β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997     

Isolation and properties of extracellular β-xylosidases from fungi <Emphasis Type="Italic">Aspergillus japonicus</Emphasis> and <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Homogeneous β-xylosidases with molecular mass values 120 and 80 kDa (as shown by SDS-PAGE), belonging to the third family of glycosyl hydrolases, were isolated by anion-exchange, hydrophobic, and gel-penetrating chromatography from enzyme preparations based on the fungi Aspergillus japonicus and Trichoderma reesei, respectively. The enzymes exhibit maximal activity in acidic media (pH 3.5–4.0), and temperature activity optimum was 70°C for the β-xylosidase of A. japonicus and 60°C for the β-xylosidase of T. reesei. Kinetic parameters of p-nitrophenyl β-xylopyranoside and xylooligosaccharide hydrolysis by the purified enzymes were determined, which showed that β-xylosidase of A. japonicus was more specific towards low molecular weight substrates, while β-xylosidase of T. reesei preferred high molecular weight substrates. The competitive type of inhibition by reaction product (xylose) was found for both enzymes. The interaction of the enzymes of different specificity upon hydrolysis of glucurono- and arabinoxylans was found. The β-xylosidases exhibit synergism with endoxylanase upon hydrolysis of glucuronoxylan as well as with α-L-arabinofuranosidase and endoxylanase upon hydrolysis of arabinoxylan. Addition of β-xylosidases increased efficiency of hydrolysis of plant raw materials with high hemicellulose content (maize cobs) by the enzymic preparation Celloviridine G20x depleted of its own β-xylosidase.     

The enzymic hydrolysis of amygdalin

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond beta-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the beta-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components.     

Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency values, k _cat/K _m, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1, 3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxside. In a memoriam of Dr. Kirill N. Neustroev. All we, his friends and colleagues, mourn for his sudden death. He was a bright and talented scientist, brilliant manager and good friend.     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Received: 22 May 1998 / Accepted: 13 August 1998     

CYP710A genes encoding sterol C22-desaturase in <Emphasis Type="Italic">Physcomitrella patens</Emphasis> as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants

We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC–MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56–60% of total sterol) and sitosterol to a lesser extent (8–12%); this sterol profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14 proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with β-sitosterol to produce stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K _m values for β-sitosterol of CYP710A13 (1.0 ± 0.043 μM) and CYP710A14 (2.1 ± 0.17 μM) were at comparable levels of those reported with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Catalytic degradation of amygdalin by extracellular enzymes from Aspergillus niger

Amygdalin is a controversial anti-tumor natural product that has been used as an alternative cancer drug for many years. The anti-tumor mechanism and metabolism of amygdalin have been the focus of many studies. However, previous studies by our group demonstrated that amygdalin itself has no anti-tumor activity, but rather the active ingredients were determined to be amygdalin degradation products. To screen novel drugs with anti-tumor activity, the extracellular enzymes from Aspergillus niger were used to degrade amygdalin. Within 4 h of the catalytic reaction at 37°, amygdalin was rapidly degraded into four products. The products were then extracted and purified by column chromatography. By comparing the HPLC chromatograms, ¹H NMR, ¹³C NMR and MS data, the products were identified as mandelonitrile, prunasin, benzaldehyde and phenyl-(3,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-acetonitrile (PTMT), a novel hydroxyl derivative of prunasin. Furthermore, pharmacology studies of these compounds demonstrated that 10 mg/kg of PTMT significantly suppressed the growth of S-18 tumor cells within 11 days in a concentration-dependent manner.     

Ectoenzymatic Activity and Uptake of Monomers in Marine Bacterioplankton Described by a Biphasic Kinetic Model

Abstract The kinetics of bacterial hydrolytic ectoenzymatic activity and the uptake of monomeric compounds were investigated in the Northwestern Mediterranean Sea. Aminopeptidase and α- and β-glucosidase activities were analyzed by using fluorogenic substrates at 15–22 concentrations ranging from 1 nM to 500 μM. Radiolabeled glucose and a mixture of amino acids were chosen as representatives of monomeric compounds, and the bacterial uptake rates (assimilation plus respiration) were determined over a wide range of substrate concentrations (from 0.2 nM to 3 μM). We found biphasic kinetics both for hydrolytic enzymes and uptake systems: high affinity enzymes at low concentrations of substrates (K _m values ranged from 48 nM to 2.7 μM for ectoenzymes and from 1.4 nM to 42 nM for uptake systems), and low affinity enzymes at high concentrations of substrates (K _m values ranged from 18 μM to 142 μM for ectoenzymes and from 0.1 μM to 1.3 μM for uptake systems). Transition between high and low affinity enzymes was observed at 10 μM for aminopeptidase and from 1 μM to 25 μM for glucosidases, and it was more variable and less pronounced for the uptake of glucose (40 nM–0.28 μM) and amino acids (10 nM–0.16 μM). Results showed that the potential rates of hydrolysis and uptake are tightly coupled only if the high affinity hydrolytic ectoenzymes and the low affinity uptake systems are operating simultaneously. Received: 5 March 1998; Accepted: 31 July 1998     

