Isolation and Characterization of Fructokinase from Pseudomonas sp. KN-21

A screening test was done to isolate microorganisms producing a highly specific fructokinase. A specific phosphorylative activity toward fructose was found in a cell homogenate of a bacterium, KN-21, newly isolated from a soil sample. The enzyme was isolated from the sonicated cells as a homogeneous preparation. The purified enzyme was composed of two identical subunits (37 kDa). The K_ms for ATP and fructose were estimated to be 7.0 x 10^?4 M and 1.0 x 10^?3 M, respectively. The enzyme specifically phosphorylated fructose. The use of the enzyme as an analytical reagent was also investigated.     

Heparin-benzyl alcohol enhancement of biofilms formation and antifungal susceptibility of vaginal Candida species isolated from pregnant and nonpregnant Saudi women

Biofilm formation by Candida species is a major contribute to their pathogenic potential.The aim of this study was to determine in vitro effects of EDTA, cycloheximide, and heparin-benzyl alcohol preservative on C. albicans (126) and non-albicans (31)vaginal yeast isolates biofilm formations and their susceptibility against three antifungal Etest strips. Results of the crystal violet-assay, indicated that biofilms formation were most commonly observed [100%] for C. kefyr, C. utilis, C. famata, and Rhodotorula mucilaginosa, followed by C. glabrata [70%], C. tropicalis [50%], C. albicans [29%], Saccharomyces cerevisiae [0.0%]. EDTA (0.3mg/ml) significantly inhibited biofilm formation in both C. albicans and non-albicans isolates (P=0.0001) presumably due to chelation of necessary metal cations for the process-completion. In contrast, heparin (-benzyl alcohol preservative) stimulated biofilm formation in all tested isolates, but not at significant level (P=0.567). Conversely, cycloheximide significantly (P=0.0001) inhibited biofilm formation in all C. albicans strains(126) and its effect was even 3 fold more pronounced than EDTA inhibition, probably due to its attenuation of proteins (enzymes) and/or complex molecules necessary for biofilm formation. Results also showed that all nonalbicans yeasts isolates were susceptible to 5-flucytosine (MIC₅₀, 0.016 µg/ml; MIC₉₀, 0.064 µg/ml), but 14% of C. albicans isolates were resistant (MIC₅₀, 0.064 µg/ml; MIC₉₀ >32 µg/ml). The MIC₅₀ value of amphotricin B for all C. albicans and non-albicans isolates was at a narrow range of 0.023 µg /ml, and the MIC₉₀ values were 0.047 µg/ml and 0.064 µg/ml respectively, thereby confirming its efficacy as a first line empiric- treatment of Candida spp infections.     

广州地区生殖系统念珠菌感染的菌型及其体外药敏分析

【摘 要】 目的 了解性传播疾病病人中生殖系统念珠菌感染的菌型及其对临床常用抗真菌药物的敏感性。 方法 采用法国梅里埃公司的ATB ID32C酵母菌鉴定反应板鉴定菌种并使用Rosco纸片扩散法进行体外药敏检测,比较不同菌型念珠菌的药敏情况。 结果 临床131例生殖系统念珠菌感染者中,白色念珠菌116例（88.55%）、近平滑念珠菌7例（5.34%）、光滑念珠菌5例（3.82%）、季也蒙念珠菌2例（1.53%）、克柔念珠菌1例（0.76%）。药敏分析结果：念珠菌属对制霉菌素（96.18%）、酮康唑（90.84%）、氟康唑（70.23%）敏感,而对咪康唑（31.30%）、嗌康唑（31.30%）、伊曲康唑（47.33%）和特比奈酚（7.63%）的中度敏感性和耐药性比较高。 结论 生殖系统念珠菌感染以白色念珠菌为主,感染的念珠菌除了对多烯类药物敏感率高外,对唑类抗真菌药有不同程度的交叉耐药,不同菌型其药敏谱存在差异。     

A Study on the Process of Lipase-catalyzed Synthesis of α-pinene Oxide in Organic Solvents

The synthesis of α-pinene oxide was studied in a three-phase system where immobilized Candida antarctica lipase B (Novozyme 435) was used to catalyze the formation of peroxyoctanoic acid from the parent carboxylic acid and hydrogen peroxide in toluene. The peroxycarboxylic acid formed was then used in situ for the oxidation of α-pinene to the corresponding epoxide. When hydrogen peroxide was added in the reaction mixture gradually over 6 h, conversions increased up to 31.6%. Initial rates of α-pinene oxidation increased from 85 to 708 mmol L^?1 h^?1 when the amount of H₂O₂ increased from 5 to 60 mmol. When the lipase was exposed to 75 mmol H₂O₂ for 0.5 h before its addition in the reaction mixture, its activity decreased to about 50%. The reusability of lipase was studied in five reaction cycles and was found to depend on the concentration of the hydrogen peroxide used.     

