Enzyme Fraction Research Articles

Enzymatic degradation of granular potato starch by Microbacterium aurum strain B8.A

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules.

Sample preparation in separation of the extracellular chitinolytic enzymes of the human intestinal bacterium Clostridium paraputrificum J4 from the culture fluids

Membrane ultrafiltration (UF) was used in sample preparation of the culture fluids of the human intestinal bacterium Clostridium paraputrificum strain J4 containing seven extracellular chitinolytic isoenzymes (38–90 kDa). The subsequent filtration of the bacteria-free supernatants was carried out through Millipore membranes with cut-off 100 and 30 kDa for separation of undigested components of the culture medium and bacterial metabolites with molecular weight higher and lower than that of the target enzymes. The chitinolytic enzymes, which were the minor components in the culture fluids, were concentrated at UF as well. The aim of the research consisted in evaluation of the effect of component composition of bacteria-free supernatants and the chemical nature of membrane active layer on partial fractionation of the chitinolytic enzymes, their recovery in retentates and purification degree. On the basis of the obtained experimental results, the sample preparation procedure of the culture fluids of C. paraputrificum J4 was established to be used further in chromatographic separations of the chitinolytic enzymes.

Unusual DNA-Binding Kinetics of the Restriction Endonuclease EcoRV

We have applied a self-cleavage assay, developed previously by us, to measure EcoRV-DNA binding in solution. Self-cleavage assay monitors only enzymatically competent complexes of the endonuclease. This technique does not have the limitations of more commonly used gel mobility shift assay while providing the same level of sensitivity. We found that the EcoRV has quite unusual kinetics of specific complex formation in the absence of divalent ions that was not reported previously. A significant fraction of the total enzyme, ∼ 45%, forms enzymatically competent complexes unusually slowly, especially at pH 7.6. This novel result can be explained by a very slow transition between two conformations of the free enzyme in solution. The equilibrium distribution of the slowly and quickly associating protein structures and their exchange kinetics may depend on many parameters including pH, salt, osmolytes, and divalent cations. The observation of at least two kinetics components in association indicates that EcoRV is an allosteric protein with at least two conformations. Allosterism is now recognized as important concept for DNA-protein complexes, offering an additional level of control over binding and activity. We are continuing our investigation into the EcoRV structures responsible for the different kinetic classes of association.

Single Molecule Processivity of SOS-cat, the Catalytic Core of the Ras Gef ‘son of Sevenless’

The Ras GEF Son of Sevenless (SOS) activates the membrane-anchored G-protein Ras by catalyzing the replacement of Ras bound GDP with GTP. We have previously shown that in addition to the catalytic site, SOS has a catalytically inactive allosteric binding site for Ras, which allows SOS to localize and up-concentrate at Ras presenting membranes, dramatically increasing the Ras-GDP turnover rate. Together, the catalytic and allosteric sites form the catalytic core of SOS (SOScat). In the present work we use TIRF fluorescence microscopy to show that in vitro, single SOScat enzymes can be highly processive, remaining surface bound via Ras in the allosteric site, while turning over hundreds to thousands of Ras with the catalytic site. By confining individual SOScat enzymes to micron-scale two-dimensional supported lipid bilayer ‘reaction chambers’ we can monitor arrays of hundreds to thousands of single SOScat enzymes, probing the variability in turnover rate and processivity within the enzyme ensemble. We show that only a small fraction of the SOScat enzymes are processive, and that this fraction is modulated by the nucleotide binding state of Ras, with Ras-GTP promoting a higher fraction of processive SOScat than Ras-GDP.

