André Morin

Production, partial purification and characterisation of lipases from Pseudomonas fragi CRDA 037

Abstract Pseudomonas fragi CRDA 037 produced extracellular (exo-) and intracellular (endo-) lipases when grown in whey media. Partially purified lipase extracts were obtained by precipitation with solid ammonium sulphate at 20–40% saturation for the exolipase and 20–60% saturation for the endolipase. Native polyacrylamide gel electrophoresis of the exolipase showed the presence of a major band with a molecular weight ( M r ) of 25·5 kDa, whereas the endolipase showed the presence of three fractions with M r of 35·5, 49 and 70 kDa. The pH optima for the exo- and endolipases were 8·75 and 9·0, respectively. Substrate specificity determinations were performed with triacetin, tributyrin, trimyristin and triolein, where exo- and endolipase demonstrated higher affinity for trimyristin, with K m values of 5·11 and 3·24 m m , respectively. Inhibition of the exo- and endolipase occurred in the presence of a range of chemicals, including sodium deoxychelate, ferrous and ferric chloride, diisopropyl fluorophosphate, N -bromosuccinimide and 5,5-dithiobis-(-2-nitrobenzoic acid).

Comparison of seven microbial D-hydantoinases

Abstract d -Hydantoinase (E.C.3.5.2.2) from Pseudomonas fluorescens (strains 1.2 and 1.9), Pseudomonas putida (strains 2.2, 2.5 and DSM 84), Serratia liquefaciens (strain 1.15) and Corynebacterium pseudodiphtheriticum (strain 14.10) were compared according to their stability to different purification procedures. With exception to C. pseudodiphtheriticum where between 75 and 85% of the enzyme was recovered after purification, yields less than 30% were observed for all other strains. These low recovery levels were not due to the removal of essential ions or to the absence of substrate during chromatography. Purification yields could not be improved by the addition of detergents and of a cryoprotectant to prevent aggregation and/or to increase hydantoinase stability. Hydantoinases from all the strains examined except C. pseudodiphtheriticum , were stable up to 37°C and at pH 5. The C. pseudodiphtheriticum enzyme was stable at temperatures up to 57°C and at pH values above 8. All hydantoinases exhibited maximum activity at temperatures between 45 and 55°C and at pH 8.0 or above. Native and SDS-PAGE gave M r estimates ranging from 160 000 to 235 000 and 60 000 ± 4000 for the native enzymes and their subunits, respectively.

ENHANCEMENT OF FRUITY AROMA PRODUCTION OF PSEUDOMONAS FRAGI GROWN ON SKIM MILK, WHEY AND WHEY PERMEATE SUPPLEMENTED WITH C3-C7 FATTY ACIDS

SummaryPseudomonas fragi strain CRDA 037 produced a fruity aroma when grown in skim milk-, whey-and whey permeate-based culture media. The production of the odour-active metabolites was related to the lipid content of these media but was not influenced by the pH of the cultures. Analysis of the fruity aroma revealed that esters of fatty acids were some of the odouractive metabolites. Addition of C3-C7 fatty acids to the culture at 0 h stimulated the production of the corresponding fatty acid esters from 12 to 1570 times compared to unsupplemented media. Supplementation of the culture media with the C3-C7 fatty acids at 48 h, resulted in a 1.4- to 932-fold increase in the ethyl ester concentration.

D-Hydantoinase from anaerobic microorganisms

SummaryOf 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl-β-alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5′-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days.

Biogeneration of ethyl valerate by whole cells of Pseudomonas fragi CRDA 037 in aqueous medium

The production of ethyl valerate by aqueous suspensions of whole cells of Pseudomonas fragi was followed over 8 h after the addition of ethanol and valeric acid. Initial quantity of esters formed ranged from 10 to 160 μmoles/100 ml under the following conditions: pH 8–9, 8–12°C, 5% (w/v) cell concentration, 0.01–0.05M valeric acid and 0.02–0.1M ethanol.

The effect of physical and chemical treatments on the esterase activity from Pseudomonas fragi CRDA 037

Abstract The effect of different treatments on the esterase activity of the cellular debris of Pseudomonas fragi , responsible for the production of natural fruity flavors in processed products, was investigated. Glass bead homogenization (GBH) decreased activity by 50%, French press homogenization (FPH) produced only a slight decrease, while ultrasonication (US) alone or with GBH increased activity by 25 and 50%, respectively. Treatment with (i) GBH (4 min) and Triton increased activity two-fold, (ii) GBH or US (4 min), Triton and EDTA produced little effect, (iii) FPH (three passes), Triton and EDTA produced a decrease of 30 to 50%, (iv) FP (three passes), US (4 min), Triton and EDTA had no effect, while (v) GBH (4 min), US (4 min), Triton and EDTA showed a 35% decrease in activity. The esterase activity of the cellular debris stored in buffer (4°C) and hexane (−80°C) decreased by 80% after 3 days, while that of whole cells stored in hexane (−80°C), and buffer at 4 and −80°C remained preserved for 3 days whereas that of the lyophilized whole cells greatly decreased. The esterase activity of whole cells stored in buffer containing 15 and 30% glycerol was preserved for three weeks.

