Gene cloning, expression in E. coli, and in vitro refolding of a lipase from Proteus sp. NH 2-2 and its application for biodiesel production.

OBJECTIVE: To obtain active lipases for biodiesel production by refolding Proteus sp. lipase inclusion bodies expressed in E. coli. RESULTS: A lipase gene lipPN1 was cloned from Proteus sp. NH 2-2 and expressed in E. coli BL21(DE3). Non-reducing SDS-PAGE revealed that recombinant LipPN1(rLipPN1) were prone to form inclusion bodies as disulfide-linked dimers in E. coli. Site-directed mutagenesis confirmed that Cys85 in LipPN1 was involved in the dimer formation. After optimizing the inclusion body refolding conditions, the maximum lipase activity reached 1662 U/L. The refolded rLipPN1 exhibited highest activity toward p-nitrophenyl butyrate at pH 9.0 and 40 °C. It could be activated by Ca2+ with moderate tolerance to organic solvents. It could also convert soybean oil into biodiesel at a conversion ratio of 91.5%. CONCLUSION: Preventing the formation of disulfide bond could enhance the refolding efficiency of rLipPN1 inclusion bodies.

Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes.

l-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic l-amino acids into α-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Although the overall fold of this enzyme resembles that of known amine or amino acid oxidases, it shows the following specific structural features: an additional novel α+ß subdomain placed close to the putative transmembrane α-helix and to the active-site entrance; an FAD isoalloxazine ring exposed to solvent; and a large and accessible active site suitable to bind large hydrophobic substrates. In addition, PmaLAAD requires substrate-induced conformational changes of part of the active site, particularly in Arg-316 and Phe-318, to achieve the correct geometry for catalysis. These studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids.

Template-based modeling of a psychrophilic lipase: conformational changes, novel structural features and its application in predicting the enantioselectivity of lipase catalyzed transesterification of secondary alcohols.

In order to fully explore the structure-function relationship of a Proteus lipase (LipK107) that was screened from the soil in our previous study, we have modeled the three-dimensional (3-D) structures of the enzyme in its active and inactive conformations on the basis of crystal structures of Burkholderia glumae and Pseudomonas aeruginosa lipases in the present study. Both homology models suggested that LipK107 possessed a catalytic triad (Ser79-Asp232-H254), an oxyanion hole (Leu13 and Gln80) which was used to stabilize the reaction tetrahedral intermediates, and a lid substructure that controlled the access of the substrate to the active site. The existence of the lid was further verified by carrying out the interfacial activation experiment. The conformational change of LipK107 which was caused by lid opening action was predicted by superimposing the two theoretical models for the first time. Finally, both 3-D structures were used to predict the enantioselectivity of LipK107 when the enzyme was used to catalyze the resolution of racemic 1-phenylethanol. Lid-open model of LipK107 identified the R-enantiomer as the preferred enantiomer, while lid-closed mode showed that the S-enantiomer was more favored. However, only the lid-open conformational model could led to predictions that agreed with the following the experimental result of real biocatalysis reaction of 1-phenylethanol.

[Clinical and molecular characterization of ESBL-producing enterobacteria isolated from bacteremia in a university hospital]. / Caracterización clínica y molecular de bacteriemias causadas por enterobacterias productoras de β -lactamasas de espectro extendido: 2004-2007.

INTRODUCTION: Extended-spectrum-ß-lactamases (ESBL) are plasmid-encoded enzymes that confer resistance to multiple antimicrobials. ESBL-producing enterobacteria that cause bacteremia limit therapeutic options and increase mortality. OBJECTIVE: To perform a clinical and molecular description of bacteremia caused by ESBL-producing enterobacteria. METHOD: We retrospectively studied the cases of bacteremia due to ESBL-producing Escherichia coli, Klebsiella pneumoniae and Proteus spp in adults admitted to a university hospital during the years 2004-2007. We reviewed the clinical records and antimicrobial susceptibility patterns. Molecular typing was performed by polymerase chain reaction and study of clonality by pulsed-field electrophoresis. RESULTS: We found a prevalence of 9.8% ESBL in enterobacteria causing bacteremia. Decreased susceptibility to quinolones and aminoglycosides was observed, without resistance to carbapenems. The predominant ESBL types were CTX-M (96%), TEM (62%) and GES (28%). 79% of the strains presented more than one type of ESBL. Clinical analysis revealed high prevalence of risk factors, previous use of antimicrobials and of invasive devices. There was no significant clonality. CONCLUSION: The presence of ESBLs in bloodstream infections is a clinical problem that must be considered when choosing empiric therapy.

