Diplogelasinospora grovesii IMI 171018 immobilized in polyurethane foam. An efficient biocatalyst for stereoselective reduction of ketones.

Diplogelasinospora grovesii has been reported as a very active biocatalyst in the reduction of ketones. Along the text, the properties of this filamentous fungus as an immobilized catalyst are described. For this purpose, several immobilization supports as agar and polyurethane foam were tested. Experimental assays were also performed to test different co-substrates for the regeneration of the required enzyme cofactor. The fungus immobilized in polyurethane foam lead to the most stable and active catalyst. This derivative, using i-PrOH as co-substrate, could be reused at least 18 times without appreciable activity loss (>90% activity remains). Kinetic runs experiments shown that the reduction of cyclohexanone, selected as model substrate, followed a pseudo-first kinetic order and that the rate controlling step was the mass transfer through the cell wall. The deactivation kinetic constants were also determined. The reduction of different chiral ketones showed that the ketone reductase activity followed the Prelog's rule.

Microbial cells as catalysts for stereoselective red-ox reactions.

Enzyme catalyzed reactions are commonly used at laboratory or industrial scale. Contrarily, the whole cell catalyzed reactions are restricted to special cases. The tremendous advances in the last years in Molecular Biology and more specifically in Metabolic Engineering and Directed Enzyme Evolution have opened the door to create tailor-made microorganisms or "designer bugs" for industrial purposes. Whole cell catalysts can be much more readily and inexpensively prepared than purified enzymes and the enzymes - inside the cells - are protected from the external environment and stabilized by the intracellular medium. Three situations have traditionally been considered convenient to select the use of whole cell catalyzed processes against the free enzyme catalyzed process: i) when the enzyme is intracellular; ii) when the enzyme needs a cofactor to carry out the catalytic act and iii) in the development of multienzymatic processes. Red-ox reactions represent the molecular basis for energy generation in the cell. These reactions are catalyzed by intracellular enzymes and are cofactor dependent as red-ox reactions need electron carriers as helpers in reduction reactions (gain of electrons) or oxidation (loss of electrons). In this review we present an overview of the state of the art of red-ox biotransformations catalyzed by whole cells - wild-type or genetically engineered microorganisms. Stereoselective reductions, hydroxylations of arenes and unfunctionalized alkanes, alkene monooxygenation, and Baeyer-Villiger reactions are among the processes described along the text, focusing in their chemo-, regio- and stereoselectivity.

Monascus kaoliang CBS 302.78 immobilized in polyurethane foam using iso-propanol as co-substrate: Optimized immobilization conditions of a fungus as biocatalyst for the reduction of ketones.

Monascus kaoliang was selected after a microbial screening as a highly active and selective whole cell catalyst for the reduction of ketones. In the present paper we describe the optimum growing conditions and an interesting immobilization procedure by adsorption in polyurethane foams (PUFs). This methodology is easy to perform and the immobilized catalyst is active, stable and reusable. The use of different co-substrates for cofactor regeneration was also tested and iso-propanol (i-PrOH) was found as the best co-substrate, as it leads to a catalyst reusable for 17 cycles, displaying better NADH regeneration properties than others e.g., glucose (10 cycles) or saccharose (6 cycles). The reduction of different prochiral ketones showed that the ketone reductase activity of this mould follows the Prelog's rule and kinetic experiments demonstrated that the process follows a pseudo-first kinetic order.

Esterification reactions catalyzed by lipases immobilized in organogels: effect of temperature and substrate diffusion.

Rhizomucor miehei lipase was immobilized in hydroxy(propylmethyl) cellulose or agar gels containing lecithin or AOT microemulsions. The effect of the diffusion of substrates and products to this catalyst was studied, as well as the effect of temperature on the initial rate of ester synthesis. The composition of the gel affects the reaction rate due to mass transport phenomena. The apparent activation energies were higher for the systems based on agar, independently of the microemulsion used, and lower for the systems based on AOT microemulsions, independently of the polymer used.

Low-temperature synthesis of 2'-deoxyadenosine using immobilized psychrotrophic microorganisms.

Selective biocatalyzed synthesis of 2'-deoxyadenosine from 2'-deoxypyrimidine nucleosides was carried out using free or immobilized whole cells. The reaction was performed at 57 degrees C without secondary reactions. Two psychrotrophic microorganisms, Bacillus psychrosaccharolyticus and Psychrobacter immobilis, are described for the first time as active and specific strains for the synthesis of 2'-deoxyadenosine. Adenosine deaminase activity was not detected. Whole cells were immobilized in different matrixes. Calcium alginate and calcium pectate gave the best biocatalysts. The synthesis of 2'-deoxyadenosine follows an apparent first order kinetic expression. External mass transfer control was negligible as deduced from k(s), N(A), and Omega values. Internal mass transfer was the rate controlling step according to eta(T) and phi values.

