Production and Stability of Protease from <Emphasis Type="Italic">Candida buinensis</Emphasis>

Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations carried out at variable temperature, pH, and soybean flour concentration, according to a 2³ full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL) when cultivated at 35 °C, pH 6.5, and using soybean flour concentrations in the range 0.1–0.5% (w/v). The cell-free supernatants showed the highest activity at 25 °C and pH 7.0, and satisfactory stability in the ranges 25–30 °C and pH 7–9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; ΔH* = 37.3 kJ/mol; ΔS* = −197.5 J/mol K; ΔG* = 101 kJ/mol).     

Xylanase Production by <Emphasis Type="Italic">Penicillium canescens</Emphasis> on Soya Oil Cake in Solid-State Fermentation

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na₂HPO₄.2H₂O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895 ± 778 U/g (Erlenmeyer flasks) and 9,300 ± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.     

Production of Mycophenolic Acid by <Emphasis Type="Italic">Penicillium brevicompactum</Emphasis> Using Solid State Fermentation

Solid-state fermentation using the microfungus Penicillium brevicompactum for the production of mycophenolic acid is reported in this paper. Of the initial substrates tested (whole wheat, cracked wheat, long grain Basmati rice, and short grain Parmal rice), Parmal rice proved to be the best. Under initial conditions, using steamed Parmal rice with 80% (w/w) initial moisture content, a maximum mycophenolic acid concentration of 3.4 g/kg substrate was achieved in 12 days of fermentation at 25 °C. The above substrate was supplemented with the following additional nutrients (g/L packed substrate): glucose 40.0, peptone 54.0, KH₂PO₄ 8.0, MgSO4?7H₂O 2.0, glycine 7.0, and methionine 1.65 (initial pH 5.0). A small amount of a specified trace element solution was also added. The final mycophenolic acid concentration was increased to nearly 4 g/kg substrate by replacing glucose with molasses. Replacing Parmal rice with rice bran as substrate further improved the mycophenolic acid production to nearly 4.5 g/kg substrate.     

Production of 6-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="SmallCaps">l</Emphasis>-Ascorbyl Palmitate by Immobilized <Emphasis Type="Italic">Candida antarctica</Emphasis> Lipase B

l-ascorbyl palmitate (ASP) is an oil-soluble derivative of ascorbic acid which is used extensively in food, cosmetics industry, and medical hygiene. Enzymatic synthesis of ascorbyl palmitate in tert-butyl alcohol was carried out using indigenously immobilized lipase preparation PyCal with ascorbic acid and palmitic acid as starting material. The developed batch process under optimized reaction conditions resulted in conversion of 90% with relatively shorter reaction time of 6 h. Continuous process in packed bed reactor gave conversion of 50% with space time yield of 15.46 g/L/h which was found to be higher than the reported literature on enzymatic synthesis of ascorbyl palmitate. The immobilized lipase used in the present work showed good reusability. Characterization of formed ascorbyl palmitate was carried out by FTIR, MS/MS, H¹-NMR, and C¹³-NMR. The enzymatic process resulted in selective synthesis of 6-O-l-ascorbyl palmitate with purity of 98.6% and no side product formation. The use of underivatized starting materials, high space time yield of 15.46 g L^?1 h^?1, high recyclability of catalyst, and no by-product formation make the overall process highly efficient and clean in terms of energy consumption and waste generation, respectively. The optimized reaction parameters for ascorbyl palmitate synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of ascorbic acid by enzymatic route.     

Enzyme Production by Solid Substrate Fermentation of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Trametes versicolor</Emphasis> on Tomato Pomace

A process of solid state fermentation (SSF) on tomato pomace was developed with the white-rot fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support. Operative parameters (humidity, water activity, and size of substrate particles) guaranteeing a good colonization of tomato pomace by both fungi were defined and conditions for production at high titers of the industrially relevant enzymes laccase, xylanase and protease were identified. Significant laccase activity levels (up to 36 U g⁻¹ dry matter) were achieved without any optimization of culture conditions, neither by nutrient addition nor by O₂ enrichment. Furthermore, protease activity levels up to 34,000 U g⁻¹ dry matter were achieved, being higher than those reported for the fungi typically considered as the best protease producers such as Aspergillus strains. Moreover, as one of the most significant results of this study, analysis of P. ostreatus tomato SSF samples by zymogram revealed two bands with laccase activity which had not been detected so far.     

Research on the Solid State Fermentation of Jerusalem Artichoke Pomace for Producing <Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">R</Emphasis>-2,3-Butanediol by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> ZJ-9

R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90 g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Transesterification of Waste Frying Oil and Soybean Oil by <Emphasis Type="Italic">Combi</Emphasis>-<Emphasis Type="Italic">lipases</Emphasis> Under Ultrasound-Assisted Reactions

This work describes the use of an ultrasound system for the enzymatic transesterification of oils using combi-lipases as biocatalyst. The reactions were carried out evaluating the individual use of waste oil and fresh soybean oil, and the immobilized lipases CALB, TLL, and RML were used as biocatalysts. It was performed in a mixture design of three factors to obtain the ideal mixture of lipases according to the composition of fatty acids present in each oil, and the main reaction variables were optimized. After 18 h of reaction, ultrasound provided a biodiesel yield of about 90% when using soybean oil and 70% using the waste oil. The results showed that ultrasound technology, in combination with the application of enzyme mixtures, known as combi-lipases, and the use of waste oil, could be a promising route to reduce the overall process costs of enzymatic production of biodiesel.     