Identification of plasmid-mediated AmpC beta-lactamases in Escherichia coli, Klebsiella spp., and proteus species can potentially improve reporting of cephalosporin susceptibility testing results.

The goal of this study was to determine if the interpretations of extended-spectrum and advanced-spectrum cephalosporins (ESCs and ASCs, respectively) for isolates of Enterobacteriaceae would be impacted by the results of aminophenylboronic acid (APBA) testing. Fifty-three isolates of Escherichia coli, 21 Klebsiella species, and 6 Proteus species that were resistant to at least one ESC were tested by disk diffusion with ceftazidime and cefotetan disks with and without APBA. Ceftazidime disks with and without clavulanic acid (CLAV) were also tested to confirm extended-spectrum beta-lactamase (ESBL) carriage. Twenty-nine (36.3%) isolates were only APBA test positive, 27 were only CLAV test positive, 2 were positive with both substrates, and 22 were negative with both substrates. Thirteen (41.9%) of the 31 APBA-test-positive isolates (all E. coli) tested susceptible to cefotaxime, ceftriaxone, or ceftazidime. Since clinical data suggest that AmpC-producing isolates should be reported as resistant to all ESCs, APBA testing can be helpful in identifying such organisms. Screening for AmpC-producing organisms using nonsusceptibility to cefoxitin and amoxicillin-clavulanate was less specific than APBA testing; it identified ESBL as well as AmpC-producing organisms. Only 18 of 31 APBA-positive isolates were positive by PCR for an AmpC beta-lactamase gene. Thus, testing with APBA could improve the accuracy of reporting ESCs, especially for E. coli. However, results of APBA and CLAV testing did not correlate well for isolates containing both AmpC beta-lactamases and ESBLs. Thus, additional data are needed before formal recommendations can be made on changing the reporting of ASC test results.

Cyclic adenosine monophosphate in bacteria.

Both cyclic AMP and a specific inducer acting in concert are required for the synthesis of many inducible enzymes in E. coli. Little enzyme is made in the absence of either. In contrast to the specific inducers which stimulate the synthesis only of the proteins required for their metabolism, cyclic AMP controls the synthesis of many proteins. Glucose and certain other carbohydrates decrease the differential rate of synthesis of inducible enzymes by lowering cyclic AMP concentrations. In the lac operon, cyclic AMP acts at the promoter site to facilitate initiation of transcription. This action requires another protein, the cyclic AMP receptor protein. The nucleotide stimulates tryptophanase synthesis at a translational level. The action of cyclic AMP in E. coli may serve as a model to understand its action on transcriptional and translational processes in eukaryotes.

Development of recombinant Escherichia coli whole-cell biocatalyst expressing a novel alkaline lipase-coding gene from Proteus sp. for biodiesel production.

A lipase-producing bacterium K107 was isolated from soil samples of China and identified to be a strain of Proteus sp. With genome-walking method, the open reading frame of lipase gene lipK107, encoding 287 amino acids, was cloned and expressed in a heterologous host, Escherichia coli BL21 (DE3). The recombinant lipase was purified and characterized, and the optimum pH of the purified LipK107 was 9, at 35 degrees C. The recombinant E. coli expressing lipK107 was applied in biodiesel production in the form of whole-cell biocatalyst. Activity of the biocatalyst increased significantly when cells were permeabilized with 0.3% (w/v) cetyl-trimethylammoniumbromide (CTAB). This transesterification was carried out efficiently in a mixture containing 5M equivalents of methanol to the oil and 100% water by weight of the substrate. It was the first time to use E. coli whole-cell biocatalyst expressing lipase in biodiesel production, and the biodiesel reached a yield of nearly 100% after 12h reaction at the optimal temperature of 15 degrees C, which was the lowest temperature among all the known catalyst in biodiesel production.

[Epidemiology of ESBL-positive Enterobacteriaceae in Mantova hospital (Italy)]. / La problematica della circolazione di Enterobacteriaceae ESBL positive: situazione epidemiologica nell'Azienda Ospedaliera di Mantova.

In a retrospective study concerning the epidemiology of extended-spectrum beta-lactamase (ESBL) positive Enterobacteriaceae during 2007-2008 in the wards of the Carlo Poma hospital in Mantova, Mercurio surveillance software was used to detect alert microorganisms. Our objective was to link the epidemiological data with the type of patient and ward, and to assess the risk factors for such infections in particular nosocomial environments. The study enabled the change in the relative epidemiological data to be detected, and showed that such bacteria can be found almost throughout the hospital.