Unexpected reaction profile observed in the synthesis of propyl laurate when using Candida rugosa lipases immobilized in microemulsions based organogels.

1-Propyl laurate synthesis should not be used as standard reaction test of immobilized enzymes in microemulsion-based organogels (MBGs) prepared using lecithin/1-propanol as surfactant when extremely active enzymes with high load are used. In these cases, an anomalous kinetic reaction constant value is observed over short reaction times. Such an anomalous profile is strongly dependent on the concentration of catalyst in the crude powder and, consequently, is not appreciated when either commercial or low activity lipase samples are employed.

High throughput screening and QSAR-3D/CoMFA: useful tools to design predictive models of substrate specificity for biocatalysts.

After a hierarchical microbial screening process, new microorganisms have been discovered that act as biocatalysts for the stereoselective oxidation of secondary alcohols or for ketone reduction. Oxidation activity is more widespread in yeasts and bacteria, while actinomycetes, filamentous fungi and yeasts present the highest reduction activities. QSAR-3D/CoMFA is an adequate technique to design predictive models of the biocatalysts' activity. In this paper CoMFA models are designed to compare the activities of the biocatalysts selected for the oxidation of alcohols and for the reduction of ketones, starting from the results obtained during the screening process. These models are useful for learning about the activity of these microorganisms and to compare the substrate specificity requirements between alcohol oxidation and ketone reduction biocatalysts.

Stereoselective effects of the enantiomers of a new local anaesthetic, IQB-9302, on a human cardiac potassium channel (Kv1.5).

1. The N-substituent of IQB-9302 has the same number of carbons as bupivacaine, but it exhibits a different spatial localization (n-butyl vs cyclopropylmethyl). Thus, the study of the effects of IQB-9302 enantiomers on hKv1.5 channels will lead to a better knowledge of the determinants of stereoselective block. 2. The effects of the IQB-9302 enantiomers were studied on hKv1.5 channels stably expressed in LTK:(-) cells using the whole-cell configuration of the patch-clamp technique. Drug molecular modelling was performed using Hyperchem software. 3. Block induced by IQB-9302 was stereoselective with the R(+) enantiomer being 3.2-fold more potent than the S(-) one (K(D) of 17.8+/-0.5 microM vs 58.6+/-4.0 microM). 4. S(-)- and R(+)IQB-9302 induced-block was time- and voltage-dependent consistent with an electrical distance from the cytoplasmic side of 0.173+/-0.022 (n=12) and 0.181+/-0.018 (n=10), respectively. 5. Potency of block of pipecoloxylidide local anaesthetics was linearly related to the length between the cationic tertiary amine and the end of the substituent. 6. Molecular modelling shows that only when S(-) and R(+) enantiomers are superimposed by their aromatic ring, their N-substituents are in opposite directions, which can explain the stereospecific block induced by bupivacaine and IQB-9302 with hKv1.5 channels. 7. These results suggest that: (a) IQB-9302 enantiomers block the open state of hKv1.5 channels, and (b) the length of the N-substituent in these local anaesthetics and not its volume determines the potency and degree of their stereoselective hKv1.5 channel block.

Novel microbial lipases: catalytic activity in reactions in organic media.

2,000 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Culture broths were initially tested with 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufinyl ester, a chromogenic substrate specific for lipases. Fourteen lipase-producing microorganisms were selected and their taxonomic identification was carried out. Hydrolysis of tributyrin or olive oil and the esterification of oleic acid with heptanol were selected to preliminary evaluate the catalytic activity of these lipases. All the selected lipases catalysed this esterification reaction with good yields. Resolution of (R,S)-2-(4-isobutylphenyl) propionic acid, (R,S)-1-phenylethanol, (R,S) 1-phenylethylamine and of (R) or (S) glycidol were performed to evaluate the stereoselectivity of these novel enzymes as biocatalysts in reactions in organic media. Lipases from the fungi Fusarium oxysporum and Ovadendron sulphureo-ochraceum gave the best yields and enantioselectivities in the resolution of racemic ibuprofen and 1-phenylethanol. Several lipases displayed a high stereoselectivity in the resolution of chiral amines by an alcoxycarbonylation reaction.

Effect of fermentation conditions in the enzymatic activity and stereoselectivity of crude lipase from Candida rugosa.

Different fed-batch cultures of Candida rugosa were carried out using oleic acid as the only carbon source. The crude lipases obtained under several operational conditions and downstream processes showed different catalytic activity and isoenzymes ratio. This fact implied that the performance of the lipase produced could be modulated by using different operational fermentation conditions. These powders were compared with commercial lipase from Sigma (St. Louis, MO) in hydrolysis and synthesis reactions. Especially interesting was the fact that the enantioselectivity of a crude lipase was higher than that observed with commercial lipase in the resolution of racemic Ketoprofen. In addition, response of both lipases in the presence of water was different.

Influence of the nature of modifier in the enzymatic activity of chemical modified semipurified lipase from Candida rugosa.

