Cloning, expression, and characterization of a new beta-agarase from Vibrio sp. strain CN41

A new agarase, AgaA(CN41), cloned from Vibrio sp. strain CN41, consists of 990 amino acids, with only 49% amino acid sequence identity with known β-agarases. AgaA(CN41) belongs to the GH50 (glycoside hydrolase 50) family but yields neoagarotetraose as the end product. AgaA(CN41) was expressed and characterized.     

Purification and characterization of a novel β-agarase, AgaA34, from Agarivorans albus YKW-34

An extracellular β-agarase (AgaA34) was purified from a newly isolated marine bacterium, Agarivorans albus YKW-34 from the gut of a turban shell. AgaA34 was purified to homogeneity by ion exchange and gel filtration chromatographies with a recovery of 30% and a fold of ten. AgaA34 was composed of a single polypeptide chain with the molecular mass of 50 kDa. N-terminal amino acid sequencing revealed a sequence of ASLVTSFEEA, which exhibited a high similarity (90%) with those of agarases from glycoside hydrolase family 50. The pH and temperature optima of AgaA34 were pH 8.0 and 40°C, respectively. It was stable over pH 6.0–11.0 and at temperature up to 50°C. Hydrolysis of agarose by AgaA34 produced neoagarobiose (75 mol%) and neoagarotetraose (25 mol%), whose structures were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy and ¹³C NMR. AgaA34 cleaved both neoagarohexaose and neoagarotetraose into neoagarobiose. The k _cat/K _m values for hydrolysis agarose and neoagarotetraose were 4.04 × 10³ and 8.1 × 10² s⁻¹ M⁻¹, respectively. AgaA34 was resistant to denaturing reagents (sodium dodecyl sulfate and urea). Metal ions were not required for its activity, while reducing reagents (β-Me and dithiothreitol, DTT) increased its activity by 30%.     

Asp271 is critical for substrate interaction with the surface binding site in β-agarase a from Zobellia galactanivorans

In the marine environment agar degradation is assured by bacteria that contain large agarolytic systems with enzymes acting in various endo- and exo-modes. Agarase A (AgaA) is an endo-glycoside hydrolase of family 16 considered to initiate degradation of agarose. Agaro-oligosaccharide binding at a unique surface binding site (SBS) in AgaA from Zobellia galactanivorans was investigated by computational methods in conjunction with a structure/sequence guided approach of site-directed mutagenesis probed by surface plasmon resonance binding analysis of agaro-oligosaccharides of DP 4-10. The crystal structure has shown that agaro-octaose interacts via H-bonds and aromatic stacking along 7 subsites (L through R) of the SBS in the inactive catalytic nucleophile mutant AgaA-E147S. D271 is centrally located in the extended SBS where it forms H-bonds to galactose and 3,6-anhydrogalactose residues of agaro-octaose at subsites O and P. We propose D271 is a key residue in ligand binding to the SBS. Thus AgaA-E147S/D271A gave slightly decreasing K_D values from 625 ± 118 to 468 ± 13 μM for agaro-hexaose, -octaose, and -decaose, which represent 3- to 4-fold reduced affinity compared with AgaA-E147S. Molecular dynamics simulations and interaction analyses of AgaA-E147S/D271A indicated disruption of an extended H-bond network supporting that D271 is critical for the functional SBS. Notably, neither AgaA-E147S/W87A nor AgaA-E147S/W277A, designed to eliminate stacking with galactose residues at subsites O and Q, respectively, were produced in soluble form. W87 and W277 may thus control correct folding and structural integrity of AgaA.     

