Majority of native enzymes are poorly applicable for practical usage: that is why different methods of enzyme modification are used to obtain the biocatalysts with appropriate characteristics. Development of genome sequencing and various modern approaches in protein engineering allow one to identify protein of interest and to improve the enzyme properties for a particular process. This review describes the results on development of novel biocatalysts based on bioinformatics and rational design. New genes encoding formate dehydrogenase (FDH) from bacterium Staphylococcus aureus, yeasts Ogataea parapolymorpha and Saccharomyces cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH and PpaFDH, respectively), have been cloned. New FDHs were produced in the active form and characterized. SauFDH was shown to have at least 2-fold higher catalytic constant than other known FDHs. OpaFDH has catalytic parameters as good as those for soy FDH mutant forms, and in addition, is more thermostable. Apo- and holo-forms of SauFDH have been crystallized. Mutation of two Cys residues in Pseudomonas sp.101 enzyme (PseFDH) yields enzyme preparations with improved kinetic parameters and enhanced thermal and chemical stability. New generation of PseFDH preparations with the coenzyme specificity changed from NAD+ to NADP+ have been obtained. The effect of ionic liquids on the catalytic properties and thermal stability of six wild-type recombinant FDHs, and a number of their mutants, have been studied. In case of D-amino acid oxidase (DAAO), single-point mutations have been combined to create multi-point mutants. The introduced amino acid replacements have been shown to exert an additive effect, improving both kinetic parameters and increasing thermal and chemical stability. DAAO genes from Hansenula polymorpha yeast have been cloned. α-Amino acid ester hydrolase (AEH) gene has been cloned and expressed in the active form in E. coli. Structural modeling has been performed and the effectiveness in amino beta-lactams synthesis studied. The structure of a single-chain penicillin acylase from Alcaligenes faecalis (scAfPA) has been modeled and two variants of scAfPA gene was generated by PCR. Both variants have been expressed in E. coli, isolated and characterized. Catalytic properties of scAfPA were slightly better than those of its natural heterodimer.

Journal of Clinical Microbiology and Biochemical Technology is the journal aspired to serve as prominent form of communication with online publishing speeding quickly to meet the needs of literature world by publishing quality articles and making aware the people with new technologies in Microbiology and Biochemical world.

Sensors are devices composed of an active sensing material with a signal transducer. Electrochemical sensors have more advantages over the others because the electrodes can sense the analyte that is present in the host without doing any damage to the host system. The immobilization of a protein on a metallic transducer can be a crucial step in the development of bionanodevices that find applications in the field of biomaterials, biocoatings, biofuel cells, etc. In the present study, we show the immobilization by electropolymerization of thioaniline functionalized glucose oxidase and Au nanoparticles on nanostructured gold films prepared by electrodeposition and compared to sputtered gold. This enzyme is employed in the preparation of biosensors of biomedical interest. The goodness of the Au film for enzyme binding is evaluated by comparison of the enzyme activity and of the interval of linearity for the determination of glucose concentration.

In this paper, we have reported an immunoassay with time-resolved revelation system for ampicillin in raw milk samples. Immunological methods appear to be a promising approach in the analysis of beta-lactam compounds, because they do not need previous sample pre-treatments. In fact, beta-lactam ring is not very stable in extensive sample pre-treatment procedures requested in conventional analytical techniques. Specimens were collected from lactating cows bred in various conditions and assayed for the fat contents. Ampicillin was assayed in samples with different fat concentrations. The assay was performed using ampicillin-specific polyclonal antibody raised in rabbit; the immunogen was synthesized using bovine thyroglobulin conjugated to ampicillin by glutaraldehyde reaction; as fluorescent marker we used goat anti-rabbit IgG conjugated with a chelating molecule complexed with Eu(3+). Bovine serum albumin (BSA) conjugated with ampicillin was synthesized and used to prepare a solid phase on polystyrene microtiter plates. The use of a lanthanide chelate as label allowed to achieve 1 ng mL(-1) sensitivity, which is four times more sensitive than limits requested from European Community. Fat contents did not affect the assay performance.

