General Characteristics of the Intracellular Myrosinase from Aspergillus niger

The enzymatic properties of intracellular myrosinase produced by Aspergillus niger AKU 3302 were investigated. Maximum activity occurred at pH 6.2, and the enzyme was stable in a pH range of 7.6 to 8.0 at 5°C for 24 hr. Optimum temperature was about 34°C. Enzyme activity was stimulated by copper (I), (II), manganese (II) and cobalt (II) and was inhibited by mercury (II) and stannous (II) ions. However, metal complexing agents and DFP had little effect, while PCMB was a strong inhibitor. In contrast to plant myrosinase, this enzyme was neither activated nor inhibited by L-ascorbic acid. Glucosides and δ-gluconolactone inhibited enzyme activity but sugars were ineffective. The Km value for sinigrin was 3.3 × 10^?3 M and that for p-nitrophenyl β-glucoside was 1.5 × 10^?3 M. The relation between fungous myrosinases and β-glucosidase is discussed in comparison to plant myrosinase.     

Studies on Myrosinase in Mustard Seed

Various sugars and glucosides inhibited both ascorbate-activated and non-activated plant myrosinases at much higher concentrations than those of the substrate. Among these, glucose and salicin were confirmed to be competitive inhibitors. The resemblance of myrosinase to β-glucosidase was pointed out from these results. The Ki values for them remained unchanged after the activation of the enzyme by l-ascorbate, whereas the Km value for the substrate increased. Glucose had no influence on the binding of ascorbate to the enzyme. Pigman’s model for β-glucosidase can be satisfactorily applied to yield an explanation of these experimental results.

β-Glucosidase activity of mustard myrosinase was confirmed using p-nitrophenyl β-glucoside ( p-NPG) as the substrate. p-NPG hydrolysis was not accelerated by 10^?3 m ascorbate but was inhibited competitively by its higher concentrations. This result forms a distinct contrast with the activating effect of ascorbate in enzymatic sinigrin hydrolysis. The previously noticed feature of the ascorbate effect on the enzyme that the Km value for sinigrin hydrolysis increased after activation is explainable by the fact that ascorbate was a competitive inhibitor as well as a specific activator. A schematic model interpreting the interaction of ascorbate with the enzyme is proposed.     

The Production and Stability of Intracellular Myrosinase from Aspergillus niger

After Screening 100 micro-organisms to detect intracellular myrosinase, only Aspergillus niger produced myrosinase.

Enzyme production was induced by the addition of ten percent of a mustard extract^* to the culture medium. The enzyme was produced in considerable amounts on the first and second day of cultivation. L-Ascorbic acid was an excellent carbon source.

The enzyme was unstable but was stabilized by coexistence with 2-mercaptoethanol (10^?2 M) and ascorbic acid (10^?3 M).     

Myrosinase: insights on structural,catalytic, regulatory,and environmental interactions

Glucosinolate–myrosinase is a substrate-enzyme defense mechanism present in Brassica crops. This binary system provides the plant with an efficient system against herbivores and pathogens. For humans, it is well known for its anti-carcinogenic, anti-inflammatory, immunomodulatory, anti-bacterial, cardio-protective, and central nervous system protective activities. Glucosinolate and myrosinase are spatially present in different cells that upon tissue disruption come together and result in the formation of a variety of hydrolysis products with diverse physicochemical and biological properties. The myrosinase-catalyzed reaction starts with cleavage of the thioglucosidic linkage resulting in release of a D-glucose and an unstable thiohydroximate-O-sulfate. The outcome of this thiohydroximate-O-sulfate has been shown to depend on the structure of the glucosinolate side chain, the presence of supplementary proteins known as specifier proteins and/or on the physiochemical condition. Myrosinase was first reported in mustard seed during 1939 as a protein responsible for release of essential oil. Until this date, myrosinases have been characterized from more than 20 species of Brassica, cabbage aphid, and many bacteria residing in the human intestine. All the plant myrosinases are reported to be activated by ascorbic acid while aphid and bacterial myrosinases are found to be either neutral or inhibited. Myrosinase catalyzes hydrolysis of the S-glycosyl bond, O-β glycosyl bond, and O-glycosyl bond. This review summarizes information on myrosinase, an essential component of this binary system, including its structural and molecular properties, mechanism of action, and its regulation and will be beneficial for the research going on the understanding and betterment of the glucosinolate–myrosinase system from an ecological and nutraceutical perspective.     

