Activities of cellulase and other extracellular enzymes during lignin solubilization by Streptomyces viridosporus

Summary Two mutant strains of the lignin degrading bacterium Streptomyces viridosporus strain T7A with enhanced abilities to produce a soluble lignin degradation intermediate, acid-precipitable polymeric lignin (APPL) and several mutants derepressed for cellulase production were compared with the wild type to examine the roles of cellulase and selected other extracellular enzymes in lignin solubilization by S. viridosporus. The two APPL-overproducing mutants, T-81 and T-138, had higher cellulase activities than the wild type. Mutants specifically derepressed for cellulase were also isolated and were found to produce more APPL than the wild type. The results are indicative of some involvement of cellulase in the lignin solubilization process. The lignin solubilized from corn (Zea mays) lignocellulose by the mutants was slightly different chemically as compared to wild type solubilized lignin in that it had a higher coumaric acid ester content. The production of extracellular coumarate ester esterase, aromatic aldehyde oxidase, and xylanase was also examined in the mutants. Xylanase and aromatic aldehyde oxidase production did not differ significantly between the mutants and the wild type. Mutant T-81 was found to have a slightly lower activity for esterase as compared with the wild type. It was concluded that xylanase, oxidase and esterase are not the enzymes directly responsible for enhanced lignin solubilization. The results, however, do implicate cellulase in the process.Paper number 86 511 of the Idaho Agricultural Experiment Station     

Production of Major Extracellular Enzymes during Lignocellulose Degradation by Two Streptomycetes in Agitated Submerged Culture

Streptomyces viridosporus T7A and S. badius 252 were grown in 1 to 2% (wt/vol) slurry cultures with mineral salts solution containing 0.6% yeast extract and 100/200 mesh ground and extracted corn lignocellulose at 37°C. Enzyme activities rapidly increased in the first 3 to 4 days and then declined and remained at a relatively constant level. Concentrations of endoglucanase and xylanase produced by S. badius were lower than those produced by S. viridosporus. However, the lignin-peroxidase peak concentration was threefold higher than with S. viridosporus and was obtained at 9 to 10 days of incubation. By polyacrylamide gel analysis, it was determined that peroxidases from both species consisted of four enzymes, with only one, the lignin peroxidase, having high activity. A culture pH of 8.5 was preferable for lignocellulose degradation by S. badius.     

Production by Streptomyces viridosporus T7A of an Enzyme Which Cleaves Aromatic Acids from Lignocellulose

The lignocellulose-degrading actinomycete Streptomyces viridosporus T7A produced an extracellular esterase when grown in a mineral salts-yeast extract medium. Extracellular esterase activity was first detected during the late stationary phase and typically followed the appearance of intracellular activity. When the organism was grown in lignocellulose-supplemented medium, esterase activity was not increased, but lignocellulose-esterified p-coumaric acid and vanillic acid were released into the medium. Polyacrylamide gels showed that several extracellular esterases differing in substrate specificity were produced. Ultrafiltration was used to concentrate the esterase prior to purification. Activity was recovered mostly in the molecular weight fraction between 10,000 and 100,000. Concentrated esterase was further purified by DEAE-Sepharose anion-exchange chromatography to a specific activity 11.82 times greater than that in the original supernatant. There were seven detectable esterase active proteins in the partially purified enzyme solution. Three were similar esterases that may be isoenzymes. The partially purified esterase had a pH optimum for activity of 9.0, a temperature optimum of 45 to 50°C, and a K_m and V_max of 0.030 mM and 0.097 μmol/min per ml, respectively, when p-nitrophenyl butyrate was the substrate. The enzyme was unstable above 40°C but retained activity when stored at 4 or −20°C. It lost some activity (20%) when lyophilized. Substrate specificity assays showed that it hydrolyzed ester linkages of p-nitrophenyl butyrate, α-naphthyl acetate, α-naphthyl butyrate, and lignocellulose. Vanillic and p-coumaric acids were identified as products released from lignocellulose. The enzyme is thought to be a component of the lignocellulose-degrading enzyme system of S. viridosporus.     

