Actinobacteria isolated from termite guts as a source of novel oxidative enzymes

A multi-faceted screening programme was designed to search for the oxidases, laccase, peroxidase and tyrosinase. Actinobacteria were selectively isolated from the paunch and colon region of the hindguts of the higher termite, Amitermes hastatus. The isolates were subjected to solid media assays (dye decolourization, melanin production and the utilization of indulin AT as sole carbon source) and liquid media assays. Eleven of the 39 strains had the ability to decolourize the dye RBBR, an indicator for the production of peroxidases in actinobacteria. Melanin production on ISP6 and ISP7 agar plates served as a good indicator for laccase and/or tyrosinase production and the ability of the strains to grow in the presence of indulin AT as a sole carbon source served as a good indicator of lignin peroxidase and/or general peroxidase production. Enzyme-producing strains were cultivated in liquid media and extracellular enzyme activities measured. Strains with the ability to produce oxidative enzymes under the conditions tested were identified to genus level by 16S rRNA gene analysis and compared to known oxidase producers. A strong relationship was observed between the environment sampled (termite guts where lignocellulose degradation occurs) and the dominant type of oxidative enzyme activity detected (laccases and peroxidases), which suggests the possibility of future targeted screening protocols linking the physical properties of the target enzymes with specific operational conditions required, such as lignocellulosic degradation in the preparation of biofuel feedstocks.     

Production of biomass and ligninolytic enzymes by Pleurotus ostreatus in submerged culture.

The production of biomass and ligninolytic enzymes by Pleurotus ostreatus was analysed in synthetic medium with yeast extract and different glucose concentrations (0.5 - 20 g/l), at different pH (3.5-6.5) and incubation temperatures (23-32 degrees C). The best culture condition were: initial glucose concentration of 5 g/l, initial pH between 5.5-6.5 and incubation temperature between 26-29 degrees C. The saturation constant for glucose (Ks) was 1.75 g/l. The biomass concentration reached 8.6 g/l with a glucose addition of 20.0 g/l to the culture medium. The control of pH allowed an increment of 0.5 g/l of biomass concentration. The birreactor produced pellets with a homogeneous distribution of diameter size of 3.4 -/+ 0.2 mm. Approximately, 307 U/l of laccase and 0.41 U/l of manganese peroxidase were obtained in extracellular liquid medium and 0.015 U/g of laccase and 0.809 U/g of manganese peroxidase were obtained in solid substrate. Lignin peroxidase activity was not detected at any condition.     

Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii

Trametes trogii, a white rot basidiomycete involved in wood decay worldwide, produces several ligninolytic enzymes, laccase being the dominant one, with higher titers than those reported for most other white rot fungi studied up to date. The effect of copper on in vitro production of extracellular ligninolytic activities was studied. CuSO(4)·5H(2)O concentrations from 1.6 μM to 1.5 mM were tested in a synthetic medium with glucose 20 g/L and asparagine 3 g/L. The addition of copper (up to 1 mM) did not affect growth but strongly stimulated ligninolytic enzyme production; faster decolorization of the polymeric dye Poly R-478 was observed as well. Maximal production of manganese peroxidase, laccase, and glyoxal oxidase [1.28 U/mL, 93.8 U/mL (with a specific activity of 720 U/mg protein), and 0.46 U/mL respectively] was attained with 1 mM CuSO(4)·5H(2)O. However, higher copper concentrations inhibited growth and notably decreased manganese peroxidase production, although they did not affect laccase secretion. Laccase activity in the culture filtrate was maximal at 50 C and pH 3.4, and the enzyme was completely stable at pH 4.4 and above, and at 30 C for up to 5 d. Denaturing polyacrylamide gel electrophoresis of extracellular culture fluids showed two laccase activity bands (mol wt 38 and 60 kDa respectively). The pattern of isoenzyme production was not affected by medium composition but differed with culture age.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

Characterization of a thermostable and solvent-tolerant laccase produced by Streptomyces sp. LAO

Objectives

Decaying wood samples were collected, and actinomycetes were isolated and screened for laccase production. The identity of the efficient laccase-producing isolate was confirmed by using a molecular approach. Fermentation conditions for laccase production were optimized, and laccase biochemical properties were studied.

