Chromobacterium paludis sp. nov., a novel bacterium isolated from a Chesapeake Bay marsh.

Two isolates of Gram-reaction-negative, motile, violet-pigmented bacteria were isolated from a small pool in marshland near the mouth of the Nanticoke River in Maryland, USA. The isolates IIBBL 257-1T and IIBBL 257-2 had identical 16S rRNA gene sequences as determined by PCR, and highly similar fatty acid and biochemical profiles. The 16S rRNA gene sequences indicated the isolates belonged to the genus Chromobacterium. Genomic sequencing of IIBBL 257-1T revealed a genome of 4.27 Mb, with a G+C content of 63.6 %. Whole genome comparisons with other members of the Chromobacterium using JSpecies and the genome blast distance phylogeny approach indicated that among described species, IIBBL 257-1T was most closely related to C. amazonense and C. phragmitis. Comparison of the IIBBL 257-1T genome with those of type strains of these species resulted in ANIb and dDDH values of ca. 85 and 30 %, respectively, for both. These results demonstrate that IIBBL 257-1T and IIBBL 257-2 represent a new taxon within the genus Chromobacterium. We propose the name Chromobacterium paludis sp. nov. for this taxon; the type strain is IIBBL 257-1T (=NRRL B-65555T=JCM 33770T).

Chromobacterium violaceum: The Deadly Sepsis.

Chromobacterium violaceum is a Gram-negative facultatively anaerobic, oxidase-positive bacterium producing a dark violet antioxidant pigment called violacein. It is an opportunistic pathogen and has an ubiquitous distribution, mainly resides in water and soil of tropical and subtropical regions.An-18-year-old man referred to the emergency room with a 5-day history of progressively worsening swelling of the right cheek. He sought consult and hospitalized at another institution for three days prior this admission; however, his condition deteriorated. He had a history of having abscesses several time. Four month before this visit, he was also admitted in our hospital due to an abscess in the right thigh. Pus and blood culture were positive for Staphylococcus haemolyticus, with a total serum IgE of 2493.0 IU/ml. He recovered completely after being treated with vancomycin in this event. He had neither diabetes mellitus nor human immunodeficiency virus infection history. In this presentation, he was in a critically ill state with septic shock. Physical examination revealed diffuse, indurated, partly fluctuant, and some deep purple area of right hemifacial swelling. It was extended anteriorly from angle of mouth to retroauricular, superiorly from superior palpebra to lower border of mandible. Laboratory studies were notable for a white-cell count of 12,970/mm3 (total lymphocyte count 778.2), platelet count 96,000/mm3. The patient got norepinephrine drip and broad-spectrum antibiotic intravenously. He also underwent superficial drainage of the abscess. Unfortunately, the patient eventually succumbed. Sample from right submandibular abscess showed no growth, but blood sample was confirmed to grow C. violaceum. It showed sensitivity to ciprofloxacin, amikacin, cotrimoxazole, chloramphenicol, tetracycline.Since it was firstly described in 1927, only a few cases of human infection with C. violaceum have been reported. As shown in our case, the classical clinical manifestation was localized soft tissue infection which rapidly progressed to fulminant sepsis with a high mortality rate. A defect in host defense system might be the predisposing factor for this kind of infection in our case. As this is such a rare infection, there is no guideline on the choice of antibiotics or duration of treatment at present. Successful treatment is most likely due to early recognition, prompt surgical drainage and appropriate antibiotic. To the best of our knowledge, this is the first reported case from Indonesia that could be identified in the literature.

Phytochemical composition, anti-biofilm and anti-quorum sensing potential of fruit, stem and leaves of Salvadora persica L. methanolic extracts.

Emergence of antibiotic resistance among pathogenic bacteria encourages us to search for new molecules as an alternative treatment. The aim of this study was to evaluate the antiquorum sensing (anti-QS) and antibiofilm potential of Salvadora persica L. methanolic extracts to prevent the infections due to Staphylococcus as an alternate to antibiotics. The methanolic extracts of S. persica L. fruit, leaves and stems was assessed for their activity in inhibiting QS-depedent phenomenon such as violacein pigment production in Chromobacterium violaceum, swarming motility of Pseudomonas aeruginosa PAO1 and biofilm formation in oral Staphylococcus strains on polymethylmetacrylate (PMMA). Methanolic fruit extract of S. persica L. showed a high degree of anti-biofilm formation on PMMA and on violacein inhibition with a percentage of reduction equal to 90% when MIC value (20 mg/ml) was used. 100 µg/ml of S. persica L. leaves exhibited inhibition in swarming motility of PAO1 at 29.17%. Because the methanolic extracts of S. persica L. demonstrated anti-QS and antibiofilm activity at very low concentrations, it could be further exploited for novel molecules to treat oral Staphylococcus infections.

