properties when acting on 4-nitrophenyl-β-d-xylopyranoside and xylobiose. k_cat pH profiles of Cx1 and Q09LZ0 acting on 4-nitrophenyl-β-d-xylopyranoside and xylobiose have patterns containing two rate increases with increasing acidity, not reported before for glycoside hydrolases. The dexylosylation step of 4-nitrophenyl-β-d-xylopyranoside hydrolysis mediated by Q09LZ0 is not rate determining for k_cat^4NPX.     

Structure–activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase

We explored both structure–activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors.     

Crystal structures of glycoside hydrolase family 51 α-L-arabinofuranosidase from Thermotoga maritima

α-L-Arabinofuranosidase from the hyperthermophilic bacterium Thermotoga maritima (Tm-AFase) is an extremely thermophilic enzyme belonging to glycoside hydrolase family 51. It can catalyze the transglycosylation of a novel glycosyl donor, 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT)-β-D-xylopyranoside. In this study we determined the crystal structures of Tm-AFase in substrate-free and complex forms with arabinose and xylose at 1.8-2.3 ? resolution to determine the architecture of the substrate binding pocket. Subsite -1 of Tm-AFase is similar to that of α-L-arabinofuranosidase from Geobacillus stearothermophilus, but the substrate binding pocket of Tm-AFase is narrower and more hydrophobic. Possible substrate binding modes were investigated by automated docking analysis.     

A novel ubiquitin carboxy1 terminal hydrolase is involved in toad oocyte maturation

P28，a 28kD protein from toad (Bufo bufo gargarizans)oocytes,was identified by using P13^suc1-agarose affinity chromatography.Sequence homology analysis of the full-length cDNA of P28(Gene Bank accession number:AF 314091)indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70%positives to encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1,and contains homological functional domains of UCH family.Anti-p28 monoclonal antibody,on injecting into the oocytes,could inhibit the progesterone-induced resumption of meiotic division in a dose-dependent manner.The recombinant protein P28 showed similar SDS/PAGE behaviors to the native one,and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases(UCHs).The results in this paper reveal that a novel protein,p28 ,exists in the toad oocytes,is a UCH Ll homolog,was engaged in the process of progesterone-induced oocyte maturation possibly through an involvement in protein turnover and degradation.     

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.     

Cholesterol ester hydrolase inhibitors reduce the production of synaptotoxic amyloid-β oligomers

The production of amyloid-β (Aβ) is the key factor driving pathogenesis in Alzheimer's disease (AD). Increasing concentrations of Aβ within the brain cause synapse degeneration and the dementia that is characteristic of AD. Here the factors that affect the release of disease-relevant forms Aβ were studied in a cell model. 7PA2 cells expressing the human amyloid precursor protein released soluble Aβ oligomers that caused synapse damage in cultured neurons. Supernatants from 7PA2 cells treated with the cholesterol synthesis inhibitor squalestatin contained similar concentrations of Aβ₄₂ to control cells but did not cause synapse damage in neuronal cultures. These supernatants contained reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers. Treatment of 7PA2 cells with platelet-activating factor (PAF) antagonists had similar effects; it reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers in cell supernatants. PAF activated cholesterol ester hydrolases (CEH), enzymes that released cholesterol from stores of cholesterol esters. Inhibition of CEH also reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers in cell supernatants. The Aβ monomers produced by treated cells protected neurons against Aβ oligomer-induced synapse damage. These studies indicate that pharmacological manipulation of cells can alter the ratio of Aβ monomer:oligomer released and consequently their effects on synapses.     

Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability—A possible regulatory switch?

Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5′-tetraphosphate (K_m = 11 μM, k_cat = 0.79 s^?1). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP₅, K_m = 0.07 μM, k_cat = 0.024 s^?1). Assayed at 26 °C, Aps had an alkaline pH optimum and required a divalent ion: Mg²⁺ (10–20 mM) or Mn²⁺ (1 mM) were preferred for nucleotide hydrolysis and Mg²⁺ (0.5–1 mM) or Co²⁺ (1–100 μM) for PP-InsP₅ hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 °C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 °C. Heat lability was restored by H₂O₂ and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP₅ to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP₅ found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP₅ during development.     

Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr−/− mice

The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR^−/− background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target.     

Synthesis and structure–activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors

A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure–activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.     

Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 β-glucosidase

Background

Glycosyl hydrolase family 1 (GH1) β-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 β-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice.

Results

Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed β-(1,4)-linked oligosaccharides of 3–6 glucose residues and laminaribiose.

Conclusion

Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 β-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism.     

Enzymatic preparation of D-β-acetylthioisobutyric acid and cetraxate hydrochloride using a stereo- and/or regioselective hydrolase, 3,4-dihydrocoumarin hydrolase from Acinetobacter calcoaceticus

3,4-Dihydrocoumarin hydrolase (DCH) from Acinetobacter calcoaceticus F46, which was previously found on screening for aromatic lactone-hydrolyzing enzymes, catalyzes the hydrolysis of several linear esters. The substrate specificity of the enzyme toward linear esters was quite characteristic, i.e., (1) it was specific toward methyl esters, (2) it recognized the configuration at the 2-position, and (3) it hydrolyzed diesters to monoesters. DCH hydrolyzed the methyl esters of beta-acetylthioisobutyrate and cetraxate. The products of these reactions were identified as D-beta-acetylthioisobutyrate and cetraxate, respectively, i.e., the hydrolysis reactions catalyzed by DCH were stereo- and/or regioselective. With recombinant Escherichia coli cells expressing the DCH gene as a catalyst, stereospecific hydrolysis of methyl beta-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate proceeded efficiently.     

A novel glycosylphosphatidylinositol-anchored glycoside hydrolase from Ustilago esculenta functions in β-1,3-glucan degradation

A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of β-1,3-glucan after its release in the extracellular medium.