Production and Characterization of Polymeric Lignin Degradation Intermediates from Two Different Streptomyces spp.

Previous investigations have identified a quantitatively major intermediate of lignin degradation by Streptomyces viridosporus. The intermediate, a modified lignin polymer, acid-precipitable polymeric lignin (APPL), is released as a water-soluble catabolite and has been recovered in amounts equivalent to 30% of the lignin originally present in a corn stover lignocellulose substrate after degradation by this actinomycete. In the present work, APPLs were collected at various time intervals from cultures of two highly ligninolytic Streptomyces sp. strains, S. viridosporus T7A and S. badius 252, growing on corn stover lignocellulose. APPL production was measured over time, and the chemistry of APPLs produced by each organism after different time intervals was compared. Chemical characterizations included assays for lignin, carbohydrate, and ash contents, molecular weight distributions by gel permeation chromatography, and chemical degradation analyses by permanganate oxidation, acidolysis, and alkaline ester hydrolysis. Differences between the organisms were observed in the cultural conditions required for APPL production and in the time courses of APPL accumulation. S. viridosporus produced APPL in solid-state fermentation over a 6- to 8-week incubation period, whereas S. badius produced as much or more APPL, but only in liquid culture and over a 7- to 8-day incubation period. The chemistry of the APPLs produced also differed. S. viridosporus APPL was more lignin-like than that of S. badius and was slowly modified further over time, although no change in molecular weight distribution over time was observed. In contrast, S. badius APPL was less lignin-like and increased substantially in average molecular weight over time. Results indicated that differing mechanisms of lignin metabolism may exist in these two Streptomyces sp. strains. S. viridosporus APPL probably originates from the heart of the lignin and is released largely as the result of β-ether cleavage and other oxidative reactions. S. badius APPL probably originates in the same manner; however, after release as a water-soluble catabolite, lower-molecular-weight intermediates of lignin degradation are repolymerized with APPL in a reaction catalyzed by an extracellular phenol oxidase. The chemical analyses and the presence of extracellular phenol oxidase in S. badius, but not in S. viridosporus, support this conclusion.     

Production of extracellular enzymes during the solubilisation of straw by Thermomonospora fusca BD25

The production of three extracellular enzymes during the solubilisation of ball-milled wheat straw by seven actinomycete strains, was examined. A general correlation was observed between the production of extracellular enzymes (xylanases, endoglucanases and peroxidases) and the formation of the solubilised lignocellulose intermediate product (APPL), with the thermophilic actinomycete Thermomonospora fusca BD25 exhibiting greatest extracellular enzyme activity and highest APPL production. Production of all three enzymes; endoxylanase, endoglucanase and peroxidase, and lignocellulose solubilisation, occured during primary growth with maximum activity at the end of the exponential phase (48–96 h). The inducibility and stability of extracellular enzymes from T. fusca were further characterised. When xylan replaced ball-milled wheat straw as the growth substrate, reduced enzyme activities were observed (28–96% reduction in enzyme activities), whereas carboxymethylcellulose was found to be a poor inducer of all three enzyme activities (80–100% reduction in enzyme activities). The pH and temperature optima for extracellular enzyme activities from T. fusca was found to be pH 7.0–8.0 and 60°C, respectively. Analysis of concentrated crude supernatant from T. fusca by native polyacrylamide gel electrophoresis revealed the existence of two non-haem peroxidases. The stability of the extracellular lignocellulose-degrading enzymes for T. fusca suggest their suitability for future biotechnological processes such as biobleaching.     