Cellulose-Organic Montmorillonite Nanocomposites as Biomacromolecular Quorum-Sensing Inhibitor.

The aim of this study was to develop simple cellulose nanocomposites that can interfere with the quorum-sensing (QS)-regulated physiological process of bacteria, which will provide a sustainable and inexpensive solution to the serious challenges caused by bacterial infections in various products like food packaging or biomedical materials. Three cellulose nanocomposites with 1-5 w% octadecylamine-modified montmorillonite (ODA-MMT) were prepared by regeneration of cellulose from ionic liquid solutions in the presence of ODA-MMT suspension. Structural characterization of the nanocomposites showed that the ODA-MMT can be exfoliated or intercalated, depending on the load level of the nanofiller. Thermal gravimetric analysis showed that the incorporation of ODA-MMT nanofiller can improve the thermal stability of the nanocomposites compared with regenerated cellulose. Evaluation of the anti-QS effect against a pigment-producing bacteria C. violaceum CV026 by disc diffusion assay and flask incubation assay revealed that the QS-regulated violacein pigment production was significantly inhibited by the cellulose nanocomposites without interfering the bacterial vitality. Interestingly, the nanocomposite with the lowest load of ODA-MMT exhibited the most significant anti-QS effect, which may be correlated to the exfoliation of nanofillers. To our knowledge, this is the first report on the anti-QS effect of cellulose nanocomposites without the addition of any small molecular agents. Such inexpensive and nontoxic biomaterials will thus have great potential in the development of new cellulosic materials that can effectively prevent the formation of harmful biofilms.

A review on lipase-catalyzed reactions in ultrasound-assisted systems.

The named "green chemistry" has been receiving increasing prominence due to its environmentally friendly characteristics. The use of enzymes as catalysts in processes of synthesis to replace the traditional use of chemical catalysts present as main advantage the fact of following the principles of the green chemistry. However, processes of enzymatic nature generally provide lower yields when compared to the conventional chemical processes. Therefore, in the last years, the ultrasound has been extensively used in enzymatic processes, such as the production of esters with desirable characteristics for the pharmaceutical, cosmetics, and food industry, for the hydrolysis and glycerolysis of vegetable oils, production of biodiesel, etc. Several works found in the open literature suggest that the energy released by the ultrasound during the cavitation phenomena can be used to enhance mass transfer (substrate/enzyme), hence increasing the rate of products formation, and also contributing to enhance the enzyme catalytic activity. Furthermore, the ultrasound is considered a "green" technology due to its high efficiency, low instrumental requirement and significant reduction of the processing time in comparison to other techniques. The main goal of this review was to summarize studies available to date regarding the application of ultrasound in enzyme-catalyzed esterification, hydrolysis, glycerolysis and transesterification reactions.

Unusual multiple production of N-acylhomoserine lactones a by Burkholderia sp. strain C10B isolated from dentine caries.

Bacteria realize the ability to communicate by production of quorum sensing (QS) molecules called autoinducers, which regulate the physiological activities in their ecological niches. The oral cavity could be a potential area for the presence of QS bacteria. In this study, we report the isolation of a QS bacterial isolate C10B from dentine caries. Preliminary screening using Chromobacterium violaceum CV026 biosensor showed that isolate C10B was able to produce N-acylhomoserine lactones (AHLs). This bacterium was further identified as a member of Burkholderia, an opportunistic pathogen. The isolated Burkholderia sp. was confirmed to produce N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL) and N-dodecanoyl-L-homoserine lactone (C12-HSL).

Biosynthesis of novel polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate by Chromobacterium sp. USM2.

