Determination of kinetics and heat of hydrolysis for non-homogenous substrate by isothermal calorimetry.

The competitiveness of the second-generation bioethanol by biotechnological process requires an effective and quantitative control of biochemical reactions. In this study, the potential of isothermal calorimetry technique to measure heat and kinetics of a non-homogeneous substrate enzymatic hydrolysis is intended. Using this technique, optimum temperature of the enzymes used for lignocellulosic molecules hydrolysis was determined. Thus, the amount of substrate-to-enzyme ratio was highlighted as an important parameter of the hydrolysis yield. Furthermore, a new enzymes' cocktail efficiency consisting of a mix of cellulases and cellobiose dehydrogenase (CDH) was qualified by this technique. The results showed that this cocktail allowed the production of a high amount of gluconic acid that could improve the attractiveness of these second-generation biofuels. From the set of experiments, the hydrolysis heat of wheat straw was derived and a meaningful value of -32.2 ± 3.2 J g-1 (gram reducing sugars product) is calculated. Then, isothermal measurements were used to determine kinetic constants of the cellulases and CDH mix on wheat straw. Results showed that this enzyme cocktail has an optimal rate at 45 °C in the range of temperatures tested (40-55 °C).

Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications.

Tyrosinases are type-3 copper proteins involved in the initial step of melanin synthesis. These enzymes catalyse both the o-hydroxylation of monophenols and the subsequent oxidation of the resulting o-diphenols into reactive o-quinones, which evolve spontaneously to produce intermediates, which associate in dark brown pigments. In fungi, tyrosinases are generally associated with the formation and stability of spores, in defence and virulence mechanisms, and in browning and pigmentation. First characterized from the edible mushroom Agaricus bisporus because of undesirable enzymatic browning problems during postharvest storage, tyrosinases were found, more recently, in several other fungi with relevant insights into molecular and genetic characteristics and into reaction mechanisms, highlighting their very promising properties for biotechnological applications. The limit of these applications remains in the fact that native fungal tyrosinases are generally intracellular and produced in low quantity. This review compiles the recent data on biochemical and molecular properties of fungal tyrosinases, underlining their importance in the biotechnological use of these enzymes. Next, their most promising applications in food, pharmaceutical and environmental fields are presented and the bioengineering approaches used for the development of tyrosinase-overproducing fungal strains are discussed.

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.

Natural and recombinant fungal laccases for paper pulp bleaching.

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l(-1). Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (Km) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).

Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application.

A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l(-1)), improved FAEA activity 24.5-fold and a yield of 1 g l(-1) of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification--close to 75%--and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector.

Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer.

AIMS: Laccase production by the monokaryotic strain Pycnoporus cinnabarinus ss3 was studied using ethanol as inducer in the culture medium. METHODS AND RESULTS: The effect of ethanol was tested at 10, 20, 30, 35 and 45 g l-1 and compared with that of ferulic acid, known until now as the most efficient inducer for laccase expression by P. cinnabarinus ss3. In the presence of 35 g l-1 ethanol, laccase activity (266 600 U l-1) and productivity (19 000 U l-1 day-1) were nine and fivefold higher compared with ferulic acid-induced cultures, and 155- and 65-fold higher compared with non-induced cultures, respectively. In vivo, ethanol added to the culture medium of P. cinnabarinus ss3 favoured a continuous and high expression of laccase gene. Under these conditions, P. cinnabarinus ss3 produced preferentially the isoenzyme LAC I. Ethanol added in vitro to the purified P. cinnabarinus ss3 laccase typically inhibited the enzymatic activity. CONCLUSIONS: In spite of an initial inhibitory effect on mycelial growth, ethanol was shown to be a very strong inducer for laccase expression by P. cinnabarinus ss3 allowing an average yield of 1-1.5 g l-1 laccase. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. cinnabarinus ss3 as an outstanding producer of laccase in the presence of ethanol as inducer. Ethanol is an inexpensive agricultural by-product and the process is simple to scale-up for industrial production.

