Biocatalyzed Synthesis of Flavor Esters and Polyesters: A Design of Experiments (DoE) Approach.

In the present work, different hydrolases were adsorbed onto polypropylene beads to investigate their activity both in short-esters and polyesters synthesis. The software MODDE® Pro 13 (Sartorius) was used to develop a full-factorial design of experiments (DoE) to analyse the thermostability and selectivity of the immobilized enzyme towards alcohols and acids with different chain lengths in short-esters synthesis reactions. The temperature optima of Candida antarctica lipase B (CaLB), Humicola insolens cutinase (HiC), and Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) were 85 °C, 70 °C, and 50 °C. CaLB and HiC preferred long-chain alcohols and acids as substrate in contrast to Thc_Cut1, which was more active on short-chain monomers. Polymerization of different esters as building blocks was carried out to confirm the applicability of the obtained model on larger macromolecules. The selectivity of both CaLB and HiC was investigated and best results were obtained for dimethyl sebacate (DMSe), leading to polyesters with a Mw of 18 kDa and 6 kDa. For the polymerization of dimethyl adipate (DMA) with BDO and ODO, higher molecular masses were obtained when using CaLB onto polypropylene beads (CaLB_PP) as compared with CaLB immobilized on macroporous acrylic resin beads (i.e., Novozym 435). Namely, for BDO the Mn were 7500 and 4300 Da and for ODO 8100 and 5000 Da for CaLB_PP and for the commercial enzymes, respectively. Thc_Cut1 led to polymers with lower molecular masses, with Mn < 1 kDa. This enzyme showed a temperature optimum of 50 °C with 63% of DMA and BDO when compared to 54% and 27%, at 70 °C and at 85 °C, respectively.

Health Potential of Clery Strawberries: Enzymatic Inhibition and Anti-Candida Activity Evaluation.

Strawberries, belonging to cultivar Clery (Fragaria × ananassa Duchesne ex Weston) and to a graft obtained by crossing Clery and Fragaria vesca L., were chosen for a study on their health potential, with regard to the prevention of chronic and degenerative diseases. Selected samples, coming from fresh and defrosted berries, submitted to different homogenization techniques combined with thermal and microwave treatments, had been previously analyzed in their polyphenolic content and antioxidant capacity. In the present work, these homogenates were evaluated in relation to their enzymatic inhibition activity towards acetylcholinesterase and butyrylcholinesterase, α-amylase, α-glucosidase and tyrosinase. All these enzymes, involved in the onset of diabetes, and neurodegenerative and other chronic diseases, were modulated by the tested samples. The inhibitory effect on tyrosinase and cholinesterase was the most valuable. Antifungal activity against Candida albicans, recently shown to play a crucial role in human gut diseases as well as diabetes, rheumatoid arthritis and Alzheimer's disease, was also shown in vitro and confirmed by the in vivo text on Galleria mellonella. Overall, the obtained results confirm once again the health potential of strawberries; however, the efficacy is dependent on high quality products submitted to correct processing flow charts.

A comparative proteomic analysis of Candida species in response to the oxidizing agent cumene hydroperoxide.

Candida genus comprises several species that can be found in the oral cavity and the gastrointestinal and genitourinary tracts of healthy individuals. Under certain conditions, however, they behave as opportunistic pathogens that colonize these tissues, most frequently when the immune system is compromised by a disease or under certain medical treatments. To colonize the human host, these organisms require to express cell wall proteins (CWP) that allowed them to adhere and adapt to the reactive oxygen (ROS) and nitrogen (RNS) species produced in the macrophage during the respiratory burst. The aim of this study was to determine how four Candida species respond to the oxidative stress imposed by cumene hydroperoxide (CHP). To this purpose, C. albicans, C. glabrata, C. krusei and C. parapsilosis were exposed to this oxidant which is known to generate ROS in the membrane phospholipids. Accordingly, both mock and CHP-exposed cells were used to extract and analyze CWP and also to measure catalase activity and the levels of protein carbonylation. Results indicated that all four species express different CWP to neutralize ROS. Most relevant among these proteins were the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase, known as moonlight proteins because in addition to participate in glycolysis they play an important role in the cell response to ROS. In addition, a thiol-specific antioxidant enzyme (Tsa) was also found to counteract ROS.