A covalent two-step immobilization technique using itaconic anhydride

β-Glucosidase from almonds (EC 3.2.1.21) was covalently immobilized by a two-step technique. In the first step, double bonds were introduced into the β-glucosidase by derivatization with itaconic anhydride. In separate studies with α-N-protected l-amino acids, it was established that itaconic anhydride acylated mainly primary amino groups of lysines and, to a much lesser extent hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. The acylated β-glucosidase showed no loss of activity and the K _m decreased from 3.6 mM to 2.6 mM when p-nitrophenyl β-d-glucopyranoside was used as the substrate. In the second step, the derivatized β-glucosidase was co-polymerized radically with N,N′-methylenebisacrylamide in buffer solution. The resulting acrylamide immobilizate possessed a much better storage stability at 30–56 °C when compared to β-glucosidase immobilized on Eupergit C. However, the specific activity was higher with the Eupergit immobilizate. Free and acrylamide-immobilized β-glucosidase were used for glucosylation of chloramphenicol by transglucosylation in 20% (v/v) acetonitrile at 37 °C. The acrylamide immobilizate demonstrated a great enhancement of stability and approximately 50% more chloramphenicol β-glucoside was obtained after 5 h. Received: 22 September 1997 / Accepted: 28 October 1997     

Development of the Potential for Cyanogenesis in Maturing Black Cherry (Prunus serotina Ehrh.) Fruits

Biochemical changes related to cyanogenesis (hydrogen cyanide production) were monitored during maturation of black cherry (Prunus serotina Ehrh.) fruits. At weekly intervals from flowering until maturity, fruits (or selected parts thereof) were analyzed for (a) fresh and dry weights, (b) prunasin and amygdalin levels, and (c) levels of the catabolic enzymes amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. During phase I (0-28 days after flowering [DAF]), immature fruits accumulated prunasin (mean: 3 micromoles/fruit) but were acyanogenic because they lacked the above enzymes. Concomitant with cotyledon development during mid-phase II, the seeds began accumulating both amygdalin (mean: 3 micromoles/seed) and the catabolic enzymes and were highly cyanogenic upon tissue disruption. Meanwhile, prunasin levels rapidly declined and were negligible by maturity. During phases II (29-65 DAF) and III (66-81 DAF), the pericarp also accumulated amygdalin, whereas its prunasin content declined toward maturity. Lacking the catabolic enzymes, the pericarp remained acyanogenic throughout all developmental stages.     

Characterization of β-glucosidases from Hanseniaspora sp. and Pichia anomala with potentially aroma-enhancing capabilities in juice and wine

The properties of intracellular β-glucosidases produced from two yeast isolates identified as Hanseniaspora sp. BC9 and Pichia anomala MDD24 were characterized. β-Glucosidase from Hanseniaspora sp. BC9 was not inhibited by both 20% w/v fructose and 20% w/v sucrose and was slightly inhibited by glucose (> 40% relative β-glucosidase activity with 10% w/v glucose). β-Glucosidase from P. anomala MDD24 was inhibited by glucose, fructose and sucrose. In the presence of 4–12% v/v ethanol, β-glucosidase from P. anomala MDD24 was stimulated in range 110–130% relative activity whereas β-glucosidase from Hanseniaspora sp. BC9 was substantially inhibited in the presence of ethanol. Finally, juice and wine of the Muscat-type grape variety, Traminette, were selected to determine sugar-bound volatile aroma release, particularly terpenes, by the activity of those β-glucosidases. The results showed that high concentration of free aroma compounds were detected from Traminette juice treated with β-glucosidase from Hanseniaspora sp. BC9 and Traminette wine treated with β-glucosidase from P. anomala MDD24. The preliminary results with proposed an application of these enzymes in commercial wine production lead to more efficient of β-glucosidase from Hanseniaspora sp. BC9 in releasing desirable aromas during an early stage of alcoholic fermentation while β-glucosidase from P. anomala MDD24 is suitable at the final stage of alcoholic fermentation.     

Regio- and Stereo-selective Hydroxylation of Abietic Acid Derivatives by Mucor circinelloides and Mortierella isabellina

Mucor circinelloides and Mortierella isabellina hydroxylated dehydroabietic acid (DehA). DehA was converted regio- and stereo-selectively by whole cells of Mr. circinelloides to give 2α-hydroxydehydroabietic acid in a 75% molar conversion yield (11 mM from 14.7 mM DehA) after 72 h in the cultivation medium containing 3% (v/v) Tween 80. With cells of Ma. isabellina, under the same conditions, 20.5 mM (6.5 g l⁻¹) 2–hydroxydehydroabietic acid (α/β=81/19) was formed from 26.4 mM DehA.     

Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-mannans, while normal GFP could not bind to β-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.     

Ecophysiological Variabilities in Ectohydrolytic Enzyme Activities of Some <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> Species, <Emphasis Type="Italic">P. citrea,P. issachenkonii</Emphasis>, and <Emphasis Type="Italic">P. nigrifaciens</Emphasis>

The ecophysiological variabilities in the ectohydrolytic enzyme profiles of the three species of Pseudoalteromonas, P. citrea, P. issachenkonii, and P. nigrifaciens, have been investigated. Forty-one bacteria isolated from several invertebrates, macroalgae, sea grass, and the surrounding water exhibited different patterns of hydrolytic enzyme activities measured as the hydrolysis of either native biopolymers or fluorogenic substrates. The activities of the following enzymes were assayed: proteinase, tyrosinase, lipase, amylase, chitinase, agarase, fucoidan hydrolase, laminaranase, alginase, pustulanase, cellulase, β-glucosidase, α- and β-galactosidases, β-N-acetylglucosaminidase, β-glucosaminidase, β-xylosidase, and α-mannosidase. The occurrence and cell-specific activities of all enzymes varied over a broad range (from 0 to 44 μmol EU per hour) and depended not only on taxonomic affiliation of the strain, but also on the source/place of its isolation. This suggests ‘specialization’ of different species for different types of polymeric substrates as, for example, all strains of P. citrea and P. issachenkonii hydrolyzed alginate and laminaran, while strains of P. nigrifaciens were lacking the ability to hydrolyze most of the algal polysaccharides. The incidence of certain enzymes such as fucoidan hydrolases, alginate lyases, agarases, and α-galactosidases might be strain specific and reflect its particular ecological habitat. Received: 15 February 2002 / Accepted: 27 March 2002     

Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds

In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two β-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.     

Production, characterization and properties of polysaccharide depolymerizing enzymes from a strain ofCurvularia inaequalis

Xylanase, β-glucosidase, β-xylosidase, endoglucanase and polygalacturonase production fromCurvularia inaequalis was carried out by means of solid-state and submerged fermentation using different carbon sources. β-Glucosidase. β-xylosidase, polygalacturonase and xylanase produced by the microorganisms were characterized. β-Glucosidase presented optimum activity at pH 5.5 whereas xylanase, poly-galacturonase and β-xylosidase activities were optimal at pH 5.0. Maximal activity of β-glucosidase was determined at 60°C, β-xylosidase at 70°C, and polygalacturonase and xylanase at 55°C. These enzymes were stable at acidic to neutral pH and at 40–45 °C. The crude enzyme solution was studied for the hydrolysis of agricultural residues.     

The de novo production of drosophilin A (tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (tetrachloro-1,4-dimethoxybenzene) by ligninolytic basidiomycetes

Ligninolytic basidiomycetes were screened for their ability to produce the tetrachlorinated hydroquinone metabolites drosophilin A (DA, tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (DAME, tetrachloro-1,4-dimethoxybenzene). Five fungal strains produced these metabolites in detectable amounts, including strains from Bjerkandera and Peniophora, which are genera not previously known for DA or DAME production. Phellinus fastuosus ATCC26.125 had the highest and most reliable production of DA and DAME in peptone medium, respectively 15–60 μM and 4–40 μM. This fungus was used to study culture conditions that could increase DAME production. A fourfold increase in DAME production was found after the addition of hydroquinone to growing cultures of P. fastuosus. Therefore, hydroquinone is postulated to be a possible biosynthetic precursor of DAME in the fungus. Antagonising P. fastuosus by adding filter-sterilised culture fluid of a competing fungus, Phlebia radiata, increased DAME production significantly by tenfold. This result suggests that DAME production is elicited by compounds present in the culture fluid of P. radiata, indicating that DAME has an antibiotic function in P. fastuosus. Received: 17 September 1996 / Received revision: 7 February 1997 / Accepted: 15 February 1997     

Permeabilization of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus with Ethanol

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus cultures were treated with ethanol and tested for viability and β-galactosidase activity. Exposure of the biomass of test cultures to 30%–55% ethanol (vol/vol) caused a 100% loss of viability and up to 15-fold increase in measurable β-galactosidase activity in both streptococci and lactobacilli. Ethanol-treated cell suspensions could be stored for up to 6 months without loss of enzyme activity. The nonviable permeabilized biomass of the more active S. thermophilus was used to achieve up to 80% hydrolysis of lactose in aqueous solutions and non-fat milk. Received: 28 July 1997 / Accepted: 30 September 1997     

Decarboxylation of Oxalic Acid during Ripening of Banana Fruit (Musa sapientum L.)

ABSTRACT

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5?- and 3?-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.