Biodiesel production from Jatropha curcas oil catalyzed by whole cells of Aureobasidium pullulans var. melanogenum SRY 14-3

The main obstacle to using lipase as a catalyst in industrial scale biodiesel production is the cost and availability of the enzyme. To overcome this obstacle, the potential of using a whole cell biocatalyst (for at least partial in situ lipase production) was evaluated as a means to reduce the cost of the lipase. The reaction conditions for biodiesel production via transesterification between Jatropha curcas (physic nut) oil and methanol when catalyzed in the presence of lipase-producing Aureobasidium pullulans yeast cells was investigated. The appropriate conditions for optimal biodiesel production were found to be 1:3 oil:methanol molar ratio at 30°C with constant stirring at 250 rpm. Under these conditions a maximum fatty acid methyl ester (biodiesel) production level of 71.8% was obtained after 72 h.Keywords: Lipase, Aureobasidium pullulans, physic nut oil, biodiesel, green energyAfrican Journal of Biotechnology Vol. 12(27), pp. 4380-438

Immobilization of the Candida rugosa lipase onto a Scirpus grossus L.f. fiber as biocatalyst for biodiesel synthesis via hydrolysis-esterification

This study aimed to immobilize the Candida rugosa lipase (C. rugosa lipase) on ground Scirpus grossus L.f. fibers by glutaraldehyde-crosslinking to form a hydrolysis-esterification catalyst for biodiesel synthesis. The effects of different glutaraldehyde concentrations and solvent for 3- aminopropyltriethoxysilane (3-APTES) activation of the fibers on the resultant immobilized lipase activity, protein loading, degree of immobilization and catalytic efficiency were investigated. The optimum condition found was to first activate the S. grossus L.f. fibers using 2% (w/v) of 3-APTES in distilled water and subsequently crosslink with 0.2% (w/v) glutaraldehyde prior to C. rugosa lipase immobilization at pH 7. The immobilized C. rugosa lipase was then evaluated as a biocatalyst for biodiesel synthesis via the hydrolysis-esterification of palm oil and bioethanol through monitoring the production of free fatty acids (FFAs) and fatty acid ethyl ester (FAEE, biodiesel). The reusability of immobilized lipase was also determined. The immobilized C. rugosa lipase yielded a higher hydrolysisesterification efficiency (that is, FFA and FAEE formation) than that of the free lipase with the immobilized form looking promising for FAEE biodiesel production. The C. rugosa lipase immobilized with 0.2% (w/v) glutaraldehyde exhibited the highest reuse stability, retaining some hydrolysis and esterification activity for up to six uses, whereas crosslinking with higher [0.5% or 0.8% (w/v)] glutaraldehyde levels resulted in a loss of both activities within four uses.Key words: Immobilization, lipase, Scirpus grossus L.f. fiber, 3-aminopropyltriethoxysilane, glutaraldehyde, biodiesel

Proteomic evaluation of free fatty acid biosynthesis in Jatropha curcas L. (physic nut) kernel development

Jatropha curcas L. is one of the economic crops that are cultivated for biodiesel production. Here, the fatty acid and protein profiles of J. curcas kernels were evaluated during their development. The fruits were divided into eight developmental stages (stages I to VIII) based on their age and morphology. The fatty acid content was analyzed at each stage using gas chromatography after conversion to methyl esters. Fatty acid levels were found to differ between all eight developmental stages, although the major fatty acid in each stage was oleic acid followed by linoleic, palmitic and stearic acids, respectively, except in stage I where linoleic acid was more common than oleic acid. All fatty acids showed a maximum content at stage III, a rapid decline at stage IV and another peak at stage VII before declining. Significant changes were found in the relative abundance of 22 proteins during seed development, of which the expression levels for transcripts encoding for four of these proteins, acetyl CoA carboxylase, phosphoenolpyruvate carboxylase, mercaptopyruvate sulfurtransferase and 4-coumarate: coenzyme A ligase, as evaluated by quantitative RT-PCR, were altered between the developmental stages of the kernels in a broadly similar pattern as the level of most fatty acids.Keywords: Jatropha curcas L., FAME, ACCase, PEPC, MST, 4CL, quantitative real time PCRAfrican Journal of Biotechnology Vol. 12(21), pp. 3132-314

