Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis

The global regulatory gene, afsR2, from Streptomyces lividans was previously reported to highly stimulate two structurally unrelated antibiotics, actinorhodin and undecylprodigiosin, in both S. lividans and its close relative S. coelicolor. Production of eight avermectin components was also improved in S. avermitilis: the use of wild-type S. avermitilis and its high-producing mutant, transformed by introduction of multiple copies of afsR2, increased the total avermectin productions by 2.3-fold and 1.5-fold, respectively.     

Pathogenic Yersinia enterocolitica 2/O:9 and Yersinia pseudotuberculosis 1/O:1 strains isolated from human and non-human sources in the Plateau State of Nigeria

Foodborne yersiniosis, caused by enteropathogenic Yersinia, especially Yersinia enterocolitica, is an important cause of diarrhea in developed countries, especially in temperate zones. Since studies concerning the presence of enteropathogenic Yersinia in humans and foods are rare in developing countries and tropical areas, human and non-human samples were studied in Plateau state of Nigeria to obtain information on the epidemiology of Y. enterocolitica and Yersinia pseudotuberculosis. Surprisingly, ail-positive Y. enterocolitica and inv-positive Y. pseudotuberculosis were isolated in Plateau state of Nigeria from several samples of human and non-human origin. Bioserotype 1/O:1 was the only Y. pseudotuberculosis type found. Y. enterocolitica belonging to bioserotype 2/O:9 was the dominating type found in most samples. Bioserotype 4/O:3 was isolated only from one pig and one sheep. Using PFGE, 5 genotypes were obtained among 45 Y. enterocolitica 2/O:9 strains with NotI, ApaI and XhoI enzymes and 3 among 20 Y. pseudotuberculosis 1/O:1 strains with NotI and SpeI enzymes. All human Y. pseudotuberculosis 1/O:1 strains were indistinguishable from pig, sheep or food strains. The dominating genotype of Y. enterocolitica 2/O:9 strains among humans was also found among strains isolated from pig, fermented cow milk and traditional intestine pepper soap samples.     

Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus

Eight strains of Lactobacillus plantarum, 6 strains of Lactobacillus casei, Lactobacillus acidophilus LA-5, and Bifidobacterium lactis BB-12 were screened for β-glucosidase activity. We then proceeded to investigate the enzymatic potential of selected strains for bioconversion of delphinidin and malvidin glycosides to their metabolites. L. plantarum and L. casei strains showed the highest cell-envelope associated β-glucosidase activity. Intracellular β-glucosidase activity from B. lactis BB-12 was up to 287-fold higher than that of the other strains. The L. acidophilus strain showed low β-glucosidase activity, both, intra and extracellularly. No aglycons were detected in bacterial extract reactions with anthocyanin glycosides. Delphinidin-3-glucoside underwent chemical degradation to form mainly gallic acid, although delphinidin-3-glucoside degradation due to B. lactis BB-12 and enzymatic activity towards chemically-formed metabolites due to L. casei LC-01 were observed. Incubation of malvidin-3-glucoside with B. lactis BB-12, L. plantarum IFPL722, and L. casei LC-01 cell-free extracts led to different patterns of gallic, homogentisic and syringic acid formation.     

Effect of oxygen concentration on nitrogen removal by Nitrosomonas europaea and Paracoccus denitrificans immobilized within tubular polymeric gel

Tubular gel reactors containing Nitrosomonas europaea and Paracoccus denitrificans, which remove nitrogen from solutions through a process of nitrification and denitrification, require oxygen for ammonia oxidation, the first and rate-limiting step in the process. To accelerate ammonia oxidation, high concentrations of oxygen were applied to the reactors instead of air. Although a 50% O₂:N₂ gas mixture and pure oxygen were both toxic to free N. europaea cells, they actually accelerated ammonia oxidation by N. europaea immobilized within the tubular gel. Indeed, the rate of ammonia oxidation by a tube exposed to pure oxygen was twice that of one exposed to 20% O₂. When the distribution of N. europaea cells within the tubes was investigated using a fluorescently-labeled antibody, colonies were found on the external surface of the tube exposed to 20% O₂, but were located at a depth of 120–300 μm from the external surface in the case of the tube exposed to pure oxygen. The region between the external surface of the gel and the colonies apparently acted as a barrier, reducing the diffusion of oxygen and thus protecting the cells from oxygen cytotoxicity.     

