Differential regulation of Pleurotus ostreatus dye peroxidases gene expression in response to dyes and potential application of recombinant Pleos-DyP1 in decolorization.

Dye-decolorizing peroxidase (DyP) from the white rot basidiomycete Pleurotus ostreatus is a heme peroxidase able to oxidize diverse substrates, including recalcitrant phenols and dyes. This study analyzed the effect of chemical dyes on P. ostreatus growth, DyP activity and the expression of four Pleos-dyp genes during the time-course of Pleurotus ostreatus cultures containing either Acetyl Yellow G (AYG), Remazol Brilliant Blue R (RBBR) or Acid Blue 129 (AB129) dyes. Additionally, Pleos DyP1 was heterologously expressed in the filamentous fungus Trichoderma atroviride in order to explore the potential of a secreted recombinant enzyme for decolorizing different dyes in cultures and plate assays. The addition of dyes had an induction effect on the enzymatic activity, with the fermentations undertaken using RBBR and AYG dyes presenting the highest total DyP activity. DyP gene expression profiles displayed up/down regulation during the culture of three Pleos-dyp genes (Pleos-dyp1, Pleos-dyp2 and Pleos-dyp4), while Pleos-dyp3 transcript was not detected under any of the culture conditions studied. A 14-fold relative induction level (log2) increase for Pleos-dyp2 and Pleos-dyp4 in AB129 and AYG, respectively, was also found. The presence of AB129 resulted in the highest Pleos-dyp1 gene induction and repression level, corresponding to 11.83 and -14.6-fold relative expression and repression levels, respectively. The lowest expression level of all genes was observed in RBBR, a response which is associated with the growth phase. The filamentous fungus Trichoderma atroviride was successfully transformed for the heterologous expression of Pleos-dyp1. The modified strains (TaDyP) were able to decolorize mono-azo, di-azo, anthraquinone and anthracenedione dyes with extracellular DyP1 activity found in the culture supernatant. After 96 h of culture, the recombinant TaDyP strains were able to degrade (decolorize) 77 and 34% of 0.05mM AB129 and 0.25mM AYG, respectively.

Functional characterization of a novel ß-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697 on structurally diverse fructans.

AIM: In this study, we describe the isolation of a gene encoding a novel ß-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697, and the characterization of the enzyme, the second one found in this strain, significantly different in primary sequence to the already reported bifidobacterial ß-fructofuranosidases. METHODS AND RESULTS: The gene, found through genome-mining was expressed in Escherichia coli C41(DE3). The recombinant enzyme (B.longum_l1) has a molecular weight of 75 kDa, with optimal activity at 50°C, pH 6·0-6·5, and a remarkable stability with a half-life of 75·5 h at 50°C. B.longum_l1 has a wide specificity for fructans, hydrolysing all substrates through an exo-type mechanism, including Oligofructose P95 (ß2-1 fructooligosaccharides (FOS), DP 2-8), Raftilose Synergy 1(ß2-1 FOS & inulin, DP 2-60), Raftiline HP (inulin, DP 2-60), bacterial inulin (3000 kDa) and levan (8·3 & 3500 kDa), Agave fructans (mixed fructans, DP 3-29) and levan-type FOS (ß2-6 FOS, DP 2-8), with the highest relative activity and turnover number found for levan-type FOS. The apparent affinity of the enzyme for levan-type FOS and Oligofructose P95 was found to be 9·2 and 4·6 mmol l(-1) (Km ) with a specific activity of 908 and 725 µmol min(-1)  mg(-1) of protein (k2 ), respectively, more than twice the activity for sucrose. CONCLUSION: B.longum_l1 is a wide substrate specificity enzyme, which may contribute to the competitiveness and persistence of this strain in the colon. SIGNIFICANCE AND IMPACT OF THE STUDY: The bifidobacterial ß-fructofuranosidase activity was evaluated with a wide variety of substrates including noncommercial fructans, such as levan-type and mixed agave fructans. Its activity on these substrates certainly strengthens their commercial prebiotic character and contributes to the understanding of bifidobacteria stimulation by structurally diverse fructans.

