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A new effective biocatalyst was prepared by coating lipase from Candida cylindracea with sorbitan esters such as span 60 and 65. This biocatalyst was characterized by employing the Scanning Electron Microscopy, X-Ray diffraction patterns and a lipase assay. The type of surfactant used during the lipase modification process has shown significant effect in the lipase activity and the protein content of the modified lipase complex. It was found that catalytic behavior of modified lipase was greatly affected by the source of the used span. Span 65-coated Candida cylindracea lipase showed the highest conversion of 50% in enzymatic esterification of glucose and lauric acid.

An enzymatic route for phosphorous–carbon bond formation was developed by discovering new promiscuous activity of lipase. We reported a new metal-free biocatalytic method for the synthesis of pharmacologically relevant β-phosphonomalononitriles via a lipase-catalyzed one-pot Knoevenagel–phospha–Michael reaction. We carefully analyzed the best conditions for the given reaction: the type of enzyme, temperature, and type of solvent. A series of target compounds was synthesized, with yields ranging from 43% to 93% by enzymatic reaction with Candida cylindracea (CcL) lipase as recyclable and, a few times, reusable catalyst. The advantages of this protocol are excellent yields, mild reaction conditions, low costs, and sustainability. The applicability of the same catalyst in the synthesis of β-phosphononitriles is also described. Further, the obtained compounds were validated as new potential antimicrobial agents with characteristic E. coli bacterial strains. The pivotal role of such a group of phosphonate derivatives on inhibitory activity against selected pathogenic E. coli strains was revealed. The observed results are especially important in the case of the increasing resistance of bacteria to various drugs and antibiotics. The impact of the β-phosphono malonate chemical structure on antimicrobial activity was demonstrated. The crucial role of the substituents attached to the aromatic ring on the inhibitory action against selected pathogenic E. coli strains was revealed. Among tested compounds, four β-phosphonate derivatives showed an antimicrobial activity profile similar to that obtained with currently used antibiotics such as ciprofloxacin, bleomycin, and cloxacillin. In addition, the obtained compounds constitute a convenient platform for further chemical functionalization, allowing for a convenient change in their biological activity profile. It should also be noted that the cost of the compounds obtained is low, which may be an attractive alternative to the currently used antimicrobial agents. The observed results are especially important because of the increasing resistance of bacteria to various drugs and antibiotics.

