Modeling Based Structural Insights into Biodegradation of the Herbicide Diuron by Laccase-1 from Ceriporiopsis subvermispora

The herbicide diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is used in many agricultural crops and non-crop areas worldwide, leading to the pollution of the aquatic environment by soil leaching. White rot fungi and its lignin modifying enzymes, peroxidases and laccases, are responsible for its degradation. Therefore, it is of interest to explore the potential use of Ceriporiopsis subvermispora laccase (CersuLac1) in the biotransformation of this herbicide by using its enzyme laccase. However, the structure of laccase from Ceriporiopsis subvermispora is still unknown. Hence, a model of laccase was constructed using homology modeling. The model was further used to dock p-methylbenzoate in the presence of four copper ions to analyze molecular basis of its binding and interaction. The ligand-protein interaction is stereo-chemically favorable in nature. The presence of the single protonated Lys457 was necessary for catalysis, being coordinated by a cupper ion. The best pose of diuron on CersuLac1 has a theoretical Ki of 2.91 mM. This is comparable to the KM values for laccases from other organisms with similar compounds. Thus, we document the insights for the potential use of laccase from Ceriporiopsis subvermispora in the biotransfrormation of diuron.     

Characterization of white zones produced on Pinus radiata wood chips by Ganoderma australe and Ceriporiopsis subvermispora

White zones produced on biodegraded Pinus radiata wood chips were characterized by micro-localized-FTIR (Fourier Transformed Infra Red) spectroscopy and scanning electron microscopy. Both techniques permitted assignment of the white zones to a selective lignin removal process. Although both fungi studied have degraded lignin selectively in these restricted superficial areas, chemical analysis of the wood chips indicated that Ganoderma australe removed 16% of the initial amount of glucan at the 20% weight loss level. Ceriporiopsis subvermispora did not remove glucan at weight loss values below 17%. Prolonged biodegradation resulted in reduction of white zones by G. australe, and increased white zones from C. subvermispora decayed samples. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.     

Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1–6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.     

Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm

An extracellular beta-glucosidase was purified from culture filtrates of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (PNPG) as the substrate, with a K(m) of 1.52 mM, and V(max) of 3.21 U min mg(-1) protein. Glucose competitively inhibited beta-glucosidase with a K(i) value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 degrees C. The enzyme was stable at pH 5.0 at temperatures up to 50 degrees C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-beta-d-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca(2+), Co(2+), Mg(2+), Mn(2+), glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg(2+). The internal amino acid sequences of D. eschscholziibeta-glucosidase have similarity to the sequences of the family 3 beta-glucosyl hydrolase.     

Purification and specificity of two alpha-glucosidase isoforms of the parasitic protist Trichomonas vaginalis

Two isoforms of alpha-glucosidase were purified from the parasitic protist Trichomonas vaginalis. Both consisted of 103 kDa subunits, but differed in pH optimum and substrate specificity. Isoform 1 had a pH optimum around 4.5 and negligible activity on glucose oligomers other than maltose, while isoform 2 with a pH optimum of 5.5 hydrolyzed also such substrates at considerable rates. Neither had activity on glycogen or starch. Isoform 1 had a specific activity for hydrolysis of maltose of 30 U/mg protein and isoform 2 101 U/mg protein. The Km values were 0.4 mM and 2.0 mM, respectively. Isoform 2 probably corresponds to the activity detected on the cell surface.     

β-Glucosidase from the cellulolytic system of Alternaria alternata autolyzed cultures

Abstract A β-glucosidase from centrifugated autolyzed cultures of Alternaria alternata has been purified 71 times by Sephadex G-200, CM-Biogel A and DEAE-Biogel A successively. The enzyme is a glycoprotein with 16% sugar and a M _r of 160 000, formed by two subunits of 60 000 and 80 000. The enzyme has optimum pH of 5 units and optimum reaction temperature of 50°C, being stable in a pH range of 3–8 and 0 to 60°C. The enzyme hydrolyzes different substrates showing maximum affinity and maximum hydrolysis velocity on cellobiose. The β-glucosidase is inhibited by gluconolactone but not by 10 mM glucose.     