Synthesis of levan in water-miscible organic solvents.

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.

Lipase-catalyzed synthesis of xylitol monoesters: solvent engineering approach.

A solvent engineering strategy was applied to the lipase-catalyzed synthesis of xylitol-oleic acid monoesters. The different esterification degrees for this polyhydroxylated molecule were examined in different organic solvent mixtures. In this context, conditions for high selectivity towards monooleoyl xylitol synthesis were enhanced from 6 mol% in pure n-hexane to 73 mol% in 2-methyl-2-propanol/dimethylsulfoxide (DMSO) 80:20 (v/v). On the contrary, the highest production of di- and trioleoyl xylitol, corresponding to 94 mol%, was achieved in n-hexane. Changes in polarity of the reaction medium and in the molecular interactions between solvents and reactants were correlated with the activity coefficients of products. Based on experimental results and calculated thermodynamic activities, the effect of different binary mixtures of solvents on the selective production of xylitol esters is reported. From this analysis, it is concluded that in the more polar conditions (100% dimethylsulfoxide (DMSO)), the synthesis of xylitol monoesters is favored. However, these conditions are unfavorable in terms of enzyme stability. As an alternative, binary mixtures of solvents were proposed. Each mixture of solvents was characterized in terms of the quantitative polarity parameter E(T)(30) and related with the activity coefficients of xylitol esters. To our knowledge, the characterization of solvent mixtures in terms of this polarity parameter and its relationship with the selectivity of the process has not been previously reported.

Enhancement of lactase activity in milk by reactive sulfhydryl groups induced by heat treatment.

The effects of heat treatments of milk and whey prior to lactose hydrolysis with Kluyveromyces lactis beta-galactosidase were studied. It was observed that heat treatment of milk significantly increases lactase activity, with a maximum activity increase found when milk was heated at 55 degrees C. In whey from 55 up to 75 degrees C, beta-galactosidase activity decreased slightly. Nevertheless, heating whey at 85 degrees C for 30 min raised the rate of hydrolysis significantly. Electrophoretic patterns and UV spectra proved that the activity change correlated with milk protein denaturation, particularly that of beta-lactoglobulin. Heating whey permeate did not increase the enzyme activity as heating whole whey; but heating whey prior to ultrafiltration also resulted in enzyme activation. Measurement of free sulfhydryl (SH) groups in both whey and heated whey permeate showed that the liberation of free SH is highly correlated to the change of the activity. Furthermore, this activation can be reversed by oxidizing the reactive sulfhydryl groups, proving that the observed effect may be related to the release of free SH to the medium, rather than to the denaturation of a thermolabile protein inhibitor.

Enzyme-mediated solvent extraction of carotenoids from marigold flower (Tagetes erecta).

Marigold flowers are the most important source of carotenoids for application in the food industry. However, the extraction gives almost 50% losses of the carotenoids depending on conditions for silaging, drying, and solvent extraction. In the past decades, macerating enzymes have been successfully applied to improve the extraction yield of valued compounds from natural products. In this work, an alternative extraction process for carotenoids is proposed, consisting of a simultaneous enzymatic treatment and solvent extraction. The proposed process employs milled fresh flowers directly as raw material, eliminating the inefficient silage and drying operations as well as the generation of hard to deal with aqueous effluents present in traditional processes. The process developed was tested at the 80 L scale, where under optimal conditions a carotenoid recovery yield of 97% was obtained.

Characterization of a cell-associated inulosucrase from a novel source: a Leuconostoc citreum strain isolated from Pozol, a fermented corn beverage of Mayan origin.

A cell-associated fructosyltransferase was extracted from a novel source, a strain of Leuconostoc citreum isolated from Pozol, a Mexican traditional fermented corn beverage, where lactic microflora are partially responsible for the transformation process. The enzyme is associated with the cell wall. It was characterized both in its cell-associated insoluble form and after separation by urea treatment. The fructosyltransferase has a molecular mass of 170 kDa, the highest reported for this type of enzyme, and in its insoluble form is highly specific for polymer synthesis, with low fructose transferred to maltose and lactose added to the reaction medium (acceptor reactions). The synthesized polymer has an inulin-like structure with beta2-1 glycosidic linkages, as demonstrated by 13C nuclear magnetic resonance (NMR). Bacterial inulosucrases have only been reported in Streptococcus mutans.