Dissertação de mestrado em Biotecnologia, Agro-industrial residues have been considered a major problem for modern society, but due to the constant scientific and technological evolution and modernization, these residues are now seen as important resources that can be valorized in full agreement with the concept of circular economy. Vegetable oil cakes represents a part of these resources, and their characteristics allow them, to be often reused in many alternative recovery and valorization processes, thus treated as by-products by the industry that produces them. Solid state fermentation is an alternative valorization approach that has attracted more and more interest over the last few years. In this biotechnological process, oil cakes are used as a substrate and solid support to microbial growth and concomitant production of added value compounds. In this work, was tested the suitability of olive pomace, together with sunflower and rapeseed cake to be applied as substrate for the production of enzymes, through solid state fermentation using the yeast Candida cylindracea and the filamentous fungus Trichoderma harzianum, varying factors such as substrate composition (by mixing oil cakes), % of moisture and fermentation time. Maximum enzymatic activity values of 12 ± 2 U/g for lipase and 35.7 ± 2.6 U/g for protease were achieved by C. cylindracea. While with T.harziamum values of lipase and protease activities were higher than for the yeast, and several other enzymes were also detected. Through a Box-Benchen experimental design, optimal values of lipase, protease, cellulase, xylanase, β-glucosidase and phytase were found as 14.2 U/g, 108.3 U/g 10.9 U/g, 40.7 U/g 7.2 U/g and 73.7 U/g, respectively. The production of various enzymes using these vegetable oil cakes mixtures enhances them nutritionally and helps to validate the idea of circular economy, of reusing agro-industrial by-products for the solid state fermentation process, facilitating the possibility of, under the correct conditions, use the olive pomace in animal diets., Os resíduos agroindústrias têm sido cada vez mais considerados um grande problema para a sociedade moderna, no entanto por causa da constante evolução e modernização científica e tecnológica, estes resíduos são agora vistos como um importante recurso que pode ser valorizado, perfeitamente de acordo com uma economia circular. Os bagaços de oleaginosas representam uma parte destes recursos, as suas características permitem que sejam reutilizados em muitos processos alternativos de recuperação e valorização, apesar de serem tratados como subprodutos pela indústria que os produz Fermentação em estado sólido é um exemplo destas alternativas de valorização, que ao longo dos últimos anos tem atraído cada vez mais interesse. Neste processo biotecnológico, os bagaços são utlizados como substrato e suporte sólido para o crescimento dos microorganismos e concomitante produção de componentes com valor acrescentado. Neste trabalho testou-se a suscetibilidade do bagaço de azeitona, juntamente com bagaços de girassol e de colza em serem aplicados como substrato para a produção de enzimas, através da fermentação em estado sólido usando a levedura Candida cylindracea e o fungo filamentoso Trichoderma harzianum, variando fatores como a composição do substrato (misturando bagaços), % de humidade e tempo de fermentação. Valores máximos de atividade enzimática de 12 ± 2 U/g, para a lípase e 35.7 ± 2.6 U/g para a protease foram os alcançados pela C. cylindracea. Por sua vez, os valores obtidos utilizando T.harziamum foram maiores do que com a levedura, e várias outras enzimas foram também detetadas. Através de um desenho experimental de Box-Benchen, valores ótimos para a lípase, protease, celulase, xilanases, β-glucosidase e fitase foram determinados como 14.2 U/g, 108.3 U/g 10.9 U/g, 40.7 U/g 7.2 U/g e 73.7 U/g, respetivamente A produção de variadas enzimas utilizando estas misturas de bagaços, possibilita a sua valorização nutricional e ajuda a validar a ideia de economia circular, de reutilização de subprodutos agroindustriais para o processo de fermentação em estado sólido, facilitando a possibilidade de, nas condições corretas, o bagaço de azeitona ser utilizado nas dietas animais.

The aim of this study was to evaluate the possibility of using several lipid-rich food industry wastes in the culture medium on the growth of Candida cylindracea DSM 2031 yeast strain. Four lipid wastes from the food industry: waste fish oil, rancid ghee, waste pork lard, and waste duck processing oil were investigated. It has been shown in the laboratory scale that the above-mentioned wastes can be used to obtain biomass and produce lipolytic enzymes by the tested strain and the C. cylindracea extracellular lipase is not constitutive. High yields of biomass (12.84, 12.75, and 12.24 g/dm3) were obtained in media containing waste duck processing oil, olive oil, and waste pork lard, respectively. The highest lipolytic activity was obtained in the media containing waste fish oil and rancid ghee (0.050 and 0.047 U/cm3). During 192-h flask cultures the highest extracellular lipase activity and biomass yield were observed in the late logarithmic phase. The study showed that there is a potential for waste management to produce lipolytic enzymes or to produce yeast biomass. The use of waste substrates may contribute to lowering the costs of commercial production, and such a solution is part of the sustainable development strategy.

EFSA is requested to assess the safety of a broad range of biological agents in the context of notification for market authorisation as sources of food and feed additives, food enzymes and plant protection products. The qualified presumption of safety (QPS) assessment was developed to provide a harmonised generic pre‐assessment to support safety risk assessments performed by EFSA's scientific Panels. The safety of unambiguously defined biological agents (at the highest taxonomic unit appropriate for the purpose for which an application is intended), and the completeness of the body of knowledge are assessed. Identified safety concerns for a taxonomic unit are, where possible and reasonable in number, reflected as ‘qualifications’ in connection with a recommendation for a QPS status. The list of QPS recommended biological agents was reviewed and updated in the current opinion and therefore becomes the valid list. The 2016 update reviews previously assessed microorganisms including bacteria, yeasts and viruses used for plant protection purposes following an Extensive Literature Search strategy. The taxonomic units related to the new notifications received since the 2013 QPS opinion, were periodically evaluated for a QPS status and the results published as Statements of the BIOHAZ Panel. Carnobacterium divergens, Lactobacillus diolivorans, Microbacterium imperiale, Pasteuria nishizawae, Pediococcus parvulus, Bacillus flexus, Bacillus smithii, Xanthomonas campestris and Candida cylindracea were recommended for the QPS list. All taxonomic units previously recommended for the 2013 QPS list had their status reconfirmed as well their qualifications with the exception of Pasteuria nishizawae for which the qualification was removed. The exclusion of filamentous fungi and enterococci from the QPS evaluations was reconsidered but monitoring will be maintained and the status will be re‐evaluated in the next QPS Opinion update. Evaluation of bacteriophages should remain as a case‐by‐case procedure and should not be considered for QPS status., This publication is linked to the following EFSA Journal article: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2017.4663/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2017.e15031/full