Efficient synthesis of cyano-containing multi-substituted indoles catalyzed by lipase.

BACKGROUND: Indoles are important bioactive compounds that have been extensively studied in organic chemistry. In this work, a green and efficient process for the synthesis of Indoles from 1,3-diketones with fumaronitrile was developed. RESULTS: Under optimal conditions (1,3-diketones (0.5 mmol), fumaronitrile (1 mmol), water (2 ml), lipase (15 mg), 30 °C, 24 h), high yields and satisfactory regioselectivity of cyano-containing multi-substituted indoles could be obtained when CRL (C. rugosa lipase) was used as the catalyst. CONCLUSION: This enzymatic method demonstrates the great potential for the synthesis of indoles and extends the application of enzyme in organic synthesis.

Heterologous Expression and Characterization of Flavinadenine Dinucleotide Synthetase from Candida famata for Flavin Adenine Dinucleotide Production.

BACKGROUND: Flavin adenine dinucleotide (FAD) is a redox-active coenzyme that regulates several important enzymatic reactions during metabolism. FAD is used in the medicinal and food industries and FAD supplements have been used to treat some inheritable diseases. FAD can be biosynthesized from flavin mononucleotide (FMN) and adenosine triphosphate (ATP), catalyzed by FAD synthetase (FADS). OBJECTIVE: The aim of this study was to heterologously express the gene encoding FADS from the flavinogenic yeast Candida famata (FADSCf) for biosynthesis of FAD. METHODS: The sequence encoding FADSCf was retrieved and heterologously expressed in Escherichia coli. The structure and enzymatic properties of recombinant FADSCf were characterized. RESULTS: FADSCf (279 amino acids) was successfully expressed in E. coli BL21 (DE3), with a theoretical molecular weight of 32299.79 Da and an isoelectric point of 6.09. Secondary structural analysis showed that the number of α-helices was 2-fold higher than the number of ß-sheets, indicating that the protein was highly hydrophilic. Under fixed ATP concentration, FADSCf had a Km of 0.04737±0.03158 mM and a Vmax of 3.271±0.79 µM/min/mg. Under fixed FMN concentration, FADSCf had a Km of 0.1214±0.07464 mM and a Vmax of 2.6695±0.3715 µM/min/mg. Enzymatic reactions in vitro showed that expressed FADSCf could form 80 mM of FAD per mg of enzyme after 21 hours under the following conditions: 0.5 mM FMN, 5 mM ATP and 10 mM Mg2+. CONCLUSION: Under optimized conditions (0.5 mM FMN, 5 mM ATP and 10 mM Mg2+), the production of FAD reached 80 mM per mg of FADSCf after a 21-hour reaction. Our results indicate that purified recombinant FADSCf can be used for the biosynthesis of FAD.

CDATA[Recent Advances in the Development of 1,2,3-Triazole-containing Derivatives as Potential Antifungal Agents and Inhibitors of Lanoster ol 14α-Demethylase.

1,2,3-Triazole, a five-membered heterocyclic nucleus, is widely recognized as a key chromophore of great value in medicinal chemistry for delivering compounds possessing innumerable biological activities, including antimicrobial, antitubercular, antidiabetic, antiviral, antitumor, antioxidants, and anti-inflammatory activities. Mainly, in the past years, diverse conjugates carrying this biologically valuable core have been reported due to their attractive fungicidal potential and potent effects on various infective targets. Hence, hybridization of 1,2,3-triazole with other antimicrobial pharmacophores appears to be a judicious strategy to develop new effective anti-fungal candidates to combat the emergence of drug-sensitive and drug-resistant infectious diseases. Thus, the current review highlights the recent advances of this promising category of 1,2,3-triazole-containing hybrids incorporating diverse varieties of bioactive heterocycles such as conozole, coumarin, imidazole, benzimidazole, pyrazole, indole, oxindole, chromene, pyrane, quinazoline, chalcone, isoflavone, carbohydrates, and amides. It underlies their inhibition behavior against a wide array of infectious fungal species during 2015-2020.