Properties of l-amino acid deaminase: En route to optimize bioconversion reactions.

Interest is rising in the agrochemical and pharmaceutical industries concerning the use of enantiomerically pure amino acids. l-Amino acids are easily produced by deracemization of D,L-mixtures or by stereoinversion of d-amino acids, employing the flavoenzyme d-amino acid oxidase. On the other hand, the production of the D-enantiomers is hampered by the lack of a suitable enzyme with reversed stereoselectivity. In recent years, the enzyme l-amino acid deaminase has been proposed as an alternative to l-amino acid oxidase. l-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a membrane-bound flavoprotein that catalyzes the deamination of l-amino acids to the corresponding α-keto acids and ammonia without producing hydrogen peroxide since the electrons are transferred from the reduced cofactor to a b-type cytochrome. For this reason, purified PmaLAAD has no significant enzymatic activity; this can be recovered by adding exogenous E. coli membranes. In order to circumvent the use of membranes, we analyzed the ability of PmaLAAD to use alternative electron acceptors, as well as detergents, to reproduce the hydrophobic environment. With phenazine methosulfate (PMS) and anionic detergents, at concentrations lower than the critical micellar concentration, higher enzymatic activity can be reached than with membranes. The effect on stability, protein conformation, oligomeric state and activity of temperature, pH, ionic strength, and detergents was also investigated. By optimizing the reaction conditions (namely, using 0.8 mM PMS and 0.1 mM SDS) the rate of l-leucine bioconversion was improved.

Engineering of L-amino acid deaminases for the production of α-keto acids from L-amino acids.

α-keto acids are organic compounds that contain an acid group and a ketone group. L-amino acid deaminases are enzymes that catalyze the oxidative deamination of amino acids for the formation of their corresponding α-keto acids and ammonia. α-keto acids are synthesized industrially via chemical processes that are costly and use harsh chemicals. The use of the directed evolution technique, followed by the screening and selection of desirable variants, to evolve enzymes has proven to be an effective way to engineer enzymes with improved performance. This review presents recent studies in which the directed evolution technique was used to evolve enzymes, with an emphasis on L-amino acid deaminases for the whole-cell biocatalysts production of α-keto acids from their corresponding L-amino acids. We discuss and highlight recent cases where the engineered L-amino acid deaminases resulted in an improved production yield of phenylpyruvic acid, α-ketoisocaproate, α-ketoisovaleric acid, α-ketoglutaric acid, α-keto-γ-methylthiobutyric acid, and pyruvate.

Occurrence of TEM, SHV and CTX-M ß Lactamases in Clinical Isolates of Proteus Species in a Tertiary Care Center.

BACKGROUND: Extended spectrum beta lactamases (ESBL) are responsible for increased resistance to third generation cephalosporins. Proteus species is an important cause of both community acquired and nosocomial infections. The Proteus species is usually susceptible to beta lactam drugs but there is progressive increase in beta lactam resistance and recently, ESBLs are also fast spreading to this species. OBJECTIVE: This study was conducted to study ESBL production and occurrence of TEM, SHV and CTX-M beta lactamases in clinical isolates of Proteus species in a tertiary care center. METHOD: This prospective hospital based study was carried out in Microbiology, Kasturba Medical College, Mangalore over 9 months. All non-duplicate consecutive Proteus isolates were identified and antibiotic susceptibility testing was done. ESBL detection was done by double disk synergy method and TEM, SHV, CTX-M genes were detected by PCR. RESULTS: 84 Proteus isolates from urine (29), blood (1), respiratory secretions (2), tissue (20) and exudates (47) were included in the study. 20.2% (17) were ESBL positive by disk synergy method. CTX-M was present in 6, TEM in 2 and both in 9 isolates. SHV was not present in any isolate. CONCLUSION: Our findings showed that 20% of clinical isolates of Proteus species were ESBL producers. 52% of ESBL positive isolates carried both TEM and CTX-M genes followed by CTX-M alone (35%) and only 11% had TEM alone. This stresses on the fact that ESBL detection should be done routinely in Proteus isolates and the genotype surveyed periodically for better management.

High-level expression and characterization of solvent-tolerant lipase.