Copolymer of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride with maleic acid as drug carrier

A copolymer of N,N-diallyl-N,N-dimethylammonium chloride with maleic acid of constant composition was prepared under the conditions of radical initiation. The possibility of the functionalization of the copolymer with drugs containing amino groups by polymer-analogous transformations was examined. Conditions were found for preparing conjugates of the copolymer with isoniazid. The structures and the quantitative compositions of the conjugates were determined by ¹³С NMR spectroscopy, and the possibility of preparing conjugates with controlled drug content was demonstrated.     

Variable Volume Fed-Batch Fermentation for Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp<Emphasis Type="Italic">. lactis W28</Emphasis>

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l⁻¹) with 100 ml sucrose solution (190 g l⁻¹) being evenly fed (9–10 ml h⁻¹) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l⁻¹ in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l⁻¹, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

Conformations and properties of <Emphasis Type="Italic">O</Emphasis>-Alkyl-<Emphasis Type="Italic">S</Emphasis>-(2-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dialkylamino)-ethylmethylthiophosphonates

Conformers of the biologically active compounds CH₃P(O)(OR)(SCH₂CH₂NR ₂ ^′ ), where (I) R = i-C₄H₉, R′ = C₂H₅ and (II) R = C₂H₅, R′ = i-C₃H₇, are calculated within the AM1 level of theory. The elongated and twisted forms with maximum and minimum distances between a nitrogen atom and those of a phosphorus tetrahedron, respectively, and bearing a syn and anti oriented alkoxy group relative to a phosphoryl oxygen, are studied. It is found that the differences between the energy, electronic, and geometric parameters of these forms are apparent in differences between their properties, e.g., the ability to participate in complexation and protonation, reactions that to some extent simulate the interaction between a substance and a biological object.     

Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> nicotinate mononucleotide adenylyltransferase mutants <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD⁺ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD⁺, especially for one of the alleles, nadD72.     

Molecular structure of <Emphasis Type="Italic">N</Emphasis>-(<Emphasis Type="Italic">o</Emphasis>-and <Emphasis Type="Italic">p</Emphasis>-hydroxybenzyl)cytisine

The molecular structures of N-(o-and p-hydroxybenzyl)cytisine were investigated by NMR spectroscopy, x-ray structure analysis, and molecular modeling. It was found that NMR resonances of the OH and aromatic protons in N-(o-hydroxybenzyl)cytisine were doubled because of the presence of two conformers in solution. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 165–168, March–April, 2008.     

Synthesis of 6-methyl-8<Emphasis Type="Italic">H</Emphasis>-dibenzo[<Emphasis Type="Italic">a</Emphasis>,<Emphasis Type="Italic">g</Emphasis>]quinolizin-8-imines <Emphasis Type="Italic">via Reissert</Emphasis> compounds

Alkylation of Reissert compounds derived from 3-methylisoquinolines with several 2-cyanobenzylbromides followed by hydrolytic cleavage provided the corresponding 1-benzyl-3-methylisoquinolines. Treatment of the latter with methylmagnesiumiodide caused cyclization to the title compounds rather than formation of 2-acetylbenzylisoquinolines.     

Production,Purification and Characterisation of a Potential Fibrinolytic Protease from Endophytic <Emphasis Type="Italic">Xylaria curta</Emphasis> by Solid Substrate Fermentation

The present investigation highlights the optimal conditions for production of a non-toxic, bi-functional fibrinolytic enzyme xylarinase produced by endophytic fungus Xylaria curta by solid substrate fermentation using rice chaff medium. The purified enzyme is a monomeric protein with a molecular mass of ～33 kDa. The enzyme exhibits cleavage of Aα and Bβ chains of fibrin(ogen) and has no effect on γ chain. The optimal fibrinolytic activity of the enzyme was observed at 35 °C and pH 8. The fibrinolytic activity was enhanced in the presence of Ca²⁺, whereas it was completely inhibited in the presence of Fe²⁺ and Zn²⁺ ions and inhibitors like EDTA and EGTA suggesting it to be a metalloprotease. The K _m and V _max of the enzyme for azocasein were 326 μM and 0.13 μM min^?1. The N-terminal sequence of the enzyme (SNGPLPGGVVWAG) was same when compared to xylarinase isolated from culture broth of X. curta. Thus, xylarinase could be exploited as a potent clot busting enzyme which could be produced on large scale using solid substrate fermentation.     

Synthesis of <Emphasis Type="Italic">N,N</Emphasis>-disubstituted (<Emphasis Type="Italic">Z</Emphasis>)-<Emphasis Type="Italic">O</Emphasis>-methylnicotinamide oximes

A number of (Z)-N,N-dialkyl- and (Z)-N-acyl-N-alkyl-O-methylnicotinamide oximes was synthesized. Their configuration was confirmed by the NOESY experiment. Evaluation of fungicidal activity of compounds obtained was performed.