A Novel Highly Thermostable Multifunctional Beta-Glycosidase from Crenarchaeon Acidilobus saccharovorans.
ABSTRACT: We expressed a putative ?-galactosidase Asac_1390 from hyperthermophilic crenarchaeon Acidilobus saccharovorans in Escherichia coli and purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity with p-nitrophenyl (pNP) substrates followed the order pNP-?-D-galactopyranoside (328?U?mg(-1)), pNP-?-D-glucopyranoside (246?U?mg(-1)), pNP-?-D-xylopyranoside (72?U?mg(-1)), and pNP-?-D-mannopyranoside (28?U?mg(-1)). Thus the enzyme was actually a multifunctional ?-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.
Project description:Acidilobus saccharovorans is an anaerobic, organotrophic, thermoacidophilic crenarchaeon isolated from a terrestrial hot spring. We report the complete genome sequence of A. saccharovorans, which has permitted the prediction of genes for Embden-Meyerhof and Entner-Doudoroff pathways and genes associated with the oxidative tricarboxylic acid cycle. The electron transfer chain is branched with two sites of proton translocation and is linked to the reduction of elemental sulfur and thiosulfate. The genomic data suggest an important role of the order Acidilobales in thermoacidophilic ecosystems whereby its members can perform a complete oxidation of organic substrates, closing the anaerobic carbon cycle.
Project description:A gene encoding an alpha-L-arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45 degrees C and was stable over the pH range of 5.0-6.5 at 30 degrees C. The enzyme hydrolyzed p-nitrophenol (PNP)-alpha-L-arabinofuranoside but did not hydrolyze PNP-alpha-L-arabinopyranoside, PNP-beta-D-xylopyranoside, or PNP-beta-D-galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside and methyl 3-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside were not. These data suggested that the enzyme only cleaves alpha-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-alpha-L-arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.
Project description:<i>Brunfelsia calycina</i> flowers lose anthocyanins rapidly and are therefore well suited for the study of anthocyanin degradation mechanisms, which are unclear in planta. Here, we isolated an anthocyanin-β-glycosidase from <i>B. calycina</i> petals. The MS/MS (Mass Spectrometry) peptide sequencing showed that the enzyme (72 kDa) was a β-xylosidase (BcXyl). The enzyme showed high activity to p-Nitrophenyl-β-d-galactopyranoside (pNPGa) and p-Nitrophenyl-β-d-xylopyranoside (pNPX), while no activity to p-Nitrophenyl-β-d-glucopyranoside (pNPG) or p-Nitrophenyl-β-D-mannopyranoside (pNPM) was seen. The optimum temperature of BcXyl was 40 °C and the optimum pH was 5.0. The enzyme was strongly inhibited by 1 mM D-gluconate and Ag⁺. HPLC (High Performance Liquid Chromatography) analysis showed that BcXyl catalyzed the degradation of an anthocyanin component of <i>B. calycina</i>, and the release of xylose and galactose due to hydrolysis of glycosidic bonds by BcXyl was detected by GC (Gas Chromatography) /MS. A full-length mRNA sequence (2358 bp) of <i>BcXyl</i> (NCBI No. MK411219) was obtained and the deduced protein sequence shared conserved domains with two anthocyanin-β-glycosidases (Bgln and BadGluc, characterized in fungi). BcXyl, Bgln and BadGluc belong to AB subfamily of Glycoside hydrolase family 3. Similar to <i>BcPrx01</i>, an anthocyanin-degradation-related <i>Peroxidase</i> (<i>POD</i>), <i>BcXyl</i> was dramatically activated at the stage at which the rapid anthocyanin degradation occurred. Taken together, we suggest that BcXyl may be the first anthocyanin-β-glycosidase identified in higher plants.
Project description:BACKGROUND:The importance of the accessory enzymes such as ?-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails. RESULTS:Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60?°C while AFase-D3 had unique properties as it showed optimal activity at 25?°C as well as the ability to maintain substantial activity at temperatures as high as 90?°C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70?°C. The maximum activity was observed at a pH profile between pH?4.0-6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH?5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31?mM and 0.43?mM, Kcat range between 131?s-?1 and 219?s-?1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175?U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-?-L-arabinopyranoside and pNP-?-L-mannopyranoside respectively, and both hydrolysed pNP-?-D-glucopyranoside. CONCLUSION:All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study.
Project description:Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 220.127.116.11), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.