Sulphate can induce differential expression of thioglucoside glucohydrolases (myrosinases)

Thioglucoside glucohydrolase (EC 3.2.3.1; myrosinase) hydrolyses glucosinolates and thereby liberates glucose and sulphur and nitrogen compounds. To examine the hypothesis that the myrosinase-glucosinolate system is influenced by environmental factors, the effect of sulphate on the expression of myrosinases was examined. On examining different plant organs at various stages, it was observed that sulphate induces a differential expression of myrosinase polypeptides in plants ofSinapis alba L. (white mustard). Specific myrosinase polypeptides, dependent on sulphate in the growth medium, were detected on immunoblots. Without sulphate a maximum of three polypeptides was detected in buds, two in cotyledons and one in stems and roots. In plants cultured on medium with sulphate up to four polypeptides could be observed in cotyledons, five polypeptides in buds, two in stems and one in roots. Expression of myrosinases was, in general, high in plants cultured on a medium supplemented with sulphate. In floweringS. alba plants, sulphate-starved plants showed a higher expression of myrosinase in cotyledons and stems compared to plants fed with sulphate. Sulphate-fed plants had a high expression in inflorescences and roots. The organ- and time-specific induction of the myrosinase expression is discussed in relation to sulphate metabolism and availability of sulphate under normal conditions of cultivation and in relation to protection of Brassicaceae species. This is the first evidence for a specific induction of individual myrosinase proteins.     

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.     

Studies on an Alkaline Proteinase of Aspergillus sydowi

An alkaline proteinase of Aspergillus sydowi (Bainier et Sartory) Thom et Church has been purified approximately 4.5-fold from a culture filtrate by fractionation with ammonium sulfate, treatment with acrynol and Alumina gel C_γ, and DEAE-Sephadex column chromatography. The purified proteinase obtained as needle crystals was monodisperse in both the ultracentrifuge and the electrophoresis on polyacrylamide gel.

The optimum pH and temperature for the activity were 8.0 and 40°C, respectively. Fifty per cent of the activity was lost at 45°C within ten minutes and 95% at 50°C. At 5°C, the enzyme was highly stable at the range of pH 6 to 9. None of metallic salts tested promoted the activity, but Zn⁺⁺, Ni⁺⁺ and Hg⁺⁺ were found to be inhibitory. Sulfhydryl reagent, reducing and oxidizing reagents tested except iodine had no effect on the activity, but potato inhibitor, DFP and NBS caused a marked inhibition.

The alkaline proteinase from Aspergillus sydowi was markedly protected from inactivation by the presence of Ca⁺⁺ in the enzyme solution. The protective effect of Ca⁺⁺ was influenced remarkably by the pH values of the enzyme solution, i.e., optimum concentrations of Ca⁺⁺ for the protective effect at pH 7.1, 7.5 and 7.8 were 10^?2, 10^?3 and 10^?4 M, respectively. Conversely, at higher pH values such as 9.0, Ca⁺⁺ accelerated the rate of inactivation. There was a parallelism between the loss in activity and the increase in ninhydrin-positive material in the enzyme solution.

The proteinase acted on various denaturated proteins, but not on native proteins. In digestion of casein by the proteinase, 92% of nitrogen was turned into soluble form in 0.2 m trichloroacetic acid solution, with 14~17% of peptide bonds being hydrolyzed. Casein hydrolyzed with the Asp. sydowi proteinase was further hydrolyzed by Pen. chrysogenum, B. subtilis or St. griseus proteinases, which further increased the free amino residues in the reaction mixtures. On the contrary, the Asp. sydowi proteinase reacted only slightly on casein hydrolyzed by the above-mentioned proteinases.     

Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties

Myrosinases (EC 3.2.1.147) are β-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1–TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS–PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation–inhibition responses towards ascorbic acid with maximal activity around 0.7–1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-β-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.     

Studies on the Myrosinase in Mustard Seed

In order to clarify whether the myrosinase containes only thioglucosidase, or thioglucosidase and sulfatase, chromatographic behaviors of the myrosinase on cellulose ion exchanger were studied. Myrosinase showed two peaks by the chromatography on TEAE-cellulose at pH 8.5, but the fractionation of the two enzymes was not accomplished. It was concluded that the two peaks was not ascribable to an artifact but the two closely resembled species of myrosinase existing in the sample solution. Chromatographic separation of the two enzymes on DEAE-cellulose resulted in failure. These results confirmed that the myrosinase was a single β-thioglucosidase originally.     

Characterization of a new myrosinase in Brassica napus

A full-length cDNA clone defining the new myrosinase gene family MC in Brassica napus was isolated and sequenced. Southern hybridization showed that the MC family probably consists of 3 or 4 genes in B. napus. MC genes are expressed in the developing seed, but not in the vegetative tissues investigated. In situ hybridizations to developing seeds showed that the MC genes are expressed in the myrosin cells of the embryo axis and the cotyledons. Complexes with myrosinase and myrosinase-binding protein (MBP) were purified and characterized. Sequencing of peptides from myrosinases occurring in the complexes showed that the 70 kDa myrosinase is encoded by the MC genes, whereas the 65 kDa myrosinase is encoded by the MB genes. This is in contrast to the 75 kDa myrosinase which occurs in free form and is encoded by the MA genes. Deglycosylations of the myrosinase complexes and the free myrosinase showed that the molecular sizes of the myrosinases could be reduced significantly by this treatment, and that the size differences between the different myrosinases are mainly due to differences in glycosylation.     

The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system

Glucosinolates are plant secondary metabolites that are hydrolysed by the action of myrosinases into various products (isothiocyanates, thiocyanates, epithionitriles, nitriles, oxazolidines). Massive hydrolysis of glucosinolates occurs only upon tissue damage but there is also evidence indicating metabolism of glucosinolates in intact plant tissues. It was originally believed that the glucosinolate–myrosinase system in intact plants was stable due to a spatial separation of the components. This has been coined as the ‘mustard oil bomb’ theory. Proteins that form complexes with myrosinases have been described: myrosinase-binding proteins (MBPs) and myrosinase-associated proteins (MyAPs/ESM). The roles of these proteins and their biological relevance are not yet completely known. Other proteins of the myrosinase enzyme system are the epithiospecifier protein (ESP) and the thiocyanate-forming protein (TFP) that divert the glucosinolate hydrolysis from isothiocyanate production to nitrile/epithionitrile or thiocyanate production. Some glucosinolate hydrolysis products act as plant defence compounds against insects and pathogens or have beneficial health effects on humans. In this review, we survey and critically assess the available information concerning the localization, both at the tissular/cellular and subcellular level, of the different components of the myrosinase enzyme system. Data from the model plant Arabidopsis thaliana is compared to that from other glucosinolate-producing Brassicaceae in order to show common as well as divergent features of the ‘mustard oil bomb’ among these species.     