A comparative study on production of extracellular hydrolytic enzymes of Candida species isolated from patients with surgical site infection and from healthy individuals and their co-relation with antifungal drug resistance.

BACKGROUND: Surgical site infection (SSI) is a crucial dilemma of surgery. Patients with SSIs not only face difficulty in treatment but also bear extra cost with high mortality rate. Resistant strains of Candida have emerged as an important nosocomial pathogen. Proteinase and phospholipase are exo- enzymes of Candida species, have importance with respect to their contribution in diseases. This study focused on prevalence of Candida species in surgical wound, their resistance to antifungal drugs, co-relation of these resistance with virulence potential of Candida species and comparison of production level of exo-enzymes of Candida species isolated from patients with SSIs and healthy individuals to highlights their role in SSIs. RESULTS: A total of (n = 555) swab samples were investigated. (n = 450) samples were collected from patients with SSIs and (n = 105) were collected from healthy individuals. Samples were subjected for the identification of Candida species which were subsequently investigated for antifungal susceptibility, MICs and enzymatic activity of Candida species. Out of 128 strains of Candida spp. isolated from SSIs, 54(42.18%) were identified as C. albicans followed by C. glabrata 32(25%), C. parapsilosis 17(13.28%), C. krusei 13(10.16%) and C. tropicalis 12(9.38%). C. albicans isolates showed 100% susceptibility to voriconazole and amphotericin B followed by itraconazole 98% and fluconazole 89%. Out of 6 fluconazole resistant C. albicans 5(83.33%) were able to produce phospholipase while out of 48 fluconazole-susceptible strains 17(35.42%) were found to be phospholipase producer. Out of 54 C. albicans isolated from surgical wound 46(85.18%) and 49(90.74%) were found to be phospholipase and proteinase producer respectively, whereas out of 20 C. albicans isolates from healthy subjects 14(70%) produce proteinase and 12(60%) produce phospholipase. There were significant statistical differences found between the level of enzyme production by C. albicans, in relation to both sites (P = 0.014). CONCLUSION: Study revealed that prevalence of Candida species is high in SSIs. Phospholipase and proteinase activity were more pronounced in Candida Species from surgical wound in contrast to species from healthy individuals suggests these enzymes may have been responsible for the severity of infection in surgical wound patients.

Enzyme production by Candida albicans and Candida dubliniensis in periodontal HIV-positive patients receiving and not receiving antiretroviral therapy / Producción enzimática de Candida albicans y Candida dubliniensis de pacientes con periodontitis VIH+ con y sin tratamiento antirretroviral

ABSTRACT Candida dubliniensis (Cd) and Candida albicans (Ca) are the most frequently isolated yeasts in HIV+ patients. Some of the enzymes produced by these yeasts are considered virulence factors since they contribute to pathogenicity of Candida spp. The aim of the present study was to compare production of enzymes such as phospholipase (Ph), proteinase (P), and hemolysin (H) by Cd and Ca strains isolated from periodontal HIV-positive patients receiving and not receiving highly active antiretroviral therapy (HAART). Subgingival biofilm samples were obtained using paper points, and a sample of oral mucosa was taken using a swab. Phenotypic and molecular methods were used to isolate 39 strains of Candida, including 25 strains of Cd and 14 strains of Ca, obtained from 33 periodontal pocket samples and 6 oral mucosa samples collected from 15 HIV+ patients (8 receiving and 7 not receiving HAART). Malt egg-yolk agar, albumin agar and blood agar were used to evaluate pH, P and H production respectively. The strains were inoculated in duplicate and incubated at 37 ºC. Colony and halo diameters were measured. A greater proportion of Ca was observed in patients not receiving HAART, and a higher proportion of Cd was observed in those under HAART, Chi2 p< 0.001. Phospholipase production was observed in 92.9% percent of isolated Ca strains but in none of the isolated Cd strains. Proteinase production was high in Ca and Cd strains isolated from patients not receiving HAART. Hemolysin production was observed in all the studied strains, though it was significantly higher (p=0.04) in Ca and Cd strains isolated from patients not receiving HAART. To sum up, the proportion of Candida dubliniensis strains was highest in the subgingival biofilm of patients receiving HAART, and Cd strains were found to express fewer virulence factors than Ca strains.