In this study, the coding sequence of the lipase from Proteus sp. SW1 was optimized via codon optimization and subjected to expression in Pichia pastoris GS115. The maximum enzyme yield was 387 mg/L in the supernatants of the shake-flask culture. The purified recombinant lipase exhibited a specific activity of 130 U/mg toward p-nitrophenyl Laurate. Its optimum pH and temperature were 8.0 and 40°C, respectively. It was highly stable and even activated in water-miscible solvents, showing over 102% residual activity after 24 h incubation in ethanol, acetone, isopropanol and acetonitrile. In addition, the enzyme showed promoted activity with the increasing concentrations of methanol/ethanol and exhibited the maximum activity at 80%. In a solvent-free system for biodiesel synthesis with a one-step addition of methanol, the recombinant lipase displayed a 87% conversion rate toward palm oil at the high water content of 80%. The highly improved expression level and activity of the recombinant lipase may contribute to enable its commercial-scale production, and the unique properties would make it a particularly promising biocatalyst for biodiesel production in the future.

Antagonism of the antibacterial action of some penicillins by other penicillins and cephalosporins.

There are many examples of two penicillins acting synergistically, usually by one competitively inhibiting beta-lactamase, thus protecting the other from inactivation. There are few reports on penicillins antagonizing each other. Eight strains of three genera (Proteus, Escherichia, Pseudomonas) isolated at Boston City Hospital or Institut Pasteur, Paris, showed antagonism of carbenicillin or ampicillin by cephaloridine, cloxacillin, or 6-aminopenicillanic acid. Broth dilution tests showed that with seven of the eight strains the minimum inhibiting concentration (MIC) of the more active antibiotic was increased by 800-6,400% by low concentrations (often one-tenth the MIC) of the antagonist, whereas higher concentrations of "antagonist" were not as antagonistic, This suggested that two or more receptor sites of action for penicillins were present; the antagonist thus blocks the antibacterial action at the more sensitive site but acts additively with the antagonized antibiotic at the less sensitive site. The possibility that the mechanism of antagonism was induction of inactivating enzymes (beta-lactamase, penicillin acylase) was studied in two strains(one Escherichia coli and one Proteus rettgeri), and two antagonists were studied in detail. With E. coli cephaloridine was a poorer inducer of beta-lactamase than were the antagonized antibiotic and 6-aminopenicillanic acid. From these organisms, the good inducers of a beta-lactamase that acted on benzylpenicillin did not induce enzymes that inactivated carbenicillin. Thus, the mechanism of antagonism was not due to beta-lactamase induction.

Breaking the mirror: l-Amino acid deaminase, a novel stereoselective biocatalyst.

Enantiomerically pure amino acids are of increasing interest for the fine chemical, agrochemicals and pharmaceutical industries. During past years l-amino acids have been produced from deracemization of dl-solution employing the stereoselective flavoenzyme d-amino acid oxidase. On the other hand, the isolation of corresponding d-isomer was hampered by the scarce availability of a suitable l-amino acid oxidase activity. On this side, l-amino acid deaminase (LAAD), only present in the Proteus bacteria, represents a suitable alternative. This FAD-containing enzyme catalyzes the deamination of l-amino acids to the corresponding α-keto acids and ammonia, with no hydrogen peroxide production (a potentially dangerous oxidizing species) since the electrons of the reduced cofactor are transferred to a membrane-bound cytochrome. Very recently the structure of LAAD has been solved: in addition to a FAD-binding domain and to a substrate-binding domain, it also possesses an N-terminal putative transmembrane α-helix (residues 8-27, not present in the crystallized protein variant) and a small α+ß subdomain (50-67 amino acids long, named "insertion module") strictly interconnected to the substrate binding domain. Structural comparison showed that LAAD resembles the structure of several soluble amino acid oxidases, such as l-proline dehydrogenase, glycine oxidase or sarcosine oxidase, while only a limited structural similarity with d- or l-amino acid oxidase is apparent. In this review, we present an overview of the structural and biochemical properties of known LAADs and describe the advances that have been made in their biotechnological application also taking advantage from improved variants generated by protein engineering studies.

Molecular epidemiology and antimicrobial susceptibility of AmpC- and/or extended-spectrum (ESBL) ß-lactamaseproducing Proteus spp. clinical isolates in Zenica-Doboj Canton, Bosnia and Herzegovina.

