Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris

A cDNA encoding a laccase enzyme was isolated from a Trametes versicolor cDNA library. The gene was subcloned into the Pichia pastoris expression vector pPIC3.5 and transformed into the P. pastoris strains KM71 and GS115. Laccase-secreting transformants were selected by their ability to oxidise the substrate ABTS. No difference in laccase activity was observed between culture supernatants from GS115 (proteolytic) and KM71 (nonproteolytic) strains. The presence of at least 200 μM copper was necessary for optimal laccase activity in the culture supernatants. During growth of P. pastoris on minimal medium the pH of the medium was reduced to <3.0. If alanine was added to the medium the pH reduction was not as pronounced and at alanine concentrations >0.6% w/v the pH was kept constant for >7 days. Cultures in which the pH was maintained by alanine metabolism produced higher levels of laccase activity than those grown in the absence of alanine. This study describes the development of a medium that allows convenient pH control of P. pastoris without the need for continuous neutralisation. Journal of Industrial Microbiology & Biotechnology (2002) 29, 55–59 doi:10.1038/sj.jim.7000268 Received 08 August 2001/ Accepted in revised form 18 April 2002     

Biotransformation of Poly R-478 by continuous cultures of PVAL-encapsulated Trametes versicolor under non-sterile conditions

Mycelia of Trametes versicolor were aseptically encapsulated in PVAL hydrogel beads of 1–2?mm diameter. The encapsulated mycelia were grown continuously in an aerated reactor under non-sterile conditions. After 65 days contamination of the PVAL hydrogel beads by bacteria was found only in the outer layer to a depth of 50?μm. The encapsulated fungi still expressed ligninolytic enzymes, as confirmed by the biotransformation of Poly R-478. Elimination of Poly R-478 by encapsulated Trametes versicolor reached an efficiency of up to 89%, which was due partially to biotransformation (65%) and partially to adsorption onto biomass (24%). PVAL-encapsulated mycelia of Trametes versicolor were viable for at least 6 months without nutrient supplementation, if stored at 7?°C in a refrigerator. By encapsulation Trametes versicolor was apparently protected against microbial contaminants and against mechanical stress, which is known to inactivate ligninolytic enzymes. Encapsulated Trametes versicolor might thus be applicable for bioremediation to serve as an inoculum for reactor systems or for field-side applications.     

Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used.     

Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor

A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57 approximately 97 % homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.     

Optimisation of a microwave-assisted method for extracting withaferin A from Withania somnifera Dunal. using central composite design

Introduction – Recently, there have been growing attention on the modification and optimisation of new extraction and quantification methods, caused by the lack of environmentally friendly methodologies for the extraction of phytochemicals from complex matrices. In the case of pharmaceutical compounds, not only the extraction procedure but also the analysis method should be efficient, precise, fast and easy. Objectives – The essential pharmaceutical characteristics and trace concentration of withanolides led us to modify and optimise the previously reported extraction and quantification procedure for withaferin A (WA) as a candidate for withanolides. Matrial and methods – The WA from the air‐dried aerial part of Withania somnifera Dunal. was extracted using a microwave‐assisted extraction (MAE) technique. Four variables affecting the extraction procedure were optimised using the central composite design approach. The method of high‐performance thin‐layer chromatography assay was validated and applied for the quantification of each experiment. Results – The optimum values of factors were: extraction time (150 s), extraction temperature (68°C) and 17 mL of methanol : water in the ratio 25 : 75 as extracting solvent. The solvent system consisted of ethyl acetate : toluene : formic acid : 2‐propanol (7.0 : 2.0 : 0.5 : 0.5, v/v/v/v), and densitometric scanning at 220 nm was applied for the analysis. The dynamic linear range, LOD, LOQ and recovery with the inter‐day, and intra‐day RSDs of the developed method indicated the validity of the method. Conclusion – A pressurised MAE method for extracting WA from the plant's aerial part was optimised using factorial‐based design. The net effect of time, temperature, solvent volume and its ratio suggests that the yield of WA increases until each factor reaches its optimum value, and decreases with further increase in temperature or solvent ratio. Copyright © 2010 John Wiley & Sons, Ltd.     

Optimisation of conditions for the extraction of casearins from Casearia sylvestris using response surface methodology

Optimal conditions for the extraction of casearins from Casearia sylvestris were determined using response surface methodology. The maceration and sonication extraction techniques were performed using a 3 x 3 x 3 full factorial design including three acidity conditions, three solvents of different polarities and three extraction times. The yields and selectivities of the extraction of casearins were significantly influenced by acidity conditions. Taking into account all variables tested, the optimal conditions for maceration extraction were estimated to involve treatment with dichloromethane saturated with ammonium hydroxide for 26 h. Similar yields and selectivities for casearins were determined for sonication extraction using the same solvent but for the much shorter time of 1 h. The best results for stabilisation of the fresh plant material were obtained using leaves that had been oven dried at 40 degrees C for 48 h.     