The expression of genes PKM2 and CAST in the muscle tissue of pigs differentiated by glycolytic potential and drip loss, with reference to the genetic group

The present studies aimed at an analysis of the expression level of genes PKM2 and CAST in Longissimus lumborum [LL] muscle tissue of pigs differing as regards the glycolytic potential [GP] and drip loss [DL] from the LL muscle, with reference to the genetic group. The studies covered a total of 65 pigs: 20 purebred Landrace [L], 22 crossbreeds of Landrace with the Yorkshire [L × Y] and 23 three-breed crosses (Landrace × Yorkshire) × Duroc [(L × Y) × D]. In the case of gene PKM2 one may observe in (L × Y) × D crossbreds, compared to L × Y crossbreds, an increased expression, closely related with the increase in dry matter content, including intramuscular fat, as well as a more favourable progress of glycolytic and energy metabolism during the early time post mortem (pH₄₅ and R₁). Compared with Landrace animals, the lower expression of the CAST gene observed in (L × Y) × D pigs is manifested by a marked improvement of meat quality (R₁ pH₄₅ pH₂₄, pH₄₈), arising from the rate of glycolytic and energy metabolism, typical for normal meat, that in effect results in its higher culinary and technological value.     

Biotransformation of (R)-(+)- and (S)-(−)-limonene to α-terpineol by Penicillium digitatum— investigation of the culture conditions

The biotransformation of (R)-(+)- and (S)-(−)-limonene by Penicillium digitatum was investigated. One strain of P. digitatum was able to convert (R)-(+)-limonene to pure (R)-(+)-α-terpineol in 8 h with a yield of up to 93%. It was found that (R)-(+)-limonene was converted much better into α-terpineol than (S)-(−)-limonene, and that no significant chemical conversion of the substrate occurred in control flasks at pH 3.5. The culture conditions involved such as the type and concentration of co-solvent applied and the sequential addition of substrate were investigated, taking into account some findings on the physical behaviour of the system. The highest bioconversion yields were obtained when the substrate was applied as a diluted solution in EtOH.     

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion, which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and R1 stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS R1 also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10–24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS R1 and with the increases in Bacteroides in cultures with both microbial EPS (R1 and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids.     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.     

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 °C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 °C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 ± 0.2 log CFU g⁻¹ to less than detectable limit (<1.0 log CFU g⁻¹).     

Biosynthesis of PP-V, a monascorubramine homologue, by Penicillium sp. AZ

The biosynthetic pathway of PP-V, a new monascorubramine homologue, was elucidated by ¹³C-labeling studies. The [1-¹³C] of acetate was incorporated into 2-, 3a-, 4a-, 6-, 8-, 9-, 11-, 13-, 15-, 17-, and 19-Cs of PP-V, and the [2-¹³C], into 3-, 4-, 5-, 8a-, 9a-, 10-, 12-, 14-, 16-, 18-, and 20-Cs. These incorporation patterns coincide with those reported in the biosynthesis of a Monascus azaphilone pigment, monascorubrin.     