Lignin Degradation by Streptomyces viridosporus: Isolation and Characterization of a New Polymeric Lignin Degradation Intermediate

A new, quantitatively significant intermediate formed during lignin degradation by Streptomyces viridosporus T7A was isolated and characterized. In Streptomyces-inoculated cultures, the intermediate, an acid-precipitable, polyphenolic, polymeric lignin (APPL), accumulated in the growth medium. The APPL was a water-soluble polymer probably consisting of a heterogeneous mixture of molecular weight components of ≥20,000. APPLs were precipitable from culture filtrates after they had been acidified to pH <3 to 5. Noninoculated controls yielded little APPL, but supernatant solutions from inoculated cultures produced quantities of APPL that correlated with the biodegradability of the lignocellulose type. Maximal recovery of APPL was obtained from corn lignocellulose, reaching 30% of the initial lignin present in the substrate. APPLs contained small amounts of carbohydrate, organic nitrogen, and inorganic materials. The lignin origin of APPLs was confirmed by chemical analyses, which included acidolysis, permanganate oxidation, elemental analyses, functional group analyses, nuclear magnetic resonance spectroscopy, and ¹⁴C isotopic techniques. Analyses of APPLs from corn lignocelluloses showed that S. viridosporus-degraded APPLs were lignin derived but significantly different in structure from APPLs derived from uninoculated controls or from a standard corn milled-wood lignin. Degraded APPLs were enriched in phenolic hydroxyl groups and, to a small extent, in carboxyl groups. Degradative changes appeared to be largely oxidative and were thought to involve substantial cleavage of p-hydroxy ether linkages and methoxyl groups in the lignin.     

Isolation from haddock tissue of psychrophilic bacteria with maximum growth temperature below 20 degrees C.

Protoplast fusion was investigated as a technique for genetically manipulating two lignin-degrading Streptomyces strains, Streptomyces viridosporus T7A and Streptomyces setonii 75Vi2. Four of 19 recombinants tested showed enhanced production of acid-precipitable polymeric lignin (APPL), producing 155 to 264% more APPL from corn stover lignocellulose than was produced by the wild-type S. viridosporus T7A. APPLs are lignin degradation intermediates known to be potentially valuable chemical products produced by bioconversion of lignin with Streptomyces spp. The prospects of utilizing protoplast fusion to construct APPL-overproducing Streptomyces strains was considered especially promising.     

Production and Characterization of Polymeric Lignin Degradation Intermediates from Two Different Streptomyces spp

Previous investigations have identified a quantitatively major intermediate of lignin degradation by Streptomyces viridosporus. The intermediate, a modified lignin polymer, acid-precipitable polymeric lignin (APPL), is released as a water-soluble catabolite and has been recovered in amounts equivalent to 30% of the lignin originally present in a corn stover lignocellulose substrate after degradation by this actinomycete. In the present work, APPLs were collected at various time intervals from cultures of two highly ligninolytic Streptomyces sp. strains, S. viridosporus T7A and S. badius 252, growing on corn stover lignocellulose. APPL production was measured over time, and the chemistry of APPLs produced by each organism after different time intervals was compared. Chemical characterizations included assays for lignin, carbohydrate, and ash contents, molecular weight distributions by gel permeation chromatography, and chemical degradation analyses by permanganate oxidation, acidolysis, and alkaline ester hydrolysis. Differences between the organisms were observed in the cultural conditions required for APPL production and in the time courses of APPL accumulation. S. viridosporus produced APPL in solid-state fermentation over a 6- to 8-week incubation period, whereas S. badius produced as much or more APPL, but only in liquid culture and over a 7- to 8-day incubation period. The chemistry of the APPLs produced also differed. S. viridosporus APPL was more lignin-like than that of S. badius and was slowly modified further over time, although no change in molecular weight distribution over time was observed. In contrast, S. badius APPL was less lignin-like and increased substantially in average molecular weight over time. Results indicated that differing mechanisms of lignin metabolism may exist in these two Streptomyces sp. strains. S. viridosporus APPL probably originates from the heart of the lignin and is released largely as the result of beta-ether cleavage and other oxidative reactions. S. badius APPL probably originates in the same manner; however, after release as a water-soluble catabolite, lower-molecular-weight intermediates of lignin degradation are repolymerized with APPL in a reaction catalyzed by an extracellular phenol oxidase. The chemical analyses and the presence of extracellular phenol oxidase in S. badius, but not in S. viridosporus, support this conclusion.     