Project description:A ?-glucosidase from Penicillium italicum was purified with a specific activity of 61.8 U/mg, using a chromatography system. The native form of the enzyme was an 88.5-kDa tetramer with a molecular mass of 354 kDa. Optimum activity was observed at pH 4.5 and 60?, and the half-lives were 1,737, 330, 34, and 1 hr at 50, 55, 60, and 65?, respectively. Its activity was inhibited by 47% by 5 mM Ni(2+). The enzyme exhibited hydrolytic activity for p-nitrophenyl-?-D-glucopyranoside (pNP-Glu), p-nitrophenyl-?-D-cellobioside, p-nitrophenyl-?-D-xyloside, and cellobiose, however, no activity was observed for p-nitrophenyl-?-D-lactopyranoside, p-nitrophenyl-?-D-galactopyranoside, carboxymetyl cellulose, xylan, and cellulose, indicating that the enzyme was a ?-glucosidase. The k(cat)/K(m) (s(-1) mM(-1)) values for pNP-Glu and cellobiose were 15,770.4 mM and 6,361.4 mM, respectively. These values were the highest reported for ?-glucosidases. Non-competitive inhibition of the enzyme by both glucose (K(i) = 8.9 mM) and glucono-?-lactone (K(i) = 11.3 mM) was observed when pNP-Glu was used as the substrate. This is the first report of non-competitive inhibition of ?-glucosidase by glucose and glucono-?-lactone.
Project description:When cells of Bacillus sp. strain GL1 were grown in a medium containing xanthan as a carbon source, alpha-mannosidase exhibiting activity toward p-nitrophenyl-alpha-D-mannopyranoside (pNP-alpha-D-Man) was produced intracellularly. The 350-kDa alpha-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzed pNP-alpha-D-Man (Km = 0.49 mM) and D-mannosyl-(alpha-1,3)-D-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway of Bacillus sp. strain GL1. The gene for alpha-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of alpha-mannosidases belonging to glycoside hydrolase family 38.
Project description:Ginsenoside compound K has been used as a key nutritional and cosmetic component because of its anti-fatigue and skin anti-aging effects. ?-Glycosidase from Sulfolobus solfataricus (SS-BGL) is known as the most efficient enzyme for compound K production. The hydrolytic pathway from ginsenoside Rb1 to compound K via Rd and F2 is the most important because Rb1 is the most abundant component in ginseng extract. However, the enzymatic conversion of ginsenoside Rd to F2 is a limiting step in the hydrolytic pathway because of the relatively low activity for Rd. A V209 residue obtained from error-prone PCR was related to Rd-hydrolyzing activity, and a docking pose showing an interaction with Val209 was selected from numerous docking poses. W361F was obtained by rational design using the docking pose that exhibited 4.2-fold higher activity, 3.7-fold higher catalytic efficiency, and 3.1-fold lower binding energy for Rd than the wild-type enzyme, indicating that W361F compensated for the limiting step. W361F completely converted Rb1 to compound K with a productivity of 843 mg l-1 h-1 in 80 min, and showed also 7.4-fold higher activity for the flavanone, hesperidin, than the wild-type enzyme. Therefore, the W361F variant SS-BGL can be useful for hydrolysis of other glycosides as well as compound K production from Rb1, and semi-rational design is a useful tool for enhancing hydrolytic activity of ?-glycosidase.
Project description:We purified from crude extracts of the hyperthermophilic crenarchaeon Sulfolobus solfataricus a protease that is able to hydrolyse proteins with a pH optimum of 7.5 and a temperature optimum of 70 degrees C. Assays in the presence of classical protease inhibitors showed that the hydrolytic activity is sensitive to thiol-blocking reagents. Fluorescence assays using synthetic peptides demonstrated that the protease has a preference for cleaving glutamic acid residues. The first 12 residues of the protease match the N-terminus residues of a hypothetical protein in the S. solfataricus genome of 95 amino acids in length and calculated molecular mass of 11072 Da. The whole sequence of the protease is not related to any known protein, but it bears a segment which is highly similar to one containing the active cysteine residue in eukaryotic peptidases known as legumains. This is the first protease isolated from S. solfataricus capable of degrading native proteins effectively. Our results add to the knowledge of the intracellular proteolytic machine in hyperthermophilic micro-organisms.
Project description:BACKGROUND: Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. RESULTS: A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with K(m) and V(max) values of 1.148 mM and 3497 ?mol?min?¹?mg?¹, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. CONCLUSIONS: The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production.