Results

Based on the 16S rRNA gene sequencing and phylogenetic analysis, the isolate coded as HWP₃ was identified as Streptomyces sp. LAO. The time-course study showed that the isolate optimally produced laccase at 84 h with 40.58?±?2.35 U/mL activity. The optimized physicochemical conditions consisted of pH 5.0, ferulic acid (0.04%; v/v), pine back (0.2 g/L), urea (1.0 g/L), and lactose (1 g/L). Streptomyces sp. LAO laccase was optimally active at pH and temperature of 8.0 and 90 °C, respectively, with remarkable pH and thermal stability. Furthermore, the enzyme had a sufficient tolerance for organic solvents after 16 h of preincubation, with laccase activity?>?70%. Additionally, the laccase maintained considerable residual activity after pretreatment with 100 mM of chemical agents, including sodium dodecyl sulphate (69.93?±?0.89%), ethylenediaminetetraacetic acid (93.1?±?7.85%), NaN₃ (96.28?±?3.34%) and urea (106.03?±?10.72%).

Conclusion

The laccase's pH and thermal stability; and robust catalytic efficiency in the presence of organic solvents suggest its industrial and biotechnological application potentials for the sustainable development of green chemistry.

     

Use of Pleurotus dryinus for lignocellulolytic enzymes production in submerged fermentation of mandarin peels and tree leaves

It has been shown that the wood-rotting mushroom Pleurotus dryinus IBB 903 is able to effectively produce cellulases, xylanase, laccase, and manganese peroxidase in submerged fermentation of mandarin peels and tree leaves. Gradual increasing of lignocellulosic substrates concentration from 1 to 4–6% enhanced enzyme accumulation in culture liquid. A simple and inexpensive medium containing mandarin peels and yeast extract as sole carbon and nitrogen sources allowed simultaneous production of high levels of both hydrolases and oxidases by P. dryinus IBB 903. Supplementation of this medium by copper and manganese caused earlier and faster accumulation of laccase and manganese peroxidase increasing their yield by 1.5 and 7.5 times, respectively. In addition, by adding manganese to the medium it is possible to regulate the ratio of laccase and MnP in enzyme preparation. The presence of lignocellulosic substrate is the requisite for MnP production by P. dryinus IBB 903 since there was no production of MnP when mushroom has been cultivated in the synthetic medium with different carbon source. Among carbon source tested only utilization of glucose resulted to 21-fold increase of fungus laccase specific activity compared to control medium without carbon source. Carboxymethyl cellulase and xylanase appeared to be inducible enzymes.     

Optimization of simultaneous production of tyrosinase and laccase by Neurospora crassa

Increasing demand for efficient and environmentally benign oxidation technologies has resulted in a focus on the use of oxidoreductases. Laccases and tyrosinases, which utilize molecular oxygen and produce water as by-product, are particularly attractive. Simultaneous production of laccase and tyrosinase was studied in Neurospora crassa FGSC #321 as the fungal strain which has the ability to produce tyrosinase intracellularly while producing laccase extracellularly. Using one-variable-at-a-time experiments and a Taguchi orthogonal L9 array demonstrated that a Vogel minimal medium containing 2.5% sucrose at pH 6.5 and 25?°C with no agitation or oxygen purging were the optimum conditions for N. crassa FGSC #321 growth. Conditions were adjusted to obtain the highest laccase and tyrosinase production. Results indicate that the control mechanisms for the production of both enzymes in N. crassa FGSC #321 are similar but not necessarily identical. Results revealed that transferring the harvested cells from the growth medium into the phosphate buffer (pH 6.8, 0.1M) containing cycloheximide (2?μM) and fluorouracil (2?mM) and increasing the temperature to 30?°C were the best conditions for simultaneous production of laccase and tyrosinase (1278 and 410?U/g of biomass, respectively). Nonetheless, starvation at 35?°C is proposed as the most cost-effective means for inducing laccase. The N. crassa laccase was characterized by using its molecular weight, pI value, optimal pH and temperature and stability.     