Production of useful modified lignin polymers by bioconversion of lignocellulose with Streptomyces

Lignin degrading strains of Streptomyces were grown on lignocelluloses from a variety of plant sources. These actinomycetes readily degraded the lignin present in the residues and released a major portion of the lignin into the growth medium as a water soluble, modified polymer. The polymer, an acid precipitable polyphenolic lignin (APPL), was recovered from spent culture media by acid precipitation or dialysis/lyophilization. APPL's were shown to be mostly free of nonlignin components. As compared to native lignin they were more oxidized, were especially enriched in phenolic hydroxyl groups, and were significantly reduced in methoxyl groups. The yield of APPL from different lignocelluloses correlated with their biodegradability. Grasses such as corn stover were the optimal lignocellulose type for APPL production by Streptomyces. In contrast white-rot fungi produced only small amounts of APPL as they decomposed lignin. A solid state bioconversion process was developed using Streptomyces viridosporus T7A to produce APPL from corn stover lignocellulose in yields >or= 30% of the initial lignin present in the substrate. APPL produced by S. viridosporus was examined for its properties and possible use as an antioxidant. The APPL was shown to have good antioxidant properties after mild chemical treatment to reduce the alpha-carbonyl groups present in the APPL. Oxidation of the APPL with hydroxyl radical (OH(*)) further improved its antioxidant properties probably as the result of aromatic ring hydroxylation reactions. As compared with currently used commercial antioxidants, the modified APPL was thought to be competitive when economics of production was considered. Native lignin on the other hand was shown to exhibit no antioxidant properties, even after reduction and/or oxidation.     

Enhancement of Lignin Degradation in Streptomyces spp. by Protoplast Fusion

Protoplast fusion was investigated as a technique for genetically manipulating two lignin-degrading Streptomyces strains, Streptomyces viridosporus T7A and Streptomyces setonii 75Vi2. Four of 19 recombinants tested showed enhanced production of acid-precipitable polymeric lignin (APPL), producing 155 to 264% more APPL from corn stover lignocellulose than was produced by the wild-type S. viridosporus T7A. APPLs are lignin degradation intermediates known to be potentially valuable chemical products produced by bioconversion of lignin with Streptomyces spp. The prospects of utilizing protoplast fusion to construct APPL-overproducing Streptomyces strains was considered especially promising.     

Lignocarbohydrate Solubilization from Straw by Actinomycetes

Actinomycetes grown on wheat straw solubilized a lignocarbohydrate fraction which could be recovered by acid precipitation. Further characterization of this product (APPL) during growth of Streptomyces sp. strain EC1 revealed an increase in carboxylic acid and phenolic hydroxyl content, suggesting progressive modification. This was also observed in dioxane-extracted lignin fractions of degraded straw, and some similarity was further suggested by comparative infrared spectroscopy. However, the molecular weight profile of APPL was relatively constant during growth of Streptomyces sp. strain EC1 on straw, while analysis of the dioxane-extracted lignin fractions appeared to show fragmentation followed by repolymerization. Lignocarbohydrate solubilization could be monitored in all cultures by routine assay of APPL-associated protein, which accounted for up to 20% of the extracellular culture protein in some cases. Interestingly, this protein fraction was found to include active hydrolytic and oxidative enzymes involved in the degradation of lignocellulose, and specific enzyme activities were often increased in the acid-insoluble fractions of culture supernatants. This was particularly important for peroxidase and veratryl oxidase activities, which could be readily detected in the acid-precipitable lignocarbohydrate complex but were virtually undetectable in untreated culture supernatants.     