Differences in cellulose digestive systems among castes in two termite lineages

Abstract.  Termites (Isoptera) are eusocial insects and express polyphenism. Soldiers have specialized morphology for colony defense, but their feeding activity is dependent on other colony members. To determine differences in cellulose degradation between soldier and worker termites, enzymatic activity and cellulase gene expression, as well digestive tract histology, are examined in two phylogenetically distant species. In Hodotermopsis sjostesti (family Termopsidae) , endo-β-1,4-glucanase activity is identified in the salivary glands, whereas β-glucosidase activity is identified in salivary glands and hindgut. The relative expression levels of endo-β-1,4-glucanase genes in soldiers are significantly lower than in workers. Thin sections of salivary gland of workers and soldiers are different in H. sjostedti . In Nasutitermes takasagoensis (family Termitidae), the endo-β-1,4-glucanase activity is restricted to the midgut in four tested castes (i.e. three types of workers and soldier). Examination of activity per termite reveals the highest activity in minor workers and the lowest activity in major workers and soldiers. The β-glucosidase activity is also concentrated on the midgut in all four castes. The relative expression level of the endo-β-1,4-glucanase gene does not correspond with its activity in the midgut. In thin sections prepared from N. takasagoensis , the folds and pulvillus in the gizzards, and cuticle structure of soldiers are less developed compared with the other three worker castes. The differences in digestive system among termite castes in terms of caste development in each species are discussed.     

Partial purification of cell wall β-galactosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

The protein extracted from the cell wall of the epicotyls of Cicer arietinum L. cv. Castellana was separated by ion exchange chromatography in four different fractions with β-D-galactosidase (EC 3.2.1.23) activity. These were called βI, βII, βIII and βIV, according to their order of elution. βII was associated with a particularly high β-D-glucosidase (EC 3.2.1.21) activity. Gel filtration chromatography of each of the fractions gave further subdivision of fractions βI and βIII. Subfractions 1 βI, 1 βII and 1 βIV have glucosidase activity and subfractions 2 βI and 2 βIII have galactosidase activity.
The studies on the hydrolytic capacity of these fractions and its relationship with the autolytic process seem to show that subfraction 2 βIII is responsible for autolysis. The release of total and reducing sugars is very similar for autolysis and hydrolysis by 2 βIII. The sugars released are mainly galactose and, to a lesser extent arabinose and glucose. Galactose is released as a monosaccharide, while arabinose remains associated to a polysaccharide component together with glucose and small amounts of galactose.     

β-Glucosidase Catalyzing Specific Hydrolysis of an Iridoid β-Glucoside from Plumeria obtusa

An iridoid β-glucoside, namely plumieride coumarate glucoside, was isolated from the Plumeria obtusa （white frangipani） flower. A β-glucosidase, purified to homogeneity from P. obtusa, could hydrolyze plumieride coumarate glucoside to its corresponding β-O-coumarylplumieride. Plumeria β-glucosidase is a monomeric glycoprotein with a molecular weight of 60.6 kDa and an isoelectric point of 4.90. The purified β-glucosidase had an optimum pH of 5.5 for p-nitrophenol （pNP）-β-D-glucoside and for its natural substrate. The Km values for pNP-β-D-glucoside and Plumeria β-glucoside were 5.04±0.36 mM and 1.02±0.06 mM, respectively. The enzyme had higher hydrolytic activity towards pNP-β-D-fucoside than pNP-β-D-glucoside. No activity was found for other pNP-glycosides. Interestingly, the enzyme showed a high specificity for the glucosyl group attached to the C-7＂ position of the coumaryl moiety of plumieride coumarate glucoside. The enzyme showed poor hydrolysis of 4-methylumbelliferyl-β-glucoside and esculin, and did not hydrolyze alkyl-β-glucosides, glucobioses, cyanogenic-β-glucosides, steroid β-glucosides, nor other iridoid β-glucosides. In conclusion, the Plumeria β-glucosidase shows high specificity for its natural substrate, plumieride coumarate glucoside.     

Evidence of different fermentation behaviours of two indigenous strains of Saccharomyces cerevisiae and Saccharomyces uvarum isolated from Amarone wine