Effects of curing sodium nitrite additive and natural meat fat on growth control of Listeria monocytogenes by the bacteriocin-producing Lactobacillus curvatus strain CWBI-B28

In realistic model meat systems, the separate and combined effects of fat content and sodium nitrite on the antilisterial activity of the bacteriocin of Lactobacillus curvatus CWBI-B28 were studied. In laboratory fermentations where Listeria monocytogenes was co-cultured at 4 °C with bacteriocin-producing CWBI-B28 in lean pork meat (fat content: 13%) without added nitrite, a strong antilisterial effect was observed after one week. The effect was maintained for an additional week, after which a slight and very gradual rebound was observed. Both added nitrite (20 ppm) and a high-fat content (43%) were found to antagonise this antilisterial effect, the Listeria cfu count reached after six weeks being 200 times as high in high-fat meat with added nitrite than in lean meat without nitrite. This antagonism could not be attributed to slower growth of the bacteriocin-producing strain, since CWBI-B28 grew optimally in fat-rich meat with 20 ppm sodium nitrite. Bacteriocin activity was also measured in the samples. The observed activity levels are discussed in relation to the degree of antilisterial protection conferred.     

Inactivation of Yersinia enterocolitica by pulsed electric fields

The influence of growth conditions, treatment medium characteristics and PEF process parameters on the lethal effect on Yersinia enterocolitica of pulsed electric fields (PEF) treatments in batch has been investigated. Growth phase, temperature of growth, pH, conductivity of the treatment medium, pulse width and frequency of pulses did not influence the sensitivity of Y. enterocolitica to PEF. However, an A_w decrease from >0.99 to 0.93 of the treatment medium increased the PEF resistance of Y. enterocolitica with 3.5 log₁₀ cycles after a treatment of 22 kV/cm, 800 μs and 880 kJ/kg. Inactivation of Y. enterocolitica increased with the field strength, treatment time and total specific energy up to a maximum of 6 log₁₀ cycles after 28 kV/cm, 2000 μs and 3559 kJ/kg. A nonlinear relationship was found among the survival fraction and the treatment time or the specific energy that was accurately described by a mathematical model based on the Weibull distribution. The inactivation of Y. enterocolitica by PEF was characterized by maximum field strength thresholds. Above these thresholds, specific energy necessary to obtain a given level of inactivation scarcely decreased by increasing the electric field strength, and inactivation of Y. enterocolitica only depended on the specific energy applied.     

Prevalence of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis in wild boars in Switzerland

Between October 2007 and March 2008, 153 wild boars shot in the Canton of Geneva in Switzerland were sampled. Fifty-one percent of the animals were males and 49% were females. The age of most (81%) animals varied between 6 months and 2 years. Prevalence of enteropathogenic Yersinia in tonsils and faeces was studied using culture and PCR methods and in tissue fluid of tonsils using an ELISA system. Prevalence of anti-Yersinia antibodies in tissue fluid was 65%. Detection rate of enteropathogenic Yersinia in tonsils of 153 wild boars by real-time PCR was 44%. Ail-positive Yersinia enterocolitica and inv-positive Yersinia pseudotuberculosis were detected in 35 and 20% of the animals, respectively. Both species were detected in 10% of the animals. Isolation rate of enteropathogenic Yersinia was low; ail-positive Y. enterocolitica and inv-positive Y. pseudotuberculosis were found in 9 and 3% of the animals, respectively. Prevalence was shown to be significantly higher in tonsils than in faeces. Furthermore, females were more commonly positive than males. This study shows that the prevalence of enteropathogenic Yersinia is high and both enteropathogenic Y. enterocolitica and Y. pseudotuberculosis are common findings in tonsils of wild boars in Switzerland.     

Role of quantity and quality of fat in meat models inoculated with Listeria innocua or Salmonella Typhimurium treated by high pressure and refrigerated stored

Several variables can influence the effects of high hydrostatic pressure processing (HPP), but the role of fat in the treated sample is still uncertain. We designed a model by which controlling the known variables we could elucidate that role. We applied 400 MPa for 2 min to minced chicken samples inoculated with Listeria innocua and Salmonella Typhimurium mixed with 10% and 20% of three fat types with different fatty acid composition. Microbial counts were performed during 60 days of refrigerated storage either at 2 °C or 8 °C.Immediately after HPP bacterial growth was independent of the type and percentage of fat content, but a possible effect of type of fat could be observed after 60 days of cold storage.     