Comparative Study on the Ability to Produce Gentiobiose in Cellulose-producing Bacteria Asaia bogorensis and Gluconacetobacter xylinus

In a cellulose-producing bacterium Gluconacetobacter xylinus, endo-β-1,4-glucanase and β-glucosidase are suggested to have an important role in cellulose biosynthesis, and genes encoding these enzymes are present adjacent to the cellulose synthesis operon. However, in other cellulose-producing bacteria including Asaia bogorensis, β-glucosidase gene is not found next to the cellulose synthesis operon, and their cellulose productivities are lower than that of G. xylinus. To investigate whether A. bogorensis produces a similar type of β-glucosidase, crude enzyme fractions were prepared from G. xylinus and A. bogorensis. The hydrolytic activities toward p-nitrophenyl-β-D-glycosides were compared. In A. bogorensis, the hydrolytic activities were found in the cytosolic fraction, but the fraction did not indicate an ability to produce gentiobiose that is suggested to be an inducer for expression of the endo-β-1,4-glucanase gene in G. xylinus. We conclude that the mechanism of cellulose biosynthesis regulated by endo-β-1,4-glucanase may be different in A. bogorensis and G. xylinus.

Characterization of filtered honey by electrophoresis of enzyme fractions

According to both the European Council’s Honey Directive 2001/110/EC and the present Codex Aliminerius Honey Standard, filtration of honey is permitted. After this filtration process, the microscopic determination of the botanical and geographical origin of honeys is no longer possible since all the pollen has been removed. In many honey countries, there is a considerable difference in the price of honey depending on the botanical and geographical origin. There is the risk of fraud if expensive unfiltered honey is mixed with cheap filtered honey. In this research project, a method was developed that allows the detection of mixtures of filtered and unfiltered honey. Comparative tests showed that enzyme activities, mainly sucrase, were influenced by this process. The protein content did not decrease. Sucrase was isolated by gel chromatography and analysed by gel electrophoresis. One of the two dominating protein bands with 40 kDa and 65 kDa decreased significantly after filtration, which led to a shift in the natural ratio between them. The quantitative densitometric analysis of these two protein bands allows the detection of 15% added filtered honey.

Production of Iron-Binding Peptides from Colostral Whey by Enzymatic Hydrolysis

Colostral whey prepared from colostrum (pooled from first six post-partum milkings) was heated for 10 min at <TEX>$100^{\circ}C$</TEX> Heated colostral whey was incubated with 1% enzymes (protein equivalent basis) for 15, 30, 60, 90, and 120 min at <TEX>$50^{\circ}C$</TEX>. Papain, pepsin, trypsin, and alcalase produced different degrees of hydrolysis (DH), 10.66%, 12.42%, 10.83%, and 25.31%, respectively, at an incubation time of 120 min. The SDS-PAGE reveals that significant amounts of bovine serum albumin (BSA), <TEX>${\beta}$</TEX>-lactoglobulin (<TEX>${\beta}$</TEX>-LG), and <TEX>${\alpha}$</TEX>-lactalbumin (<TEX>${\alpha}$</TEX>-LA) survived papain digestion. In contrast, pepsin completely removed BSA but not <TEX>${\beta}$</TEX>-LG present in heated colostral whey. Alcalase completely eliminated BSA, <TEX>${\beta}$</TEX>-LG, and <TEX>${\alpha}$</TEX>-LA. This differential hydrolysis was confirmed by reversed-phase HPLC analysis. Using ion-exchange chromatography, fraction-1 (F-1) was obtained from alcalase hydrolysate at a NaCl gradient concentration of 0.25 M. Reversed-phase HPLC chromatograms of alcalase F-1 showed numerous small peaks, which probably indicate that a variety of new peptides were produced. Iron content of alcalase F-1 was 28.94 ppm, which was the highest among all enzyme fractions, whereas iron content of colostral whey was 36.56 ppm. Main amino acids contained in alcalase F-1 were Thr (15.45%), Glu (14.12%), and Ser (10.39%). Therefore, alcalase can be used to generate good iron-binding peptides in heated colostral whey, and the resulting iron-binding peptides could be suitable as a value-added food ingredient for food supplements.