Beta-agarases I and II from Pseudomonas atlantica. Substrate specificities

Beta-Agarase I and II were characterised by their action on agar-type polysaccharides and oligosaccharides. Beta-Agarase I, an endo-enzyme, was specific for regions containing a minimum of one unsubstituted neoagarobiose unit [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose], hydrolysing at the reducing side of this moiety. Yaphe demonstrated that agar was degraded by this enzyme to neoagaro-oligosaccharides limited by the disaccharide but with a predominance of the tetramer [Yaphe, W. (1957) Can. J. Microbiol. 3, 987-993]. Beta-Agarase I slowly degraded neoagarohexaose but not the homologous tetrasaccharide. [1-3H]Neoagarohexaitol was cleaved to neoagarotetraose and [1-3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to methylated, sulphated and unsubstituted neoagaro-oligosaccharides which were invariably terminated at the reducing end by unsubstituted neoagarobiose. The novel enzyme, beta-agarase II, was shown to be an endo-enzyme. Preliminary evidence indicated this enzyme was specific for sequences containing neoagarobiose and/or 6(1)-O-methyl-neoagarobiose. It degraded agar to neoagaro-oligosaccharides of which the disaccharide was limiting and predominant. Beta-Agarase II rapidly degraded isolated neogarotetraose and neoagarohexaose to the disaccharide. With [1-3H]neoagarohexaitol, exo-action was observed, the alditol being cleaved to neoagarobiose and [1-3H]neoagarotetraitol. Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol. Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24% of the starting carbohydrate. This fraction was almost exclusively disaccharides (22.4%) containing neoagarobiose (7.4%) and 6(1)-O-methyl-neoagarobiose (15%). Beta-Agarase II is probably the 'beta-neoagarotetraose hydrolase' reported by Groleau and Yaphe as an exoenzyme against neoagaro-oligosaccharides [Groleau, D. and Yaphe, W. (1977) Can. J. Microbiol. 23, 672-679].     

Improvement in the catalytic activity of β-agarase AgaA from Zobellia galactanivorans by site-directed mutagenesis

In this study, site-directed mutagenesis was performed on the β-agarase AgaA gene from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutant enzymes, S63K, C253I, and S63K-C253I, were 126% (1,757.78 U/mg), 2.4% (33.47 U/mg), and 0.57% (8.01 U/mg), respectively, relative to the wildtype beta-agarase AgaA (1,392.61 U/mg) at 40°C. The stability of the mutant S63K enzyme was 125% of the wild-type up to 45°C, where agar is in a sol state. The mutant S63K enzyme produced 166%, 257%, and 220% more neoagarohexaose, and 230%, 427%, and 350% more neoagarotetraose than the wild-type in sol, gel, and nonmelted powder agar, respectively, at 45°C over 24 h. The mutant S63K enzyme produced 50% more neoagarooligosaccharides from agar than the wild-type beta-agarase AgaA from agarose under the same conditions. Thus, mutant S63K β-agarase AgaA may be useful for the production of functional neoagarooligosaccharides.     

alpha-Galactosidase A from Pseudomonas fluorescens subsp. cellulosa: cloning, high level expression and its role in galactomannan hydrolysis

A library of Pseudomonas fluorescens subsp. cellulosa genomic DNA, constructed in lambda ZAPII, was screened for alpha-D-galactosidase activity. The DNA inserts from six galactosidase-positive clones were rescued into plasmids. Restriction digestion and Southern analysis revealed that each of the plasmids contained a common DNA sequence. The sequence of the Pseudomonas DNA in one of the plasmids revealed a single open reading frame (aga27A) of 1215 bp encoding a protein of M(r) 45900, designated alpha-galactosidase 27A (Aga27A). Aga27A exhibited extensive sequence identity with alpha-galactosidases in glycoside hydrolase 27, and appeared to be a single domain protein. The recombinant alpha-galactosidase was expressed at high levels in Escherichia coli and the biophysical properties and substrate specificity of the enzyme were evaluated. The data showed that Aga27A was a mesophilic neutral acting non-specific alpha-galactosidase. Both P. fluorescens subsp. cellulosa mannanase A (ManA) and Aga27A hydrolyse the polymeric substrate, carob galactomannan. Sequential hydrolysis with AgaA followed by ManA, or ManA followed by AgaA enhanced product release. The positive effects of sequential hydrolysis are discussed.     

Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by beta-agarase and HCl in liquid chromatography systems

A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by beta-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Profiles of the oligomer length in the crude product mixtures were monitored by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) and NH2-column chromatography (NH2-HPLC), coupled with an evaporative light-scattering detector (ELSD). Calibration curves were established separately to identify the DP and quantify the amount of the oligomer products analyzed in the two systems. Each system was optimized to generate a spectrum of saccharide oligomers with various DP, where the reaction yield for NAOS was 52.7% by 4U/mg beta-agarase and for AOS was 45.6% by 0.4M HCl. SEC resolved the product in size ranges consisting of DP 1-22 for NAOS and DP 1-14 for AOS. NH2-HPLC clearly resolved both distinct saccharide product sizes within DP 12. The optimized system was connected with a fraction collector to isolate and quantify these individually separated products. The total product yields of the recovered NAOS of DP 1-22 and AOS of DP 1-14 by the SEC system were 84.7% and 82.9%, respectively. NH2-HPLC recovered NAOS and AOS, both with a DP of 1-10 with total product yields of 48.9% and 90.0%, respectively. Isolated NAOS and AOS product fractions were inspected by (1)H NMR spectroscopy and ESIMS spectrometry to confirm structure, molecular mass, and purity. This study established feasible systems for the preparation and qualitative and quantitative measurements, as well as for the isolation of various sizes of oligomers generated from agarose.     

Expression of a novel hyaluronidase from Streptococcus zooepidemicus in Escherichia coli and its application for the preparation of HA oligosaccharides

Hyaluronan (HA) oligosaccharides which can stimulate angiogenesis and suppress the growth of tumors have attracted more and more attention. In order to prepare pure and well-defined oligosaccharides from high-molecular-weight HA in a rapid and simple manner, an enzymatic degradation method was developed, which included degradation with a novel recombinant hyaluronan lyase (HA lyase, hyaluronidase, or HAase) and gel permeation chromatography. The HAase protein was expressed in Escherichia coli with the expression vector pBV220. The HAase was purified and refolded, and specific activity of the enzyme solution was 3800 U/mg. HA was degraded with HAase at the optimized conditions, yielding 46% and 31% of HA disaccharides and HA tetrasaccharides, respectively. These HA oligosaccharides were conveniently separated by consecutive column chromatography on Bio-gel P6 and were identified by HPLC–MS.     

The regulation of agarase production by resting cells of Cytophaga flevensis

The regulation of the synthesis of extracellular agarase by Cytophaga flevensis was studied in resting-cell suspensions. Enzyme synthesis was strictly dependent on the presence of a suitable inducer. Enzyme production was maximal at 20 C in phosphate buffer pH 6.9 in the presence of 1.3mm calcium chloride, 0.03% casamino acids and inducer. Enzyme production was virtually the same at 15 and 20 C, reduced to 50% at 25 C and was not detectable at 30 C. It was highly stimulated by the presence of 0.03% of casamino acids in the incubation mixture and was also favoured by the presence of 1.3mm calcium ions. Of a variety of compounds tested, only melibiose or neoagaro-oligosaccharides were effective inducers. Among the neoagaro-oligosaccharides, neoagarotetraose was the best inducer. At higher concentrations of inducer compounds catabolite repression of enzyme synthesis was apparent. This was also found when glucose was added to the incubation mixture. This repression was not relieved by the addition of cyclic AMP. Indications were found that the excretion process was limiting the rate of production of extracellular enzyme.     

Hepatic catabolism of serum amyloid A during an acute phase response and chronic inflammation

