Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70 degrees C, respectively. The activity was strongly enhanced in the presence of Cu(2+), Mn(2+), and Mg(2+) and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.     

Solubilisation and mineralisation of [14C]lignocellulose from wheat straw by Streptomyces cyaneus CECT 3335 during growth in solid-state fermentation

Nine Streptomyces strains were screened for their ability to solubilise and mineralise ¹⁴C-labelled lignin during growth in solid-state fermentation. Streptomyces viridosporus was confirmed as an active lignin-degrading organism along with a new isolate, Streptomyces sp. UAH 15, further classified as Streptomyces cyaneus CECT 3335. This organism was able to solubilise and mineralise the [¹⁴C]lignin fraction of lignocellulose (44.96 ± 1.77% and 3.41 ± 0.48% respectively) after 21 days of incubation. Cell-free filtrates from Streptomyces sp. grown in solid-state fermentation were capable of solubilising up to 20% of the [¹⁴C]lignin after 2 days incubation, with most of the product detected in the acid-soluble rather than in the water-soluble fraction. Identification of the extracellular enzymes produced during growth of S. cyaneus CECT 3335 revealed that extracellular peroxidase and phenol oxidase activities were present, with the activity of phenol oxidase being 100 times greater than peroxidase activity. The activity of these two enzymes was found to correlate with both solubilisation and mineralisation rates. This is the first report of phenol oxidase activity produced by a Streptomyces strain during growth in solid-state fermentation. A role for the enzyme in the solubilisation and mineralisation of lignocellulose by S. cyaneus is suggested. Received: 12 May 1997 / Accepted: 19 May 1997     

Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp

Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50–80°C. It also showed broad pH adaptability (>35% activity at pH 4.0–9.0) and alkali-tolerance (>80% activity after incubation at pH 5–10 for 1 h at 37°C), and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0). SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.     

Manganese Is Not Required for Biobleaching of Oxygen-Delignified Kraft Pulp by the White Rot Fungus Bjerkandera sp. Strain BOS55

The white rot fungus Bjerkandera sp. strain BOS55 extensively delignified and bleached oxygen-delignified eucalyptus kraft pulp handsheets. Biologically mediated brightness gains of up to 14 ISO (International Standards Organization units) were obtained, providing high final brightness values of up to 80% ISO. In nitrogen-limited cultures (2.2 mM N), manganese (Mn) greatly improved manganese-dependent peroxidase (MnP) production. However, the biobleaching was not affected by the Mn nutrient regimen, ranging from 1,000 (mu)M added Mn to below the detection limit of 0.26 (mu)M Mn in EDTA-extracted pulp medium. The lowest Mn concentration tested was at least several orders of magnitude lower than the K(infm) known for MnP. Consequently, it was concluded that Mn is not required for biobleaching in Bjerkandera sp. strain BOS55. Nonetheless, fast protein liquid chromatography profiles indicated that MnP was the predominant oxidative enzyme produced even under culture conditions in the near absence of manganese. High nitrogen (22 mM N) and exogenous veratryl alcohol (2 mM) repressed biobleaching in Mn-deficient but not in Mn-sufficient culture medium. No correlation was observed between the titers of extracellular peroxidases and the biobleaching. However, the decolorization rate of the polyaromatic dye Poly R-478 was moderately correlated to the biobleaching under a wide range of Mn and N nutrient regimens.     

Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32

Streptomyces cyaneus SN32 was used in this study to produce extracellular xylanase, an important industrial enzyme used in pulp and paper industry. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by anion exchange chromatography using DEAE-Sepharose column, with 43.0% yield. The enzyme was found to be a monomer of 20.5 kDa as determined by SDS gel electrophoresis and has a pI of 8.5. The optimum pH and temperature for purified xylanase activity was 6.0 and 60-65 degrees C, respectively. The half-lives of xylanase at 50 and 65 degrees C were approximately 200 and 50 min, respectively. The xylanase exhibited K(m) and V(max) values of 11.1 mg/ml and 45.45 micromol/min/mg. The 15 residue N-terminal sequence of the enzyme was found to be 87% identical up to that of endoxylanases from Steptomyces sp. Based on the zymogram analysis, sequence similarity and other characteristics, it is proposed that the purified enzyme from S. cyaneus SN32 is an endoxylanase and belongs to Group 1 xylanases (low molecular weight - basic proteins). The purified enzyme was stable for more than 20 week at 4 degrees C. Easy purification from the fermentation broth and its high stability will be highly useful for industrial application of this endoxylanase.     

Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane, a nonphenolic dimeric lignin model compound. The spectrum of the peroxidase showed a soret band at 405 nm, which disappeared after reduction with sodium dithionite, indicating that the enzyme is a hemoprotein. Testing the effects of various inhibitors on the enzyme activity showed that it is a bifunctional enzyme having catalase and peroxidase activities.     

Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane, a nonphenolic dimeric lignin model compound. The spectrum of the peroxidase showed a soret band at 405 nm, which disappeared after reduction with sodium dithionite, indicating that the enzyme is a hemoprotein. Testing the effects of various inhibitors on the enzyme activity showed that it is a bifunctional enzyme having catalase and peroxidase activities.     

Degradation of Kraft Indulin Lignin by Streptomyces viridosporus and Streptomyces badius

Crawford and collaborators have studied extensively the solubilization of lignocellulose by two Streptomyces species, S. badius and S. viridosporus. Using a condensed industrial lignin essentially devoid of carbohydrates, Indulin AT, as the sole source of carbon, similar results were obtained: (i) the growths of the bacteria were optimum at pH 7.5 to 8.5; (ii) yeast extract was a better source of nitrogen than NH(4)Cl; (iii) the products of the depolymerization of Indulin were soluble, acid-precipitable polymers. When d-glucose was added as a secondary carbon source, it was used preferentially and the production of acid-precipitable polymers began only after the complete depletion of the sugar. On the assumption that the degradation of Indulin was catalyzed by enzymes, proteins found in the culture media and soluble and insoluble intracellular proteins were incubated with Indulin at pH 7.0 at 37 degrees C. Proteins in all fractions from S. badius had ligninolytic activities which, with the exception of those in the intracellular soluble fraction, were increased in the presence of H(2)O(2). In S. viridosporus, both extra- and intracellular soluble activities were found which were not increased by H(2)O(2). The extracellular activity of S. viridosporus was not affected by heat, resisted partially an exposure to pH 1.0, and was completely destroyed by proteolysis.     

Production of Cellulase-Free Xylanase from Bacillus megaterium by Solid State Fermentation for Biobleaching of Pulp

Xylanase production from B. megaterium was enhanced using solid state fermentation with respect to the use of solid substrate, moistening solution, moisture content, inoculum, sugars, soyabean meal, amino acids, and extraction with surfactant. An increase of ≈423-fold in xylanase production and complete suppression of CMCase production was achieved over submerged liquid fermentation. Biobleaching using this cellulase-free xylanase, 8 U/g of oven dried pulp of 10% consistency, showed 8.12% and 1.16% increase in brightness and viscosity, 13.67% decrease in kappa number, and 31% decrease in chlorine consumption at the CD stage.     

Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae

Laccase oxidation of phenolic azo dyes was examined with a commercially available laccase from Pyricularia oryzae as the model. Methyl-, methoxy-, chloro-, and nitro-substituted derivatives of 4-(4(prm1)-sulfophenylazo)-phenol were examined as substrates for this laccase. Only the substituents on the phenolic ring were changed. Among the dyes examined, only 2-methyl-, 2-methoxy-, 2,3-dimethyl-, 2,6-dimethyl-, 2,3-dimethoxy-, and 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol served as substrates. Preliminary kinetic studies suggest that 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol is the best substrate. Laccase oxidized the 2,6-dimethyl derivative of 4-(4(prm1)-sulfophenylazo)-phenol to 4-sulfophenylhydroperoxide (SPH) and 2,6-dimethyl-1,4-benzoquinone. The 2-methyl- and 2-methoxy-substituted dyes were oxidized to SPH and either 2-methyl- or 2-methoxy-benzoquinone. Six products were formed from laccase oxidation of the 2,6-dimethoxy-substituted dye. Three of them were identified as SPH, 4-hydroxybenzenesulfonic acid, and 2,6-dimethoxybenzoquinone. A mechanism for the formation of benzoquinone and SPH from laccase oxidation of phenolic azo dyes is proposed. This study suggests that laccase oxidation can result in the detoxification of azo dyes.     

Characterization of Streptomyces D3 derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9

Streptomyces D3, derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9, has the ability to produce high levels of xylose isomerase when grown on hemicellulosic materials such as xylan as the carbon source. Comparison between the partial nucleotide sequences of the 16S ribosomal RNA genes from S. cyaneus 190-1, S. griseoruber 42-9, and fusant D3 showed that the 16S rRNA gene of fusant D3 was identical to that of S. cyaneus 190-1. Partial sequence analysis of the xylose isomerase genes also indicated that the gene of fusant D3 was identical to that of S. cyaneus 190-1. The partial DNA fragments for the xylanase genes (xlnA and xlnB) of fusant D3 were amplified by PCR, and subjected to Southern hybridization analysis. The results revealed that the xlnB gene of fusant D3 was similar to that of S. cyaneus 190-1, but that the xlnA gene of fusant D3 was similar to that of S. griseoruber 42-9. These results suggest that the majority of the genome of fusant D3 may be derived from S. cyaneus 190-1.     