Bioassay based screening of steroid derivatives in animal feed and supplements

Receptor binding transcription activation bioassays are valuable tools for the screening of steroid hormones in animal feed and supplements. However, steroid derivatives often lack affinity for their cognate receptor and do not show any direct hormonal activity by themselves. These compounds are thus not detected by these kinds of bioassays and need a bioactivation step in order to become active, both in vivo and in vitro. In this study a comparison was made between different in vitro activation methods for hormone esters and hormone glycosides. Testosterone acetate and testosterone decanoate were chosen as model compounds for the hormone esters, representing the broad range of steroid esters of varying polarities, while genistin was used as a substitute model for the steroid-glycosides. Concerning bioactivation of the steroids esters, the efficiency for alkaline hydrolysis was 90–100% and much better as compared to enzymatic deconjugation by esterase. As a result 1 μg testosterone ester per gram of animal feed could easily be detected by a yeast androgen bioassay. When comparing different enzyme fractions for deglycosilation, genistin was shown to be deconjugated most efficiently by β-glucuronidase/aryl sulfatase from Helix pomatia, resulting in a significant increase of estrogenic activity as determined by a yeast estrogen bioassay. In conclusion, chemical and enzymatic deconjugation procedures for ester and glycoside conjugates respectively, resulted in a significant increase in hormonal activity as shown by the bioassay readouts and allowed effective screening of these derivatives in animal feed and feed supplements.

Biochemical study for the effect of henna (Lawsonia inermis) on Escherichia coli

Leaf samples of Lawsonia inermis (Li) were examined for their antimicrobial potential. Broth extracts in different concentrations were prepared and bioassayed in vitro for the growth of Escherichia coli. The growth of E. coli pathogen was inhibited to various degrees by increasing the concentration of the herbal powder. In addition to the observed alterations which were detected electrophoretically in the protein pattern, were activities of the amylase enzyme and glycoprotein fractions. The protein pattern has one common band of Rf 0.47 and two characteristic bands of Rf 0.36 and Rf 0.42 for E. coli sample. The quantitative mutation was observed in the bacteria with different concentrations of L. inermis compared with the control. Some types of proteins in E. coli completely disappeared upon being S.I affected. The amylase pattern showed one common band with Rf 0.037 and two characteristic bands with Rf 0.18 and Rf 0.37 for E. coli sample. The obvious quantitative mutation observed in bacteria with different concentrations of L. inermis compared with E. coli. The glycoprotein pattern recorded one common band at R1 with Rf 0.94 for E. coli sample and bacteria inoculated with different concentrations of L. inermis. These results confirmed the antibacterial activity of henna leaves and supported the traditional use of the plant in therapy of bacterial infections and disturbances that occurred at the biochemical level. The broth extract of the L. inermis leaves showed obvious antibacterial activity against E. coli.

Salicylic acid increased aldose reductase activity and sorbitol accumulation in tomato plants under salt stress

Increased aldose reductase (ALR) activities were detected in the leaf tissues of tomato plants grown for 3 weeks in culture medium containing 10−7 or 10−4 M salicylic acid (SA), and in the roots after the 10−4 M SA pretreatment. The ALR activity changed in parallel with the sorbitol content in the leaves of the SA-treated plants. Salt stress elicited by 100 mM NaCl enhanced the accumulation of sorbitol in the leaves of control plants and as compared with the untreated control the sorbitol content in the SA-pretreated leaves remained elevated under salt stress. DEAE cellulose anionexchange column purification of the protein precipitated with 80 % (NH4)2SO4 revealed two enzyme fractions with ALR activity in both the leaf and the root tissues. The fraction of the leaf extract that was not bound to the column reacted with glucose and glucose-6-P as substrates, whereas glucose was not a substrate for the bound fraction or for root isoenzymes. The root enzyme was less sensitive to salt treatment: 50 mM NaCl caused 30 % inhibition in the leaf extract, whereas the enzyme activity of the root extract was not affected. It is suggested that increased ALR activity and sorbitol synthesis in the leaves of SA-treated tomato plants may result in an improved salt stress tolerance.

ChemInform Abstract: Mechanisms of Mammalian Selenocysteyl-tRNA Synthesis

Abstract The mechnisms of synthesis of mammalian selenocysteyl-tRNA were studied with murine active enzyme fractions. (75Se)Selenocysteyl-tRNA was synthesized in vitro by 500kDa protein from seryl-tRNA and (75Se)HSe-, which should be activated with ATP by 20kDa protein. This and other factors in mamalian systems are compared with those in Escherichia coli.