Aim To investigate prevalence, antimicrobial susceptibility, molecular characteristics, and genetic relationship of AmpC- and/or extended spectrum beta lactamase (ESBL)- producing Proteus spp. clinical isolates in Zenica-Doboj Canton, Bosnia and Herzegovina. Methods Antibiotic susceptibility was determined by disc diffusion and broth microdilution methods according to CLSI guidelines. Double-disk synergy test was performed in order to screen for ESBLs, and combined disk test with phenylboronic acid to detect AmpC ß -lactamases. PCR was used to detect blaESBL/blacarb genes. Genetic relatedness of the strains was determined by pulsed-fieldgel-electrophoresis (PFGE). Results Eleven ESBL-producing isolates were included in the study (six inpatients and five outpatients). Susceptibility rate to amoxicillin-clavulanic acid, imipenem and meropenem was 100%. Resistance rate to cefuroxime was 100%, gentamicine 90.9%, piperacillin/tazobactam 81.8%, cefotaxim, ceftriaxone and ceftazidime 72.7%, cefoxitine and ciprofloxacine 63.6% and to cefepime 45.5%. In five (out of 11) isolates multi-drug resistance (MDR) to cephalosporins, cefamicines, amynocligosides and fluoroquinolones was detected. Besides TEM-1 which was detected in all isolates, CTX-M+OXA-1 ß-lactamases were detected in seven (out of 11; 63.6%) isolates (five blaCTX-M-1 and two blaCTX-M-15 genes), and CMY-2 ß-lactamase in two isolates. PFGE showed no genetic relatedness. Conclusion Because of high prevalence of MDR strains in epidemiologically unrelated patients with AmpC- and/or ESBL producing Proteus spp. infection, further surveillance is needed. Molecular characterization and strain typing, or at least phenotypic test for AmpC/ESBL production is important for appropriate therapy and the detection of sources and modes of spread, which is the main step in order to design targeted infection control strategies.

Insights into copper effect on Proteus hauseri through proteomic and metabolic analyses.

This is the first-attempt to use liquid chromatography coupled with tandem mass (LC-MS-MS) in deciphering the effects of copper ion on Proteus hauseri. Total 941 proteins in copper-addition (+Cu) group and 898 proteins in non-copper-addition (-Cu) group were found, which containing 221 and 178 differential proteins in +Cu and -Cu group, respectively. Differential proteins in both groups were defined into 14 groups by their functional classification which transport/membrane function proteins were the major different part between the two groups, which took 19.5% and 7.7%, respectively. The result of BioCyc and KEGG analyses on metabolic pathway indicated that copper could interrupted the pathway of chemotaxis CheY and inhibited the swarming of P. hauseri, which provided a potential in controlling the pathogenicity of this strain.

Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.

The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.

The tryptophanase from Proteus rettgeri, improved purification and properties of crystalline holotryptophanase.

The inducible tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) was crystallized in holoenzyme from the cell extract of Proteus rettgeri. The purification procedure included ammonium sulfate fractionation, heat treatment at 60 degrees C, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystallization was performed by the addition of ammonium sulfate to the purified enzyme solution containing 20% (v/v) glycerol, 0.1 mM pyridoxal phosphate and 10 mM mercaptoethanol. The crystallized enzyme was yellow and showed absorption maxima at 340 and 420 nm. The crystalline holotryptophanase preparation was homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight of the enzyme was calculated as approx. 222 000. The amount of pyridoxal phosphate bound to the enzyme was determined to be 4 mol per mol of the enzyme. The enzyme is composed of four subunits of identical molecular size (mol. wt 55 000) and irreversibly dissociates into these subunits in the presence of a high concentration of sodium dodecylsulfate or guanidine hydrochloride. The NH2-terminal amino acid of the enzyme was identified as alanine.

Membrane ATPase of Proteus L-forms. Solubilization and molecular properties.

The Mg2+ -dependent ATPase (EC 3.6.I.3) of Proteus L-form membrane has been solubilized according to various procedures (Tris - HCL shock-wash with or without MG2+, EDTA, Triton X-100). The best results were obtained by the same 33mM Tris-HCL (pH 7.5) shock-wash without MG2+ used for ATPase of protoplasts from Streptococcus faecalis. The solubilized enzyme after 105 000 times g centrifugation was purified on acrylamide/agarose. The molecular weight was established to be 360 000 by gel filtration and by sedimentation coefficient (12.5 S). Polyacrylamide disc-gel electrophoresis in sodium dodecylsulphate revealed two classes or subunit of mol. wt. 64 000 (alpha) and 58 000 (beta), associated in ratio 1:1. We propose a formula alpha-3beta-3 for the native ATPase of Proteus L-forms. Structural similarities to ATPase of various origins are discussed.

The apparent conservation of the internal low efficiency promoter of the tryptophan operons of several species of Enterobacteriaceae.

The specific activities of the tryptophan biosynthetic enzymes were determined for eleven different species of Enterobacteriaceae grown under repressing and non-repressing conditions. In all the bacteria examined the multipliciy of derepression for the first two enzymes of the pathway was at least twofold greater than the multiplicity of derepression for the last three enzymes.