Characterization of a Novel β-thioglucosidase CpTGG1 in Carica papaya and its Substrate-dependent and Ascorbic Acid-independent O-β-glucosidase Activity

Plant thioglucosidases are the only known S-glycosidases in the large superfamily of glycosidases.These enzymes evolved more recently and are distributed mainly in Brassicales.Thioglucosidase research has focused mainly on the cruciferous crops due to their economic importance and cancer preventive benefits.In this study,we cloned a novel myrosinase gene,CpTGG1,from Carica papaya Linnaeus.and showed that it was expressed in the aboveground tissues in planta.The recombinant CpTGG1 expressed in Pichia pastoris catalyzed the hydrolysis of both sinigrin and glucotropaeolin(the only thioglucoside present in papaya),showing that CpTGG1 was indeed a functional myrosinase gene.Sequence alignment analysis indicated that CpTGG1 contained all the motifs conserved in functional myrosinases from crucifers,except for two aglycon-binding motifs,suggesting substrate priority variation of the non-cruciferous myrosinases.Using sinigrin as substrate,the apparent Km and Vmax values of recombinant CpTGG1 were 2.82 mM and 59.9 μmol min-1 mg protein-1,respectively.The Kcat IKm value was 23 s-1 mM-1.O-β-glucosidase activity towards a variety of substrates were tested,CpTGG1 displayed substrate-dependent and ascorbic acid-independent O-β-glucosidase activity towards 2-nitrophenyl-βD-glucopyranoside and 4-nitrophenyl-β-D-glucopyranoside,but was inactive towards glucovanillin and n-octyl-β-D-glucopyranoside.Phylogenetic analysis indicated CpTGG1 belongs to the MYR II subfamily of myrosinases.     

Characterization of a novel β-glucosidase from a Stachybotrys strain

An improved mutant was isolated from the cellulolytic fungus Stachybotrys sp. after nitrous acid mutagenesis. It was fed-batch cultivated on cellulose and its extracellular cellulases (mainly the endoglucanases and β-glucosidases) were analyzed. One β-glucosidase was purified to homogeneity after two steps, MonoQ and gel filtration and shown to be a dimeric protein. The molecular weight of each monomer is 85 kDa. Besides its aryl β-glucosidase activity towards salicin, methyl-umbellypheryl-β-d-glucoside (MUG) and p-nitrophenyl-β-d-glucoside (pNPG), it showed a true β-glucosidase activity since it splits cellobiose into two glucose monomers. The V_max and the K_m kinetics parameters with pNPG as substrate were 78 U/mg and 0.27 mM, respectively. The enzyme shows more affinity to pNPG than cellobiose and salicin whose apparent values of K_m were, respectively, 2.22 and 37.14 mM. This enzyme exhibits its optimal activity at pH 5 and at 50 °C. Interestingly, this activity is not affected by denaturing gel conditions (SDS and β-mercaptoethanol) as long as it is not pre-heated. The N-terminal sequence of the purified enzyme showed a significant homology with the family 1 β-glucosidases of Trichoderma reesei and Humicola isolens even though these two enzymes are much smaller in size.     

Characterization of a novel β-glucosidase-like activity from a soil metagenome

We report the cloning of a novel β-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgllC and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known β-glucosidases. The putative β-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacI; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant BgllC protein hydrolyzed d-glucosyl-β-(l–4)-d-glucose to glucose. The maximum activity for BgllC protein occurred at pH 8.0 and 42°C using p-nitrophenyl-β-d-glucoside as the substrate. A CaCl₂ concentration of 1 mM was required for optimal activity. The putative β-glucosidase had an apparent K _m value of 0.19 mM, a V _max value of 4.75 U/mg and a k _cat value of 316.7/min under the optimal reaction conditions. The biochemical characterization of BgllC has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.     

Purification and Characterization of β-d-Galactosidase and α-d-Mannosidase from Papaya (Carica papaya) Seeds

β-D-Galactosidase was purified 115-fold from a saline extract of papaya seeds by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel-filtration on Sephadex G-75, G-150, and G-100. The purified β-D-galactosidase (MW, 56,000 daltons) had an isoelectric point (pI) at pH 8.4 and the optimal pH for its activity was 3.5 to 4.5. The enzyme activity was inhibited by Cu²⁺,Ag⁺,Hg²⁺,Pb²⁺,NaAsO₂ and р-chloromercuribenzoate at concentrations of 1x10^-3 M. Among the various mono- and oligosaccharides tested, D-galactose, D-galacturonic acid, D-galactono-γ-lactone and melibiose significantly inhibited the enzyme activities at concentrations of 2xl0^-3 to 1X10^-2M. The purified enzyme hydrolyzed β-nitrophenyl β-D-galactoside (Km = 1.0X10^-3M), methyl β-D-galactoside (Km=1.6x10^-2M), aminoethyl β-D-galactoside (Km =3.3X10^-2M) and lactose (Km = 9.1X10^-2M). β-(l→3)-Linked galactotetraosyl-eryth itol and asialo-glycopeptide isolated from fetuin were also hydrolyzed to the extent of 78 and 75%, 4respectively, on the basis of their galactose contents.