Design and scale up of a process for manganese peroxidase production using the hypersecretory strain Phanerochaete chrysosporium I-1512.

Manganese peroxidase (MnP) production was performed in a airlift bioreactor in which Phanerochaete chrysosporium I-1512, an MnP hypersecretory strain, was immobilized on a stainless steel mesh. Production was scaled up from a 2.5-L bench scale to a 100-L bioreactor. The yield of MnP was increased 2-fold and reached 6600 U L(-1). These results indicate the feasibility of MnP production on a medium scale, which promises sufficient MnP availability for its use in pulp bleaching at industrial scale.

Isolation from coastal sea water and characterization of bacterial strains involved in non-ionic surfactant degradation.

A bacterial community degrading branched alkylphenol ethoxylate (APE) was selected from coastal sea water intermittently polluted by urban sewage. This community degraded more than 99% of a standard surfactant, TRITON X 100, but I.R. analysis of the remaining compound showed the accumulation of APE2 (alkylphenol with a two units length ethoxylated chain) which seemed very recalcitrant to further biodegradation. Twenty-five strains were isolated from this community, essentially Gram negative and were related to Pseudomonas, Oceanospirillum or Deleya genera. Among these strains, only four were able to degrade APE9-10 (TRITON X 100). They were related to the Pseudomonas genus and were of marine origin. Pure cultures performed with these strains on TRITON X 100 gave APE5 and APE4 as end products. These products were further degraded to APE2 by two other strains unable to degrade the initial surfactant.

Complete oxidation of linear alkylbenzene sulfonate by bacterial communities selected from coastal seawater.

Anionic surfactants, especially alkylbenzene sulfonates, are discharged into marine areas in great quantities. Because of their poor biodegradability, linear alkylbenzene sulfonates accumulate in seawater and sediments. Bacterial communities that can degrade surfactants were selected from coastal seawater contaminated by urban sewage. All the isolated strains consisted of gram-negative, strictly aerobic rods or helical bacteria. Some of these, though isolated from coastal seawater, did not need sodium for growth and appeared to be related to the genera Alcaligenes and Pseudomonas. Complete surfactant biodegradation was achieved by three important steps: terminal oxidation of the alkyl chain, desulfonation, and aromatic-ring cleavage. Only a few strains were able to carry out the first two steps. The aromatic ring was then cleaved by other strains that possess very specific enzymatic activities. Finally, a number of strains grew on short acids that were end-of-metabolism products of the others.

[Degradation of a commercial surface-active agent, in the presence of a complementary source of carbon, by a selected bacterial colony in a marine environment]. / Dégradation d'un tensio-actif commercial, en présence d'une source complémentaire de carbone, par une communauté bactérienne sélectionnée en milieu marin.

Bacterial communities that can degrade surfactants have been selected from coastal seawaters contaminated by urban sewages. Only the linear fraction of commercial anionic surfactants was quickly degraded, and a residual fraction representing 10% of the initial concentration always remained. The highest concentrations of surfactant tolerated by these communities depend on the nature of the co-substrate and on the degree of adaptation of the selected bacteria.

Determination of sterol and triterpene alcohol acetates in natural products by reversed-phase liquid chromatography and gas chromatography-mass spectrometry.

A partial separation of nine sterol acetates and seven triterpene alcohol acetates by reversed-phase liquid chromatography is described. Good results are obtained using acetonitrile-water (90:10, v/v) as mobile phase with an UV detector at 205 nm. The variation in sterol sensitivity shows that this technique is not suitable for quantitative analyses. A combination of this technique for the fractionation of the natural sterol mixture, gas-liquid chromatography for quantitation and gas chromatography-mass spectrometry for identification is necessary for the determination of sterol compounds contained in natural products. An example of the separation, identification and quantitation of sterol acetates from sunflower seed oil is given.