Three proteins belonging to the thaumatin-like proteins family were compared in this study from a structural point of view: zeamatin, a new recently isolated PR-5 from Cassia didymobotrya and the commercial sweet-thaumatin. The former two proteins possess antifungal activities while commercial thaumatin is well known to be a natural sweetener. Intrinsic fluorescence studies have evidenced that the three proteins behave differently in unfolding experiments showing different structural rigidity. All the three proteins are more stable at slight acidic buffers, but sweet-thaumatin has a major tendency to destructurate itself. Similar observations were made from circular dichroism studies where a structural dependence relationship from the pH and the solvent used confirmed a hierarchic scale of stability for the three proteins. These structural differences should be considered to be significant for a functional role.

A 1905‐Da cationic proline‐rich peptide, named SP‐B, was recently isolated by our group as the main component of salivary gland granules, and its primary sequence fully characterized by means of automated Edman sequencing and LC‐MS/MS tools. In the present study SP‐B is shown to possess antifungal activity when challenged with strains of Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, while only negligible antibacterial activity was detected. Furthermore, SP‐B was found to be non‐cytotoxic when tested on fibroblast cell lines. To obtain information regarding its structure affinity, capillary electrophoresis (CE), circular dichroism (CD) and attenuated total reflection (ATR)‐FT/IR experiments were performed. CE revealed a pH dependence of the hydrodynamic radial dimensions both in aqueous and 2,2,2‐trifluoroethanol solutions. CD and ATR‐FT/IR measurements confirmed the structure–pH relationship, revealing a secondary structure composed of mixed proportions of polyproline‐II, unordered and turn motifs, the last being more evident in the zwitterionic form of the peptide. From these findings SP‐B peptide could be classified as a new member of the proline‐rich antimicrobial peptide family. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

Synthetic beidellite clays with variable Si/Al ratio in the tetrahedral layers were prepared by hydrothermal crystallization in an acid fluoride medium. The clay materials were characterized by powder-XRD, MAS NMR, BET specific area surfaces and chemical analysis. These samples were used as support for the immobilization of lipase B from Candida antarctica and lipase from Burkholderia cepacia by adsorption. The amount of lipase immobilized on the clay surface increased with decreasing level of aluminium substitution in the tetrahedral layer. The immobilization percentage was significantly higher for CALB (even for three times in the case of high siliceous clays) than those of BCL, when a sample of clay with the same composition is used as support. The catalytic properties of supported enzymes were investigated using the transesterification of vinyl acetate with 1-octanol as test reaction. The specific activity of the immobilized lipases depends on both the clay composition – the samples with low content of aluminum exhibited the highest specific activity-, and the amount of adsorbed enzyme – it increases as the amount of enzyme decreases. Also, the catalytic activity of lipase deposited on H+-beidellite is higher than those of enzyme deposited on the Na+ form.

C‐terminal binding proteins (CtBPs) are moonlighting proteins involved in nuclear transcriptional corepression and in Golgi membrane tubule fission. Structural information on CtBPs is available for their substrate‐binding domain, responsible for transcriptional repressor recognition/binding, and for the nucleotide‐binding domain, involved in NAD(H)‐binding and dimerization. On the contrary, little is known about the structure of CtBP C‐terminal region (∼90 residues), hosting sites for post‐translational modifications. In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small‐angle X‐ray scattering, and we show that the CtBP C‐terminal region is intrinsically unstructured in the full‐length CtBP and in constructs lacking the substrate‐ and/or the nucleotide‐binding domains. The flexible nature of this protein region, and its structural transitions, may be instrumental for CtBP recognition and binding to diverse molecular partners.