AIMS: Polyhydroxyalkanoate (PHA) with enhanced physicochemical properties will be ideal for a wide range of practical applications. The incorporation of 3-hydroxy-4-methylvalerate (3H4MV) into the polymer backbone is known to improve the overall properties of the resulting polymer. However, the most suitable micro-organism and PHA synthase that can synthesize this monomer efficiently still remain unknown at present. Therefore, we evaluated the abilities of a locally isolated Chromobacterium sp. USM2 to produce PHA containing 3H4MV. METHODS AND RESULTS: The ability of Chromobacterium sp. USM2 to synthesize poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) [P(3HB-co-3H4MV)] was evaluated under different culture conditions. It was found that Chromobacterium sp. USM2 can synthesize P(3HB-co-3H4MV) when glucose and isocaproic acid were fed as carbon source. However, the highest molar fraction of 3H4MV, 22 mol% was detected in Chromobacterium sp. USM2 when isocaproic acid was provided as the sole carbon source. In addition, aeration was identified as a crucial factor in initiating the accumulation of high 3H4MV molar fractions. CONCLUSIONS: Chromobacterium sp. USM2 was able to synthesize broad comonomer compositional distribution of P(3HB-co-3H4MV). SIGNIFICANCE AND IMPACT OF THE STUDY: Compared with Cupriavidus necator and Burkholderia sp., Chromobacterium sp. USM2 was found to have better ability to bioconvert isocaproic acid to form 3H4MV unit.

Highly efficient production of chiral amines in batch and continuous flow by immobilized ω-transaminases on controlled porosity glass metal-ion affinity carrier.

In this study, two stereocomplementary ω-transaminases from Arthrobacter sp. (AsR-ωTA) and Chromobacterium violaceum (Cv-ωTA) were immobilized via iron cation affinity binding onto polymer-coated controlled porosity glass beads (EziG™). The immobilization procedure was studied with different types of carrier materials and immobilization buffers of varying compositions, concentrations, pHs and cofactor (PLP) concentrations. Notably, concentrations of PLP above 0.1 mM were correlated with a dramatic decrease of the immobilization yield. The highest catalytic activity, along with quantitative immobilization, was obtained in MOPS buffer (100 mM, pH 8.0, PLP 0.1 mM, incubation time 2 h). Leaching of the immobilized enzyme was not observed within 3 days of incubation. EziG-immobilized AsR-ωTA and Cv-ωTA retained elevated activity when tested for the kinetic resolution of rac-α-methylbenzylamine (rac-α-MBA) in single batch experiments. Recycling studies demonstrated that immobilized EziG3-AsR-ωTA could be recycled for at least 16 consecutive cycles (15 min per cycle) and always affording quantitative conversion (TON ca. 14,400). Finally, the kinetic resolution of rac-α-MBA with EziG3-AsR-ωTA was tested in a continuous flow packed-bed reactor (157 µL reactor volume), which produced more than 5 g of (S)-α-MBA (>49% conversion, >99% ee) in 96 h with no detectable loss of catalytic activity. The calculated TON was more than 110,000 along with a space-time yield of 335 g L-1 h-1.

Chumacin-1 and Chumacin-2 from Pseudomonas aeruginosa strain CGK-KS-1 as novel quorum sensing signaling inhibitors for biocontrol of bacterial blight of rice.

The in vitro inhibition of quorum sensing signal, xanthan gum secretion, biofilm formation in different Xanthomonas pathovars and biological control of bacterial blight of rice by the two bioactive extrolites produced by Pseudomonas aeruginosa strain CGK-KS-1 were explored. These extrolites were extracted from Diaion HP-20 resin with methanol and purified by preparative-thin layer chromatography. Further, spectroscopic structural elucidation revealed the tentative identity of these extrolites to be (R,3E,5E,9Z,11E)-13-((3S,5R)-5-acetyl-2,6-dimethylheptan-3-yl)-10-hydroxy-4-methyl-1,8-diazabicyclo[9.3.1]pentadeca-3,5,9,11(15),13-pentaen-2-one and (R,3E,5E,8E,11E)-13-((3S,5R)-5-acetyl-2,6-dimethylheptan-3-yl)-4-methyl-1,8-diazabicyclo[9.3.1]pentadeca-3,5,8,11(15),13-pentaene-2,10-dione, named as Chumacin-1 and Chumacin-2, respectively. Antimicrobial assay showed Chumacin-1 and Chumacin-2 exhibited a strong in vitro growth inhibition against various Xanthomonas pathovars. Quorum sensing overlay assay using a reporter strain Chromobacterium violaceum strain CV026 showed that Chumacin-1 and Chumacin-2 inhibited quorum sensing signaling. The mechanistic studies revealed that these extrolites inhibited the production of quorum sensing signaling factor, cis-11-methyl-2-dodecenoic acid; suppressed the xanthan gum secretion and also inhibited the biofilms formed by various Xanthomonas pathovars. Both Chumacin-1 and Chumacin-2 showed ROS generation in the test Xanthomonas strains, resulting in in vitro cell membrane damage was revealed through CSLM and FE-SEM micrographs. Further, greenhouse experiments using Samba Mashuri (BPT-5204) revealed that seed treatment with Chumacin-1 and Chumacin-2 along with foliar spray groups showed up to ˜80% reduction in bacterial blight disease in rice. To the best of our knowledge, this is the first report on new quorum sensing inhibitors, Chumacin-1 and Chumacin-2 produced by Pseudomonas aeruginosa strain CGK-KS-1 exhibiting DSF inhibition activity in Xanthomonas oryzae pv. oryzae.