Effect of inducers and process parameters on laccase production by Streptomyces psammoticus and its application in dye decolourization

The process parameters influencing the production of extracellular laccases by Streptomyces psammoticus MTCC 7334 were optimized in submerged fermentation. Coffee pulp and yeast extract were the best substrate and nitrogen source respectively for laccase production by this strain. The optimization studies revealed that the laccase yield was maximum at pH 7.5 and temperature 32 degrees C. Salinity of the medium was also observed to be influencing the enzyme production. An agitation rate of 175 rpm and 15% inoculum were the other optimized conditions for maximum laccase yield (5.9 U/mL). Pyrogallol and para-anisidine proved to be the best inducers for laccase production by this strain and the enzyme yield was enhanced by 50% with these inducers. S. psammoticus was able to decolourize various industrial dyes at different rates and 80% decolourization of Remazol Brilliant Blue R (RBBR) was observed after 10 days of incubation in dye based medium.     

Extracellular ligninolytic enzymes by Lentinus polychrous Lév. under solid-state fermentation of potential agro-industrial wastes and their effectiveness in decolorization of synthetic dyes

Six agro-industrial wastes were evaluated as a support for ligninolytic enzyme production by the white-rot fungus Lentinus polychrous Lév. under solid-state fermentation. Enzyme production was markedly different according to the substrate used. Rice bran (RB) yielded the highest laccase activity of 1,449 U/L (after 21 days of culture) with specific activity of 4.4 U/g substrate. Rice bran supplemented with rice husk (RH) (2:1 by wt) showed high laccase activity of 1,425 U/L with specific activity of 10.0 U/g substrate (after 17 days of culture). The crude enzyme of the RH-RB culture also contained manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities in relative proportions of 1.9:1.4:1 of laccase:MnP:MIP, respectively. Zymogram studies showed the same isoenzyme pattern with these ligninolytic enzymes. The high enzyme production level and low substrate cost of SSF-L. polychrous Lév. suggest that it has potential for industrial applications. Our studies showed that the crude enzyme from this culture exhibited in vitro decolorization of Indigo Carmine. The highest efficiency of dye decolorization was observed under alkaline conditions (pH 9.0) at an initial dye concentration of 10 mg/L. The rather high pH conditions and high efficiency in Indigo Carmine decolorization make the enzyme further interest for the applications in treatment of waste water from the textile industry, which contains synthetic dyes.     

Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications

AIMS: Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. METHODS AND RESULTS: Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8-10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45.4 and 163.6 U g(-1) protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35-38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4.5-5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6-7, in a large temperature range (30-70 degrees C), and was stable below 60 degrees C. The main kinetic constants were determined. The tyrosinase was able to convert p-tyrosol and p-coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. CONCLUSIONS: Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg(-1) protein for monophenolase and diphenolase respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking.     

Production of laccase by a newly isolated strain of Trametes modesta

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by Trametes modesta were evaluated using the one-factor-at-a-time method. The final optimisation was done using a central composite design resulting in a four-fold increase of the laccase activity to 178 nkat ml(-1). Response-surface analysis showed that 7.34 g l(-1) wheat bran, 0.87 g l(-1) glucose, 2.9 g l(-1) yeast extract, 0.25 g l(-1) ammonium chloride, an initial pH of 6.95 and an incubation temperature of 30.26 degrees C were the optimal conditions for laccase production. Laccase produced by T. modesta was fully active at pH 4 and at 50 degrees C. The laccase was very stable at pH 4.5 and at 40 degrees C but half-lives decreased to 120 and 125 min at higher temperature (60 degrees C) and lower pH (pH 3).     