The protein ataxin-3 is responsible for Machado-Joseph disease/spinocerebellar ataxia type 3, a neurodegenerative disorder caused by the presence of an expanded polyglutamine tract. A previous investigation [Bevivino, A. E., and Loll, P. J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 11955-11960] showed that a nonexpanded ataxin-3 (Q27) was fully soluble, whereas an expanded form (Q78) gave rise to amyloid fibrils. Here, we report investigations on three forms of ataxin-3 (i.e., human nonexpanded (Q26), moderately expanded (Q36) ataxins-3, and the murine protein (Q6)). Far-UV circular dichroism spectra at room temperature were substantially similar, with a relatively high helical content. On heating to 96 degrees C, human Q26 and murine proteins did not display large structural changes, nor did they undergo any precipitation, which highlights their amazing heat-resistance. In contrast, human Q36 ataxin-3 underwent a progressive increase in the beta-sheet and a concomitant decrease in helical content when the temperature was shifted from 37 to 80 degrees C, followed by the irreversible formation of aggregates above 80 degrees C. They were shown to consist of amyloid fibrils, as supported by both electron microscopy images and the typical spectral shift displayed by Congo red when it was added to the protein at growing temperatures. We also found that protein precipitation could be prevented by mixing the dye with Q36 ataxin-3 prior to heating, which also confirms that the precipitates do represent authentic amyloid fibrils. In contrast, other compounds structurally related to Congo red did not exert significant effects. Our observations suggest that the temperature of the observed transition is inversely related to the length of the expansion. Finally, we suggest that antiamyloidogenic compounds might be selected on the basis of their ability to block or retard human Q36 ataxin-3 precipitation on heat-treatment.

The activity of different formulations of Candida antarctica lipase B (CALB), such as crude CALB, purified CALB, purified CALB lyophilized with PEG (CALB + PEG) or oleic acid (CALB + OA), and the commercial formulation Novozym 435, was determined in toluene, carbon tetrachloride, and 1,4-dioxane at various water activities (a(w)). The reaction between vinylacetate and 1-octanol was used as the model reaction and both transesterification (formation of 1-octylacetate) and hydrolytic (formation of acetic acid from vinylacetate) activities were determined. For equal amounts of lipase protein, CALB + PEG (and to a lesser extent CALB + OA) displayed higher activity than that of the other formulations; for instance, in toluene (a(w) < 0.1), it was 260-, 13-, and 1.8-fold more active than crude CALB, purified CALB, and Novozym 435, respectively. Moreover, the transesterification activity of CALB + PEG was of the same order of magnitude (51%) of the activity shown by the enzyme in the hydrolysis of vinylacetate in aqueous buffer. These results suggest that PEG and oleic acid could act as lyoprotectants, preventing the formation of intermolecular interactions during the lyophilization process that might be responsible for protein denaturation. No diffusional limitation was observed for CALB + PEG-catalyzed reactions. Purified CALB, in contrast to the other formulations, showed a marked activity increase (2.1 to 7.8-fold) as a function of a(w) and, in 1,4-dioxane, it was 3.5-fold more active when it was added to the solvent after previous dissolution of the lyophilized powder in water.

A procedure for enzyme entrapment into matrices suitable for biocatalytic applications is reported. The method, which takes advantage of the stable formation of polyvinyl alcohol (PVA) hydrogels by freezing and thawing PVA aqueous solutions, was assayed using lipase as model enzyme. The leakage of lipase was minimised by using high molecular weight PVA and by previous conjugation of the enzyme to PEG. The immobilised PEG enzyme maintained its catalytic activity in organic solvents also, thus allowing enzymatic activity towards water insoluble substrates. The activity was largely increased reducing the diffusional constrain by cutting the matrices into slices of micron size. Matrix-entrapped lipase-PEG, when used in the hydrolysis of acetoxycoumarins, showed a conversion rate of about 10 times lower than the enzyme-PEG in the free form, and maintained regioselectivity when a diacetylated product was used as substrate.