RESUMEN Las levaduras más aisladas en pacientes VIH+ son Candida dubliniensis (Cd) y Candida albicans (Ca). Algunas de sus enzimas constituyen factores de virulencia ya que favorecen la diseminación tisular. El objetivo fue comparar la producción de enzimas como fosfolipasa (F), proteinasa (P) y hemolisina (H) en cepas de Cd y Ca aisladas de pacientes VIH+ tratados y no tratados con antirretrovirales (TARGA). Se realizó la toma del biofilm de placa subgingival con conos de papel y la muestra de la mucosa bucal con hisopo. Se aislaron y tipificaron por métodos fenotípicos y moleculares 39 cepas: 25 de Cd y 14 Ca, obtenidas 33 de bolsas periodontales y 6 de mucosa bucal de 15 pacientes VIH+ (8 con y 7 sin tratamiento). Se utilizó agar malta con yema de huevo, agar albúmina y agar sangre para demostrar la producción de F, P y H, respectivamente. Se inocularon por duplicado e incubaron a 37°C. Se midieron los diámetros de las colonias y los de hidrólisis alrededor de las mismas. Se observó mayor proporción de Ca en los pacientes sin tratamiento y mayor proporción de Cd en los con tratamiento; Chi2 p< 0.001. El 92,9% de las Ca estudiadas, fueron productoras de fosfolipasa. En tanto que ninguna Cd produjo la enzima. En cuanto a la producción de proteinasa se observa una alta producción tanto en las cepas de Ca, como en las Cd aisladas en los pacientes no tratados. Todas las cepas estudiadas produjeron hemolisina, observándose una diferencia estadísticamente significativa (p=0,04) en ambas especies a favor de la alta producción de la enzima en las cepas obtenidas de pacientes no tratados. Podemos concluir que en el biofilm subgingival, en los pacientes bajo TARGA, se aíslan mayor proporción de Candida dubliniensis las cuales expresan menos factores de virulencia.

Integration of metal organic frameworks with enzymes as multifunctional solids for cascade catalysis.

Enzymes exhibit a large degree of compatibility with metal-organic frameworks (MOFs) which allows the development of multicomponent catalysts consisting of enzymes adsorbed or occluded by MOFs. The combination of enzymes and MOFs in a multicomponent catalyst can be used to promote cascade reactions in which two or more individual reactions are performed in a single step. Cascade reactions take place due to the cooperation of active sites present on the MOF with the enzyme. A survey of the available data establishes that often an enzyme undergoes stabilization by association with a MOF and the system exhibits notable recyclability. In addition, the existence of synergism is observed as a consequence of the close proximity of all the required active sites in the multicomponent catalyst. After an introductory section describing the specific features and properties of enzyme-MOF assemblies, the main part of the present review focuses on the description of the cascade reactions that have been reported with commercial enzymes associated with MOFs, paying special attention to the advantages derived from the multicomponent catalyst. Related to the catalytic activity to metabolize glucose, generating reactive oxygen species (ROS) and decreasing the solution pH, an independent section describes the recent use of enzyme-MOF catalysts in cancer therapy. The last paragraphs summarize the current state of the art and provide our view on future developments in this field.

Ultrastructural investigation of acidocalcisomes and ATPase activity in yeast Candida guilliermondii NP-4 as 'complementary' stress-targets.

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25-150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.

Biochemical Convergence of Mitochondrial Hsp70 System Specialized in Iron-Sulfur Cluster Biogenesis.