Biodecolorization of the azo dye Orange G was investigated using a new strain of Candida cylindracea SJL6, isolated from freshwater samples of the Subaé river in Bahia state, Brazil. Strain SJL6 was identified as C. cylindracea on the basis of 26S rDNA region. The various parameters of dye decolorization and cell growth were studied, including the Orange G dye concentration (100 to 500 ppm), temperature (20 to 40 °C), glucose concentration (0 to 5%), and initial pH (3 to 8). Biotoxicity tests were performed using shrimp (Artemia salina) to determine the lethal concentration (LC50) and onion bulbs (Allium cepa) to determine the cytotoxic and genotoxic effects of both Orange G dye and metabolites formed after decolorization. Up to 90% of decolorization of the Orange G dye at 500 ppm was achieved by C. cylindracea SJL6 at 30 °C, pH 3, and 1% glucose. However, the biotoxicity tests showed that there was increased toxicity after decolorization, suggesting partial Orange G dye degradation and production of toxic metabolites. A biodescoloração do corante Alaranjado G foi investigada utilizando um novo isolado de Candida cylindracea SJL6, isolado de amostras de água do Rio Subaé, Bahia, Brasil. A linhagem SJL6 foi identificada como Candida cylindracea com base na região 26S do rDNA. Os parâmetros estudados na descoloração do corante e crescimento celular foram: concentração do Alaranjado G (100 a 500 ppm), temperatura (20 a 40 ºC), concentração de glicose (0 a 5%) e pH inicial (3 a 8). Os testes de biotoxicidade foram realizados utilizando o microcrustáceo Artemia salina para determinar a concentração letal (L50) e bulbos de cebola (Alium cepa) para determinar os efeitos citotóxicos e genotóxicos tanto do corante alaranjado G quanto dos metabólitos produzidos após a descoloração. Uma taxa de descoloração acima de 90% foi atingida a 500 ppm por C. cylindracea SJL6 a 30 ºC, pH 3 e 1% de glicose. Entretanto, os testes de biotoxicidade mostraram que ocorreu um aumento da toxicidade após a descoloração, o que sugere uma degradação parcial da molécula do corante Alaranjado G e produção de metabólitos tóxicos.

A method for purification of cis-9,trans-11 isomer of conjugated linoleic acid (CLA) is described. Lipase from Candida cylindracea (CCL) was highly selective towards cis-9,trans-11 isomer and was chosen to catalyze esterification of a mixture of two CLA isomers. The effect of various parameters on the esterification process of an almost equimolar mixture of cis-9,trans-11 (44.0%), trans-10,cis-12 (42.5%) and other CLA isomers (13.5%) was studied. The influence of alcohol substrate, CLA/alcohol molar ratio, lipase amount, reaction temperature, pH and time of the reaction on the total esterification and cis-9,trans-11 isomer purity in ester fraction were examined. The highest isomeric excess of cis-9,trans-11 isomer on trans-10,cis-12 CLA and good esterification degree was observed when the esterification condition were as follows: CLA/2-propanol molar ratio, 1:5; water, 200% wt of CLA; CCL, 24 μg of CCL/mg CLA; temperature 30 C; time of reaction 6 h and when mixture enriched in cis-9,trans-11 CLA was used as a substrate for esterification. A preparative scale purification combining crystallization and one step enzymatic process were made to afford purified cis-9,trans-11 (94.2%) with total recovery of 31%.