Background: The phospholipase D (PLD) superfamily includes enzymes that are involved in phospholipid metabolism, nucleases, toxins and virus envelope proteins of unknown function. PLD hydrolyzes the terminal phosphodiester bond of phospholipids to phosphatidic acid and a hydrophilic constituent. Phosphatidic acid is a compound that is heavily involved in signal transduction. PLD also catalyses a transphosphatidylation reaction in the presence of phosphatidylcholine and a short-chained primary or secondary alcohol.Results: The first crystal structure of a 54 kDa PLD has been determined to 1.9 Angstrom resolution using the multiwavelength anomalous dispersion (MAD) method on a single WO4 ion and refined to 1.4 Angstrom resolution. PLD from the bacterial source Streptomyces sp. strain PMF consists of a single polypeptide chain that is folded into two domains. An active site is located at the interface between these domains. The presented structure supports the proposed superfamily relationship with the published structure of the 16 kDa endonuclease from Salmonella typhimurium.Conclusions: The structure of PLD provides insight into the structure and mode of action of not only bacterial, plant and mammalian PLDs, but also of a variety of enzymes as diverse as cardiolipin synthases, phosphatidylserine synthases, toxins, endonucleases, as well as poxvirus envelope proteins having a so far unknown function. The common features of these enzymes are that they can bind to a phosphodiester moiety, and that most of these enzymes are active as bi-lobed monomers or dimers.

Crystals of purified phospholipase D (E.C. 3.1.4.4) from Streptomyces sp. strain PMF have been grown under two different crystallization conditions using vapour diffusion. Both conditions gave monoclinic crystals in space group P2(1). The unit-cell parameters were a = 57.28, b = 57.42, c = 68.70 A, beta = 93.17 degrees. The crystals diffract at 110 K to a resolution beyond 1.4 A using synchrotron radiation.

Lipases from different sources were tested in the kinetic resolution of 2-hydroxy-3-butenyl butanoate [(R, S)-2] carried out by transesterification of the secondary alcohol. The influence of organic solvent, acyl donor and temperature on the enantioselectivity and activity of lipases was also investigated. Our study showed that both R- (+)-2 and S-(-)-2 could be obtained in high enantiomeric purity (ee ≥ 99%) and satisfactory yield (29% and 27%, respectively). Among the enzymes tested, lipase from Candida antarctica B (CALB) showed the highest preference for the (R)-enantiomer (E=26 at -13°C), whereas lipase from Pseudomonas fluorescens (lipase AK) acylated the (S)-enantiomer preferentially (E= 18 at -9°C).

Fourier-transform infrared (FT-IR) spectroscopy was employed to investigate potential lyophilization-induced changes in the secondary structure of lipases from Candida antarctica B and Pseudomonas cepacia. The secondary structure elements were determined by curve fitting of the amide III bands of the two lipases in the lyophilized state in KBr pellets and in solution. It was found that lyophilization decreased the alpha-helix and increased the beta-sheet content. However, FT-IR analysis of crosslinked enzyme crystals of Pseudomonas cepacia lipase also indicated an increase in the beta-sheet content, which appears despite the fact that the enzyme, being in the crystallized state, should possess native conformation. This result partially questions the suitability of FT-IR for analysis of the structure of solid proteins, at least as far as the beta-sheet content is concerned, because it is possible that the method overestimates the beta-sheets by measuring other hydrogen-bonded nonperiodic intermolecular structures. No significant modification was observed when lipase from Pseudomonas cepacia was lyophilized in the presence of methoxypoly(ethylene glycol). Copyright 1999 John Wiley & Sons, Inc.

Lipase from Pseudomonas cepacia was made soluble in 1,4-dioxane by lyophilization of the enzyme from aqueous solutions containing methoxypoly(ethylene glycol) (PEG). The solubility of the enzyme–PEG complex depended both on protein concentration and PEG protein ratio. Intrinsic protein fluorescence and far- and near-UV circular dichroism revealed that not only did the enzyme not unfold in the organic solvent, but rather became more compact. This was seen by the slight quenching of fluorescence intensity and by the enhancement of the near-UV circular dichroism negative signals, which are indicative of stronger interactions of tryptophanyl and/or tyrosyl residues among themselves or with other parts of the enzyme molecule. The specific activity of the lipase–PEG complex in the organic solvent was at least 2 orders of magnitude higher than that of the enzyme powder. This can be attributed both to the maintenance of native conformation and to enzyme dissolution in the reaction medium which should minimize possible limitations to enzyme–substrate interactions. © 1999 John Wiley & Sons, Inc., Biotechnol Bioeng 64: 624–629, 1999.