Antimicrobial activity of violacein against oral bacteria associated with halitosis: an in vitro study

Introduction: violacein is a natural purple pigment produced by environmental bacteria that presents antimicrobial activity, particularly against Gram-positive bacteria. Intraoral halitosis (IOH) is a condition defined by the unpleasant odor emanating from the mouth, whose main source are volatile sulfur compounds, produced by Gram-negative oral bacteria on the tongue coating. In IOH treatment, antimicrobials have been indicated as chemical adjuncts, including natural products. Objective: thus, this study tested the antimicrobial activity of a violacein extract on key IOH-related bacteria (Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum, Prevotella intermedia, Solobacterium moorei). Materials and Methods: bacteria were cultured in fastidious anaerobe blood agar in anaerobiosis, and 109 cells/ml suspensions were plated. Crude extract of violacein obtained from Chromobacterium violaceum was diluted in a 25% ethanol aqueous solution to 8, 4, 2, 1, 0.5 and 0.25 mg/ml. Using the disk agar diffusion method, 10 µl aliquots of each dilution were deposited on the seeded plates. Chlorohexidine (0.1%) and 25% ethanol solution were used as controls. Plates were incubated in anaerobiosis at 37°C for 72h, and the inhibition halos were recorded. Results: although chlorhexidine showed higher inhibition halos than the violacein extract, most species were inhibited at 4 and 8 mg/ml concentrations (p<0.05). P. gingivalis followed by F. nucleatum were the most affected species in relation to the other bacteria, although statistical significance was only reached for P. gingivalis (p<0.05). Conclusion: crude violacein extract from C. violaceum demonstrated antimicrobial activity against IOH-associated oral bacteria, being a potential antimicrobial to be studied as an adjunct in the control of IOH.

Introdução: a violaceína é um pigmento roxo natural produzido por bactérias ambientais que apresenta ação antimicrobiana, particularmente contra bactérias Gram-positivas. A halitose intraoral (HIO) é uma condição definida pelo odor desagradável que emana da boca, cuja principal fonte são os compostos sulfurados voláteis produzidos por bactérias Gram-negativas da saburra lingual. No tratamento da HIO, antimicrobianos têm sido indicados como adjuvantes, incluindo produtos naturais. Objetivo: assim, este estudo avaliou o potencial antimicrobiano de um extrato de violaceína em patógenos-chave da HIO (Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum, Prevotella intermedia, Solobacterium moorei). Materiais e Métodos: bactérias foram cultivadas em meio ágar sangue para fastidiosos, em anaerobiose, e suspensões de 109 células/ml foram semeadas. O extrato bruto de violaceína obtido de Chromobacterium violaceum foi diluído em solução aquosa com 25% de etanol nas concentrações de 8, 4, 2, 1, 0,5 e 0,25 mg/ml. Através do método de disco difusão, 10 µl de cada diluição foram depositados nas placas semeadas. A clorexidina (0,1%) e a solução etanólica a 25% foram usadas como controles. As placas foram incubadas em anaerobiose a 37°C por 72h, e os halos de inibição foram registrados. Resultados: embora a clorexidina tenha apresentado os maiores halos de inibição do do que o extrato, a maioria das espécies foi inibida nas concentrações de 4 e 8 mg/ml (p<0,05). P. gingivalis e F. nucleatum foram as espécies mais afetadas em relação às outras bactérias, porém só foi observada significância estatística para P. gingivalis (p<0,05). Conclusão: o extrato bruto de violaceína de C. violaceum demonstrou atividade antimicrobiana contra bactérias orais associadas a HIO, sendo um potencial antimicrobiano a ser estudado como adjuvante no controle da HIO.

Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation.

N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.

Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics.

Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaC Cs -CAT. The structure shows that PhaC Cs -CAT forms an α/ß hydrolase fold comprising α/ß core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaC Cn -CAT). Structural comparison showed PhaC Cn -CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaC Cs -CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaC Cn -CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.