Characterization of dextran-producing Weissella strains isolated from sourdoughs and evidence of constitutive dextransucrase expression

The study of exopolysaccharide production by heterofermentative sourdough lactic acid bacteria has shown that Weissella strains isolated from sourdoughs produce linear dextrans containing α-(1→6) glucose residues with few α-(1→3) linkages from sucrose. In this study, several dextran-producing strains, Weissella cibaria and Weissella confusa, isolated from sourdough, were characterized according to carbohydrate fermentation, repetitive element-PCR fingerprinting using (GTG)(5) primers and glucansucrase activity (soluble or cell-associated). This study reports, for the first time, the characterization of dextransucrase from Weissella strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ polymer production (after incubation with sucrose) from enzymatic fractions harvested from both sucrose and glucose culture media. Results demonstrate that dextransucrase activity was mainly soluble and associated with a constitutive 180-kDa protein. In addition, microsequencing of the active dextransucrase from W. cibaria LBAE-K39 allowed the design of specific primers that could detect the presence of glucansucrase encoding genes similar to GTFKg3 of Lactobacillus fermentum Kg3 and to DSRWC of W. cibaria CMU. This study hence indicates that sourdough Weissella strains synthesize original dextransucrase.

Analysis of a thermophilic lignocellulose degrading microbial consortium and multi-species lignocellulolytic enzyme system

Degradation of lignocellulosic agro-industrial residues by means of complex microbial community is a promising approach providing efficient biomass decomposition for subsequent conversion to value-added products. In this study, an active thermophilic lignocellulose degrading microbial consortium was bred from high-temperature sugarcane bagasse compost by successive subcultivation under aerobic static conditions. The microbial consortium showed efficient degradation activity on potential biorefinery cellulosic substrates, including bagasse, rice straw, corn stover and industrial eucalyptus pulp sludge. The consortium was structurally stable with the co-existence of eight major microbes, comprising anaerobic bacterial genera Clostridium and Thermoanaerobacterium along with an aerobic/facultative anaerobic Rhodocyclaceae bacterium, bacilli, and uncultured bacteria. Majority of the lignocellullolytic activities including endo-glucanase, xylanase and β-glucanase was present in the crude culture supernatant compared to the cell-bound fraction. Proteomic analysis of cellulose bound fraction of the crude extracellular enzyme revealed a multi-species lignocellulolytic enzyme system composed mainly of cellulosomal components and extracellular cellulases of clostridia along with hemicellulases and a β-glucanase from Clostridium, Bacillus, and Thermobacillus related origins. This work presents the first report on analysis of the complex structurally stable lignocellulose degrading microbial consortium together with the characterization of its lignocellulolytic enzyme system applicable for biomass degradation and conversion in biotechnological industry.

A modified spectrophotometric assay to estimate deglycosylation of steroidal saponin to sapogenin by mixed ruminal microbes

The lack of a method for measuring deglycosylation of saponins in ruminal fluid has limited the ability to investigate the impact of these compounds on rumen microorganisms. A simple spectrophotometric assay was adapted and a protocol developed to enable measurement of steroidal saponin and sapogenin in ruminal fluid. The procedure was used for in vitro determination of deglycosylation activity of rumen bacteria obtained from cattle fed or not fed Yucca schidigera saponin, and to determine the relative deglycosylase activities of extracellular and cell-associated enzymes from ruminal content. Modifications to the spectrophotometric assay (i.e. heating time shortened to 10 min and 0.5 mL dH(2)O added to the reaction mixture) improved the stability of the optical density (425 nm) of the chromophore for up to 24 h post-reaction. Centrifugation (12 000 x g, 20 min) enabled differential estimations of steroidal saponin and sapogenin in ruminal fluid. Steroidal saponin added to defaunated ruminal fluid (dRF) or clarified ruminal fluid (cRF) was recovered completely from the mixture as saponin + sapogenin (99.1% and 100.6%, respectively), whereas saponin recovery from the supernatant of dRF was greatly reduced (P < 0.001) compared to that from supernatant of cRF (58.5 vs. 98.7%). Saponin recoveries from the supernatants of dRF and cRF did not differ between donor cattle fed or not fed Yucca schidigera saponin (59.2 vs. 57.3% and 98.4 vs. 99.3%, respectively). The majority (89-90%) of saponin added to a ruminal extracellular enzyme preparation was recoverable in supernatant after 24 h, compared with only 26-32% remaining in supernatant from incubation with a cell-associated enzymes fraction. Mixed rumen bacteria deglycosylate steroidal saponin to sapogenin, at activity levels unaffected by prior exposure to saponin, but they were unable to degrade the sapogenin core structure. Deglycosylation activity occurred primarily in the cell-associated enzyme fraction of ruminal content.