The Potential of Cellulosic Ethanol Production from Grasses in Thailand

The grasses in Thailand were analyzed for the potentiality as the alternative energy crops for cellulosic ethanol production by biological process. The average percentage composition of cellulose, hemicellulose, and lignin in the samples of 18 types of grasses from various provinces was determined as 31.85–38.51, 31.13–42.61, and 3.10–5.64, respectively. The samples were initially pretreated with alkaline peroxide followed by enzymatic hydrolysis to investigate the enzymatic saccharification. The total reducing sugars in most grasses ranging from 500–600 mg/g grasses (70–80% yield) were obtained. Subsequently, 11 types of grasses were selected as feedstocks for the ethanol production by simultaneous saccharification and cofermentation (SSCF). The enzymes, cellulase and xylanase, were utilized for hydrolysis and the yeasts, Saccharomyces cerevisiae and Pichia stipitis, were applied for cofermentation at 35°C for 7 days. From the results, the highest yield of ethanol, 1.14 g/L or 0.14 g/g substrate equivalent to 32.72% of the theoretical values was obtained from Sri Lanka ecotype vetiver grass. When the yields of dry matter were included in the calculations, Sri Lanka ecotype vetiver grass gave the yield of ethanol at 1,091.84 L/ha/year, whereas the leaves of dwarf napier grass showed the maximum yield of 2,720.55 L/ha/year (0.98 g/L or 0.12 g/g substrate equivalent to 30.60% of the theoretical values)

NirK and nirS Nitrite reductase genes from non-agricultural forest soil bacteria in Thailand

The genetic heterogeneity of the nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in a non-agricultural forest soil in Thailand was investigated using soil samples from the Plant Germplasm-Royal Initiation Project area in Kanchanaburi Province, Thailand. Soil bacteria were screened for denitrification activity and 13 (from 211) positive isolates were obtained and further evaluated for their ability to reduce nitrate and to accumulate or reduce nitrite. Three species with potentially previously unreported denitrifying activities were recorded. Analysis of the partial nirK and nirS sequences of these 13 strains revealed a diverse sequence heterogeneity in these two genes within the same environment and even potentially within the same host species, the potential existence of lateral gene transfer and the first record of both nirK and nirS homologues in one bacterial species. Finally, isolates of two species of bacteria (Corynebacterium propinquum and Micrococcus lylae) are recorded as denitrifiers for the first time

Antioxidant and anticancer activities of freshwater green algae, Cladophora glomerata and Microspora floccosa, from Nan River in northern Thailand

Organic solvent and hot water extracts of freshwater macroalgae, Cladophora glomerata and Microspora floccosa, harvested from Nan River in northern Thailand were screened for antioxidant and anticancer activities using DPPH free radical scavenging assay and inhibition of proliferation of the KB human oral cancer cell lines respectively. The ethyl acetate extract of C. glomerata showed the highest total phenol content (18.1±2.3 mg GAE/g), radical scavenging activity (49.8±2.7% DPPH scavenging at 100 g/ml) and in vitro growth inhibition (IC50=1420.0±66 g/g) of the KB cell lines. These results indicate that C. glomerata could be a source of valuable bioactive materials

Chemical composition and antibacterial activity of extracts of freshwater green algae, Cladophora glomerata Kützing andMicrospora floccosa (Vaucher) Thuret

Freshwater macroalgae, Cladophora glomerata Kützing and Microspora floccosa (Vaucher) Thuret, harvested from Nan River in northern Thailand, were extracted with hexane, ethyl acetate, methanol and hot water. The extracts were screened for antibacterial activities. Hexane and ethyl acetate extracts of both algae showed the activities against Bacillus cereus and Vibrio parahaemolyticus. The extracts were further separated using column chromatography and chemically characterized by GC–MS in order to be tentative identify the compounds responsible for such activities. The main compositions were fatty acid and other organic compounds, in which have not been reported in these algae. These results indicate that extracts of C. glomerata and M. floccosa exhibited appreciable antimicrobial activity and could be a source of valuable bioactive materials for health products

Expression et évolution des lipases de Candida rugosa et Yarrowia lipolytica pour modifier leurs activités et spécificités