Laccase activity in melanin-producing strains of Sinorhizobium meliloti

Laccase-like activity was detected in melanin-producing strains of Sinorhizobium meliloti mainly in cells at the stationary growth phase when copper was added to the medium. The laccase showed both syringaldazine and ABTS (2,2'-azino-bis-ethylbenzthiazoline-6-sulfonic acid) oxidase activities and was activated by the addition of 1.7 mM sodium dodecyl sulfate. Activity was totally inhibited by the addition of 1.0 mM EDTA, suggesting that the enzyme is a metal-dependent one. The enzyme was found to be cytosolic having an optimum pH of 5.0, an estimated molecular mass of 95 kDa and a K(m) of 4 microM for syringaldazine. Both laccase and tyrosinase activities were detected in melanin-producing S. meliloti strains. Plant growth-promoting (PGP) effect in rice by a laccase-producing S. meliloti strain when co-inoculated with Azospirillum brasilense Cd was observed. PGP effect by co-inoculation significantly increased plant yield compared to A. brasilense by itself. To the best of our knowledge this is the first report on laccase production in rhizobia and cooperation between Azospirillum and Sinorhizobium in rice.     

Evaluation of Argentinean white rot fungi for their ability to produce lignin-modifying enzymes and decolorize industrial dyes

The decolorizing capacity of 26 white rot fungi from Argentina was investigated. Extracellular production of ligninolytic enzymes by mycelium growing on solid malt extract/glucose medium supplemented with different dyes (Malachite Green, Azure B, Poly R-478, Anthraquinone Blue, Congo Red and Xylidine), dye decolorization and the relationship between these two processes were studied. Only ten strains decolorized all the dyes, all ten strains produced laccase, lignin peroxidase and manganese peroxidase on solid medium. However, six of the strains could not decolorize any of the dyes; all six strains tested negative for lignin peroxidase, and produced less than 0.05 U/g agar of manganese peroxidase. Comparing the isolates with the well-known dye-degrader Phanerochaete chrysosporium, a new fungus was identified: Coriolus versicolor f. antarcticus, potentially a candidate for use in biodecoloration processes. Eighteen day-old cultures of this fungus were able to decolorize in an hour 28%, 30%, 43%, 88% and 98% of Xylidine (24 mg/l), Poly R-478 (75 mg/l), Remazol Brilliant Blue R (9 mg/l), Malachite Green (6 mg/l) and Indigo Carmine (23 mg/l), respectively. Laccase activity was 0.13 U/ml, but neither lignin peroxidase nor manganese peroxidase were detected in the extracellular fluids for that day of incubation.     

Effect of carbon,nitrogen sources and inducers on ligninolytic enzyme production by Morchella crassipes

The effect of different carbon, nitrogen sources and inducers on growth and ligninolytic activity by Morel mushroom Morchella crassipes was investigated. The maximum growth was observed in mineral salts broth containing glucose as the carbon source and sodium nitrate as the nitrogen source. Among the inducers, chemical inducers inhibited the growth whereas in natural substrates, growth was not affected much. Manganese peroxidase and lignin peroxidase activity were not detected in the medium with different carbon and nitrogen sources, whereas laccase activity varied depending on carbon source (0.7–3.48 U/ml). Among the inducers, natural inducers resulted in an increase in the enzyme activities. Maximum laccase activity was observed in rice straw (12. 6 U/ml) followed by ABTS (11.6 U/ml); Manganese peroxidase activity was maximum in rice straw (14.32 U/l) wheat straw (12.16 U/l) and phenol red (15 U/l) as the inducers, whereas for Lignin peroxidase activity, rice straw (22 U/l), wheat straw (16 U/l) and veratrylalcohol (20 U/l) served as the best inducers.     

Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi

White rot fungi were collected from Chirinda and Chimanimani hardwood forests in Zimbabwe and studied with respect to growth temperature optima and dye decolorization. Temperature optima were found to vary (between 25-37 degrees C) amongst the isolates. The isolates were screened for their ability to degrade the polymeric dyes; blue dextran and Poly R478 and the triphenylmethane dyes; cresol red, crystal violet and bromophenol blue. Semi-quantitative determination of the hydrolytic enzyme activities possessed by the white rot fungi was determined using the API ZYM system. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in the fungi were also determined. No LiP was detected in any of the isolates but all isolates showed manganese peroxidase and laccase activities. Time related decolorization studies and optimum pH determinations for Poly R478 degradation by the isolates were carried out in liquid cultures. The most significant rates of Poly R478 decolorization in liquid cultures were found with the following isolates: Trametes cingulata, Trametes versicolor, Trametes pocas, DSPM95 (a species to be identified), Datronia concentrica and Pycnoporus sanguineus.     

Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by the endophytic fungus Monotospora sp. were evaluated. The optimal temperature and initial pH for laccase production by Monotospora sp. in submerged culture were found to be 30 degrees C and 8.5, respectively. Maltose (2 g l(-1)) and ammonium tartrate (10 g l(-1)) were the most suitable carbon and nitrogen source for laccase production. Under optimal culture medium, the maximum laccase activity was determined to be 13.55 U ml(-1), which was approximately four times higher than that in basal medium. This is the first report on laccase production by an endophytic fungus.     

Utilization of rice straw for laccase production by <Emphasis Type="Italic">Streptomyces psammoticus</Emphasis> in solid-state fermentation

Laccase production from a novel actinobacterial strain, Streptomyces psammoticus, MTCC 7334 was optimized in solid-state fermentation. The process parameters were initially optimized by the conventional “one factor at a time” approach, and the optimal levels of the factors that had considerable influence on enzyme production were identified by response surface methodology. Rice straw was identified as a suitable substrate for laccase production (17.3 U/g), followed by coffee pulp (15.8 U/g). Other optimized conditions were particle size, 500–1,000 μm (21.2 U/g); initial moisture content, 65% (26.8 U/g); pH of moistening solution, 8.0 (26.9 U/g); incubation temperature, 32°C (27.6 U/g) and inoculum size, 1.5 × 10⁷ CFU (33.8 U/g). Yeast extract served as the best nitrogen source (34.8 U/g). No enhancement in enzyme yield was observed with carbon supplementation. The level of yeast extract, inoculum size and copper sulphate were optimized statistically. Statistical optimization performed using a central composite design resulted in threefold increase in laccase activity (55.4 U/g) as compared to the unoptimized medium (17.3 U/g). The upgrading of fermented rice straw for fodder enhancement is also discussed briefly.     

Uricase production by a recombinant <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> strain harboring <Emphasis Type="Italic">Candida utilis</Emphasis> uricase gene

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae alpha-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.     

Candida albicans cell wall lytic enzyme produced by Streptomyces thermodiastaticus

The production of lytic enzyme by Streptomyces thermodiastaticus was found to be affected by some growth conditions and nutritional factors. The highest enzyme production was obtained after 18 h of incubation at pH 5.5 and at 50 degrees C. The carbon source influenced the lytic enzyme production. A higher enzyme yield was obtained when Candida albicans cell wall (1 g/100 ml) was used as the sole carbon source. NaNO3 at 0.1 g/100 ml was the best nitrogen source for enzyme production. From all phosphorous sources, microelements, and growth factors tested, KH2PO4 (1 g/l), ZnSO4 (1 mg/I) and Tween 80 (0.1%), respectively, were found to favour the highest production of lytic enzymes by S. thermodiastaticus. The lytic enzymes mainly produced chitinolytic and proteolytic activities.