Degradation of serum amyloid A (SAA) was studied in isolated perfused livers of mice treated with either a single injection of casein to induce an acute phase response or with 14 daily casein injections to maintain chronic inflammation. Littermates administered sterile saline served as controls. Radioiodinated SAA and apolipoprotein A-I, reconstituted with high-density lipoproteins in vivo, were studied in parallel. Degradation was monitored by appearance of acid-soluble radioactivity in the perfusate. Induction of an acute phase response reduced hepatic catabolism of SAA by 14% (from 8.6 +/- 1.2% to 7.4 +/- 1.1%/g liver in 3 hr, P less than 0.05, n = 16). The acute phase response had no effect on apolipoprotein A-I degradation or bile production. Livers from animals receiving 14 daily injections of casein were 31% less active than control livers at degrading SAA (8.1 +/- 1.6%/g/3 hr for treated group vs. 11.7 +/- 2.3%/g/3 hr for control group, P less than 0.025). Apolipoprotein A-I degradation was decreased but differences were not statistically significant and bile production was the same in both treatment groups. However, livers from treated animals were larger (mean weight 1.8 g) than those from controls (1.5 g) (P less than 0.05), although amyloid fibrils were not detected by Congo red stain. The size of the degradation products was analyzed by column chromatography. Elution profiles of perfusates from livers of chronically inflamed animals contained a peak corresponding to the molecular weight of amyloid A which was not present in perfusates from control liver. We conclude that hepatic catabolism of SAA is decreased both early and late in an inflammatory response and intermediate degradation products corresponding in size to amyloid A are released into the circulation following prolonged inflammation.     

Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine alpha(s1)-, beta-, and kappa-Casein

Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine alpha(s1)-, beta-, and kappa-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM(1), was concluded. The action of the HP-type proteinase P(1) (also detectable in strains Wg(2), C(13), and TR) was established by electrophoretic methods to be directed preferentially towards beta-casein. The AM(1)-type proteinase P(III) (also detectable in strain SK(11)) was also able to degrade beta-casein, but at the same time split alpha(s1)- and kappa-casein more extensively than did P(I). Strain FD(27) exhibited mainly P(I) activity but also detectable P(III) degradation characteristics. The cell wall proteinase preparation of strain E(8) showed low P(I) as well as low P(III) activity. All proteinase preparations produced from kappa-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between P(I) and P(III) in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-C-labeled beta-casein and by the effect of alpha(s1)- plus kappa-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.     

Analysis of keystone enzyme in Agar hydrolysis provides insight into the degradation (of a polysaccharide from) red seaweeds

Agars are abundant polysaccharides from marine red algae, and their chemical structure consists of alternating D-galactose and 3,6-anhydro-L-galactose residues, the latter of which are presumed to make the polymer recalcitrant to degradation by most terrestrial bacteria. Here we study a family 117 glycoside hydrolase (BpGH117) encoded within a recently discovered locus from the human gut bacterium Bacteroides plebeius. Consistent with this locus being involved in agarocolloid degradation, we show that BpGH117 is an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase that removes the 3,6-anhydrogalactose from the non-reducing end of neoagaro-oligosaccharides. A Michaelis complex of BpGH117 with neoagarobiose reveals the distortion of the constrained 3,6-anhydro-L-galactose into a conformation that favors catalysis. Furthermore, this complex, supported by analysis of site-directed mutants, provides evidence for an organization of the active site and positioning of the catalytic residues that are consistent with an inverting mechanism of catalysis and suggests that a histidine residue acts as the general acid. This latter feature differs from the vast majority of glycoside hydrolases, which use a carboxylic acid, highlighting the alternative strategies that enzymes may utilize in catalyzing the cleavage of glycosidic bonds.     

Purification of cytochrome P450 and ferredoxin, involved in bisphenol A degradation, from Sphingomonas sp. strain AO1

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450bisd), ferredoxin (Fd(bisd)), and ferredoxin reductase (Red(bisd)). We demonstrated that P450bisd and Fd(bisd) are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd(bisd) revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450(bisd), in the presence of Fd(bisd), Red(bisd), and NADH, was able to convert BPA. The K(m) and kcat values for BPA degradation were 85 +/- 4.7 microM and 3.9 +/- 0.04 min(-1), respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450bisd might exhibit strict specificity. Two BPA degradation products of the P450(bisd) system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.     