Aims:  To explain the role of Saccharomyces cerevisiae and Saccharomyces uvarum strains (formerly Saccharomyces bayanus var. uvarum ) in wine fermentation.
Methods and Results:  Indigenous Saccharomyces spp. yeasts were isolated from Amarone wine (Italy) and analysed. Genotypes were correlated to phenotypes: Melibiose⁻ and Melibiose⁺ strains displayed a karyotype characterized by three and two bands between 225 and 365 kb, respectively. Two strains were identified by karyotype analysis (one as S. cerevisiae and the other as S. uvarum ). The technological characterization of these two strains was conducted by microvinifications of Amarone wine. Wines differed by the contents of ethanol, residual sugars, acetic acid, glycerol, total polysaccharides, ethyl acetate, 2-phenylethanol and anthocyanins. Esterase and β-glucosidase activities were assayed on whole cells during fermentation at 13° and 20°C. Saccharomyces uvarum displayed higher esterase activity at 13°C, while S. cerevisiae displayed higher β-glucosidase activity at both temperatures.
Conclusions:  The strains differed by important technological and qualitative traits affecting the fermentation kinetics and important aroma components of the wine.
Significance and Impact of the Study:  The contribution of indigenous strains of S. cerevisiae and S. uvarum to wine fermentation was ascertained under specific winemaking conditions. The use of these strains as starters in a winemaking process could differently modulate the wine sensory characteristics.     

Endogenous beta-glucosidase and beta-galactosidase activities from selected probiotic micro-organisms and their role in isoflavone biotransformation in soymilk

AIM: To compare endogenous beta-glucosidases and beta-galactosidases for hydrolysis of the predominant isoflavone glycosides into isoflavone aglycones in order to improve biological activity of soymilk. METHODS AND RESULTS: beta-glucosidase and beta-galactosidase activities of probiotic organisms including Lactobacillus acidophilus ATCC 4461, Lactobacillus casei 2607 and Bifidobacterium animalis ssp. lactis Bb12 in soymilk were evaluated and correlated with the increase in concentration of isoflavone aglycones during fermentation. The concentrations of isoflavone compounds in soymilk were monitored using a Varian model high-performance liquid chromatography (HPLC) with an amperometric electrochemical detector. In all micro-organisms, beta-glucosidase activity was found greater than that of beta-galactosidase. There was an increase in the aglycone concentration with incubation time because of the apparent hydrolytic action on isoflavone glycosides. Aglycone concentration in the soymilk with L. acidophilus 4461, L. casei 2607 and B. animalis ssp. lactis Bb12, increased by 5.37-, 5.52- and 6.10-fold, respectively, after 15 h of fermentation at 37 degrees C. The maximum hydrolytic potential was also observed at 15 h of fermentation for the three micro-organims coinciding with peak activities of the two enzymes. CONCLUSIONS: beta-glucosidase activity was more than 15 times higher than beta-galactosidase activity in soymilk for each of the micro-organisms during fermentation. beta-glucosidase played a greater role in isoflavone glycoside hydrolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening for beta-glucosidase and beta-galactosidase activities among probiotics in soymilk is important for the improvement of biological activity of soymilk and in the selection of micro-organisms for use in the growing industry of functional foods and beverages.     

Purification and characterization of β-amylase from leaves of potato (Solanum tuberosum)

Amylolytic activity is widely distributed in plants. In potato leaves ( Solanum tuberosum L.) the abundant amylolytic activity was found to be β-amylase (EC 3.2.1.2, a-1,4-D-glucan maltohydrolase). β-Amylase from potato leaves was purified to homogeneity for study of enzyme characteristics. The purification steps included ammonium sulphate precipitation, anion exchange chromatography, affinity chromatography and gel filtration. The end product of α-1,4-glucan degradation was maltose. The protein is a 111-kDa homo-dimer with a subunit molecular mass of 56 kDa and a pl of 5.6. The pH-optimum is 6.5 using p -nitrophenylmaltopentaoside (PNPG5) as substrate. The optimal temperature for hydrolysis is at 40°C. The enzyme is unstable at temperatures above 40°C. The K_nt-value for PNPG5 is 0.73 m M and the activity is inhibited by cyclodextrins. At a concentration of 1 m M , β-cyclodextrin is a stronger inhibitor than α-cyclodextrin (68 and 20% inhibition, respectively). Branched glucans (e.g. starch and amylopectin) are superior substrates as compared to long, essentially unbranched glucans (e.g. amylose). This study of the catalytic properties of β-amylase from potato leaves indicates the importance of β-amylase as a starch degrading enzyme.     