Mitochondria play a central role in the biogenesis of iron-sulfur cluster(s) (FeS), protein cofactors needed for many cellular activities. After assembly on scaffold protein Isu, the cluster is transferred onto a recipient apo-protein. Transfer requires Isu interaction with an Hsp70 chaperone system that includes a dedicated J-domain protein co-chaperone (Hsc20). Hsc20 stimulates Hsp70's ATPase activity, thus stabilizing the critical Isu-Hsp70 interaction. While most eukaryotes utilize a multifunctional mitochondrial (mt)Hsp70, yeast employ another Hsp70 (Ssq1), a product of mtHsp70 gene duplication. Ssq1 became specialized in FeS biogenesis, recapitulating the process in bacteria, where specialized Hsp70 HscA cooperates exclusively with an ortholog of Hsc20. While it is well established that Ssq1 and HscA converged functionally for FeS transfer, whether these two Hsp70s possess similar biochemical properties was not known. Here, we show that overall HscA and Ssq1 biochemical properties are very similar, despite subtle differences being apparent - the ATPase activity of HscA is stimulated to a somewhat higher levels by Isu and Hsc20, while Ssq1 has a higher affinity for Isu and for Hsc20. HscA/Ssq1 are a unique example of biochemical convergence of distantly related Hsp70s, with practical implications, crossover experimental results can be combined, facilitating understanding of the FeS transfer process.

Hydrophilic Glyceryl Ferulates Preparation Catalyzed by Free Lipase B from Candida antartica.

Ferulic acid (FA), 4-hydroxyl-3-methoxy-2-benzylacrylic acid, has antioxidant, anticancer and ultraviolet absorption activities. However, the low hydrophilicity of FA has limited its application. Glyceryl ferulate (FG), which is an all-natural hydrophilic derivative of FA, can be used as an antioxidant and UV filter in food and cosmetic formulations. However, the applications of FG in these fields are limited due to its low content in nature. In this work, free liquid lipase was firstly used as a catalyst for FG preparation. Several different free liquid lipases (Candida antartica lipase-B, Candida antartica lipase-A, Thermomyces lanuginosus (Lipozyme TL 100L)) were screened and compared. The effects of the transesterification parameters (time, temperature, enzyme load and substrate ratio) were optimized and evaluated by response surface methodology. A reaction thermodynamic investigation was also performed. The results showed that, among the tested free lipases, the maximum FG yield (84.8±1.5%) was achieved using free Candida antartica lipase-B. Under the optimized conditions (an atmospheric system, an enzyme load of 11.1% and a 20:1 molar ratio of glycerol to EF at 70°C for 39.5 h), the FG yield and EF conversion were 84.8±1.5% and 95.7±1.2%, respectively. The activation energies of FG formation and EF conversion were 56.4 and 58.0kJ/mol, respectively.

Copper-only superoxide dismutase enzymes and iron starvation stress in Candida fungal pathogens.

Copper (Cu)-only superoxide dismutases (SOD) represent a newly characterized class of extracellular SODs important for virulence of several fungal pathogens. Previous studies of the Cu-only enzyme SOD5 from the opportunistic fungal pathogen Candida albicans have revealed that the active-site structure and Cu binding of SOD5 strongly deviate from those of Cu/Zn-SODs in its animal hosts, making Cu-only SODs a possible target for future antifungal drug design. C. albicans also expresses a Cu-only SOD4 that is highly similar in sequence to SOD5, but is poorly characterized. Here, we compared the biochemical, biophysical, and cell biological properties of C. albicans SOD4 and SOD5. Analyzing the recombinant proteins, we found that, similar to SOD5, Cu-only SOD4 can react with superoxide at rates approaching diffusion limits. Both SODs were monomeric and they exhibited similar binding affinities for their Cu cofactor. In C. albicans cultures, SOD4 and SOD5 were predominantly cell wall proteins. Despite these similarities, the SOD4 and SOD5 genes strongly differed in transcriptional regulation. SOD5 was predominantly induced during hyphal morphogenesis, together with a fungal burst in reactive oxygen species. Conversely, SOD4 expression was specifically up-regulated by iron (Fe) starvation and controlled by the Fe-responsive transcription factor SEF1. Interestingly, Candida tropicalis and the emerging fungal pathogen Candida auris contain a single SOD5-like SOD rather than a pair, and in both fungi, this SOD was induced by Fe starvation. This unexpected link between Fe homeostasis and extracellular Cu-SODs may help many fungi adapt to Fe-limited conditions of their hosts.