∝-D-Mannosidase from papaya seeds was also purified 130-fold by ammonium sulfate fractionation, DEAE-Sephadex chromatography, gel-filtration on Sephadex G-150 and hydroxylapatite chromatography. The purified enzyme (MW, 156,000 daltons), consisting of two subunits (78,000x2), was inhibited by Hg²⁺,Ag⁺,Cu²⁺, р-chloromercuribenzoate, D-glucose, D-glucosamine and D-mannose at concentrations of lx10^-3 to 1x10^-2M. The ∝-D-mannosidase hydrolyzed р-nitrophenyl ∝-D-mannoside (Km=5.6x10^-3M), methyl ∝-D-mannoside (Km=2.8X10^-2M), ∝-D-mannosyl-D-mannitol (Km=2.2X10^-2M), ∝-(l→2)linked D-mannobiosyl-D-mannitol (Km=6.3x10^-3M) and D-mannotriosyl-D-mannitol (Km=5.3x10^-3 M).     

Purification and characterization of a novel β-glucosidase from Aspergillus flavus and its application in saccharification of soybean meal

Abstract

Aspergillus flavus has been regarded as a potential candidate for its production of industrial enzymes, but the details of β-glucosidase from this strain is very limited. In herein, we first reported a novel β-glucosidase (AfBglA) with the molecular mass of 94.2?kDa from A. flavus. AfBglA was optimally active at pH 4.5 and 60?°C and is stable between pH 3.5 and 9.0 and at a temperature of up to 55?°C for 30?min remaining more than 90% of its initial activity. It showed an excellent tolerance to Trypsin, Pepsin, Compound Protease, and Flavourzyme and its activity was not inhibited by specific certain cations. AfBglA displayed broad substrate specificity, it acted on all tested pNP-glycosides and barley glucan, indicating this novel β-glucosidase exhibited a β-1, 3-1, 4-glucanase activity. Moreover, the AfBglA could effectively hydrolyze the soybean meal suspension into glucose and exhibit a strong tolerance to the inhibition of glucose at a concentration of 20.0?g/L during the saccharification. The maximum amount of the glucose obtained by AfBglA corresponded to 67.0?g/kg soybean meal. All of these properties mentioned above indicated that the AfBglA possibly attractive for food and feed industry and saccharification of cellulolytic materials.     

Characterization of a novel steviol-producing β-glucosidase from Penicillium decumbens and optimal production of the steviol

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing β-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3–6.0 and at 40–60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and ¹H- and ¹³C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other β-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 μl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel β-glucosidase, which may be useful for the pharmaceutical and agricultural areas.     

Purification and partial characterization of β-glucosidase from papaya fruit

β-Glucosidase (I) was isolated from Carica papaya fruit pulp and purified ca 1000-fold to electrophoretic homogeneity. The procedure used ammonium sulphate fractionation followed by chromatography on Phenyl-Sepharose CL-4B and Sephacryl S-200 to separate α-mannosidase (II) and, in part, β-galactosidase (III) from (I). Final separation of (III) from (I) was achieved by preparative isoelectric focusing (PIEF). The glycosidases had pI of 5.2 (I), 4.9 (II) and 6.9 (III). M,s of 54 000 (I), 260 000 (II) and 67 000 (III) were determined by gel filtration. The M, of (I) estimated by SDS-PAGE was 27 000 suggesting that (I) consisted of two subunits. The optimum pH and optimum temperature of (I) were 5.0 and 50°, respectively, and the enzyme followed typical Michaelis kinetics with K_m and V_max of 1.1 × 10⁻⁴ M and 1.8 × 10⁻⁶ mol/hr, respectively, for p-nitrophenyl-β-d-glucoside (40°).     