Semipurified lipase of Candida rugosa (CRSL) was subjected to chemical modification, and the activities of the modified lipase, in hydrolysis and esterification reactions, were examined. The esterification reactions were carried out in the absence and presence of isooctane. When the enzyme was modified with polyethylene glycol (PEG), two methodologies were studied. The activation of PEG with p-NO(2)-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG. The chemical modification with PEG increases the stability of pure lipases in isooctane at 50 degrees C (extreme conditions). The chemically modified enzymes are useful for biotransformations in organic solvents. In addition the nitration of tyrosines with tetranitromethane was also studied. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 252-260, 1997.

Contribution to the study of the alteration of lipase activity of Candida rugosa by ions and buffers.

A semipurified C. rugosa lipase (LS) has been prepared from commercial lipase (LC) using an economical procedure. The presence of sugars and glycopeptides has been detected in LS and LC. Pure lipase only has covalently bonded sugars. The hydrolysis of olive oil catalyzed by LS and commercial lipase (LC) is sensitive to the presence of cations Na(I), Mg(II), Ca(II), and Ba(II) and to the nature of buffer. Highest enzyme activity is obtained with 0.1M Tris/HCl buffers and the combination of NaCl 0.11M and CaCl2 0.11M. Fluorescence spectroscopy analysis of LC, LS, and both pure isoenzymes lipases A and B, was used to analyze the interaction of the lipase with these effectors. Inorganic cations Na or Ca do not interact with pure enzyme LA but do interact with LC and LS and do so slightly with LB. The organic cations (morfolinium or tris) interact with pure lipases. We postulate that the increase in the lipase activity produced by Na(I) or Ca(II) is related with interfacial phenomena, but the increase might be more specific in the hydrolysis of olive oil in the presence of Tris-HCl or morfoline-HCl buffer, owing to enzyme-buffer interaction.

Application of ultrasound to biotechnology: an overview.

Application of ultrasound to biotechnology is relatively new, but several processes that take place in the presence of cells or enzymes are activated by ultrasonic waves. High intensity ultrasonic waves break the cells and denaturize the enzymes. Low intensity ultrasonic waves can modify cellular metabolism or improve the mass transfer of reagents and products through the boundary layer or through the cellular wall and membrane. In the case of enzymes, the increase in the mass transfer rate of the reagents to the active site seems to be the most important factor. Immobilized enzymes are more resistant to thermal deactivation produced by ultrasound than native enzymes. Reverse micelles can be used to carry out synthesis using enzymes. Several applications of ultrasound to the biotechnology are discussed.

Hydrolysis of nucleic acids in single-cell protein concentrates using immobilized benzonase.

Hydrolysis of nucleic acids for single-cell protein concentrates has been carried out in one step using immobilized benzonase on corn cob. The immobilization is carried out by tosylation of primary alcohols of cellulose of corn cob. The immobilized benzonase is more stable vs pH changes than native benzonase, but the same optimum values of [Mg(II)] and temperature are obtained. The DNase activity is greater than the RNase activity. The percentage of DNA is reduced to 3-6% and that of RNA to 50%. The protein loss is negligible (1%). The enzymatic activity per weight unit of enzyme is greater in the case of benzonase that in reported data for other nucleases insolubilized on corn cob by the same procedure.

New insolubilized derivatives of ribonuclease and endonuclease for elimination of nucleic acids in single cell protein concentrates.

Two derivatives of pancreatic ribonuclease and endonuclease of Staphylococcus aureus, insolubilized on corn cob, have been used to reduce the percentage of nucleic acids in single cell protein (SCP) concentrates from yeasts. These derivatives are thermostable and active at 45 degrees C. At these temperatures the contamination by bacteria is negligible. The thermostability is remarkable, since the native nuclease is deactivated at above 39 degrees C. The hydrolysis of the nucleic acids in SCP is carried out first with the ribonuclease derivative followed by the endonuclease derivative. The catalytic activity of the insolubilized derivatives is similar to that of the native enzymes in the hydrolysis of RNA but not of DNA. The percentage of nucleic acids is reduced from 5-15 to 0.5%, with a loss of protein of 6%. These percentages are lower than those previously reported.

DNase activity of micrococcal endonuclease insolubilized on corn cob.

The endonuclease from S. aureus has been immobilized on ground maize cob, previously activated with tosyl chloride. Pretreatment of the support on acid before tosylation yielded the best insoluble enzyme derivatives. The catalytic activity has been evaluated as percent of total hydrolysis attained in a batch reactor using DNA as a model substrate. The derivatives prepared are very resistant to high temperatures under conditions of catalysis (24 h at 45 degrees C). For these long reaction times, the extent of hydrolysis in the presence of small amounts of organic solvent (dimethyl sulfoxide at 2 percent) is larger than in plain buffer (Tris). This type of derivative could be very useful for the removal of nucleic acids from single-cell protein concentrates.