Effect of operative variables and kinetic study of butyl butyrate synthesis by Candida rugosa lipase activated by chitosan-reinforced nanocellulose derived from raw oil palm leaves.

Currently, the chemically-assisted esterification to manufacture butyl butyrate employs corrosive homogeneous acid catalyst and liberates enormous quantities of hazardous by-products which complicate downstream treatment processes. This study aimed to identify the optimized esterification conditions, and the kinetic aspects of the enzyme-assisted synthesis of butyl butyrate using immobilized Candida rugosa lipase activated by chitosan-reinforced nanocellulose derived from raw oil palm leaves (CRL/CS-NC). The best process variables that gave the maximum conversion degree of butyl butyrate by CRL/CS-NC (90.2%) in just 3 h, as compared to free CRL (62.9%) are as follows: 50 °C, 1:2 M ratio of acid/alcohol, stirring rate of 200 rpm and a 3 mg/mL enzyme load. The enzymatic esterification followed the ping pong bi-bi mechanism with substrate inhibition, revealing a ˜1.1-fold higher Ki for CRL/CS-NC (55.55 mM) over free CRL (50.68 mM). This indicated that CRL/CS-NC was less inhibited by the substrates. Butanol was preferred over butyric acid as reflected by the higher apparent Michaelis-Menten constant of CRL/CS-NC for butanol (137 mM) than butyric acid (142.7 mM). Thus, the kinetics data conclusively showed that CRL/CS-NC (Vmax 0.48 mM min-1, Keff 0.07 min-1 mM-1) was catalytically more efficient than free CRL (Vmax 0.35 mM min-1, Keff 0.06 min-1 mM-1).

Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105-/His224+ rather than a concerted process.

Lipase-catalysed synthesis of palm oil-omega-3 structured lipids.

In this work, Candida antarctica lipase A was applied to selectively remove saturated fatty acids from palm oil to prepare palm oil acylglycerol concentrate (POAC), where palmitic acid decreased from 40.0 to 28.7% and oleic acid increased from 40.0 to 50.5% after 3 h of hydrolysis. Lipozyme RMIM from Rhizomucor miehei was then used to incorporate either alpha linolenic acid (ALA) or eicosapentaenoic acid (EPA) into the resulting POAC. Optimum omega-3 incorporation was achieved when POAC to omega-3 ratio was 6 : 3, reaction temperature was 40 °C and reaction time was 18 h. Under these conditions, the ALA content in the separated ALA incorporated structured lipid (POAC-ALA) was 27.1%, and the EPA content in the EPA incorporated structured lipids (POAC-EPA) was 30.9%. The formed structured lipids had lower levels of saturated fatty acids, and significantly lower melting points, in both cases below 8 °C. The enzymatic process developed produces new structured lipids, with lower saturated fat and higher omega-3, with potential as a healthy palm oil derived lipid ingredient.

Enzyme cocktail containing NADH regeneration system for efficient bioremediation of oil sludge contamination.