Crystal structure at 1.1 Angstroms resolution of an insect myrosinase from Brevicoryne brassicae shows its close relationship to beta-glucosidases

The aphid Brevicoryne brassicae is a specialist feeding on Brassicaceae plants. The insect has an intricate defence system involving a beta-D-thioglucosidase (myrosinase) that hydrolyses glucosinolates sequestered from the host plant into volatile isothiocyanates. These isothiocyanates act synergistically with the pheromone E-beta-farnesene to form an alarm system when the aphid is predated. In order to investigate the enzymatic characteristics of the aphid myrosinase and its three-dimensional structure, milligram amounts of pure recombinant aphid myrosinase were obtained from Echerichia coli. The recombinant enzyme had similar physiochemical properties to the native enzyme. The global structure is very similar to Sinapis alba myrosinase and plant beta-O-glucosidases. Aphid myrosinase has two catalytic glutamic acid residues positioned as in plant beta-O-glucosidases, and it is not obvious why this unusual enzyme hydrolyses glucosinolates, the common substrates of plant myrosinases which are normally not hydrolyzed by plant beta-O-glucosidases. The only residue specific for aphid myrosinase in proximity of the glycosidic linkage is Tyr180 which may have a catalytic role. The aglycon binding site differs strongly from plant myrosinase, whereas due to the presence of Trp424 in the glucose binding site, this part of the active site is more similar to plant beta-O-glucosidases, as plant myrosinases carry a phenylalanine residue at this position.     

Myrosinase in Brassicaceae: I. LOCALIZATION OF MYROSINASE IN CELL FRACTIONS OF ROOTS OF Sinapis alba L.

Myrosinases (thioglucoside glucohydrolases E.C. 3.2.3.1 [EC] .), whichcatalyse the hydrolysis of glucosinolates present in Brassicaceae,were isolated from Sinapis alba L. seeds. The crude enzyme extractwas purified using gel and ion-exchange chromatography, isoelectricfocusing, and polyacrylamide gel electrophoresis. The separationof two myrosinase isoenzymes was obtained after gel chromatographyon Sephadex G-100. Further purification of the main myrosinasecomponents was achieved when the combined isoenzymes were separatedon the anion-exchanger DEAE-Sephadex A-50 followed by polyacrylamidegel electrophoresis. A similar purification was obtained when the crude extract wasgroup-fractionated on Sephadex G-50 followed by DEAE-cellulosechromatography on Whatman DE-52 and gel chromatography on SephadexG-200. The enzyme from the last step was further separated byisolectric focusing into two isoenzymes with isoelectric points4.9 and 6.2. In order to clarify where the myrosinase was localized in theroot tip cells, cell fractionation studies were performed usingaldehydes as pre-fixatives to stabilize the enzymes and thecell organelles. Biochemical tests of crude and purified samplesof the isolated myrosinases showed that when glutaraldehydeor formaldehyde were used as pre-fixatives at a final concentrationof 1% (w/v), they did not inhibit the enzyme activity. Relativelyhomogeneous cell organelle fractions were obtained using ultracentrifugationand stepwise sucrose gradients. The myrosinase activity expressedon the basis of the protein content was found to be highestin the dictyosome and smooth endoplasmic reticulum fractions     

Characterization and evolution of a myrosinase from the cabbage aphid Brevicoryne brassicae.

The aphid myrosinase gene has been elucidated using Rapid Amplification of cDNA Ends-PCR. Sequencing has shown that aphid myrosinase has significant sequence similarity (35%) to plant myrosinases and other members of glycosyl hydrolase family 1 (GHF1). The residues acting as proton donor and nucleophile, in the hydrolysis of glucosinolates by aphid myrosinase, are identified as Glu 167 and Glu 374 respectively. The equivalent residues in plant myrosinase are Gln 187 and Glu 409 and for the cyanogenic beta-glucosidase Glu 183 and Glu 397. Thus it would appear that the absence of a proton donor is not necessary for the hydrolysis of glucosinolates as was thought to be the case for the plant myrosinases. Aphid myrosinase appears to be more similar to animal beta-O-glucosidases than to plant myrosinases, as assessed by sequence similarity and phylogenetic techniques. These results strongly suggest that myrosinase activity has twice arisen from beta-O-glucosidases in plants and animals. Comparison of aphid myrosinase with plant myrosinase has highlighted Lys 173 and Arg 312 as possibly playing a crucial role in the hydrolysis of glucosinolates by aphid myrosinase.     