Starch-based plastic polymer degradation by the white rot fungus Phanerochaete chrysosporium grown on sugarcane bagasse pith: enzyme production

In this study, starch metabolites and enzymes were determined during starch-based plastic polymer biodegradation by the white rot fungus Phanerochaete chrysosporium, grown in sugarcane bagasse pith in tubular reactors. Various metabolites, amylase, ligninase and cellulase production were measured during P. chrysosporium growth on sugarcane bagasse pith with added glucose and starch polymer. On-line respirometric analyses followed during 32 days confirmed the P. chrysosporium capability of growing on sugarcane bagasse pith with starch polymer degradation. Enzyme activity during secondary metabolism increased, and a 70% and 74% starch degradation was reached with and without glucose addition, generating low molecular weight metabolites (e.g.) dextrin, maltotriose, maltose and glucose that were detected by high performance liquid chromatography.     

Pathways of bradykinin degradation in blood and plasma of normotensive and hypertensive rats

Kinins are vasoactive peptide hormones that can confer protection against the development of hypertension. Because their efficacy is greatly influenced by the rate of enzymatic degradation, the activities of various kininases in plasma and blood of spontaneously hypertensive rats (SHR) were compared with those in normotensive Wistar-Kyoto rats (WKY) to identify pathogenic alterations. Either plasma or whole blood was incubated with bradykinin (10 microM). Bradykinin and kinin metabolites were measured by high-performance liquid chromatography. Kininase activities were determined by cumulative inhibition of angiotensin I-converting enzyme (ACE), carboxypeptidase N (CPN), and aminopeptidase P (APP), using selective inhibitors. Plasma of WKY rats degraded bradykinin at a rate of 13.3 +/- 0.94 micromol x min(-1) x l(-1). The enzymes ACE, APP, and CPN represented 92% of this kininase activity, with relative contributions of 52, 25, and 16%, respectively. Inclusion of blood cells at physiological concentrations did not extend the activities of these plasma kininases further. No differences of kinin degradation were found between WKY and SHR. The identical conditions of kinin degradation in WKY and SHR suggest no pathogenic role of kininases in the SHR model of genetic hypertension.     

Effect of rhamnolipid on biodegradation of hydrocarbons in non-aqueous-phase liquid (NAPL)

Aliphatic and aromatic hydrocarbons are environmental pollutants of serious concern. Their bioavailability is the major limiting factor that makes the bioremediation process slow. Therefore, the present study focuses on biodegradation of non-aqueous-phase liquids (NAPL) by a halophilic consortium (Pseudomonas aeruginosa and Escherichia fergusonii) in presence of rhamnolipid as well as a rhamnolipid-producing Pseudomonas aeruginosa AMB AS7. The study was performed in microcosms, and the residual hydrocarbons after degradation were estimated by gas chromatography. It was found that the degradation of hydrocarbons in NAPL was more in presence of rhamnolipid in comparison with their biotic controls. However, among NAPL, the degradation of phenanthrene (37.5%) and octadecane (47.8%) was found to be more by co-culture of halophilic consortium and rhamnolipid-producing P. aeruginosa AMB AS7. Denaturing gradient gel electrophoresis was performed to determine the viability of different bacterial strains (halophilic and rhamnolipid-producing bacterial strain). Besides, the results also revealed that during NAPL degradation, the cell surface hydrophobicity (CSH) of halophilic consortium increased from 9.12% to 69.55% when added with 100 mg/L of rhamnolipid, whereas CSH of rhamnolipid-producing P. aeruginosa AMB AS7 was constant at 31.9%, even though it produced about 271.8 mg/L of rhamnolipid.     

A unique β-agarase,AgaA, from a marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. strain PO-303

The agaA gene encoding β-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40°C and pH 7.5, with a specific activity of 16.4 units mg⁻¹, a K _m of 1.10 mg ml⁻¹, and a V _max of 22.5 μmol min⁻¹ mg⁻¹ for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.     