Identification of a new polyhydroxyalkanoate (PHA) producer Aquitalea sp. USM4 (JCM 19919) and characterization of its PHA synthase.

Aquitalea sp. USM4 (JCM 19919) was isolated from a freshwater sample at Lata Iskandar Waterfall in Perak, Malaysia. It is a rod-shaped, gram-negative bacterium with high sequence identity (99%) to Aquitalea magnusonii based on 16S rRNA gene analysis. Aquitalea sp. USM4 also possessed a PHA synthase gene (phaC), which had amino acid sequence identity of 77-78% to the PHA synthase of Chromobacterium violaceum ATCC12472 and Pseudogulbenkiania sp. NH8B. PHA biosynthesis results showed that wild-type Aquitalea sp. USM4 was able to accumulate up to 1.5 g/L of poly(3-hydroxybutyrate), [P(3HB)]. The heterologous expression of the PHA synthase gene of Aquitalea sp. USM4 (phaCAq) in Cupriavidus necator PHB-4 had resulted in PHA accumulation up to 3.2 g/L of P(3HB). It was further confirmed by 1H nuclear magnetic resonance (NMR) analysis that Aquitalea sp. USM4 and C. necator PHB-4 transformant were able to produce PHA containing 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB) and 3-hydroxy-4-methylvalerate (3H4MV) monomers from suitable precursor substrates. Interestingly, relatively high PHA synthase activity of 863 U/g and 1402 U/g were determined in wild-type Aquitalea sp. USM4 and C. necator PHB-4 transformant respectively. This is the first report on the member of genus Aquitalea as a new PHA producer as well as in vitro and in vivo characterization of a novel PHA synthase from Aquitalea sp. USM4.

Efficient poly(3-hydroxypropionate) production from glycerol using Lactobacillus reuteri and recombinant Escherichia coli harboring L. reuteri propionaldehyde dehydrogenase and Chromobacterium sp. PHA synthase genes.

Poly(3-hydroxypropionate), P(3HP), is a polymer combining good biodegradability with favorable material properties. In the present study, a production system for P(3HP) was designed, comprising conversion of glycerol to 3-hydroxypropionaldehyde (3HPA) as equilibrium mixture with 3HPA-hydrate and -dimer in aqueous system (reuterin) using resting cells of native Lactobacillus reuteri in a first stage followed by transformation of the 3HPA to P(3HP) using recombinant Escherichia coli strain co-expressing highly active coenzyme A-acylating propionaldehyde dehydrogenase (PduP) from L. reuteri and polyhydroxyalkanoate synthase (PhaCcs) from Chromobacterium sp. P(3HP) content of up to 40% (w/w) cell dry weight was reached, and the yield with respect to the reuterin consumed by the cells was 78%. Short biotransformation period (4.5h), lack of additives or expensive cofactors, and use of a cheap medium for cultivation of the recombinant strain, provides a new efficient and potentially economical system for P(3HP) production.

Stimulation of Chromobacterium lipase activity and prevention of its adsorption to palmitoyl cellulose by hydrophobic binding of fatty acids.

Fatty acids prevented adsorption of purified Chromobacterium lipase [triacylglycerol acylhydrolase, EC 3.1.1.3] onto palmitoyl cellulose (Pal-C) and also increased the activity of the purified lipase. These effects increased with increase in the concentration and chainlength (up to 16 carbon atoms) of the fatty acids, and long-chain unsaturated fatty acids, such as oleic acid, linoleic acid and erucic acid, were most effective. When the lipase was adsorbed (immobilized) on Pal-C, its activity was elevated to 20 times that of the free lipase in detergent-free reaction mixture (olive oil-buffer system). Thus lipase was adsorbed to Pal-C through a hydrophobic site distinct from its catalytic site and the binding of fatty acids to the hydrophobic site seems to result in stimulation of the lipase activity.

Purification of lipase from Chromobacterium viscosum by chromatography on palmitoyl cellulose.

Palmitoyl cellulose was used to adsorb the extracellular lipase [triacylglycerol acyl-hydrolase EC 3.1.1.3] of Chromobacterium viscosum from crude enzyme solution, and the adsorbed enzyme was eluted with a suitable detergent, such as Adekatol 45-S-8 or Triton X-100. The enzyme was then purified by chromatography on a palmitoylated gauze column with an overall recovery of 71% and an increase in the specific activity of 11-fold from the supernatant fluid of bacterial cultures. Further purification procedures included fractionation with acetone, and chromatography on Sephadex G-150 and G-75 columns. Two isoenzymes were obtained, each in a homogeneous state on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: one had a molecular weight of 120,000 and pI of 3.7 and the other a molecular weight of 30,000 with a pI of 7.3.