An improved regioselective preparation of methyl 2,3-di-O-acetyl-α,β-d-xylofuranoside

Abstract

Methyl 2,3-di-O-acetyl-α,β-d-xylofuranoside was prepared as the sole regioisomer in 63–72% yield, according to the applied mass of substrate, through a Candida antarctica lipase B catalysed alcoholysis of methyl 2,3,5-tri-O-acetyl-α,β-d-xylofuranoside. The product is a potential synthetic precursor for 5-modified xylofuranosides and 5′-modified xylonucleosides.     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

Effects of Alcohol and Buffer Treatments on the Activity and Enantioselectivity of Candida rugosa Lipase

Abstract

Several treatments were employed on Candida rugosa lipase (CRL) to improve its biocatalytic performance. Besides conventional alcohol treatment conditions, the effects of pH of the buffer solution used in the treatment as well as the changes in stirring, dialysis, and centrifugation steps of the treatment procedure were investigated for the first time for the resolution of racemic naproxen methyl ester. The highest enantioselectivity and conversion in S-naproxen production were achieved by CRL treated with pH 7.5 buffer solution. The elimination of the centrifugation step resulted in an increase in the enantioselectivity, whereas alcohol treatment of CRL was found to be inconvenient for S-naproxen production. Higher activity for p-nitrophenyl acetate was achieved when 20% butanol and pH 4 buffer solution were used, and when dialysis and stirring times were shortened.     

EXPLORING THE OPTIMUM CONDITIONS FOR MAXIMIZING THE MICROBIAL GROWTH OF Candida intermedia BY RESPONSE SURFACE METHODOLOGY

Exploring optimum and cost-efficient medium composition for microbial growth of Candida intermedia Y–1981 yeast culture growing on whey was studied by applying a multistep response surface methodology. In the first step, Plackett–Burman (PB) design was utilized to determine the most significant fermentation medium factors on microbial growth. The medium temperature, sodium chloride and lactose concentrations were determined as the most important factors. Subsequently, the optimum combinations of the selected factors were explored by steepest ascent (SA) and central composite design (CCD). The optimum values for lactose and sodium chloride concentrations and medium temperature were found to be 18.4 g/L, 0.161 g/L, and 32.4°C, respectively. Experiments carried out at the optimum conditions revealed a maximum specific growth rate of 0.090 1/hr; 42% of total lactose removal was achieved in 24 h of fermentation time. The obtained results were finally verified with batch reactor experiments carried out under the optimum conditions evaluated.     

Increased susceptibility to Candida infection following cecal ligation and puncture

Secondary infection following septic insult represents a significant cause of morbidity and mortality in hospitalized patients. Sepsis induced immunosuppression is a major factor in the host’s susceptibility to nosocomial infections and Candida albicans accounts for a growing number of these. Given the importance of improving our understanding of the immune response to sepsis and the increasing rates of C. albicans infections, we sought to develop a murine model of double injury consisting of primary peritonitis, i.e., cecal ligation and puncture (CLP), followed by a secondary challenge of C. albicans. As observed in previous work, after primary injury the immune profile of the host changes over time. Therefore, while keeping the mortality rates from the respective individual injuries low, we altered the timing of the secondary injury between two post-CLP time points, day two and day four. Mice subjected to C. albicans infection following CLP have significantly different survival rates dependent upon timing of secondary injury. Animals challenged with C. albicans at two days post CLP had 91% mortality whereas animals challenged at four days had 47% mortality. This improvement in survival at four days was associated with restoration of innate cell populations and as evidenced by stimulated splenocytes, increases in certain inflammatory cytokines. In addition, we show that susceptibility to C. albicans infection following CLP is dependent upon the depth of immunosuppression. Although at four days post-CLP there is a partial reconstitution of the immune system, these animals remain more susceptible to infection compared to their single injury (C. albicans alone) counterparts. Collectively, these studies demonstrate that immunosuppression following initial septic insult changes over time. This novel, two hit model of CLP followed by Candida provides additional insight into the immune compromised state created by primary peritonitis, and thereby opens up another avenue of investigation into the causes and possible cures of an emerging clinical problem.