Ornithine-containing lipids of some Pseudomonas species

Ornithine-containing lipids purified by thin-layer chromatography were found to represent 2-15% of the total extractable cellular lipids in two or three strains each of four Pseudomonas species: P. aeruginosa, P. fluorescens, P. stutzeri and P. cepacia. The structures of the ornithine-containing lipids were elucidated by chemical analysis, thin-layer chromatography, gas-liquid chromatography, gas-liquid chromatography/mass spectrometry (electron impact or secondary ion) and infrared absorption spectroscopy. At least six molecular species of ornithine-containing lipids were present in common in all of the preparations of the four Pseudomonas species. The structure which was the most abundantly in P. fluorescens (about 60% of the total amount of the ornithine-containing lipid) was 3-hydroxyhexadecanoic acid amide-linked to ornithine and esterified to hexadecanoic acid. In addition to this structure, 3-hydroxyoctadecenoic acid amide-linked to ornithine and esterified to hexadecanoic acid was a dominant structure in the ornithine-containing lipids of P. aeruginosa, P. stutzeri or P. cepacia. In P. cepacia, another ornithine-containing lipids with a terminal polar fatty acid, 3-hydroxyhexadecanoic acid amide-linked to ornithine and esterified to 2-hydroxynonadecacyclopropanoic acid or 2-hydroxyoctadecenoic acid, was found; its content, which represented 8-11% of the total extractable cellular lipids, was higher than that of the ornithine-containing lipids with a terminal nonpolar fatty acid. These ornithine-containing lipids exhibited hemagglutinating activity. Additionally, it was very interesting that hydroxy fatty acids included in the ornithine-containing lipids were not found in the phospholipids which represented more than 80% of the total extractable cellular lipids.     

Laboratory composting of extruded poly(lactic acid) sheets

Composting of extruded poly(lactic acid) (PLA) in combination with pre-composted yard waste in a laboratory composting system was studied. Yard waste and PLA mixtures containing 0%, 10%, or 30% PLA (dry weight basis) were placed in composting vessels for four weeks. Exhaust gases were analyzed for carbon dioxide concentration twice per week. After the first week, significantly greater (P < 0.05) amounts of carbon dioxide were generated in vessels with 10% or 30% PLA than in control (0% PLA) vessels. Data indicated that microbial degradation of PLA occurred. There was no significant difference (P > 0.05) in carbon dioxide emission between 10% and 30% PLA mixtures. Compost pH dropped (from 6.0 to 4.0) after 4 weeks of composting for 30% PLA, but remained unchanged (6.3) for 0% or 10% PLA. Most likely, in the case of 30% PLA, substantial chemical hydrolysis and lactic acid generation lowered the compost pH. The lowered pH likely suppressed microbial activity, thus explaining the lack of difference in carbon dioxide emissions between 10% and 30% PLA mixtures. Gel permeation chromatography showed a notable decrease in PLA molecular weight as a result of composting. It was demonstrated that PLA can be efficiently composted when added in small amounts (<30% by weight) to pre-composted yard waste.     

Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants

Aflatoxin B1 (AFB1) is a highly toxic fungal metabolite having carcinogenic, mutagenic and teratogenic effects on human and animal health. Accidental feeding of aflatoxin-contaminated rice straw may be detrimental for ruminant livestock and can lead to transmission of this toxin or its metabolites into the milk of dairy cattle. White-rot basidiomycetous fungus Pleurotus ostreatus produces ligninolytic enzymes like laccase and manganese peroxidase (MnP). These extracellular enzymes have been reported to degrade many environmentally hazardous compounds. The present study examines the ability of P. ostreatus strains to degrade AFB1 in rice straw in the presence of metal salts and surfactants. Laccase and MnP activities were determined spectrophotometrically. The efficiency of AFB1 degradation was evaluated by high performance liquid chromatography. Highest degradation was recorded for both P. ostreatus MTCC 142 (89.14 %) and P. ostreatus GHBBF10 (91.76 %) at 0.5 µg mL^?1 initial concentration of AFB1. Enhanced degradation was noted for P. ostreatus MTCC 142 in the presence of Cu²⁺ and Triton X-100, at toxin concentration of 5 µg mL^?1. P. ostreatus GHBBF10 showed highest degradation in the presence of Zn²⁺ and Tween 80. Liquid chromatography-mass spectrometric analysis revealed the formation of hydrated, decarbonylated and O-dealkylated products. The present findings suggested that supplementation of AFB1-contaminated rice straw by certain metal salts and surfactants can improve the enzymatic degradation of this mycotoxin by P. ostreatus strains.