Mestrado em Biomedicina Molecular A Candida cylindracea constitui um caso particular do grupo de leveduras CTG-clade - apresenta uma total conversão do codão CUG de leucina (standard) em serina, em vez de o fazer de forma ambígua como os restantes membros do grupo. Para além disso, após a sequenciação e anotação do seu genoma completo e do seu mRNA, verificou-se que a Candida cylindracea possuí uma frequência consideravelmente elevada de genes iniciados pelos codões alternativos CTG e TTG relativamente às outras espécies filogeneticamente próximas, cuja grande maioria dos genes é iniciada por ATG (standard). Durante este trabalho foi validada a anotação do genoma desta espécie de modo a descartar possíveis artefactos, utilizando o MAKER como ferramenta. As sequências anotadas foram introduzidas na plataforma ANACONDA para desvendar algumas das principais características do genoma e do transcriptoma desta espécie. A análise destes dados basou-se em encontar diferenças significativas entre os diferentes tipos de sequências, de acordo com o seu codão de iniciação, tanto no genoma como no transcriptoma. A notória diferença entre a frequencia dos codões de iniciação das sequências de DNA e RNA, por sua vez, abriu portas à especulação acerca da presença de fenómenos de RNA editing. Ao reunir as peças deste puzzle tão singular, espera-se conseguir dar um passo em frente na compreensão do funcionamento do genoma de acordo com a relevância deste fenómeno. Resta para isso entender de que forma estas diferenças poderão estar conectadas e influenciar o genoma. Estudos posteriores com recurso a novas técnicas da era ómica poderão fornecer novos discernimentos nesta materia. Candida cylindracea yeast is a peculiar case within the CTG clade – its total conversion of the CUG leucine codon into serine contrasts with the ambiguous way that the rest of the yeasts belonging to this group decode the CUG codon. Furthermore, after the sequencing and annotation of its complete genome and its mRNA sequences, it was yet ascertained that Candida cylindracea has a substantial frequency of alternative initiation codons, when compared to other phylogenetically close species, where the majority of the genes is started with the standard ATG codon. MAKER was used as annotation tool to validate the previous annotation of Candida cylindracea’s genome and transcriptome in order to forgo possible artifacts. The sequences produced were introduced in the ANACONDA platform to unveil some of the main features of the genome and transcriptome of this species. The analysis of this data was based in finding the significant differences between the distinct types of sequences according to their initiation codon, in both genome and transcriptome levels. The considerable differences between the DNA and the RNA sequences regarding their initiation codon allowed instigating the presence of RNA editing phenomena. Putting it all together, these singular events are expected to yield a better comprehension of the genome functioning. It is, therefore, necessary to understand in which ways these differences may be connected and if they influence the genome. Posterior studies resorting to new techniques of the omics era can provide new insights on this matter. .

Mestrado em Biomedicina Molecular Submitted by Cristina Santos (cmaria@ua.pt) on 2017-04-04T14:25:09Z No. of bitstreams: 1 Tese de Mestrado - Carolina Lobato - Biomedicina Molecular - UA.pdf: 1916505 bytes, checksum: 50ac7e0ef097a90ce500af9b55c8d666 (MD5) Made available in DSpace on 2017-04-04T14:25:10Z (GMT). No. of bitstreams: 1 Tese de Mestrado - Carolina Lobato - Biomedicina Molecular - UA.pdf: 1916505 bytes, checksum: 50ac7e0ef097a90ce500af9b55c8d666 (MD5) Previous issue date: 2016

Sequential optimization strategy based on statistical experimental design including one-factor-at-a-time (OFAT) method was used to enhance the production of lipase by Candida cylindracea ATCC 14830 using palm oil mill effluent as a basal medium in shake flask cultures. The two-level Plackett–Burman (PB) design was implemented to screen the medium components that significantly influence the production. Following the OFAT method, three significant components influencing lipase production were identified as peptone, Tween-80 and inoculum. The optimum values of these three components were determined by response surface methodology (RSM) based on the face-centered central composite design (FCCCD). The validity of the model developed was verified, and the optimum medium containing 0.45% (w/v) peptone, 0.65% (v/v) Tween-80 and 2.2% (v/v) inoculum led to amaximumlipase production of 20.26 U/ml, which was 5.19-fold higher than the unoptimized medium. The analysis of variance indicated that the established model was significant (P < 0.05). This study innovatively developed a fermentation medium utilizing renewable palm oil mill effluent (POME) to produce lipase at considerable level.