Les lipases, protéines ubiquitaires, sont les enzymes les plus étudiées et les plus utilisées dans l industrie. Elles catalysent un très grand nombre de réactions, d hydrolyse et de synthèse, conduisant à une grande diversité de molécules, acides, esters, amides . Les domaines d applications sont nombreux : les bio-énergies, les arômes, bio-lubrifiants, bio-plastifiants, émulsifiants, produits phytosanitaires et détergents, cosmétiques, synthons pour la chimie fine, produits pharmaceutiques Aujourd hui, grâce aux outils génétiques, il est possible de modifier leur activité, spécificité et thermostabilité pour les adapter idéalement aux contraintes industrielles. Dans ce travail de doctorat, nous nous sommes intéressés à quatre lipases d intérêt industriel. Les 3 premières appartiennent à la famille des lipases de Candida rugosa (Lip1, Lip3 et Lip4). Bien que très homologues, leurs spécificités sont très différentes. Elles se distinguent de toutes les autres lipases par un site actif composé d un long tunnel avec la triade catalytique à l entrée de celui-ci. Cela en fait une enzyme particulièrement intéressante pour la conversion et la purification d acides gras à longue chaîne. La quatrième est une nouvelle lipase identifiée chez la levure oléagineuse, Yarrowia lipolytica. Elle est très active sur les acides gras à longue chaîne, active à pH acide et présentant une grande énantiosélectivité sur des molécules d intérêt pharmaceutique, les esters d acide 2- halogéno-aryl acide acétique. Dans un premier temps, un nouveau système d expression, une souche spécifique de Yarrowia lipolytica, a été étudié pour l expression de variants construits par mutagenèse dirigée. Cette souche JMY1212 permet une intégration ciblée dans le génome de Y. lipoytica. Nous avons démontré qu il s agissait du premier système d expression permettant de comparer statistiquement l activité de variants directement à partir du surnageant de culture. Trois des lipases de Candida rugosa ont été clonées avec succès dans cette souche et leurs activités et spécificités vis-à-vis de la longueur de chaines des acides gras ont été étudiées. Lip1 et Lip3 présentent une spécificité pour les acides gras à longueur de chaine moyenne (C8-C10) alors que Lip4 préfère les C18:1. De, plus, pour la première fois, la purification, à partir d un mélange d esters éthyliques issu d huile de poissons, d acides gras poly-insaturés (PUFAs); acides cis-5, 8, 11, 14, 17-eicosapentaenoic (EPA) et cis-4, 7, 10, 13, 16, 19-docosahexaenoic (DHA), molécules bonnes pour la santé, a été réalisée avec les trois lipases séparées de C. rugosa. Quelle que soit l enzyme, le rendement de récupération du DHA est supérieur à 93 % (97, 100 et 93 % pour Lip1, Lip3 et Lip4 respectivement. Une pureté maximale en DHA de ~60 % a été obtenue avec Lip3 et Lip4, à partir d un mélange initial d esters éthyliques contenant 25% de DHA. Une différence remarquable entre ces trois enzymes est que Lip4 est capable de mieux hydrolyser l ester d EPA (60% contre 14 et 16% pour Lip1 et Lip3). Lip4 est même capable d hydrolyser le DHA (7% contre 3 et 0 % pour Lip1 et Lip3). La deuxième partie de ce travail a été consacrée à l amélioration de l énantiosélectivité des deux enzymes étudiées vis-à-vis de synthons d intérêt dans l industrie pharmaceutique, les esters de 2-bromo aryl acide acétique. La construction raisonnée d un double variant de la lipase Lip2 de Y. lipolytica, D97AV232F, a permis d obtenir une enzyme totalement énantiosélective (E >200). Celle-ci reconnaît l énantiomère R alors que la lipase sauvage avait une faible préférence pour l énantiomère S (E=5). Par ailleurs, cette exceptionnelle augmentation de l énantiosélectivité s accompagne d une amélioration de l activité de l enzyme qui est ainsi multipliée par 4,5. Sur ce même mélange d énantiomères, les 3 lipases de C. rugosa se sont avérées remarquables. Malgré leur grande homologie, leur spécificité est différente. Lip1 et Lip3 sont totalement S spécifiques (E>200), alors que Lip4 est R spécifique (E=15). Le docking moléculaire des énantiomères S et R dans le site actif des lipases Lip1 et Lip4 a permis de mieux comprendre ces différences de spécificité et de proposer des cibles de mutagenèse dirigée. L encombrement et la nature de l acide aminé présent en position 296 sont cruciaux pour la discrimination de l enzymeLipases, ubiquitous proteins, are the most studied enzymes and the most used in industry. They catalyse a great number of reactions, hydrolysis and synthesis, leading to a great diversity of molecules, acids, esters, amides. There are numerous fields of applications: bio-energies, flavours, bio-lubricants, bio-plasticizers, emulsifiers, detergents, cosmetics, synthons for fine chemistry, and pharmaceutical products. Nowadays, thanks to genetic tools, it is possible to modify their activity, specificity and thermostability to ideally adapt enzymes for the industrial constraints. In this work, we were interested in four lipases of industrial interest. The third ones belong to the lipase family of Candida rugosa (Lip1, Lip3 and Lip4). Although they present high homology, their specificities are very different. They are distinct from the other lipases by the active site composed of a long tunnel with the catalytic triad at the entry of the tunnel. It leads to enzymes particularly interesting for the conversion and the purification of long chain fatty-acids. The fourth one is a new lipase identified from oleaginous yeast, Yarrowia lipolytica. It is one of the most active lipase on long chain fatty-acids; it is very active and stable at acid pH and presents a high enantioselectivity on molecules of pharmaceutical interest, the esters of 2- halogeno-aryl acetic acid. In this work, we first tested a new expression system, a specific strain of Y. lipolytica, for expression of variants obtained by site-directed mutagenesis. This strain JMY1212 enables integration to be targeted to a special locus of the Y. lipoytica genome. We demonstrated that it is the first expression system in which it is possible to compare statistically variant activities directly from the supernatant of the culture. Secondly, three lipases of C. rugosa were cloned successfully in this strain and their activities and specificities with respect to fatty acid chain lengths were studied. Lip1 and Lip3 have specificity for the fattyacids of medium chain (C8-C10) whereas Lip4 prefers C18: 1. Moreover, for the first time, purification, from a mixture of ethyl esters issued from fish oil, polyunsaturated fatty acids (PUFAs); cis-5, 8, 11, 14, 17- eicosapentaenoic acid (EPA) and cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA), molecules with health benefits, was realised with the three C. rugosa lipases, separately. Whatever the enzyme the recovery of DHA is superior to 90 % (97, 100 and 93 % for Lip1, Lip3 and Lip4 respectively. The maximal DHA purity ~60 % was obtained with Lip3 and Lip4, with an initial ethyl ester mixture containing 25% DHA. A remarkable difference between these enzymes lies in the fact that Lip4 is able to better hydrolyse the EPA esters (60% against 13% and 16% respectively for Lip1 and Lip3). Lip4 is also able to hydrolyse DHA (7% against 3 and 0 % for Lip1 and Lip3 respectively). The third part of this work was devoted to the improvement of the enantioselectivity of the two enzymes studied with respect to the resolution of a racemic mixture of pharmaceutical industry, the R, S esters of 2-bromo aryl acetic acid. The rational construction of a double variant of Lip2 lipase from Y. lipolytica, D97A V232F was realized to obtain a total enantioselective enzyme (E > 200). This variant recognizes the enantiomer R whereas wild-type lipase had a weak preference for the enantiomer S (E=5). In addition, this exceptional increase in the enantioselectivity is accompanied by a 4.5 fold improvement of the activity. With the same mixture of enantiomers, the 3 lipases of C. rugosa proved to be remarkable from the point of view of enantioselectivity. In spite of their high homology, their specificity is different. Lip1 and Lip3 are completely specific for the S enantiomer, whereas Lip4 is R specific (E=15). The molecular docking of the S and R enantiomers in the active site of Lip1 and Lip4 lipases enables the observed differences in specificity to be better understood and targets for site-directed mutagenesis to be proposed. We demonstrated that the nature of the amino acid present in position 296 is crucial for the discrimination of these enzymesTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Biotransformation of lauric acid into 1,12-dodecanedioic acid using CYP52A17 expressed in Saccharomyces cerevisiae and its application in refining coconut factory wastewater