Optimization of gene delivery to HEK293T cells by microporation using a central composite design methodology

Microporation is an efficient method for delivering plasmid DNA molecules into cultured cells. Herein, we present the optimization of gene delivery by microporation using a Central Composite Design methodology. It was given relevance not only to the transfection efficiency but also to the cell recovery. Different amounts of DNA (1 and 3 μg) mainly affected cell viabilities and cell recoveries, which decrease from 93 to 76% and from 47 to 25% respectively, when higher DNA quantity is used. With this work we suggest an easy methodology to improve transfection of mammalian cells underlining the feasibility to achieve 60% of gene delivery efficiencies whilst recovering 50% of cells, with 90% of viability.     

Amelioration of the ability to decolorize dyes by laccase: relationship between redox mediators and laccase isoenzymes in Trametes versicolor

The effect of redox mediators in the dye decolorization by two laccase isoenzymes from Trametes versicolor cultures supplemented with barley bran has been investigated. All the redox mediators tested, 1-hydroxybenzotriazole (HBT), promazine (PZ), para-hydroxybenzoic acid (pHBA) and 1-nitroso-2-naphthol-3,6-disulfonic acid (NNDS), led to higher dye decolorization than those obtained without mediator addition. Among the different tested mediators, PZ was the most effective one at a low range of concentration (0.5–50 μM) and the natural mediator employed, pHBA did not improve significantly the degree of decolorization, and was slightly inhibitory.The two laccase isoenzymes, LacI and LacII, showed different decolorization capability depending on the mediator used. No significant differences were detected for NNDS, however LacII was more effective than LacI in the presence of PZ, while in the presence of HBT LacI was the fastest and the most effective isoenzyme.     

Carbon mass balance methodology to characterize the growth of pigmented marine bacteria under conditions of light cycling

A carbon mass balance methodology employing minimal measurements was applied to heterotrophic and photoheterotrophic marine bacteria grown under constant dilution and exposed to 12-h intervals of light or darkness. Carbon mass balance calculations using measurements taken every 3 h closed to within 93–103% using dissolved organic carbon, biomass carbon and CO₂ production data only, indicating that background interference from dissolved inorganic carbon variations in the amended seawater medium was not significant. Neither strain was observed to sustain a net CO₂ fixation using paramagnetic measurement of oxygen uptake rates (OUR), indicating a need for more sensitive on-line measurement techniques for OUR. Photoheterotrophic growth demonstrated lower carbon-mole biomass yields (0.41±0.026 vs. 0.64±0.013 mol mol^–1) despite higher specific glucose uptake rates (0.025 vs. 0.02 mol mol^–1 h^–1), suggesting that bioreactor-based study of marine bacteria can present growth modes that are different from those encountered in the marine environment.     

Bacterial degradation of pyridine, indole, quinoline, and their derivatives under different redox conditions

Bacteria have evolved a diverse potential to transform and even mineralize numerous organic compounds of both natural and xenobiotic origin. This article describes the occurrence of N-heteroaromatic compounds and presents a review of the bacterial degradation of pyridine and its derivatives, indole, isoquinoline, and quinoline and its derivatives. The bacterial metabolism of these compounds under different redox conditions – by aerobic, nitrate-reducing, sulfate-reducing and methanogenic bacteria – is discussed. However, in natural habitats, various environmental factors, such as sorption phenomena, also influence bacterial conversion processes. Thus, both laboratory and field studies are necessary to aid our understanding of biodegradation in natural ecosystems and assist the development of strategies for bioremediation of polluted sites. Occurring predominantly near (former) wood-treatment facilities, creosote is a frequent contaminant of soil, subsoil, groundwater, and aquifer sediments. In situ as well as withdrawal-and-treatment techniques have been designed to remediate such sites, which are polluted with complex mixtures of aromatic and heterocyclic compounds. Received: 26 September 1997 / Received revision: 23 December 1997 / Accepted: 27 December 1997     

Optimisation of medium and culture conditions for the production of antifungal substances to Colletotrichum musae by Trametes elegans SR06

