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.

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.

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.

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.

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.

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.

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.

A method to rationally increase protein stability based on the charge-charge interaction, with application to lipase LipK107.

We report a suite of enzyme redesign protocol based on the surface charge-charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with ∼10°C higher half-inactivation temperature than wild-type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14-fold increase in the half-inactivation time at 50°C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.

Gene cloning and characterization of a novel highly organic solvent tolerant lipase from Proteus sp. SW1 and its application for biodiesel production.

Proteus sp. SW1 was found to produce an extracellular solvent tolerant lipase. The gene, lipA, encoding a bacterial lipase, was cloned from total Proteus sp. SW1 DNA. lipA was predicted to encode a 287 amino acid protein of 31.2 kDa belonging to the Group I proteobacterial lipases. Purified His-tagged LipA exhibited optimal activity at pH 10.0 and 55°C. It was highly stable in organic solvents retaining 112% of its activity in 100% isopropanol after 24 h, and exhibited more than 200% of its initial activity upon exposure to 60% acetone, ethanol, and hexane for 18 h. Biodiesel synthesis reactions, using a single step addition of 13% an acyl acceptor ethanol, showed that LipA was highly effective at converting palm oil into biodiesel.

Copper ion-stimulated McoA-laccase production and enzyme characterization in Proteus hauseri ZMd44.

The novel bioelectricity-generating bacterium of Proteus hauseri ZMd44 has been first identified to produce McoA-laccase (EC 1.10.3.2) induced by copper sulphate. The optimal concentration of copper is 3 mM as supplementation at the beginning of culture or early exponential growth phase, during which laccase is predominantly synthesized. Moreover, the whole cellular and intracellular activities of laccase increase in the degrees of inducible copper concentrations. A possible mechanism for this phenomenon is that copper ions enhance the laccase genetic transcription level during the laccase synthesis thus granting this strain in copper tolerance. McoA-laccase belongs to typical type 1 (T1) Cu site laccase by electron paramagnetic resonance (EPR) analysis of intracellular enzyme. From our results, the optimal temperature and pH are 60°C and pH 2.2, respectively. The kinetic profiles show that this enzyme is stable under 50°C and in the slightly acidic environment, making it a potentially useful enzyme in dye decolorization, paper-pulp bleaching and bioremediation industries.

Crystal structures of lysine-preferred racemases, the non-antibiotic selectable markers for transgenic plants.

Lysine racemase, a pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr) from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar) structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/ß)(8) barrel and a C-terminal ß-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.

Development of an "early warning" sensor for encrustation of urinary catheters following Proteus infection.

Biofilm formation in long-term urinary catheterized patients can lead to encrustation and blockage of urinary catheters with serious clinical complication. Catheter encrustation stems from infection with urease-producing bacteria, particularly Proteus mirabilis. Urease generates ammonia from urea, and the elevated pH of the urine results in crystallization of calcium and magnesium phosphates, which block the flow of urine. The aim of this research is to develop an "early warning" silicone sensor for catheter encrustation following bacterial infection of an in vitro bladder model system. The in vitro bladder model was infected with a range of urease positive and negative bacterial strains. Developed sensors enabled catheter blockage to be predicted ~17-24 h in advance of its occurrence. Signaling only occurred following infection with urease positive bacteria and only when catheter blockage followed. In summary, sensors were developed that could predict urinary catheter blockage in in vitro infection models. Translation of these sensors to a clinical environment will allow the timely and appropriate management of catheter blockage in long-term catheterized patients.

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

Introduction: Extended-spectrum-β-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 percent 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 percent), TEM (62 percent) and GES (28 percent). 79 percent 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.

Introducción: β-lactamasas de espectro extendido (BLEE) son enzimas plasmidiales que confieren resistencia a múltiples antimicrobianos. Las bacteriemias por enterobacterias productoras de BLEE restringen las opciones terapéuticas y aumentan la mortalidad. Objetivo: Realizar una descripción clínica y molecular de las bacteriemias causadas por enterobacterias productoras de BLEE. Método: Se estudiaron retrospectivamente los casos de bacteriemia por Escherichia coli, Klebsiella pneumoniae y Proteus spp. confirmadas para BLEE, en adultos ingresados en un hospital universitario durante los años 2004-2007. Se revisaron los registros clínicos y de susceptibilidad. Se realizó tipificación molecular por reacción de polimerasa en cadena y estudio de clonalidad por electroforesis de campo pulsado. Resultados: Se identificó una prevalencia de BLEE de 9,8 por ciento en enterobacterias causantes de bacteriemias. Se observó susceptibilidad disminuida a quinolonas y aminoglucósidos, sin resistencia a carbapenémicos. Los tipos de BLEE predominantes fueron CTX-M (96 por ciento), TEM (62 por ciento) y GES (28 por ciento). El 79 por ciento de las cepas presentó más de un tipo de BLEE. El análisis clínico reveló alta frecuencia de patologías de riesgo, uso previo de antimicrobianos y uso de dispositivos invasores. No se encontró clonalidad significativa. Conclusión: La presencia de BLEE en bacteriemias constituye un problema clínico que debe ser considerado al elegir la terapia empírica.

[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.

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.

Ureases as a target for the treatment of gastric and urinary infections.

Urease is known to be a major contributor to pathologies induced by Helicobacter pylori and Proteus species. In H pylori, urease allows the bacteria to survive in an acidic gastric environment during colonisation, playing an important role in the pathogenesis of gastric and peptic ulcers. Ureolytic activity also results in the production of ammonia in close proximity to the gastric epithelium, causing cell damage and inflammation. In the case of Proteus species (notably Proteus mirabilis) infection, stones are formed due to the presence of ammonia and carbon dioxide released by urease action. In addition, the ammonia released is able to damage the glycosaminoglycan layer, which protects the urothelial surface against bacterial infection. In this context, the administration of urease inhibitors may be an effective therapy for urease-dependent pathogenic bacteria. This is a review of the role of ureases in H pylori and Proteus species infections, focussing on the biochemical and clinical aspects of the most promising and/or potent urease inhibitors for the treatment of gastric and urinary tract infections.

[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.