Enzymatic extraction and transformation of glucovanillin to vanillin from vanilla green pods.

Glucovanillin was extracted from green pods and simultaneously transformed to vanillin by a combination of enzyme activities involving cell wall degradation and glucovanillin hydrolysis. The reaction is best carried out with 47.5% v/v aqueous ethanol solution during 8 h at 70 degrees C, in a two-step enzymatic reaction using Viscozyme followed by Celluclast, two commercial enzymatic products containing mainly pectinase and cellulase activities, respectively. The extractive reaction proceeded with high efficiency with an amount of extracted vanillin 3.13 times higher than the one obtained with the Soxhlet method. The classical curing/extraction process results in 1.1-1.8 g of vanillin/100 g of dry pods. It is concluded that the enzymatic reaction may substitute the microbial process involved in tissue fermentation previous to vanillin extraction with the simultaneous hydrolysis of glucovanillin.

Effect of beta-galactosidase hydration on alcoholysis reaction in organic one-phase liquid systems.

Alcoholysis reactions were performed in organic one-phase liquid systems with E. coli beta-galactosidase to produce heptyl-beta-galactoside from lactose and 1-heptanol. The reaction rate was highly dependent on the amount of water solubilized in the alcohol. A larger amount of water led to a system of two liquid phases in which the alcoholysis rate was 73% faster than in the one-phase system. No hydrolysis reaction of either lactose or product was observed in one-phase liquid systems up to 20 h, independent of the water content. Solubility of lactose in the organic phase increased with the water content in the system and the reaction followed the Michaelis-Menten model. Water activity was calculated for heptanol containing different amounts of water and the obtained values were used to estimate the hydration of beta-galactosidase from known models. Enzyme activity correlated with sorbed water, similar to the behavior reported for lysozyme in low water environments. It is concluded that water contribution to enzyme hydration dominates the rate of reaction compared to its effect on lactose solubilization.

Solubility of purified lutein diesters obtained from Tagetes erecta in supercritical CO(2) and the effect of solvent modifiers.

Purified lutein diesters deposited on commercial nonporous glass beads were solubilized in supercritical CO(2) in a computerized batch extractor, and their solubilities were compared to their solubilities in hexane. Densities of 0.7, 0.8, and 0.9 g/mL were evaluated without modifiers. Both pressure and temperature increased solubility, although temperatures >50 degrees C promoted carotenoid loss as determined by mass balance. Solubility was enhanced by the use of modifiers and was related to their log P. Chloroform (log P = 2) increased 2.8 times the amount of solubilized lutein diesters compared to pure CO(2) at the same extraction conditions (0.9 g/mL and 40 degrees C) to yield 65% of the amount extracted with hexane. Supercritical CO(2) extraction of lutein diesters could represent a cleaner technology as compared to the current industrial use of hexane with important ecological and health-related implications.

Selective enzyme-mediated extraction of capsaicinoids and caratenoids from chili guajillo puya (Capsicum annum L.) using ethanol as solvent.

The selective extraction of capsaicinoids and carotenoids from chili guajillo "puya" flour was studied. When ethanol was used as solvent, 80% of capsaicinoids and 73% of carotenoids were extracted, representing an interesting alternative for the substitution of hexane in industrial processes. Additionally, when the flour was pretreated with enzymes that break the cell wall and then dried, extraction in ethanol increased to 11 and 7% for carotenoid and capsaicinoid, respectively. A selective two-stage extraction process after the treatment with enzymes is proposed. The first step uses 30% (v/v) ethanol and releases up to 60% of the initial capsaicinoids, and the second extraction step with industrial ethanol permits the recovery of 83% of carotenoids present in the flour.