An endophytic fungus SR06 was isolated from a leaf of Amomum villosum Lour., which had a high antagonistic effect on Colletotrichum musae with an inhibition ratio of 41.20%. The antifungal substances could be secreted into fermentation broth, which had a high inhibitory activity. Strain SR06 was identified as Trametes elegans according to internal transcribed spacer sequence analysis. Response surface methodology (RSM) was used to optimise the process parameters of antifungal substances production. Using the Plackett–Burman design, three variables (glucose, yeast extract and MgSO₄·7H₂O) exerted significant effects on antifungal substances production. Then RSM experiments were conducted to further optimise the three variables. The optimal medium components were 26.45?g/L glucose, 10?g/L peptone, 14.96?g/L yeast extract and 1.49?g/L MgSO₄·7H₂O, and the optimal initial pH was 6.0, with a culture temperature of 28°C and a shaking speed of 180?rpm. Under the optimised conditions, a significant improvement in the production of antifungal substances by T. elegans SR06 was accomplished, and the inhibition zone diameter was up to 29.2?mm after culturing for 7d. The average control efficacy of the fermentation supernatant of SR06 against C. musae was 51.29% on banana fruits, which was significantly higher than that of the fungicide carbendazim.     

Modeling of polyphenols extraction from pomegranate by-product using rotatable central composite design of experiments

There is a growing request to find an effective method of polyphenols extraction from agro-industry by-product as pomegranate. In this study, response surface methodology (RSM) was used to explore the effect of three factors on ultrasonic assisted extraction (UAE) of total polyphenols (TP), total flavonoids (TF) and condensed tannins (CT) from pomegranate peels. The optimal conditions were determined for each phenolic compound using regression model equations and 3-D plots. The high TP, TF and CT content were obtained with, respectively, liquid/solid ratio of 20.00, 9.77, 9.77, extraction time of 36.38, 41.82, 30.39 min and 36.00, 83.64, 59.26% of ethanol percentage. In fact, liquid/solid ratio of 20, extraction time of 30.94 min and 59.26% of ethanol gives the highest contents of TP, TF and CT simultaneously. The experimental values using optimal conditions agreed with the predicted values. The pomegranate peels extract obtained under optimum conditions have an effective antioxidant activity as determined by ABTS and DPPH assays.     

Developement of serum-free media in CHO-DG44 cells using a central composite statistical design

A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM’s F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM’s F12: IMDM (1:1). CHO-DG44 cells expressing S25 antibody grew from 2 × 10⁵ cells to maximum cell density of 1.04 × 10⁶ cells/ml after 5 days in this control medium. A central composite design was used to identify optimal levels and interaction among five groups of medium components. These five groups were glutamine, Essential Amino Acids (EAA), Non Essential Amino Acids (NEAA), Insulin, Transferrin, Selenium (ITS), and lipids. Fifty experiments were carried out in four batches, with two controls in each batch. There was little effect of ITS and Lipid concentrations over the range studied, and glutamine concentration showed a strong interaction with EAA. The optimal concentrations of the variables studied were 2.5 mM Glutamine, 7.4 mM (2×) EAA, 1.4 mM (0.5×) NEAA, 1× ITS supplement, 0.7× Lipids supplement. The maximum viable cell density attained in the optimized medium was 1.4 × 10⁶ cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 ± 3.4 μg/mL, a 50% improvement.     

Optimization of cellulase-free xylanase production by alkalophilic Cellulosimicrobium sp. CKMX1 in solid-state fermentation of apple pomace using central composite design and response surface methodology

Microbial xylanases and associated enzymes degrade the xylans present in lignocellulose in nature. Xylanase production by Cellulosimicrobium sp. CKMX1, isolated from mushroom compost, produced a cellulase-free extracellular endo-1, 4-β-xylanase (EC 3.2.1.8) at 35 °C and pH 8.0. Apple pomace—an inexpensive and abundant source of carbon—supported maximal xylanase activity of 500.10 U/g dry bacterial pomace (DBP) under solid state fermentation. Culture conditions, e.g., type of medium, particle size of carbon source, incubation period, temperature, initial pH, and inoculum size, were optimized and xylanase activity was increased to 535.6 U/g DBP. CMCase, avicelase, FPase and β-glucosidase activities were not detected, highlighting the novelty of the xylanase enzyme produced by CKMX1. Further optimization of enzyme production was carried out using central composite design following response surface methodology with four independent variables (yeast extract, urea, Tween 20 and carboxymethyl cellulose), which resulted in very high levels of xylanase (861.90 U/g DBP). Preliminary identification of the bacterial isolate was made on the basis of morphological and biochemical characters and confirmed by partial 16Sr RNA gene sequencing, which identified CKMX1 as Cellulosimicrobium sp. CKMX1. A phylogenetic analysis based on the 16Sr RNA gene sequence placed the isolate within the genus Cellulosimicrobium, being related most closely to Cellulosimicrobium cellulans strain AMP-11 (97% similarity). The ability of this strain to produce cost-effective xylanase from apple pomace on a large scale will help in the waste management of apple pomace.     