Effects of pH on Lignin and Cellulose Degradation by Streptomyces viridosporus

Lignocellulose degradation by Streptomyces viridosporus results in the oxidative depolymerization of lignin and the production of a water-soluble lignin polymer, acid-precipitable polymeric lignin (APPL). The effects of the culture pH on lignin and cellulose metabolism and APPL production by S. viridosporus are reported. Dry, ground, hot-water-extracted corn (Zea mays) lignocellulose was autoclaved in 1-liter reagent bottles (5 g per bottle) and inoculated with 50-ml volumes of S. viridosporus cells suspended in buffers of specific pH (pH 6.0 to 9.2 at 0.4 pH unit intervals). Four replicates of inoculated cultures and of uninoculated controls at each pH were incubated as solid-state fermentations at 37°C. After 6 weeks of incubation the percent loss of lignocellulose, lignin, and carbohydrate and the amount of APPL produced were determined for each replicate. Optimal lignocellulose degradation, as shown by substrate weight loss, was observed in the pH range of 8.4 to 8.8. Only minor differences were seen in the Klason lignin, carbohydrate, protein, and ash contents of the APPLS produced by cultures at each pH. The effects of pH on the degradation of a spruce (Picea pungens) [¹⁴C-lignin]lignocellulose and a Douglas fir (Pseudotsuga menziesii) [¹⁴C-glucan]-lignocellulose were also determined at pH values between 6.5 and 9.5 (0.5 pH unit intervals). The incubations were carried out for 3 weeks at 37°C with bubbler-tube cultures. The percentage of initial ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-labeled water-soluble products, and [¹⁴C]APPL was then determined. The mineralization of lignin and cellulose to CO₂ was optimal at pHs 6.5 and 7.0, respectively. However, the optimum for lignin and cellulose solubilization was pH 8.5, which correlated with the pH 8.5 optimum for APPL production. Overall, the data show that, whereas lignin mineralization is optimal at neutral to slightly acidic pHs, lignocellulose degradation with lignin solubilization and APPL production is promoted by alkaline pHs. These findings indicate that lignin-solubilizing actinomycetes may play an important role in the metabolism of lignin in neutral to alkaline soils in which ligninolytic fungi are not highly competitive.     

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.     

The isozymic similarity of indoleacetic Acid oxidase to peroxidase in birch and horseradish

The relationship of indoleacetic acid oxidase activity to peroxidase activity is complicated by numerous multiple forms of this enzyme system. It is not known if all isozymes of this complex system contain both types of activity. Isozyme analysis of commercial horseradish peroxidase and leaf extracts of yellow birch (Betula alleghaniensis) by isoelectric focusing in polyacrylamide gels was used to examine this problem. Horseradish and birch exhibited 20 and 13 peroxidase isozymes, respectively, by staining with benzidine or scopoletin. Guaiacol was less sensitive. Indoleacetic acid oxidase staining (dimethylaminocinnamaldehyde) generally showed fewer bands, and left doubt as to the residence of both types of activity on all isozymes. Elution of the isozymes from the gels and wet assays verified that all peroxidase isozymes contained indoleacetic acid oxidase activity as well. Estimation of oxidase to peroxidase ratios for the major bands indicated small differences in this parameter. A unique isozyme for one or the other type of activity was not found.     

Modification of wheat straw lignin by solid state fermentation with white-rot fungi

The potential of crude enzyme extracts, obtained from solid state cultivation of four white-rot fungi (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa), was exploited to modify wheat straw cell wall. At different fermentation times, manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), laccase, carboxymethylcellulase (CMCase), avicelase, xylanase and feruloyl esterase activities were screened and the content of lignin as well as hydroxycinnamic acids in fermented straw were determined. All fungi secreted feruloyl esterase while LiP was only detected in crude extracts from B. adusta. Since no significant differences (P > 0.05) were observed in remaining lignin content of fermented straw, LiP activity was not a limiting factor of enzymatic lignin removal process. The levels of esterified hydroxycinnamic acids degradation were considerably higher than previous reports with lignocellulosic biomass. The data show that P. rufa, may be considered for more specific studies as higher ferulic and p-coumaric acids degradation was observed for earlier incubation times.     