The operational stability of the lipase enzyme from Candida rugosa (formerly known as Candida cylindracea) has been investigated. The activity was found to be dependent on shear rate, temperature and emulsifier concentration. The enzyme remained active (the activity was about 80% of the original) up to a shear rate of 122 s−1 for 2 hours at 40dC. Concentrated enzyme was more resistant to shear rate than the diluted enzyme. It was also found that the presence of emulsifier increased the enzyme inactivation. The enzyme lost almost all its activity in 45 minutes at an emulsifier concentration of 0.5%.

Mestrado em Biomedicina Molecular The non-standard decoding of the CUG codon in Candida cylindracea raises a number of questions about the evolutionary process of this organism and other species Candida clade for which the codon is ambiguous. In order to find some answers we studied the transcriptome of C. cylindracea, comparing its behavior with that of Saccharomyces cerevisiae (standard decoder) and Candida albicans (ambiguous decoder). The transcriptome characterization was performed using RNA-seq. This approach has several advantages over microarrays and its application is booming. TopHat and Cufflinks were the software used to build the protocol that allowed for gene quantification. About 95% of the reads were mapped on the genome. 3693 genes were analyzed, of which 1338 had a non-standard start codon (TTG/CTG) and the percentage of expressed genes was 99.4%. Most genes have intermediate levels of expression, some have little or no expression and a minority is highly expressed. The distribution profile of the CUG between the three species is different, but it can be significantly associated to gene expression levels: genes with fewer CUGs are the most highly expressed. However, CUG content is not related to the conservation level: more and less conserved genes have, on average, an equal number of CUGs. The most conserved genes are the most expressed. The lipase genes corroborate the results obtained for most genes of C. cylindracea since they are very rich in CUGs and nothing conserved. The reduced amount of CUG codons that was observed in highly expressed genes may be due, possibly, to an insufficient number of tRNA genes to cope with more CUGs without compromising translational efficiency. From the enrichment analysis, it was confirmed that the most conserved genes are associated with basic functions such as translation, pathogenesis and metabolism. From this set, genes with more or less CUGs seem to have different functions. The key issues on the evolutionary phenomenon remain unclear. However, the results are consistent with previous observations and shows a variety of conclusions that in future analyzes should be taken into consideration, since it was the first time that such a study was conducted. A descodificação não-standard do codão CUG na Candida cylindracea levanta uma série de questões sobre o processo evolutivo deste organismo e de outras espécies do subtipo Candida para as quais o codão é ambíguo. No sentido de encontrar algumas respostas procedeu-se ao estudo do transcriptoma de C. cylindracea, comparando o seu comportamento com o de Saccharomyces cerevisiae (descodificador standard) e de Candida albicans (descodificador ambíguo). A caracterização do transcriptoma foi realizada a partir de RNA-seq. Esta metodologia apresenta várias vantagens em relação aos microarrays e a sua aplicação encontra-se em franca expansão. TopHat e Cufflinks foram os softwares utilizados na construção do protocolo que permitiu efectuar a quantificação génica. Cerca de 95% das reads alinharam contra o genoma. Foram analisados 3693 genes, 1338 dos quais com codão start não-standard (TTG/CTG) e a percentagem de genoma expresso foi de 99,4%. Maioritarimente, os genes têm níveis de expressão intermédios, alguns apresentam pouca ou nenhuma expressão e uma minoria é altamente expressa. O perfil de distribuição do codão CUG entre as três espécies é muito diferente, mas pode associar-se significativamente aos níveis de expressão: os genes com menos CUGs são os mais altamente expressos. Porém, o conteúdo em CUG não se relaciona com o nível de conservação: genes mais e menos conservados têm, em média, igual número de CUGs. Os genes mais conservados são os mais expressos. Os genes de lipases corroboram os resultados obtidos para os genes de C. cylindracea em geral, sendo muito ricos em CUGs e nada conservados. A quantidade reduzida de codões CUG que se observa em genes altamente expressos pode dever-se, eventualmente, a um número insuficiente de genes de tRNA para fazer face a mais CUGs sem comprometer a eficiência da tradução. A partir da análise de enriquecimento foi possível confirmar que os genes mais conservados estão associados a funções básicas como tradução, patogénese e metabolismo. Dentro destes, os genes com mais e menos CUGs parecem ter funções diferentes. As questões-chave sobre o fenómeno evolutivo permanecem por esclarecer. No entanto, os resultados são compatíveis com as observações anteriores e são apresentadas várias conclusões que em futuras análises devem ser tidas em consideração, já que foi a primeira vez que um estudo deste tipo foi realizado.

This work deals with the resolution of DL-menthol with propionic acid by Candida cylindracea lipase (Ccl) in organic solvent reaction systems and a reverse micelles system of sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (AOT). The activity and stability as well as enantioselectivity of the lipase in two systems were studied. The results indicate that the lipase showed higher stability in reverse micelles than in organic solvent, which proved that the reverse micelles system has potential application for maintaining the activity of the enzyme for a long time. This is because lipase molecules can be entrapped in water-containing micro-drops of reverse micelles, avoiding direct-contract with unfavorable organic medium. The enantioselectivity (E > 30, eep = 92.5) in the two systems is relatively high, although the conversion is moderate. The influence of the characteristic parameters of the two systems, such as pH, temperature, w0 (molar ratio of water to AOT in reverse micelles systems) and water content (organic solvent) on the conversion of DL-menthol was also investigated. Copyright © 2005 Society of Chemical Industry

Porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL) were immobilized on Celite and Amberlite IRA 938 by deposition from the aqueous solution by the addition of hexane. The influence of the immobilization on the activities of the immobilized lipase derivatives has been studied. The immobilized lipases were used in synthesis of pentyl isovalerates. Various reaction parameters affecting the synthesis of pentyl isovalerates were investigated. The reaction rates were compared with the rates of esterification with free lipases. The immobilized lipases were found to be very effective in the esterification reaction. The lipases immobilized on Celite 545 exhibited better operational stabilities than that of immobilized on Amberlite IRA‐938.

Extensive screenings using commercially available enzymes were performed in relation to the asymmetric hydrolysis reactions of the (±)- cis- ( 1 , 3 and 5 ) and (±)- trans -1,2,3,4,6,7,8,8a-octahydro-8a-methyl-6- oxo -naphthyl acetates ( 7 , 9 and 11 ). The combinations of enzymes from plants and cis -equatorial compounds as well as the combinations of lipases from microorganisms and trans -axial-5-methoxycarbonyl acetate showed high enantiomeric ratio in asymmetric hydrolysis. However, the enantiomeric ratio of the combinations of lipases from microorganisms and trans -axial isomers except trans -axial-5-methoxycarbonyl derivative was less satisfactory. High E values (>200) were observed when β-amylase from wheat and lipase from Candida cylindracea were used for the (±)- cis -acetate 1 and the (±)- trans -isomer 7 , respectively, to obtain the corresponding chiral products with high enantiomeric ratios (>99:1).

Racemic 1,1'-methylene[(1RS, 1'RS, 3RS, 3'RS, 5RS, 5'RS)-8-oxabicyclo[3.2.1]oct-6-en-3-ol] ((+/-)-6) derived from 2,2'-methylenedifuran has been resolved kinetically with Candida cylindracea lipase-catalysed transesterification giving 1,1'-methylenedi[(1R, 1'R, 3R, 3'R, 5R, 5'R)-8-oxabicyclo[3.2.1]oct-6-en-3-ol] (-)-6 (30% yield, 98% ee) and 1,1'-methylenedi[(1S, 1'S, 3S, 3'S, 5S, 5'S)-8-oxabicyclo[3.2.1]oct-6-en-3-yl] diacetate (+)-8, (40% yield, 98% ee). These compounds have been converted into 1,1'-methylenedi[(4S, 4'S, 6S, 6'S)- and (4R, 4'R, 6R, 6'R)-cyclohept-1-en-4,6-diyl] derivatives. (C) 2001 Published by Elsevier Science Ltd.

Enzymatic kinetic resolution of racemic aryl β - hydroxy ketones in organic media catalyzed by Candida cylindracea lipase is reported.

A modified chromatographic process was used to determine the enantiomeric ratio (E) in enantioselective biocatalysis. Theoretical results indicated that the established relationship, E=ln(1-x S )/ln(1-x R ), was valid for this modified operation. In addition, the Candida cylindracea lipase-catalyzed enantioselective esterification of racemic naproxen was selected to verify experimentally the effectiveness of the proposed method. The ease of operation and simplicity of data analysis of the proposed method makes it a promising alternative for determining the enantioselectivity of enzymatic chiral resolution.

enantio-3-Hydroxyambrox derivatives were obtained through combined chemical and microbiological methods from ent-12-o×o-13-epi-manoyl oxides. Regioselcctive and stereoselcctive reduction ofthe ¢~bonyl group at C-3 of ent-3,12- dioxo- 13 -epi-manoyl oxide was card ed out with Baker "s yeast. Regioselcctive acetyl ation of ent- 3,12 -dihydxoxy- 13 -epi-manoyl oxide was accomplished with Candida cylindracea lipase or Novozym 435 and vinyl acetate. Microbial bioa'ansfonnation of ent-3(~-acetoxy-12-oxo- 13-epi-manoyl oxide with Nectria ochroleuca yielded new ent- l13-hydroxy- and ent-7~-hydroxy derivatives, Baeyer-Villiger oxidation at C-12 was observed in the bioa-ansformation with baker's yeast and G. ro.Teun to obt~n norambreinolide lactones which were converted in to enantio-Ambrox ® derivatives. © 1999 Elsevier Science Ltd. All fights reserved.

Tricarbonylchromium complexes of 1-indanol, 1-tetralol, 2-tetralol and the uncomplexed counterparts were kinetically resolved by asymmetric esterification with the Candida cylindracea lipase. We observed a beneficial effect of the tricarbonyl chromium group on the enantioselectivity and we have studied the implication of the relative positions of the tricarbonylchromium group and the alcohol function.

Two microbial lipases from Geotrichum spp and Rhizopus spp isolated previously in this laboratory and a commercial lipase from Candida cylindracea were evaluated as biocatalysts for the enrichment of gamma‐linolenic acid (GLA, 18:3 n‐6) from a fungal oil derived from Mucor spp. Lipase from Geotrichum spp, as compared to lipases from Candida cylindracea and Rhizopus spp was found to be most effective in the enrichment of GLA in unesterified fatty acids upon esterification of the fungal oil fatty acids with n‐butanol. The level of GLA in the unesterified fatty acid could be raised from 10.9 % in the starting material to about 30 % in the unesterified fatty acids after a reaction period of 4 h. Selective hydrolysis of the fungal oil using lipase from Rhizopus spp resulted in about 1.5‐fold enrichment of gamma‐linolenic acid in the unhydrolysed acylglycerols. The other Upases tested were ineffective in the enrichment of GLA by selective hydrolysis of the fungal oil triacylglycerols.

A new practical carrier for an interface bioreactor has been developed. This carrier consists of two different crosslinks, i.e., polyvinyl alcohol (PVA)-glutaraldehyde (GA) and alginate (ALG)-Ca2+ crosslinks. This combined crosslinking leads to high gel strength, heat tolerance, and water holding capacity. A pad-packed interface bioreactor using the crosslinked gel pads was applied to the hydrolysis of 2-ethylhexyl acetate using Candida cylindracea ATCC 14830. The pad-packed interface bioreactor with the crosslinked gel pads gave satisfactory accumulation of 2-ethyl-1-hexanol compared to that with agar pads.

The enzymatic kinetic resolution of (±)-4-acetoxy[2.2]paracyclophane by Candida cylindracea lipase was investigated in water and in a two-phase aqueous organic system. The (+)-(R)-4-hydroxy- and (+)-(S)-4-acetoxy[2.2]-paracyclophanes were isolated in excellent yields and high enantiomeric excesses. The resolution was carried out on multi-gram scale in hexane-water at 40° C.

Candida cylindracea lipase (CCL) and porcine pancreatic lipase (PPL) have been used for deacetylation of peracetates of methyl and ethyl esters of six different polyphenolic acids in organic solvents. Exclusive de-esterification of the ester groups derived from the phenolic hydroxy and aliphatic acid over the ester group of the aromatic acid and aliphatic alcohol has been achieved affording the corresponding esters of phenolic acids in as high yields as 90–97%. The results have been corroborated with the mechanism of lipase action.