Characterization and identification of SFDC-1, a novel AmpC-type β-lactamase in Serratia fonticola

The clinical and environmental infections caused by AmpC β-lactamases have been increasingly reported recently. In this study, we characterize the novel chromosome-encoded AmpC β-lactamase SFDC-1 identified in Serratia fonticola strain R28, which was isolated from a rabbit raised on a farm in southern China. SFDC-1 shared the highest amino acid identity of 79.6% with the functionally characterized AmpC β-lactamase gene bla_YRC-1, although it had highly homologous functionally uncharacterized relatives in the same species from different sources, including some of the clinical significance. The cloned bla_SFDC-1 exhibited resistance to a broad spectrum of β-lactam antibiotics, including most cephalosporins with the highest resistance to ampicillin, cefazolin and ceftazidime, with increased MIC levels ≥128-fold compared with the control strains. The purified SFDC-1 showed catalytic activities against β-lactams with the highest catalytic activity to cefazolin. The genetic context of bla_SFDC-1 and its relatives was conserved in the chromosome, and no mobile genetic elements were found surrounding them.     

A novel β-glucosidase with lipolytic activity from a soil metagenome

Moonlighting proteins have two different functions within a single polypeptide chain. Exploring moonlighting enzymes from the environment using the metagenomic approach is interesting. In the present study, a novel β-glucosidase gene, designated as bgl1D, with lipolytic activity (renamed Lip1C) was cloned through function-based screening of a metagenomic library from uncultured soil microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis also indicated that Lip1C and other putative lipases are closely related. Biochemical characterization demonstrated that the maximum activity of the recombinant Lip1C protein occurs at pH 8.0 and 30°C using 4-nitrophenyl butyrate as substrate. The putative lipase had an apparent K _m value of 0.88 mmol/L, a k _cat value of 212/min, and a k _cat/K _m value of 241 L/mmol/min. Lip1C exhibited habitat-specific characteristics with 5 mmol/L AlCl₃, CuCl₂, and LiCl. The characterization of the biochemical properties of Lip1C enhances our understanding of this novel moonlighting enzyme isolated from a soil metagenome.     

Characterization of a novel endo-β-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Armillaria gemina KJS114 based on its morphology and internal transcribed spacer rDNA gene sequence. A. gemina EG (AgEG) was purified using a single-step purification by gel filtration. The relative molecular mass of AgEG by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 65 kDa and by size exclusion chromatography was 66 kDa, indicating that the enzyme is a monomer in solution. The pH and temperature optima for hydrolysis were 5.0 and 60 °C, respectively. Purified AgEG had the highest catalytic efficiency with carboxymethylcellulose (k _cat/K _m?=?3,590 mg mL^?1 s^?1) unlike that reported for any fungal EG, highlighting the significance of the current study. The amino acid sequence of AgEG showed homology with hydrolases from the glycoside hydrolase family 61. The addition of AgEG to a Populus nigra hydrolysate reaction containing a commercial cellulase mixture (Celluclast 1.5L and Novozyme 188) showed a stimulatory effect on reducing sugar production. AgEG is a good candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.     

Characterization and solvent engineering of wheat β-amylase for enhancing its activity and stability

The kinetic and thermodynamic parameters of wheat β-amylase (WBA) were characterized and various additives were evaluated for enhancing its activity and thermostability. WBA activity was examined by neocuproine method using soluble starch as substrate. The Michaelis constant (K(m)) and molecular activity (k(cat)) were determined to be 1.0±0.1% (w/v) and 94±3s(-1), respectively, at pH 5.4 and at 25°C. The optimum reaction temperature (T(opt)) for WBA activity was 55°C and the temperature (T(50)) at which it loses half of the activity after 30-min incubation was 50±1°C. Modifications of the solvent with 182mM glycine and 0.18% (w/v) gelatin have increased the T(50) by 5°C. Glycerol, ethylene glycol, dimethylformamide (DMF) and dimethyl sulfoxide have also slightly enhanced the thermostability plausibly through weakening the water structure and decreasing the water shell around the WBA protein. Ethanol and DMF activated WBA by up to 24% at 25°C probably by inducing favorable conformation for the active site or changing the substrate structure by weakening the hydrogen bonding. Its half-life in the inactivation at 55°C was improved from 23 to 48min by 182mM glycine. The thermodynamic parameters indicate that WBA is thermo-labile and sufficient stabilization was achieved through solvent modification with additives and that the heat inactivation of WBA is entropic-driven. It is suggested that WBA could be applied more widely in starch-saccharification industries with employing suitable additives.     

Antimicrobial activity and structure of a consensus human β-defensin and its comparison to a novel putative hBD10

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human β-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical β-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in ¹H NMR spectra and structural studies were not pursued. The evaluation of different β-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.     

Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization

A β-glucosidase gene (bglI) from Trichoderma reesei was cloned into the pPIC9 vector and integrated into the genome of Pichia pastoris GS115. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter and using Saccharomyces cerevisiae secretory signal peptide (α-factor), the recombinant β-glucosidase was expressed and secreted into the culture medium. The maximum recombinant β-glucosidase activity achieved was 60 U/ml, and β-glucosidase expression reached 0.3 mg/ml. The recombinant 76 kDa β-glucosidase was purified 1.8-fold with 26% yield and a specific activity of 197 U/mg. It was optimally active at 70°C and pH 5.0.     