Biodegradation of lignin-carbohydrate complexes

Covalent lignin-carbohydrate (LC) linkages exist in lignocellulose from wood and groups herbaceous plants. In wood, they consist of ester and ether linkages through sugar hydroxyl to the -carbanol of phenylpropane subunits in lignin. In grasses, ferulic and p-coumaric acids are esterified to hemicelluloses and lignin, respectively. Hemicelluloses also contain substitutents and side groups that restrict enzymatic attack. Watersoluble lignin-carbohydrate complexes (LCCs) often precipitate during digestion with polysaccharidases, and the residual sugars are more diverse than the bulk hemicellulose. A number of microbial esterases and hemicellulose polysaccharidases including acetyl xylan esterase, ferulic acid esterase, and p-coumaric esterase attack hemicellulose side chains. Accessory hemicellulases include -l-arabinofuranosidase and -methyl-glucuranosidase. Both of these side chains are involved in LC bonds. -Glucosidase will attach sugar residues to lignin degradation products and when carbohydrate is attached to lignin, lignin peroxidase will depolymerize the lignin more readily.Abbreviations APPL acid precipitable polymeric lignin - CBQase cellobioquinone oxidoreductase - LC lignincarbohydrate - LCC(s) lignin-carbohydrate complex - DHP Dehydrogenative polymerisate - DMSO dimethylsulfoxide - DP degree of polymerisation - MWEL milled wood enzyme lignin - MWL milled wood lignin (not digested with carbohydrases)     

Characterization of an extracellular lignin peroxidase of the lignocellulolytic actinomycete Streptomyces viridosporus

Previously we reported production of an extracellular lignin-inducible peroxidase by Streptomyces viridosporus (M. Ramachandra, D.L. Crawford, and A.L. Pometto III, Appl. Environ. Microbiol. 53:2754-2760, 1987). This peroxidase was shown to oxidize 3,4-dihydroxyphenylalanine, 2,4-dichlorophenol, homoprotocatechuic acid, caffeic acid, and N,N,N',N'-tetramethylphenylenediamine and was found in higher than normal levels in strains enhanced for lignocellulose degradation. In the present study, we used a pure extracellular enzyme preparation with high peroxidase isoform P3 activity to oxidize lignin substructure model compounds of both the 1,2-diaryl propane and arylglycerol-beta-aryl ether types and containing C alpha-carbonyl and C alpha-hydroxyl groups. The reactions were monitored by gas chromatography-mass spectrometry and high-pressure liquid chromatography techniques. In the presence, but not the absence, of hydrogen peroxide, the enzyme preparation catalyzed C alpha-C beta bond cleavage in the side chains of the diaryl ethers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propan-1-one (II) and the diaryl ethane 1-(4-methoxyphenyl)-2-(phenyl)ethan-1-one (III). Rapid hydrogen peroxide consumption was observed when the enzyme preparation was added to either milled corn lignin or lignocellulose. Additional characterizations showed that this enzyme is a heme protein (Soret band, 408 nm) and a major component of the ligninolytic system of S. viridosporus T7A. This is the first report of a lignin peroxidase in a bacterium. We have designated this new lignin peroxidase as ALiP-P3.     

Characterization of an extracellular lignin peroxidase of the lignocellulolytic actinomycete Streptomyces viridosporus.

Previously we reported production of an extracellular lignin-inducible peroxidase by Streptomyces viridosporus (M. Ramachandra, D.L. Crawford, and A.L. Pometto III, Appl. Environ. Microbiol. 53:2754-2760, 1987). This peroxidase was shown to oxidize 3,4-dihydroxyphenylalanine, 2,4-dichlorophenol, homoprotocatechuic acid, caffeic acid, and N,N,N',N'-tetramethylphenylenediamine and was found in higher than normal levels in strains enhanced for lignocellulose degradation. In the present study, we used a pure extracellular enzyme preparation with high peroxidase isoform P3 activity to oxidize lignin substructure model compounds of both the 1,2-diaryl propane and arylglycerol-beta-aryl ether types and containing C alpha-carbonyl and C alpha-hydroxyl groups. The reactions were monitored by gas chromatography-mass spectrometry and high-pressure liquid chromatography techniques. In the presence, but not the absence, of hydrogen peroxide, the enzyme preparation catalyzed C alpha-C beta bond cleavage in the side chains of the diaryl ethers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propan-1-one (II) and the diaryl ethane 1-(4-methoxyphenyl)-2-(phenyl)ethan-1-one (III). Rapid hydrogen peroxide consumption was observed when the enzyme preparation was added to either milled corn lignin or lignocellulose. Additional characterizations showed that this enzyme is a heme protein (Soret band, 408 nm) and a major component of the ligninolytic system of S. viridosporus T7A. This is the first report of a lignin peroxidase in a bacterium. We have designated this new lignin peroxidase as ALiP-P3.     