Effect of an additionally introduced degQ gene on di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system

Di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.     

Fumigant properties of physical preparations from mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) beetle for stored grain insects

Vapors released from foliage of mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) Beetle, through a patented process, were hypothesized to have an insecticidal time of action (24 h or less after time of exposure) similar to the fumigant methyl bromide. Patented preparations were more effective from plants harvested from a relatively wet site in mid to late summer (5 July to 11 September). Bioassays with the lesser grain borer, Rhyzopertha dominica (F.), 0–3 days after adult emergence indicated an LT₅₀ of 7.0±1.2 h for the volatiles generated from only 30 mg dry processed plant material (=0.56 mg active ingredients) per ml headspace. Hatching of eggs of the Indian meal moth, Plodia interpunctella (Hübner), was completely suppressed when exposed 4–20 h after oviposition to a concentration of 7 mg processed plant material per ml headspace (=0.14 mg active ingredients) in a container that allowed passive diffusion and from which the terpenes disappeared by 48 h. Adult red flour beetle, Tribolium castaneum (Herbst), had an LT₅₀ of 40.7±1.2 h when exposed to 29 mg processed plant material per ml headspace. Gas chromatography/mass spectrometry (GC/MS) analyses of the headspace above this processed plant material revealed five major peaks, all non-chlorinated and non-brominated. The two main volatiles, 1,8-cineole and camphor, occurred initially in a mean ratio of 1:3.2, gradually shifting to 1:2.4 over 24 h. The μg/ml headspace of each detectable compound in a sealed container was followed intensely (0.25, 1, 2, 12, 24, 48, and 72 h) for 72 h and at less frequent intervals for 60 days. The active compounds released by the plant material in a closed, but not airtight container, were no longer detectable after 24 h based on GC/MS analysis. Fumigative studies with the same ratio of the two main compounds generated synthetically indicated that embryos of P. interpunctella and adults of R. dominica were as sensitive to the synthetic mixture as they were to the processed plant material. Although one could apply the precise commercial terpenes in the same ratio, the plant material provides a natural formulation that is conveniently diluted (formulated) to levels safe for handling. Therefore, this preparation method and plant material shows good potential as an alternative to methyl bromide for protection of stored grain, commodity, and space fumigations. No residues are detectable in the headspace of aerated commodity, milled product, or in fumigated space.     

The influence of precultivation parameters on the catabolism of branched-chain amino acids by Staphylococcus xylosus and Staphylococcus carnosus

The influence of precultivation parameters on the ability of Staphylococcus xylosus and Staphylococcus carnosus to convert branched-chain amino acids—leucine, isoleucine and valine—into volatile flavour compounds was investigated using resting cells in a defined reaction medium. The studied precultivation parameters were: growth phase, temperature, NaCl concentration and the concentration of leucine, isoleucine and valine (only for S. xylosus). Flavour compounds were sampled by automatic static headspace collection and separated/quantified using gas chromatography/flame ionization detection (GC/FID).Main catabolic products from degradation of leucine, isoleucine and valine were the flavour intensive branched-chain acids: 2- and 3-methylbutanoic and 2-methylpropanoic acids. The precultivation parameters altered the production of the branched-chain acids significantly, but to various degrees for S. xylosus and S. carnosus.Production of branched-chain acids by S. carnosus was only influenced slightly by the growth phase and not by changing the NaCl concentration between 4.0% and 10.0% (w/w). Lowering the temperature from 28°C to 18°C significantly decreased S. carnosus’ generation of branched-chain acids. In contrast, S. xylosus was significantly influenced by all precultivation parameters, in particular by the growth phase. Cells taken from growing cultures had a much higher production of branched-chain acids compared to cells taken from stationary cultures. Addition of leucine and valine to the precultivation medium enhanced the production of branched-chain acids whereas addition of isoleucine had the opposite effect.     

