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°C, respectively. The activity was strongly enhanced in the presence of Cu²⁺, Mn²⁺, and Mg²⁺ 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     

An alkalophilic laccase from Rheinheimera species isolate: Production and biobleaching of kraft pulp

Medium optimization was carried out to enhance laccase production from a novel Rheinheimera species, isolated from industrial effluent. Out of the 15 variables tested by Placket–Burman design (PBD)—yeast extract, soyabean meal, and peptone were the positively significant ones, enhancing laccase production. Both simple and complex sugars showed a negative effect on laccase production. Central composite design (CCD) of experiments, using the three positively significant variables in combinations, showed that laccase production was not affected by molar carbon, molar nitrogen levels or molar C/N ratio. Maximum laccase yield of 2.5 × 10⁵ nkat L^?1, 31 fold enhancement over the unoptimized medium, was achieved when soyabean meal (0.6%) was used alone as medium showing that laccase production was substrate dependent. Laccase was used, in the presence of 2 mM ABTS, for the biobleaching of eucalyptus kraft pulp resulting in kappa number reduction by 20% and brightness increase by 2.9%. Biobleaching improved further by sequential application of an alkalophilic xylanase (X) and laccase‐ABTS system (LAS) that decreased kappa number by 10, 15, and 35%, increased brightness by 2.7, 3.2, and 5.9% as compared to X treated, LAS treated and untreated control, respectively. XLAS treatment resulted in 15, 13, 10.9% increase in burst factor, tear factor, and viscosity with a 20% reduced consumption of elemental chlorine and hypochlorite. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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).     

Xylanase and Mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase and mannanase, singly or in combination, either sequentially or simultaneously. Enzymes were obtained from Streptomyces galbus NR that had been cultivated in a medium, containing either xylan of sugar cane bagasse or galactomannan of palm-seeds, when they were used as sole carbon sources from local wastes in fermentation media. No cellulase activity was detected. Incubation period, temperature, initial pH values and nature of nutritive constituents were investigated. Optimum production of both enzymes was achieved after 5 days incubation on a rotary shaker (200 rpm) at 35 degrees C and initial pH 7.0. Partial purification of xylanase and mannanase in the cultures supernatant were achieved by salting out at 40-60 and 60-80% ammonium sulphate saturation with a purification of 9.63- and 8.71-fold and 68.80 and 62.79% recovery, respectively. The xylanase and mannanase from S. galbus NR have optimal activity at 50 and 40 degrees C, respectively. Both enzymes were stable at a temperature up to 50 degrees C. Xylanase and mannanase showed highest activity at pH 6.5 and were stable from 5.0 to 8.0 and from 5.5 to 7.5, respectively. The partial purified enzymes preparations of xylanase and mannanase enzymes showed high bleaching activity, which is an important consideration for industry. Xylanase was found to be more effective for paper-bleaching than mannanase. When xylanase and mannanase were dosed together (simultaneously), both enzymes were able to enhance the liberation of reducing sugars and improve pulp bleachability, possibly as a result of nearly additive interactions. The simultaneous addition of both enzymes was more effective in pulp treatment than their sequential addition.     

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.     

Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp

A thermostable and cellulase-free xylanase has been produced from Streptomyces sp. QG-11-3 in solid substrate fermentation using wheat bran and eucalyptus kraft pulp as the prime solid substrates. The maximum xylanase yield obtained using these two substrates were 2360 U/g and 1200 U/g dry solid substrate at substrate:moisture ratios of 1:3 and 1:2.5, respectively. In immobilized cell system using polyurethane foam (PUF) and three nonwoven fabrics, namely, polyester, silk, and cotton, the xylanase yields were enhanced by 2.5-fold (203 U/ml), 1.91-fold (155 U/ml), 1.54-fold (125 U/ml), and 1.47-fold (119 U/ml), respectively, compared to the xylanase yield in liquid-batch fermentation (81 U/ml). In the biobleaching experiments, the xylanase dose of 3.5 U/g moisture free pulp exhibited the optimum bleach boosting of eucalyptus kraft pulp at pH 8.5 and 50 degrees C after 2 h of treatment. When xylanase treated pulp was subsequently treated with 4.5% chlorine, it resulted in reduction of kappa number by 25%, enhanced the brightness (%ISO) by 20% and improved the pulp properties such as tensile strength and burst factor by up to 63% and 8%, respectively.     

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.     