Modeling and optimization of lipase-catalyzed production of succinic acid ester using central composite design analysis

Esterification of succinic acid with oleyl alcohol catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was investigated in this study. Response surface methodology (RSM) based on a five-level, four-variable central composite design (CCD) was used to model and analyze the reaction. A total of 21 experiments representing different combinations of the four parameters including temperature (35–65°C), time (30–450 min), enzyme amount (20-400 mg), and alcohol:acid molar ratio (1:1-8:1) were generated. A partial cubic equation could accurately model the response surface with a R² of 0.9853. The effect and interactions of the variables on the ester synthesis were also studied. Temperature was found to be the most significant parameter that influenced the succinate ester synthesis. At the optimal conditions of 41.1°C, 272.8 min, 20 mg enzyme amount and 7.8:1 alcohol:acid molar ratio, the esterification percentage was 85.0%. The model can present a rapid means for estimating the conversion yield of succinate ester within the selected ranges.     

Statistical optimization of culture conditions for 1,3-propanediol by Klebsiella pneumoniae AC 15 via central composite design

A central composite design was used to study the effect of glycerol, rate of stirring, air aeration and pH on the synthesis of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae AC 15. Among the four variables, glycerol and rate of stirring significantly affected 1,3-PD productivity, whereas air aeration and pH were not effective. A quadratic polynomial equation was obtained for 1,3-PD productivity by multiple regression analysis using response surface methodology. The validation experimental confirmed with the predicted model. The optimum combinations for 1,3-PD productivity was glycerol, rate of stirring, air aeration, and pH of 50 g/l, 318 rpm, 0.6 vvm, 6.48, respectively. The subsequent fed batch experiments produced 1,3-PD of 70 g/l at a fermentation of 30 h.     

Optimization of flocculation conditions for kaolin suspension using the composite flocculant of MBFGA1 and PAC by response surface methodology

The response surface methodology (RSM) was employed to study the treatment of kaolin suspension by the composite flocculant of MBFGA1 and PAC. And the two quadratic models of the five factors were established with the flocculating rate and floc size as the target responses. The optimal flocculating conditions are MBFGA1 99.75 mg/L, PAC 121 mg/L, pH 7.3, CaCl₂ 27 mg/L and the top speed of stir 163 rpm, respectively. That was obtained from the compromised results of two desirable responses, flocculating rate as 100% and floc size as 0.7 mm which were deduced from the frequency of responses. By means of Zeta potential measurement and experiment of flocculating process, it could be concluded that PAC has more capability on changing the potential of colloid and MBFGA1 is good at absorption and bridge effect. The composite of two kinds of predominance makes a significant sense on enhancing flocculating rate, reducing flocculent costs and decreasing secondary pollution.     

A method for the decrease of phenolic content in commercial canola meal using an enzyme preparation secreted by the white-rot fungus Trametes versicolor

An enzymatic process for upgrading the quality of canola meal (CM) by decreasing its phenolic content was investigated. The new method was based on the addition of the enzyme preparation from white-rot fungus Trametes versicolor to the meal-buffer slurry. A 98% decrease in the concentration of SAE was observed after 1 h of the treatment. The following process variables were considered for optimizing the process: pH, temperature, enzyme, meal, and oxygen concentrations. It was found that: (1) the natural buffering capacity of CM resulted in a negligible effect of the pH of the buffer, which was used as the continuous phase in the process, on the extent of decrease in sinapic acid esters (SAE); (2) the system was saturated with the enzyme when its concentration was 4 nkat/mL of the continuous phase; and (3) the optimum temperature was 50 degrees C. The process could be carried out even at higher temperatures due to the protective action of CM, which resulted in an increase in the thermal stability of the enzyme. The particle size influenced the extraction of the SAE from the meal, indicating that, at lower SAE concentrations, the process became diffusion limited. This result, together with those showing no effect of the intensity of agitation, indicated that the enzymatic process can be characterized by high Biot numbers. During the enzymatic process, the molar concentration of available oxygen can become a limiting factor when it is more than four times lower than the molar concentration of phenolics in the treated meal. The new enzymatic method was compared with other methods reported in the literature for the decrease in the phenolic content of rapeseed meals. It was found that, among the methods tested, the enzymatic treatment was the most effective, followed by the lime treatment. The enzymatic process did not reduce the quality of the protein isolates prepared from the CM. After the addition of a simple acetone-washing step, the isolate from the enzymatically treated meal had even better properties. Copyright 1999 John Wiley & Sons, Inc.