Effect of auxin on cell wall glycanhydrolytic enzymes in epicotyls of Cicer arietinum

Cell wall glycanhydrolytic enzymes have been related to cell wall loosening and cell growth, although the mechanism of this relationship has not been clarified. Since auxins are plant hormones that stimulate growth in elongating organs, in the present work we studied the effect of auxin on cell wall glycanhydrolytic enzymes, which were extracted with LiCl. Our results show that incubation of sections of Cicer arietinum epicotyls with indoleacetic acid elicit some minor changes in electrophoretic patterns of cell wall proteins when compared with control sections. This indicates that there is no appearance of a specific polypeptide synthesized de novo in response to the hormone, although there are increases in the intensity of some of the polypeptides, which could indicate an enhancement of wall protein biosynthesis. Brief incubation with IAA led to a general increase in the specific activities of these different cell wall enzyme fractions separated by chromatography, with the exception of the α-fraction, with α-galactosidase activity. Longer incubation resulted in an increase in the amount of protein associated with some of the enzyme fractions. In particular, it induced a large increase in the amount of protein associated with the β111-galactosidase fraction that is involved in the autolytic process of cell walls of chick-pea epicotyls. Our results indicate that auxin-enhanced growth could be the result of the action of the hormone al the level of the cell wall glycanhydrolytic proteins that have been related to the wall-loosening process.     

Isolates of Microdochium nivale and M. majus Differentiated by Pathogenicity on Perennial Ryegrass (Lolium perenne L.) and in vitro Growth at Low Temperature

Pink snow mould is a serious disease on grasses and winter cereals in cold and temperate zones during winter. To better understand the basis for the variation in pathogenicity between different isolates of Microdochium nivale and M. majus and to simplify selection of highly pathogenic isolates to use when screening for resistance to pink snow mould in perennial ryegrass, we sought traits correlated with pathogenicity. Isolates of M. nivale were more pathogenic on perennial ryegrass than isolates of M. majus, as measured by survival and regrowth of perennial ryegrass after infection and incubation under simulated snow cover. Pathogenicity as measured by relative regrowth was highly correlated with fungal growth rate on potato dextrose agar (PDA) at 2°C. Measuring fungal growth on PDA therefore seems to be a relatively simple method of screening for potentially highly pathogenic isolates. In a study of a limited number of isolates, highly pathogenic isolates showed an earlier increase and a higher total specific activity of β‐glucosidase, a cell wall‐degrading enzyme, compared with less pathogenic isolates. None of the M. majus isolates was highly pathogenic on perennial ryegrass. Our results indicate biological differences between M. nivale and M. majus and thus strengthen the recently published sequence‐based evidence for the elevation of these former varieties to species status.     

Distribution and morphology of extrafloral nectaries in some Cucurbitaceae

A study of extrafloral nectaries has been made in the Cucurbitaceae to ascertain their structure and assess their taxonomic potential. Nineteen species representing nine Old World genera and one New World genus were examined. These included Telfairia occidentalis, Telfairia pedata, Momordica charantia, Lagenaria siceraria, Citrullus lanatus, Luffa aegyptiaca, Cucurbita moschata and Trichosanthes cucumerina , which are of economic importance and cultivated in Nigeria for their leaves and/or fruits.
Observation of the regularity of ant and insect-visitors, along with tests for glucose and β-glucosidase enzymes, revealed the presence of extrafloral nectaries in nine species. Considerable variation exists in the distribution and morphology of nectaries between genera, especially in the tribe Benincaseae. The nutritional and ecological significance of the occurrence of extrafloral nectaries in Telfairia occidentalis is discussed.     

毁灭柱孢菌中一种人参皂苷Rb1水解酶的纯化及酶学性质研究

通过DEAE-纤维素阴离子交换层析、30％～80％（NH3）2SO3盐析、Sepharose CL-6B凝胶过滤层析和Mono Q HR5／5阴离子交换层析,从毁灭枉孢菌培养液中部分纯化出一种能够水解人参皂苷Rb,的β-葡萄糖苷酶F-I。F—I具有较好的pH稳定性和热稳定性,在pH4．0～11．0范围内和55℃以下表现出良好的β-葡萄糖苷酶活性,其最适pH为5．0,最适温度为55℃。EDTA、Cu^2＋和Zn^2＋对该酶活性有较强的抑制作用。底物专一性分析表明,F—I能高特异性水解人工合成的底物pNPG,还能水解β-葡萄糖苷键连接的二糖如纤维二糖和龙胆二糖,说明此酶为一种β-葡萄糖苷酶。F—I对人参皂苷Rb1表现了较强的水解活性,而对人参皂苷Rb2和Rc的水解活性较低。该酶水解人参皂苷Rb1的路径为Rb1→Rd→F2→C—K。F—I对人参皂苷Rb1的这种高效水解为稀有人参皂苷的工业制备奠定了基础。