Oxygen Activation during Oxidation of Methoxyhydroquinones by Laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH₂ more efficiently than it oxidized MBQH₂; both the affinity and maximal velocity of oxidation were higher for DBQH₂ than for MBQH₂. Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q^·− + O₂ ↔ Q + O₂^·−). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O₂^·− on quinone formation rates. Then, the production of H₂O₂ in laccase reactions, as a consequence of O₂^·− dismutation, confirmed that semiquinones autoxidized. The highest H₂O₂ levels were obtained with DBQH₂, indicating that DBQ^·− autoxidized to a greater extent than did MBQ^·−. Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O₂^·− consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O₂^·−, besides undergoing dismutation, reacts with Mn²⁺, Fe³⁺, and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 μM concentrations of MBQ^·− and DBQ^·− from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ^·− autoxidation rose to 13.8% and that of DBQ^·− increased to 39.9%.     

Bleaching of wheat straw-rich soda pulp with xylanase from a thermoalkalophilic Streptomyces cyaneus SN32

An alkalistable endoxylanase from Streptomyces cyaneus SN32 was applied in bleaching of wheat straw enriched soda pulp. The xylanase dose of 10 IUg(-1) moisture free pulp exhibited maximum bleach boosting of soda pulp (pH 9.5-10.0) optimally at 65 degrees C after 2 h of reaction time. Pre-treatment of pulp with xylanase and its subsequent treatment with 6% hypochlorite reduced the kappa number by 8.7%, enhanced the brightness index by 3.56% and improved other paper properties such as tear index and burst index. The enzymatically-prebleached pulp when treated with 10% reduced level of hypochlorite (5.4%) gave comparable brightness of resultant hand sheets to the fully bleached pulp (6% hypochlorite).     

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.     

Use of Xylanase and Rabinofuranosidase for Arabinose Removal from Unbleached Kraft Pulp

Preparations of arabinofuranosidase and xylanase, respectively from Aureobasidium pullulans and Trichoderma longibrachiatum, were used to remove selectively xylose and arabinose from kraft pulp. The equilibrium moisture content of pulps treated with both enzymes, at varying relative humidities, revealed a consistently lower percent moisture content at all humidity set points. Shorter fiber lengths indicated some deterioration when pulp was exposed to high concentrations of both enzymes.     

Manganese Peroxidase, Produced by Trametes versicolor during Pulp Bleaching, Demethylates and Delignifies Kraft Pulp

Previous work has shown that Trametes (Coriolus) versicolor bleaches kraft pulp brownstock with the concomitant release of methanol. In this work, the fungus is shown to produce both laccase and manganese peroxidase (MnP) but not lignin peroxidase during pulp bleaching. MnP production was enhanced by the presence of pulp and/or Mn(II) ions. The maximum level of secreted MnP was coincident with the maximum rate of fungal bleaching. Culture filtrates isolated from bleaching cultures produced Mn(II)- and hydrogen peroxide-dependent pulp demethylation and delignification. Laccase and MnP were separated by ion-exchange chromatography. Purified MnP alone produced most of the demethylation and delignification exhibited by the culture filtrates. On the basis of the methanol released and the total and phenolic methoxyl contents of the pulp, it appears that MnP shows a preference for the oxidation of phenolic lignin substructures. The extensive increase in brightness observed in the fungus-treated pulp was not found with MnP alone. Therefore, either the MnP effect must be optimized or other enzymes or compounds from the fungus are also required for brightening.     

The analysis of handsheets from wheat straw following solid substrate fermentation by Streptomyces cyaneus and soda cooking treatment

The recent interest in the utilisation of agricultural fibres has promoted research into their potential as raw materials for the pulp and paper industry. In the current study, we report on the effect of biological pretreatment of wheat straw by Streptomyces cyaneus on the performance of the handsheets produced from the treated pulps. The pre-treatment of wheat straw with S. cyaneus had a positive effect on both the burst and tear indexes of the pulps but had a negative impact on tensile index. No significant variation in permeability and in folding endurance was observed. Manipulation of handsheets from wheat straw through biological treatment may therefore result in improved quality traits.     

Purification and partial characterization of an extracellular serine-proteinase of Streptomyces cyaneus isolated from Brazilian cerrado soil

Streptomyces cyaneus, a micro-organism isolated from Brazilian cerrado soil, produces an extracellular proteinase (SCP), which was purified 22-fold to homogeneity from culture supernatant fluid, using a single aprotinin-agarose affinity chromatography step. It is produced at a level corresponding to approximately 15% of total protein, but its physiological function has yet to be determined. The molecular mass of this S. cyaneus proteinase was estimated to be 120 kDa by gel filtration high performance liquid chromatography, and it migrates by SDS-PAGE as a single band of 30 kDa. It was optimally active at 25 degrees C and pH 9.0, and was fully inhibited by the serine-proteinase inhibitors PMSF and TPCK. A Km value of 1. 86 x 10-5 mmol l-1, and Vmax of 2.0 x 10-2 mmol l-1 (Abs247 nm microg-1 min-1), were calculated for alpha-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate.