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This work describes an efficient, simple, and green bioprocess for obtaining 5-halogenated pyrimidine nucleosides from thymidine by transglycosylation using whole cells. Biosynthesis of 5-fluoro-2'-deoxyuridine (floxuridine) was achieved by free and immobilized Aeromonas salmonicida ATCC 27013 with an 80% and 65% conversion occurring in 1 h, respectively. The immobilized biocatalyst was stable for more than 4 months in storage conditions (4 °C) and could be reused at least 30 times without loss of its activity. This microorganism was able to biosynthesize 2.0 mg L(-1) min(-1) (60%) of 5-chloro-2'-deoxyuridine in 3 h. These halogenated pyrimidine 2'-deoxynucleosides are used as antitumoral agents., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Different green synthesis of alkyl esters of acyclovir (acyclovir prodrugs) is described. Hexanoic, decanoic, dodecanoic and tetradecanoic acyclovir esters were synthesized reacting acyclovir and the respective acid anhydride in dimethyl sulfoxide (DMSO), in solvents from renewable sources and without solvent (T=30 °C). Yields in prodrugs after 10 min of reaction were >95% using DMSO as solvent. The purification methodology was very simple, shorter and greener than previously described. The biosolvent, N,N-dimethylamide of decanoic acid, let us to obtain >95% yield at 24 h. This oily biosolvent is not dermotoxic and the reaction crude can directly be used in topic formulations. Syntheses without solvent proceeded successfully for acyclovir esters. Indeed, dodecanoate and tetradecanoate yielding >98% conversion of reactants in 30 min. In spite of requiring mild temperature (65 °C), substrate molar ratios were lowered to 1 : 1, thus conducing to a more efficient use of raw materials. The synthetic procedures were scaled up to a 300 g batch (yield 98-99% isolated ester). These esters can be used as acyclovir prodrugs in topic formulations. The esters release from an oil/water micro-emulsion and a hydrogel formulation were tested with good results.

A practical methodology to obtain alpha-diazoketones through an improved Arndt-Eistert synthesis is described. The method allows the efficient transformation of acid halides using a stoichiometric amount of diazomethane in the presence of calcium oxide, without concomitant ketene or haloketone formation. The obtained alpha'-brominated-alpha-diazoketones were employed as suitable substrates for the synthesis of interesting alpha-arylamino-alpha'-halomethylketones.

The definite interest in implementing sustainable industrial technologies has impelled the use of biocatalysts (enzymes or cells), leading to high chemo-, regio- and stereoselectivities under mild conditions. As usual substrates are not soluble in water, the employ of organic solvents is mandatory. We will focus on different attempts to combine the valuable properties of green solvents with the advantages of using biocatalysts for developing cleaner synthetic processes.

Functionalized fluorescent glycans have the potential to act as tools to detect and analyze protein-carbohydrate interactions. We present here a facile strategy for immobilization of functionalized lactose as a model disaccharide. Bioactivity was tested with three members of the adhesion/growth-regulatory galectins family in different types of assay, i.e. matrix in surface plasmon resonance (SPR), free ligand in solution by STD/trNOESY and docking measurements. In all cases, the activity of the disaccharide was maintained. The attachment of this new fluorescent glycoconjugate to the surface results in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The results indicate that this new glycoconjugate exhibits binding affinity to galectin-1, 3 and CG-16. Kinetic analysis of the interaction between these galectins and immobilized glycoconjugate by SPR yielded a K(D) of 1.01 mM for galectin-1, 83.5 microM for galectin-3 and 0.28 mM for CG-16. No major contacts to the aglyconic part were detected, which might compromise the specificity of the binding process with other headgroups. Thus, testing these proteins offers the potential for medical applications to detect these endogenous effectors or further derivatives and characterize their carbohydrate specificity.

A novel type II nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% alpha-helix, 10% beta-strand, 16% beta-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T(m)) of 64 degrees C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40 degrees C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2'-fluorodeoxyribonucleosides was carried out.

The development of efficient syntheses for enantiomerically enriched alpha-hydroxy ketones is an important research focus in the pharmaceutical industry. For example, alpha-hydroxy ketones are found in antidepressants, in selective inhibitors of amyloid-beta protein production (used in the treatment of Alzheimer's), in farnesyl transferase inhibitors (Kurasoin A and B), and in antitumor antibiotics (Olivomycin A and Chromomycin A3). Moreover, alpha-hydroxy ketones are of particular value as fine chemicals because of their utility as building blocks for the production of larger molecules. They can also be used in preparing many other important structures, such as amino alcohols, diols, and so forth. Several purely chemical synthetic approaches have been proposed to afford these compounds, together with some organocatalytic strategies (thiazolium-based carboligations, proline alpha-hydroxylations, and so forth). However, many of these chemical approaches are not straightforward, lack selectivity, or are economically unattractive because of the large number of chemical steps required (usually combined with low enantioselectivities). In this Account, we describe three different biocatalytic approaches that have been developed to efficiently produce alpha-hydroxy ketones: (i) The use of thiamine diphosphate-dependent lyases (ThDP-lyases) to catalyze the umpolung carboligation of aldehydes. Enantiopure alpha-hydroxy ketones are formed from inexpensive aldehydes with this method. Some lyases with a broad substrate spectrum have been successfully characterized. Furthermore, the use of biphasic media with recombinant whole cells overexpressing lyases leads to productivities of approximately 80-100 g/L with high enantiomeric excesses (up to >99%). (ii) The use of hydrolases to produce alpha-hydroxy ketones by means of (in situ) dynamic kinetic resolutions (DKRs). Lipases are able to successfully resolve racemates, and many outstanding examples have been reported. However, this approach leads to a maximum theoretical yield of 50%. As a means of overcoming this problem, these traditional lipase-catalyzed kinetic resolutions are combined with racemization of remnant substrate, which can be done in situ or in separate compartments. Examples showing high conversions (>90%) and enantiomeric excesses (>99%) are described. (iii) Whole-cell redox processes, catalyzed by several microorganisms, either by means of free enzymes (applying a cofactor regeneration system) or by whole cells. Through the use of redox machineries, different strategies can lead to high yields and enantiomeric excesses. Some enantiopure alpha-hydroxy ketones can be formed by reductions of diketones and by selective oxidations of vicinal diols. Likewise, some redox processes involving sugar chemistry (involving alpha-hydroxy ketones) have been developed on the industrial scale. Finally, the redox whole-cell concept allows racemizations (and deracemizations) as well. These three strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can assist practical synthetic processes, and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and enantioselectivities.

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.

Suitable glycan derivatives will find immediate application in the study of their interactions. Here, we present an efficient synthetic strategy to introduce a fluorescent tag functionalized with an amino group into a model disaccharide structure (lactose). This strategy led to the maintenance of bioactivity, checked by the study of the interaction of this bioconjugate with a plant lectin (mistletoe lectin 1) by NMR spectroscopy, computational docking, and surface plasmon resonance (SPR). To demonstrate the versatility of this approach, we immobilized the new glycan derivatives on different surfaces, and a comparative analysis is presented and can be successfully used for biomimetic carbohydrate-protein interaction studies on the SPR biochip.

Several strains of Thermus thermophilus were tested in order to detect purine nucleoside synthase activity using pyrimidine nucleosides as the sugar-donor and adenine or hypoxanthine as bases. High productivity values (t =1 hr) were obtained while completely avoiding adenosine-deaminase degradation of the products. N-2-deoxy-ribosyltransferase activity is described for the first time in hyperthermophilic bacteria.

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.

The emerging role of glycans as versatile biochemical signals in diverse aspects of cellular sociology calls for establishment of sensitive methods to monitor carbohydrate recognition by receptors such as lectins. Most of these techniques involve the immobilization of one of the binding partners on a surface, e.g. atomic force microscopy, glycan array and Surface Plasmon Resonance (SPR), hereby simulating cell surface presentation. Here, we report the synthesis of fluorescent glycoconjugates, with a functionalization strategy which avoids the frequently occurring ring opening at the reducing end for further immobilization on a surface or derivatization with biotin. In order to improve the versatility of these derivatized glycans for biological studies, a new approach for the synthesis of biotinylated and fluorescent glycans has also been realized. Finally, to illustrate their usefulness the neoglycoconjugates were immobilized on different surfaces, and the interaction analysis with a model lectin, the toxin from mistletoe, proved them to act as potent ligands, underscoring the merit of the presented synthetic approach.

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.

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.

A mild and straightforward monoallylation procedure for different anilines is described using the efficient, inexpensive, noncorrosive, and environmentally friendly reagent KF-Celite. By using only a 1/1.2 stoichiometric ratio of electrophilic reagent to aniline, in very short reaction times, the monoallylated products are obtained in high isolated yields via this procedure, which works very effectively regardless of the electronic nature of the substituent on the ring, although electron withdrawing groups make the reactions go even faster.

The synthesis of some noncommercial racemic 1,2-diaryl-2-hydroxyethanones (benzoins) is described, optimizing the previously reported methodologies. In a further step, the kinetic resolution of these substrates is reported, obtaining conversions of around 50% and ee(p) higher than 99% in very short reaction times. As enzymatic catalyst, after screening of several enzymes, the lipase TL (from Pseudomonas stutzeri) was the most efficient, working in an organic solvent with a very low log P value, such as THF. Finally, the dynamic-kinetic resolution of different benzoins using a lipase-ruthenium-catalyzed transesterification in organic solvents is described for the first time, obtaining conversions up to 90% maintaining the excellent enantioselectivity in all cases.

In recent years, the Carica papaya lipase (CPL) is attracting more and more interest. This hydrolase, being tightly bonded to the water-insoluble fraction of crude papain, is thus considered as a "naturally immobilized" biocatalyst. To date, several CPL applications have already been described: (i) fats and oils modification, derived from the sn-3 selectivity of CPL as well as from its preference for short-chain fatty acids; (ii) esterification and inter-esterification reactions in organic media, accepting a wide range of acids and alcohols as substrates; (iii) more recently, the asymmetric resolution of different non-steroidal anti-inflammatory drugs (NSAIDs), 2-(chlorophenoxy)propionic acids, and non-natural amino acids. Taking into account the novelty and the current interest of the topic, this review aims to highlight the origin, features, and applications of the C. papaya lipase, with the objective to prompt research groups to further investigate the spectra of applications that this emerging and versatile CPL could have in the future.

Candida rugosa lipase (CRL) is one of the enzymes most frequently used in biotransformations. However, there are some irreproducibility problems inherent to this biocatalyst, attributed either to differences in lipase loading and isoenzymatic profile or to other medium-engineering effects (temperature, a(w), choice of solvent, etc.). In addition, some other properties (influence of substrate and reaction conditions on the lid movement, differences in the glycosylation degree, post-translational modifications) should not be ruled out. In the present paper the recent developments published in the CRL field are overviewed, focusing on: (a) comparison of structural and biochemical data among isoenzymes (Lip1-Lip5), and their influence in the biocatalytical performance; (b) developments in fermentation technology to achieve new crude C. rugosa lipases; (c) biocatalytical reactivity of each isoenzyme, and methods for characterising them in crude CRL; (d) state-of-the-art of new applications performed with recombinant CRLs, both in CRL-second generation (wild-type recombinant enzymes), as well as in CRL-third generation, (mutants of the wt-CRL).

Candida rugosa was cultured using different inducers (oleic acid, olive oil, sunflower oil, n-dodecanol and glycerol) as the only carbon source in batch conditions, as well as in several fed-batch fermentations (oleic acid as inducer) at variable feed rate conditions. The N-terminal analysis of each crude lipase revealed that, while the isoenzymes Lip2 and Lip3 are always secreted (at different proportions depending on the inducer), Lip1 was produced only using n-dodecanol (batch conditions) or oleic acid (fed-batch at high feed rate). The nature of the inducer controls the isoenzyme percentage; when this is fixed, as well as the feed rate in fed-batch fermentation, the isoenzymatic profile remained unaltered and the samples differed only in the activity of the lipases, as determined by heptyl oleate synthesis.

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.

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.

Different supports, such as alginate, agar, agarose, and polyacrylamide, were used to immobilize Escherichia coli BL 21 by entrapment techniques. The transglycosylation reaction involved in the synthesis of adenosine from uridine and adenine was chosen as a model system to study the characteristics of these biocatalysts. Whole cells immobilized on agarose proved to be optimal and could be used up to 30 times without significant loss of activity. This biocatalyst was further employed to test its ability in the synthesis of other adenine and hypoxanthine nucleosides. Ribo-, 2'-deoxyribo-, and arabinonucleosides could be prepared in high yields starting from the corresponding pyrimidine nucleosides and purine bases. Similar product yields were obtained with both free and immobilized cells, though, in the latter case, a longer reaction time was necessary.

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.

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.

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.

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.

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 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 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.

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.

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.