Characterization of a recombinant thermostable β-glucosidase from Putranjiva roxburghii expressed in Saccharomyces cerevisiae and its use for efficient biomass conversion

A β-glucosidase gene from Putranjiva roxburghii (PRGH1) was heterologously expressed in Saccharomyces cerevisiae to enable growth on cellobiose. The recombinant enzyme was secreted to the culture medium, purified and biochemically characterized. The enzyme is a glycoprotein with a molecular weight of ∼68 kDa and exhibited enzymatic activity with β‐linked aryl substrates like pNP-Fuc, pNP-Glc, pNP-Gal and pNP-Cel with catalytic efficiency in that order. Significant enzyme activity was observed for cellobiose, however the enzyme activity was decreased with increase in chain length of glycan substrates. Using cellobiose as substrate, the enzyme showed optimal activity at pH 5.0 and 65 °C. The enzyme was thermostable up to 75 °C for 60 min. The enzyme showed significant resistance towards both glucose and ethanol induced inhibition. The recombinant S. cerevisiae strain showed advantages in cell growth, glucose and bio-ethanol production over the native strain with cellobiose as sole carbon source. In simultaneous saccharification and fermentation (SSF) experiments, the recombinant strain was used for bio-ethanol production from two different cellulosic biomass sources. At the end of the SSF, we obtained 9.47 g L⁻¹ and 14.32 g L⁻¹ of bio-ethanol by using carboxymethyl cellulose and pre-treated rice straw respectively. This is first report where a β-glucosidase gene from plant origin has been expressed in S. cerevisiae and used in SSF.     

Location and contribution of individual β-glucosidase from Neurospora crassa to total β-glucosidase activity

This study investigated the cellular location and the contribution of individual β-glucosidase (BGL) to total BGL activity in Neurospora crassa. Among the seven bgl genes, bgl3, bgl5, and bgl7 were transcribed at basal levels, whereas bgl1, bgl2, bgl4, and bgl6 were significantly up-regulated when the wild-type strain was induced with cellulose (Avicel). BGL1 and BGL4 were found to be contributors to intracellular BGL activity, whereas the activities of BGL2 and BGL6 were mainly extracellular. Sextuple bgl deletion strains expressing one of the three basally transcribed bgls did not produce any detectable BGL activity when they were grown on Avicel. BGL6 is the major contributor to overall BGL activity, and most of its activity resides cell-bound. The sextuple bgl deletion strain containing only bgl6 utilized cellobiose at a rate similar to that of the wild type, while the strain with only bgl6 deleted utilized cellobiose much slower than that of the wild type.     

Immobilization of β-glucosidase in fixed bed reactor and evaluation of the enzymatic activity

Adsorption of β-glucosidase from almonds, an enzyme with big molecular size (130?kDa, 6.7?nm molecular diameter), on mesoporous SBA-15 silica in fixed bed column was studied. Previously, zeta potential analysis confirmed that the electrostatic interactions between β-glucosidase and SBA-15 were the driving force of the immobilization process. The maximum difference in the zeta potential was 25?mV at pH 3.5. Adsorption isotherm was classified as an L3 (Langmuir type 3) curve according to the Giles classification and fitted to a double Langmuir equation. The adsorbed amount in a fixed bed column was around 3.5 times higher than the amount reached in the adsorption in batch. In addition, the β-glucosidase was strongly immobilized on SBA-15 with only 7?% of leaching in the washing step with buffer solution. Immobilized β-glucosidase was catalytically active in a continuous process, reaching 100?% substrate conversion and maintaining this activity level for more than 10?h without deactivation of the enzyme. Adsorption-desorption isotherms at 77?K before and after the adsorption were carried out, concluding that the adsorption of β-glucosidase was produced blocking the pore mouth, so that a part of the enzyme penetrates inside and another part stays outside the pore.     

Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus

The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0–6.0 and 55–65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0–10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K_M = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V_max for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V_max was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.     

Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosisH37Rv demonstrating β-lactamase activity

The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus β-lactamase motif ‘SXXK’ in addition to a conserve pentapeptide –GXSXG-, characteristic of lipolytic enzymes, at the C-terminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and β-lactamase activities. A serine located within consensus β-lactamase motif ‘SXXK’ was identified as catalytic residue in both esterase and β-lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for β-lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly up-regulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing β-lactamase activity.     

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 β-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (r _s/r _h) from 0.33 to 1.45–2.71. Further increase in r _s/r _h was achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5–10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.