Alcoholysis and reverse hydrolysis reactions in organic one-phase system with a hyperthermophilic beta-glycosidase.

Alcoholysis and reverse hydrolysis reactions were performed enzymatically in one-phase water-saturated 1-heptanol systems. Lactose or glucose was used as substrate to produce heptyl-beta-galactoside and/or heptyl-beta-glucoside, respectively. When alcoholysis of lactose was performed at 37 degrees C with beta-galactosidase from Escherichia coli, the initial rate was 14 nmol/mL min, and the limiting factors were the poor solubility of the substrate in 1-heptanol and low thermal stability of the enzyme. When a hyperthermophilic beta-glycosidase was used at 90 degrees C, the rate was 3.14-fold higher; in this case a higher concentration of soluble lactose in the water-saturated heptanol was available to the enzyme due to the higher temperature. The hyperthermophilic beta-glycosidase was also able to use glucose and galactose as substrates to achieve the reverse hydrolysis reaction. As a consequence, when lactose was used as substrate, heptyl-beta-galactoside was formed by alcoholysis, while the released glucose moiety was used in a secondary reverse hydrolysis reaction to produce heptyl-beta-glucoside. Both reactions followed Michaelis-Menten kinetics behavior. Neither lactose nor heptyl glycosides were hydrolyzed by this enzyme in water-saturated heptanol. However, the conversion was limited by a strong product inhibition and the formation of oligosaccharides, especially at high substrate concentrations, reducing the final glycoside yield.

Induction and transcription studies of the dextransucrase gene in Leuconostoc mesenteroides NRRL B-512F.

Dextransucrase production by Leuconostoc mesenteroides NRRL B-512F in media containing carbon sources other than sucrose is reported for the first time. Dextransucrases were analyzed by gel electrophoresis and by an in situ activity assay. Their polymers and acceptor reaction products were also compared by (13)C nuclear magnetic resonance and high-performance liquid chromatography techniques, respectively. From these analyses, it was found that, independently of the carbon source, L. mesenteroides NRRL B-512F produced dextransucrases of the same size and product specificity. The 5' ends of dextransucrase mRNAs isolated from cells grown under different culture conditions were identical. Based on this evidence, we conclude that dextransucrases obtained from cells grown on the various carbon sources result from the transcription of the same gene. The control of expression occurs at this level. The low dextransucrase yields from cultures in D-glucose or D-fructose and the enhancement of dextransucrase gene transcription in the presence of sucrose suggest that an activating phenomenon may be involved in the expression mechanism. Dextransucrase mRNA has a size of approximately 4.8 kb, indicating that the gene is located in a monocistronic operon. The transcription start point was localized 34 bp upstream from the ATG start codon. The -10 and -35 sequences found, TATAAT and TTTACA, were highly homologous to the only glycosyltransferase promoter sequence reported for lactic acid bacteria.

Proteolytic processing of dextransucrase of Leuconostoc mesenteroides.

Various dextransucrase molecular mass forms found in enzyme preparations may sometimes be products of proteolytic activity. Extracellular protease in Leuconostoc mesenteroides strains NRRL B-512F and B-512FMC dextransucrase preparations was identified. Protease had a molecular mass of 30 kDa and was the predominant form derived from a high molecular mass precursor. The production and activity of protease in culture medium was strongly dependent on pH. When L. mesenteroides dextransucrase (173 kDa) was hydrolyzed by protease, at pH 7 and 37 degrees C, various dextransucrase forms with molecular masses as low as 120 kDa conserving dextransucrase activity were obtained.

Introducing transglycosylation activity in a liquefying alpha-amylase.

By mutating Ala-289 by Phe or Tyr in the Bacillus stearothermophilus alpha-amylase, we induced this enzyme to perform alcoholytic reactions, a function not present in the wild-type enzyme. This residue was selected from homology analysis with neopullulanase, where the residue has been implicated in the control of transglycosylation [Kuriki et al. (1996) J. Biol. Chem. 271, 17321-173291. We made some inferences about the importance of electrostatic and geometrical modifications in the active site environment of the amylase to explain the behavior of the modified enzyme.