Use of a polymerase-chain-reaction-amplified DNA probe from Pseudomonas putida to detect d-hydantoinase-producing microorganisms by direct colony hybridization

Pseudomonas putida strain DSM 84 produces N-carbamyl-d-amino acids from the corresponding d-5-monosubstituted hydantoins. The sequence of the d-hydantoinase gene from this strain (GenBank accession number L24157) was used to develop a DNA probe of 122 base pairs (bp) that could detect d-hydantoinase genes in other bacterial genera by DNA and by colony hybridization. Under conditions tolerating 32% mismatch, the probe was specific for all strains that expressed d-hydantoinase activity. These include Pseudomonadaceae of all rRNA groups, and bacteria belonging to the genera Agrobacterium, Serratia, Corynebacterium, and Arthrobacter. Environmental sampling was simulated by screening a mixture of unknown microorganims from commercial inocula for the biodegradation of industrial, municipal and domestic wastes. The 122-bp probe was specific for microorganisms that subsequently demonstrated d-hydantoinase activity. Bacterial species from four different genera were detected, which were Pseudomonas, Klebsiella, Enterobacter, and Enterococcus.

Production of fatty acid ethyl esters by Pseudomonas fragi under conditions of gas stripping

Abstract Pseudomonas fragi biomass was produced on whey and whey permeate with and without supplementation with various concentrations of butyric or valeric acid. The biomass produced in whey supplemented with 0·1% butyric acid possessed the highest ethylating activity of valeric acid in the presence of ethanol. The performance of the ethylating activity of P. fragi biomass was studied in aqueous medium under conditions of in-situ product removal using gas stripping. From 1000 ppm valeric acid and 2000 ppm ethanol, resting cells of P. fragi produced more than 800 ppm total fatty acid ethyl esters of which valeric acid ethyl ester accounted for more than 90%. The use of a gas stripping technique resulted in a 3.6-fold and 4.4-fold increase in the formation of total fatty acid and valeric acid ethyl esters, respectively, as compared to bioconversions conducted under conditions of product accumulation.

Extraction of cyclic amide amidohydrolase from green hulls of Pisum sativum and its use as biocatalyst for N-carbamyl amino acids

Abstract This work evaluates the potential of different varieties of legumes, Phaseolus vulgaris and Pisum sativum , as a source of a biocatalyst, cyclic amide amidohydrolase, which is used for the in vitro synthesis of N-carbamyl amino acids precursors. For most legumes evaluated, the substrate specificity of the amide amidohydrolase favored hydantoin hydrolysis, as compared to dihydropyrimidine and 5-monosubstituted hydantoins. The green hulls from P. sativum showed hydantoinase activity against hydantoin (34·9 U/g), d,l -5-methylmercaptoethylhydantoin (18·1 U/g) and d,l -5-isopropylhydantoin (5·3 U/g), while the feed culls of P. vulgaris had the highest d,l -5-methylhydantoin-hydrolysing activity (15·1 U/g). Conversion of up to 100% of hydantoin to N-carbamylglycine and of d,l -5-methylhydantoin to N-carbamylalanine was observed within 12 h when the crude hydantoinase from P. sativum variety green field pea was used in a batch-operated bioreactor. Hydantoinase extracted from pea hulls has been shown to be more stable and active at higher temperatures than the microbial hydantoinase from the bacterium Pseudomonas putida . An economic analysis indicates that legume surplus is a cheaper source of hydantoinase than microbial material.

Continuous production of N-carbamyl-D-alanine byPeptococcus anaerobius adsorbed on activated charcoal

SummaryThe strict anaerobic bacteriumPeptococcus anderobius produced a D-hydantoinase which was used to catalyse the formation of N-carbamyl-D-alanine from DL-5-methylhydantoin. Whole cells ofP. anaerobius adsorbed on granular activated charcoal were stable for up to seven days when used in two bioreactor systems (percolation and fluidization) operated continuously under aerobic conditions at 50°C.