1,12-Dodecanedioic acid (DDA), a primary compound and an intermediate precursor for various chemicalproducts, is normally produced by chemical synthesis, which presents potential disadvantages. Instead, thebiosynthesis of 1,12-DDA by recombinant (r) microorganisms may offer a viable production route. CytochromeP450 (CYP) can terminally oxidize fatty acids to ω-hydroxy-fatty acids and further to dicarboxylic acids (ω-oxidation). The wild type (r) CYP52A17LL and its engineered L261S/L490S form, in which the two leucineresidues were changed into serine (rCYP52A17SS), were expressed in Pichia pastoris and a strain of Saccharomycescerevisiae coexpressing the yeast NADPH cytochrome P450 reductase gene. Both organisms produced 12-hydroxydodecanoicacid (HDDA) and 1,12-DDA from lauric acid. In vitro, microsomes containing rCYP52A17SSextracted from the S. cerevisiae strain BY(2R)/pYeDP60-CYP52A17SS were able to convert lauric acid to 12-HDDA quite efficiently. Biotransformation of lauric acid using S. cerevisiae BY(2R)/pYeDP60-CYP52A17SS inculture gave the highest level of 12-HDDA (45.8 μM) at 24 h, which was oxidized to yield 20.8 μM of 1,12-DDA at72 h. The recombinant S. cerevisiae BY(2R)/pYeDP60-CYP52A17SS, which was initially cultured in YPGE, producedthe highest yield of 1,12-DDA from coconut milk wastewater at 24 h. Hence, our designed S. cerevisiaestrain BY(2R)/pYeDP60-CYP52A17SS can potentially produce 1,12-DDA for industrial applications.1. Introductionα,ω-Dicarboxylic acids (DCAs) are important versatile chemicalintermediates for the preparation of nylon and other polyamides,polyesters, perfumes, polymers, lubricants, cosmetic ingredients andcoatings. For example, dodecanedioic acid (DDA) is the precursor ofpolyamide 6,12 (nylon 6,12) (Funk et al., 2017; Huf et al., 2011;Waché, 2013). Currently, DCA production is mainly performed viachemical synthesis, but this has limitations and drawbacks, such as thesynthesis of unwanted by-products, the cost of removal of these alternatemolecules produce