Transformation of humic acids by two-domain laccase from Streptomyces anulatus

The properties of two-domain laccase of Streptomyces anulatus (SaSL) and its role in transformation of humic acids (HA) from chernozem, sod-podzolic soil and peat at alkaline pH values were studied. The SaSL was cloned, expressed in E. coli and obtained in an electrophoretically homogeneous state. SaSL is an oligomeric protein with a molecular weight of 235-300 kDa and six or eight subunits in the molecule. The molecular weight of the subunit is 37.7 kDa. Its catalytic properties are similar to those of the previously described two-domain laccase. The enzyme catalyzed oxidation of electron donors (K₄[Fe(CN)₆], ABTS) at acidic pH and phenolic substrates (2-methoxyphenol, 2,6-dimethixyphenol) at alkaline pH values. The efficiency of catalysis was higher in the case of electron donors than phenolic substrates. The enzyme showed a high thermal stability and was more stable at neutral and alkaline pH values. The enzyme effectively transformed humic acids at alkaline pH values. It was found that polymerization reactions took place during interaction of SaSL with HA, as well as with their high-molecular weight (>80 kDa) and low-molecular weight (<5 kDa) fractions. Our results suggest a possible involvement of the two-domaim laccases in humification processes in alkaline soils.     

Laccase for biobleaching of eucalypt kraft pulp by means of a modified industrial bleaching sequence

Biobleaching of kraft pulp is a possible application of laccase, but it has not been described in detail for complete industrial bleaching sequences yet. Therefore, in this work, the biobleaching of Eucalyptus globulus kraft pulp was performed using a modified industrial totally chlorine‐free sequence. The modification consisted in the substitution of an enzymatic delignification stage, based on the application of laccase from Trametes villosa, for the first alkaline extraction one. The enzymatic stage was performed with several synthetic and natural mediators, namely 1‐hydroxybenzotriazole (HBT), violuric acid (VA), methyl syringate, and syringaldehyde. Several pulp properties were analyzed after each stage of the bleaching process—kappa number, ISO brightness, viscosity, and optical properties of CIEL*a*b* system. The new biobleaching sequence improved the pulp properties, in comparison to the conventional bleaching sequence, if HBT or VA was used as mediators. VA was selected as the best mediator of those tested and the effect of its concentration in the enzymatic stage was subsequently studied. Reducing the initial concentration by 30%, the same pulp quality was obtained, but if the reduction attained 60%, an important decrease in pulp integrity was detected. The modified bleaching sequence could improve the bleached pulp properties (kappa number 10%, ISO brightness 1%, and viscosity 5%) in comparison to the mill sequence. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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.     

漆酶催化苯甲醇氧化制备苯甲醛

以自制的高活性漆酶为催化剂,考察漆酶催化苯甲醇制备苯甲醛的工艺条件(底物浓度、介质体系、溶剂体系、氢受体、酶的用量、通氧方式等)对氧化反应的影响。结果发现:优化反应条件为以2,2,6,6-四甲基哌啶-1-氧基(TEMPO)为介质体系且TEMPO与苯甲醇的摩尔比为1∶4、60 mmol/L的丙酮为氢受体、漆酶比酶活80 U/mL、60mmol/L的苯甲醇,反应体系通O20.5 h后密闭反应36 h,苯甲醛的产率达98%。     

Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase

A high molecular model compound of polycyclic aromatic hydrocarbon was synthesised by coupling pyrene to PEG(5000). The pyrene-PEG was used for the study of a laccase-mediator-system. To prevent direct contact between the substrate and the enzyme the two were kept in their own compartments separated by a membrane. The low molecular mediators, 1-hydroxybenzotriazole and 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), which were oxidised by laccase to the corresponding radicals or cations permeated the membrane and reacted with the pyrene-PEG model compound. Oxidation of the model compound resulted in an alpha-oxidation of the alkyl-chain leading to two main oxidation products. The same oxidation products were obtained in the reaction system without a membrane.     

Production of a recombinant xylanase in plants and its potential for pulp biobleaching applications

The purpose of this study was to produce recombinant xylanase in transgenic plants and to test its potential application for pulp bleaching. The xynII xylanase gene from Trichoderma reesei was inserted into the Arabidopsis genome. Many transgenic plants produced biologically active XYNII and accumulated in leaves at level of 1.4-3.2% of total soluble proteins. The bleaching ability of XYNII on Kraft pulp was demonstrated by a reduction in the kappa number and the residual lignin contents. The bleaching efficiency of transgenic plant produced XYNII was similar to commercial xylanase on unbleached Kraft pulp. The effect of xylanase treatment on Kraft pulp was also investigated by SEM. Clear physical change on the pulp fiber surface was observed and was related to the amount xylan removed and microfibrils were visible on the fiber surface. This report demonstrates the potential application of plant produced recombinant xylanase for pulp and paper bleaching.