Preliminary study on hydrophobic interaction chromatography of Chromobacterium viscosum lipase on polypropylene glycol immobilized on Sepharose.

The purification of Chromobacterium viscosum lipase was performed using a polypropylene glycol-Sepharose gel. The influence of the mobile phase composition on the chromatographic behaviour of Chromobacterium viscosum lipase was studied and it was found that the retention of lipase depends on the salt used and increased with ionic strength. Using 20% (w/v) ammonium sulphate in the eluent, a total retention of lipase on the column was obtained.

Hydrophobic interaction chromatography of Chromobacterium viscosum lipase on polypropylene glycol immobilised on Sepharose.

The fractionation of Chromobacterium viscosum lipase was performed using a polypropylene glycol-Sepharose gel. The influence of mobile phase composition on the adsorption of lipase on the gel was studied and it was found that the retention of lipase depends on the salt used and increased with increasing the ionic strength. The retention was not strongly affected by changing the pH value of the mobile phase. By using 20% (w/v) ammonium sulphate in phosphate buffer a total retention of lipase on the column was obtained and by simply decreasing the ionic strength of the buffer, desorption of lipase could be achieved. The chromatographic purification of Chromobacterium viscosum lipase by hydrophobic interaction chromatography on Sepharose CL-6B modified by covalent immobilisation of 1,4-butanediol diglycidyl ether, polyethylene glycol and polypropylene glycol was also compared.

Biodegradation of poly(tetramethylene succinate-co-tetramethylene adipate) and poly(tetramethylene succinate) through water-soluble products.

Poly(tetramethylene succinate-co-tetramethylene adipate) (PBSA) and poly(tetramethylenesuccinate) (PBS) were hydrolyzed experimentally into water-soluble oligomers and monomers by Chromobacterium extracellular lipase. The oligomers were identified by high-performance liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance, which indicated that a total of 28 oligomer species were liberated from PBSA, and that 13 of them were identical to the hydrolysates from PBS. Moreover, 20 of the species were polyester-based compounds of monomer units, and the other 8 species were small amounts of diurethane compounds. Bis(hydroxybutyl) succinate (BSB) and bis(hydroxybutyl) hexamethylene dicarbamate (BHB) were the typical oligomers and were chemically synthesized. Biodegradability of BSB and BHB was examined for 28 d in the activated sludge, and analysis of the results of this study indicated that the final conversion rate of constituent carbon to carbon dioxide was estimated at 80 mol% for BSB and 10 mol% for BHB. The remaining amount of carbon in the undegraded BHB was 20 mol%. In the presence of BSB, the biodegradability of BHB was increased by about 1.5 times. The suggestion was made that BSB induced a growth of microorganisms and helped BHB degradation. This is consistent with the observation that the biodegradation of BHB in native soil for 60 d reached > 60%.

Improvement in enzyme activity and stability by addition of low molecular weight polyethylene glycol to sodium bis(2-ethyl-L-hexyl)sulfosuccinate/isooctane reverse micellar system.

The activity and stability of Chromobacterium viscosum lipase (glycerolester hydrolase, EC 3.1.1.3)-catalyzed olive oil hydrolysis in sodium bis (2-ethyl-l-hexyl)sulfosuccinate (AOT)/isooctane reverse micelles is increased appreciably when low molecular weight polyethylene glycol (PEG 400) is added to the reverse micelles. To understand the effect of PEG 400 on the phase behavior of the reverse micellar system, the phase diagram of AOT/ PEG 400/water/isooctane system was studied. The influences of relevant parameters on the catalytic activity in AOT/PEG 400 reverse micelles were investigated and compared with the results in the simple AOT reverse micelles. In the presence of PEG 400, the linear decreasing trend of the lipase activity with AOT concentration, which is observed in the simple AOT reverse micelles, disappeared. Enzyme entrapped in AOT/PEG reverse micelles was very stable, retaining >75% of its initial activity after 60 d, whereas the half-life in simple AOT reverse micelles was 38 d. The kinetics parameter maximum velocity (Vmax) exhibiting the temperature dependence and the activation energy obtained by Arrhenius plot was suppressed significantly by the addition of PEG 400.