Comparison of antimicrobial resistance in Salmonella and Campylobacter isolated from turkeys in the Midwest USA

The current study was carried out to determine the antimicrobial resistance profiles and evaluate some molecular characteristics of a set of Salmonella and Campylobacter isolates recovered from production line turkeys in the Midwest region of the United States. A total of 94 birds identified as being positive for both Salmonella and Campylobacter spp. were selected for study. All Salmonella isolates were examined for antimicrobial resistance using the methods employed in the National Antimicrobial Resistance Monitoring System (NARMS). Campylobacter isolates were subjected to similar analysis using the Etest^®. In addition, polymerase chain reaction (PCR) was carried out to determine the presence of the antimicrobial resistance associated genes, integrase (int1), class 1 integrons (Salmonella and Campylobacter) and a multidrug efflux pump (Campylobacter spp.). Results from the study showed that the Salmonella and Campylobacter isolates examined displayed resistance to a number of antimicrobials, with Salmonella and Campylobacter isolates being resistant to at least three antimicrobials while some isolates showed resistances to 6 or 8 different antimicrobials. In addition, 68.1% of the Salmonella isolates tested were found to be positive for the class I integrase gene (int1), 28.7% possessed a 1000 bp gene cassette and 17% possessed an 800 bp gene cassette. All Campylobacter isolates were negative for int1, but 36.2% tested positive for the Campylobacter multidrug efflux pump (CmeB). A considerable number of Salmonella and Campylobacter isolates tested displayed varying degrees of antimicrobial resistance as well as the presence of some factors associated with the carriage and persistence of antimicrobial resistance. Similarities in the types of antimicrobial resistance observed in Campylobacter and Salmonella strains was evident. The results of this study suggest that prescribing practice at the farm level may be a factor in promoting antimicrobial resistance in more than one species of organism. Such practices may, therefore, contribute to the potential health risk for consumers should micro-organisms carrying multiple antimicrobial resistances enter the food chain. This study may be one of the first to report on the incidence of the multidrug efflux pump (CmeB) in Campylobacters recovered from processed turkeys. The antimicrobial resistance and molecular characteristics of Salmonella and Campylobacter is discussed.     

Fe2+-催化臧红T褪色光度法测定过氧化氢酶活性

基于Fe²⁺-H₂O₂-臧红T褪色体系完成过氧化氢酶活性测定。H₂O₂氧化臧红T引起吸光度变小,褪色反应又受酶分解反应的抑制,加酶前后吸光度变化(ΔA)与酶活性有相关性。取酶分解反应和褪色反应的最佳条件,CAT活性在0.00~0.02U/mL之间与ΔA呈现良好的线性关系:ΔA=-0.0012+9.5528E(U/mL)(r=0.9990),检出限为6.28×10^-3U/mL;采用酶失活样品液作空白,可有效排除样品共存还原性物质的干扰。方法用于新鲜大米样品检测,结果令人满意。     

Modulator-mediated synthesis of active lipase of Pseudomonas sp. 109 by Escherichia coli cell-free coupled transcription/translation system

Catalytically active lipase was synthesized using Escherichia coli S30 extract from the signal-deleted lipL gene (lipL) in the presence of its N-terminal hydrophobic fragment-truncated modulator (rLimL) that was purified from the overexpressing E. coli cells. The specific activity of the lipase thus synthesized was 125 times higher than that of the purified one from Pseudomonas sp. 109. No lipase activity was detected in the absence of rLimL, even though the lipase protein itself was synthesized. Active lipase was also produced in vitro by coexpression of rlipL and the modulator gene (rlimL), although a much smaller amount of the lipase was formed. In the absence of rLimL, aggregates of the lipase were formed during its folding process. The addition of rLimL proportionally raised both lipase solubility and enzyme activity. An unstable but high activity peak of the lipase was found during its folding process.