三种白腐菌生物学特性与木质纤维素酶基因遗传多样性

以采自东北林业大学帽儿山实验林场的3种白腐真菌——木蹄层孔菌(Fomes fomentarius)、鲍姆鲍姆木层孔菌(Phellinus baumiibaumii)和火木层孔菌(Phellinus igniarius)为材料,用菌落直径测量法比较3种白腐菌在马铃署葡萄糖固体培养基上的生长速度,采用菌丝体干重法比较其在马铃署葡萄糖液体培养基中的生物量变化。结果显示:马铃薯葡萄糖固体培养基上3种白腐菌均为快速生长类型,其生长速度木蹄层孔菌火木层孔菌鲍姆鲍姆木层孔菌;马铃署葡萄糖液体培养基中生物量增长速度木蹄层孔菌鲍姆鲍姆木层孔菌火木层孔菌。用比色法测量其木质纤维素酶活性,结果显示:木蹄层孔菌产锰过氧化物酶和漆酶量较高,鲍姆鲍姆木层孔菌和火木层孔菌产木质素过氧化物酶量较高;木蹄层孔菌、鲍姆鲍姆木层孔菌和火木层孔菌3种白腐菌的3种主要木质素酶(锰过氧化物酶、漆酶和木质素过氧化物酶)的表达量,种间差异显著(F=3.75*、5.20**、3.01*),白桦木屑诱导处理与对照间差异显著(F=3.84*、4.19*、5.28*);两种主要纤维素酶(葡聚糖内切酶、葡聚糖外切酶)的表达量,种间差异不显著,受碳源影响作用显著(F=3.99*、4.04*)。筛选29对引物组合,对3种白腐菌几种主要木质纤维素酶基因进行TRAP-PCR分子标记检测,比较3菌种间遗传差异,扩增总条带数为357条,多态性条带数为255条,多态性条带的比例为71.43%,其中木质素降解酶基因总多态位点比率为73.77%,纤维素降解酶基因总多态位点比率为68.97%。3种白腐菌的木质纤维素降解酶基因在种间均存在较高的遗传差异。因此,特定基因的TRAP分子标记可以用于木腐菌的遗传变异分析。     

Characterization of a new rhamnogalacturonan acetyl esterase from Bacillus halodurans C-125 with a new putative carbohydrate binding domain

BH1115 is a gene from Bacillus halodurans strain C-125 that hypothetically encodes a rhamnogalacturonan acetyl esterase (RGAE) of the CE-12 family. As confirmation, this gene was cloned, and the product was expressed in Escherichia coli strain Rosetta (DE3) cells and purified. The enzyme obtained was monomeric, with a molecular mass of 45 kDa, and exhibited alkaliphilic properties. A study of the inhibition of the activity by some modulators confirmed that the catalytic triad for the esterase activity was Ser-His-Asp. This enzyme also presents broad substrate specificity and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, β-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, RGAE from B. halodurans achieves a synergistic effect with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common α/β hydrolase fold. The homology between the folds of RGAE from Aspergillus aculeatus and the hypothetical YxiM precursor from Bacillus subtilis, which both belong to the SGNH family, illustrates the divergence of such proteins from a common ancestor. Furthermore, the enzyme possesses a putative substrate binding region at the N terminus of the protein which has never been described to date for any RGAE.