Purification and properties of two β-glucosidases isolated from Aspergillus niger

The cellulase complex of the fungus Aspergillus niger (strain CBS 554.65 = ATCC 16 888) was fractionated by gel filtration yielding six pronounced peaks. Only proteins from the fraction corresponding to the first peak (96 kDa) showed β-glucosidase activity vs. the substrate 4-nitrophenyl-β-D-glucopyranoside (pNPG). These proteins have been fractionated by chromatofocusing, yielding two β-glucosidases (I and II) which are shown to be homogeneous in isoelectric focusing experiments (pI = 4.6 and 3.8, respectively). Kinetic experiments with pNPG, MU-glucopyranoside and cellobiose revealed that both types of β-glucosidases behave like aryl-β-glucosidases. β-Glucosidase-I acting on pNPG exhibits a split kinetics characterized by high and low substrateconcentration kinetics which are differentiated by different values of V and of K_m. In addition, β-glucosidase-II is shown to be an exo-glucohydrolase as deduced from experiments with MU-cellobiopyranoside. Experimental features should be emphasized; usual soft-gel ion-exchange materials did not work in the chromatofocusing separation of the two β-glucosidases, in contrast to the 10μ-Si 500 = DEAE exchange material (Serva) typically used in HPLC-experiments. Furthermore, protein content determinations based on different procedures yielded widely differing values.     

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.     

Studies on the Decomposition of Sinalbin

In the mustard paste, sinalbin is hydrolyzed by myrosinase to p-hydroxybenzyl isothiocyanate (I), sinapine acid sulfate and glucose. It was found that the three decomposition products were formed from sinalbin, and two of them were isolated from the mustard paste and identified as p-hydroxybenzyl alcohol (II) and di-(p-hydroxybenzyl)-disulfide (IV), respectively. II was a major product and IV was a minor product.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Myrosinase in Brassicaceae (Cruciferae): III. EFFECT OF ASCORBIC ACID ON MYROSINASES FROM SINAPIS ALBA L. SEEDLINGS

The effect of L-ascorbic acid (AA) on myrosinase activity wasstudied in white mustard, Sinapis alba L. Enzyme extracts wereprepared from different organs of dark- and light-grown seedlings.The highest activation caused by AA was found for myrosinasefrom light-grown primary roots. The activation level was morethan 12 times higher than that of the control. The maximum activationgenerally occurred at 1–10 mM AA, and in the case of myrosinasefrom light-grown hypocotyls even at 50 mM AA. The myrosinasesfrom cotyledons were least affected by addition of AA. To studythe effect of AA on different isoenzymes of myrosinases, theisoenzymes were separated on polyacrylamide gels. When the gelswere incubated in an AA solution, after electrophoresis differenceswere found in the activation of isoenzymes of different myrosinases.     

Characterization of a Brassica napus myrosinase expressed and secreted by Pichia pastoris

In Brassica napus three different gene families with different temporal and tissue-specific expression and distribution patterns encode myrosinases (thioglucoside glucohydrolases, EC 3.2.3.1). Myrosinases encoded by the MA gene family are found as free and soluble dimers, while myrosinases encoded by the MB and MC gene families are mainly found in large insoluble complexes associated with myrosinase-binding proteins and myrosinase-associated proteins. These large complexes impede purification and characterization of MB and MC myrosinases from the plant. We used Pichia pastoris to express and secrete functional recombinant MYR1 myrosinase from B. napus to allow further characterization of myrosinase belonging to the MB gene family. The purified recombinant myrosinase hydrolyzes sinigrin with a K(m) of 1.0 mM; the specific activity and calculated k(cat)/K(m) were 175 U/mg and 1.9 x 10(5) s(-1) M(-1), respectively. A novel in-gel staining method for myrosinase activity is presented.