Deciphering the physiological blueprint of a bacterial cell

During the last few months, several pioneer genome-wide transcriptomic, proteomic and metabolomic studies have revolutionised the understanding of bacterial biological processes, leading to a picture that resembles eukaryotic complexity. Technological advances such as next-generation high-throughput sequencing and high-density oligonucleotide microarrays have allowed the determination, in several bacteria, of the entire boundaries of all expressed transcripts. Consequently, novel RNA-mediated regulatory mechanisms have been discovered including multifunctional RNAs. Moreover, resolution of bacterial proteome organisation (interactome) and global protein localisation (localizome) have unveiled an unanticipated complexity that highlights the significance of protein multifunctionality and localisation in the cell. Also, analysis of a complete bacterial metabolic network has again revealed a high fraction of multifunctional enzymes and an unexpectedly high level of metabolic responses and adaptation. Altogether, these novel approaches have permitted the deciphering of the entire physiological landscape of one of the smallest bacteria, Mycoplasma pneumoniae. Here, we summarise and discuss recent findings aimed at defining the blueprint of any prokaryote.

Prodrug Activation by Cryptosporidium Thymidine Kinase

Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k(cat) = 0.28 s(-1) and K(m)(,ATP) = 140 microm are similar to those of human TK1, the value of K(m)(thymidine) = 48 microm is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.

Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers

Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers ( Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase, and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000–400,000 units) of SSS fractions and 187% (40,000–50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50 K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate.

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5'-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

Cross-talk between Mitochondrial Malate Dehydrogenase and the Cytochrome bc1 Complex

The interactions between the mitochondrial cytochrome bc(1) complex and matrix-soluble proteins were studied by a precipitation pulldown technique. Purified, detergent-dispersed bc(1) complex was incubated with mitochondrial matrix proteins followed by dialysis in the absence of detergent. The interacting protein(s) was co-precipitated with bc(1) complex upon centrifugation. One of the matrix proteins pulled down by bc(1) complex was identified as mitochondrial malate dehydrogenase (MDH) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and confirmed by Western blotting with anti-MDH antibody. Using a cross-linking technique, subunits I, II (core I and II), and V of the bc(1) complex were identified as the interacting sites for MDH. Incubating purified MDH with the detergent dispersed bc(1) complex results in an increase of the activities of both the bc(1) complex and MDH. The effect of the bc(1) complex on the activities of MDH is unidirectional (oxaloacetate --> malate). These results suggest that the novel cross-talk between citric acid cycle enzymes and electron transfer chain complexes might play a regulatory role in mitochondrial bioenergetics.

In-vitro depolymerization of Scutia buxifolia leaf-litter by a dominant Ascomycota Ciliochorella sp.

Scutia buxifolia leaf-litter is recalcitrant to degradation but hosts an assemblage of Ascomycota fungi among which Ciliochorella sp. is the most frequent one. The aim of this work was to analyze the role Ciliochorella sp. plays in degradation of S. buxifolia leaf-litter in-vitro. Decay was assessed based on mass loss and Fourier transform infrared (FT-IR) spectroscopy analysis. After 60 days of fungal growth, there was a 4.7% mass reduction that was associated with degradation of polysaccharides, proteins and lignin-related molecules. Ciliochorella sp. synthetized and released to the water-soluble fraction of leaf-litter enzymes with β-1,4 endoglucanase activity, that increased 3-fold over a 30 day period, and no laccase and peroxidase activity. We conclude that Ciliochorella sp. transformed S. buxifolia litter by degrading polysaccharides and also aromatic structures at early stages of decay.