Oil sludge is one kind of toxic and persistent contamination to ecology system from petroleum industry. In order to recycle contaminated sands and reduce environmental impacts at a lower operating cost, enzyme cocktail 21/CbFDH including NADH regeneration system for oily sludge bioremediation was constructed for the first time. The intracellular enzymes of oil-degrading strain Acinetobacter calcoaceticus 21 were prepared and the formate dehydrogenase gene Cbfdh from Candida boidinii was cloned and functionally expressed in E.coli BL21 induced by lactose. The activity and stability of CbFDH was enhanced through self-induction medium optimization using Box-Behnken design. The CbFDH activity was 12.2 times increased and was only decreased 3.9% upon storage at 30 °C for 5 d. The CbFDH increased the degradation rate of oil in high concentration. For the sludge with 10% oil (w/w), the degradation rate achieved 35.6% after 12 h using enzyme 21/CbFDH with the protein ratio of 1:4. The results will provide novel perspectives for creation and operation of petroleum-degrading enzymes involving formate dehydrogenase with higher efficiency and lower cost comparing to current microbial strains or consortium.

Virulence factors of Candida spp. obtained from blood cultures of patients with candidemia attended at tertiary hospitals in Northeast Brazil.

BACKGROUND: Yeasts of the Candida genus are one of the most common causes of bloodstream infections associated with high rates of morbidity and mortality, mainly affecting immunocompromised patients. We aimed to identify yeasts obtained from blood cultures of patients interned at tertiary hospitals in Brazil. METHODS: We evaluated some of the major virulence factors of Candida spp., including the ability to adhere to human buccal epithelial cells, biofilm formation, hemolytic and phospholipase activity. RESULTS: We analyzed 70 isolates of Candida spp. obtained from March 2011 and March 2015. Candida spp. showed different peculiarities in terms of expression of virulence factors evaluated in vitro. C. albicans strains were more adherent to HBEC than all the other Candida species. C. tropicalis strains were considered strong biofilm producers. Strains belonging to the C. parapsilosis species complex were able to produce hemolysins, while C. glabrata was also able to lyse erythrocytes and to produce phospholipase. CONCLUSION: These results suggest that Non-Candida albicans Candida species are also able to express virulence factors which play an important role in bloodstream infectious caused by these yeasts.

Effect of selenium on growth and antioxidative system of yeast cells.

Selenium exhibits health-promoting properties in humans and animals. Therefore, the development of selenium-enriched dietary supplements has been growing worldwide. However, it may also exhibit toxicity at higher concentrations, causing increased oxidative stress. Different species of yeasts may exhibit different tolerances toward selenium. Therefore, in this study, we aimed to determine the effect of selenium on growth and on the antioxidative system in Candida utilis ATCC 9950 and Saccharomyces cerevisiae ATCC MYA-2200 yeast cells. The results of this study have demonstrated that high doses of selenium causes oxidative stress in yeasts, thereby increasing the process of lipid peroxidation. In addition, we obtained an increased level of GSSG from aqueous solutions of yeast biomass grown with selenium supplementation (40-60 mg/L). Increased levels of selenium in aqueous solutions resulted in an increase in the activity of antioxidant enzymes, including glutathione peroxidase and glutathione reductase. These results should encourage future research on the possibility of a thorough understanding of antioxidant system functioning in yeast cells.

Proteolytic activity of non-albicans Candida and Candida albicans in oral cancer patients.

Oral Candida infections can be life-threatening in medically compromised patients. In particular non-albicans Candida strains are virulent. However, our knowledge is sparse on how proteolytic these strains are in patients with oral cancer. Our study aimed to investigate differences in proteolytic activity of non-albicans Candida and Candida albicans isolated from oral cancer patients. The hypothesis was based on anticipated different invasive capacity of the strains. Clinical and reference yeast samples from our laboratory were used for analyses. Candida strains were grown in yeast peptone glucose and the activity of Candida proteinases of broken cell fractions were analysed by MDPF-gelatin zymography. Fluorometric assay was used to compare activities of proteolytic enzymes and degradation assays were performed using CLDN 4 and plasma fibronectin. Clear differences were seen in the proteolytic activity between the studied non-albicans Candida and C. albicans strains. C. tropicalis had the highest proteolytic activity followed by strains of C. krusei and C. glabrata. The results confirmed our study hypothesis by showing differences between the non-albicans Candida and Candida albicans strains studied. Higher proteolytic activity may thus have an effect on the virulence of non-albicans Candida strains in oral cancer patients.