Selectivity of methyl-fructoside synthesis with beta-fructofuranosidase.

Enzyme synthesis of methyl fructoside was studied using beta-fructofuranosidase from Sacharomyces cerevisiae and sucrose and methanol as substrates. Taking into account the inhibition and deactivation effects of methanol on the enzyme, a system with 4.9M (20%, v/v) methanol was selected. At this alcohol level, 35% of sucrose is converted to fructoside at low or high substrate concentrations. The effect of enzyme concentration, pH, and temperature on both the synthesis and the hydrolysis of the fructoside was investigated. It was found that if the reaction proceeds at pH 6.0, 4 degree C and/or 0.014 mg/mL (3 U/mL) of beta-fructofuranosidase at varying sucrose concentrations, methyl fructoside may be obtained with a minimum loss of the fructoside at the end of the reaction.

Exponentially fed-batch cultures as an alternative to chemostats: the case of penicillin acylase production by recombinant E. coli.

Exponentially fed-batch cultures (EFBCs), fed with medium containing a highly concentrated carbon source, are commonly employed for attainment of high cell densities. However, large variations in environmental conditions occur, and quasi-steady-state is usually achieved only for the limiting substrate concentration, restricting the use of such cultures in kinetic characterization studies. In this work we report the production of recombinant penicillin acylase (PA) in EFBC of an E. coli JM101 transformed with the pPA102 plasmid, which includes the PA gene under regulation of the lacZ gene promoter and using isopropyl-beta-thio-galactopyranoside (IPTG) as inducer. The culture was fed with nonconcentrated complete medium, resulting in the attainment of quasi-steady-state conditions not only in substrate concentration, but also in cell concentration, and in the specific rates of growth, product production, and substrate consumption. Similar transient behavior was observed between EFBC and chemostat results. At quasi-steady-state, the dilution rate in the EFBC equaled the growth rate. Specific PA production rate during the fed-batch phase remained relatively constant at each dilution rate and followed typical Luedeking-Piret kinetics, with growth-associated and non-growth-associated constants of 142 U gDCW-1 and 7.2 U gDCW-1 h-1, respectively. Specific glucose consumption rate linearly increased from 0.025 to 0.6 g gDCW-1 h-1 as the dilution rate increased from 0.01 to 0.35 h-1. The maximum specific PA activity increased with decreasing dilution rate, reaching its highest value of 2.0 U mg-1 at a dilution rate of 0.01 h-1, the lowest dilution tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and use of glucosyltransferases from Leuconostoc mesenteroides NRRL B-1299 for the synthesis of oligosaccharides containing alpha-(1-->2) linkages.

Glucosyltransferase activities, produced by batch culture of Leuconostoc mesenteroides NRRL B-1299, were recovered both in the culture supernatant (SGT) and associated with the insoluble part of the culture (IGT). A total glucosyltransferase activity of 3.5 U/mL was measured in batch culture. The enzymes from the supernatant were purified 313 times using aqueous two-phase partition between dextran and PEG phases, yielding a preparation with 18.8 U/mg protein. It was shown that both SGT and IGT preparations catalyze acceptor reactions and transfer the glucose unit from sucrose onto maltose to produce glucooligosaccharides. Some of the glucooligosaccharides synthesized (Ln series) contain alpha-(1-->6) osidic linkages and a maltose residue at the reducing end. They were completely hydrolyzed by glucoamylase and dextranase. The other glucooligosaccharides synthesized (Bn series) resisted the action of these enzymes. The tetrasaccharide of this series has been characterized by 13C NMR. Its structure was determined as 2-O-alpha-D-glucosylpanose. The oligosaccharides synthesized by the maltose acceptor reaction with the SGT and IGT preparations only differed in the relative amounts in which they were produced. The difference may arise from diffusional limitations appearing when the insoluble catalyst is used. Under the assay conditions, the glucanase resistant oligosaccharide yield was 35% with both glucosyltransferase preparations.