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Screening of Lipase Production Among Different Microorganisms
New Biotechnology · Volume 31S · July 2014
BIOCATALYSIS
PA-35 Screening of Lipase Microorganisms
Production
Among
Different
¸ ın 2 , Nihat Alpagu Sayar 1 Duygu Elif Yılmaz 1,∗ , H. Tansel Yalc 1
2
Marmara University Ege University
The design and manifecture of single enantiomers of chiral intermediates have been attracting attention in the pharmaceutical industry during the last decades. Single enantiomers that are potential biocatalysts due to their higher degree of enantioselectivity and regioselectivity are usually prepared by chemical catalysis. L-amino acid esters are promising intermediates for various active drug components and their catalytic biosythesis through transesterification reactions could be a rational alternative way. Besides, they can be effectively producted by using lipase from various microorganisms as a biocatalyst. The selection of the most appropriate organism among different strains can be a valuable starting point for this study. Various isolates from various potantial microorganisms which differ significantly from each other, may exhibit different catalytic effectiveness. The microbial isolates for this study are maintained by monthly transfers on MGYP agar slants and stored at + 5◦ C. For the production stage, microorgansims are transferred to MGYP preculture medium. After overnight incubation, this culture is inoculated to production medium including different types of oil. The culture is incubated in appropriate conditions. Samples are taken at different time intervals to determine the growth and lipase activity. The enzyme activity is shown by p-nitrophenyl palmitate (pNPP) as substrate. P-nitrophenol is obtained from pNPP and absorbance is measured spectrophotometrically against an enzyme free sample. One unit of lipase activity (U) is defined as the amount of enzyme which liberates 1 mol p-nitrophenol/min under assay conditions. Protein is measured by the Coomassie Blue G-250 binding method using bovine serum albümin as standard. http://dx.doi.org/10.1016/j.nbt.2014.05.1821
PA-36 A cutinase from Fusarium oxysporum with potential for PET surface modification Evangelos Topakas ∗ , Efstratios Nikolaivits, Maria Kanelli, Paul Christakopoulos National Technical University of Athens
Cutinases are small extracellular serine hydrolases whose natural function is the hydrolysis of the polyester cutin. Their ability to hydrolyze a wide range of substrates (from low molecular weight esters to high molecular weight polymers) makes cutinases very useful biocatalysts for various applications. In the present study, a cutinase (FoCut5a) from the ascomycete fungus Fusarium oxysporum was functionally overexpressed in Escherichia coli BL21, harboring pET22b(+)-cut16606. In order to improve enzyme sta-
S90
www.elsevier.com/locate/nbt
bility, the folding of the expressed protein took place also in the periplasm in addition to cytosolic protein production by cloning cut16606 gene downstream of the pelB signal peptide. The heterologous expression was induced with IPTG in 16о C for 20 h of incubation. The recombinant cutinase was purified from the cytosol or culture supernatant using immobilized-metal affinity chromatography (IMAC), resulting in a monomeric protein of ca. 23 kDa that is optimally active at 40 ◦ C. FoCut5a cutinase was tested for its potential use in surface modification of PET fabrics, with the intention to increase their hydrophilicity and to improve their properties. Towards this direction, the enzymatic hydrolysis of two model substrates bis(benzoyloxyethyl)terephthalate (1) and commercial bis(2-hydroxyethyl)terephthalate (2) was attempted, in order to evaluate the capability of the recombinant cutinase in PET modification. FoCut5a succeeded in hydrolyzing both models, releasing bis(2-hydroxyethyl)terephthalate and benzoic acid from substrate 1 and a bis(2-hydroxyethyl)terephthalate derivative in case of substrate 2. Experiments are in progress for the surface modification and functionalization of PET fibers. http://dx.doi.org/10.1016/j.nbt.2014.05.1822
PA-37 Synthesis of biological active compounds using carbohydrate esterases as biocatalysts Paul Christakopoulos 1,∗ , Io Antonopoulou 1 , Evangelos Topakas 2 1
2
Luleå University of Technology National Technical University of Athens
Various fungal and bacterial carbohydrate esterases represent appealing biocatalysts that have the ability not only to deconstruct plant biomass but also to modify compounds with a potential use in food, cosmetic and pharmaceutical industries. Feruloyl esterases (FAEs, E.C. 3.1.1.73) have been proved promising candidates for the enzymatic synthesis of antioxidants allowing more flexible process configurations. Among the advantages they provide are use of lower temperatures (50-60 ◦ C) comparing to the counterpart chemical process (150о C), one step production of one product instead of mixtures and no need of by-product and catalyst residues removal in order to produce clean and high quality substances. Glucuronoyl esterase (GE) synthetic ability needs to be explored towards the production of alkyl branched glucuronic acid derivatives which are non-ionic surfactants and have good surface properties, including biodegradability. In addition, due to their tastelessness, non skin-irritation and non toxicity, these bioactive compounds find diverse uses in the cosmetic and pharmaceutical industries. Aim of this work is the development of competitive and eco-friendly bioconversions based on transesterification reactions catalyzed by FAEs and GEs, for the production of molecules with antioxidant activity, such as phenolic fatty and sugar esters. The synthesis of four biological active compounds (prenyl ferulate, prenyl caffeate, 5-O-(trans-feruloyl)-arabinofuranose, and glyceryl ferulate) was evaluated using recombinant FAEs from Myceliopthora thermophila and Fusarium oxysporum, while the
BIOCATALYSIS
PA-35 Screening of Lipase Microorganisms
Production
Among
Different
¸ ın 2 , Nihat Alpagu Sayar 1 Duygu Elif Yılmaz 1,∗ , H. Tansel Yalc 1
2
Marmara University Ege University
The design and manifecture of single enantiomers of chiral intermediates have been attracting attention in the pharmaceutical industry during the last decades. Single enantiomers that are potential biocatalysts due to their higher degree of enantioselectivity and regioselectivity are usually prepared by chemical catalysis. L-amino acid esters are promising intermediates for various active drug components and their catalytic biosythesis through transesterification reactions could be a rational alternative way. Besides, they can be effectively producted by using lipase from various microorganisms as a biocatalyst. The selection of the most appropriate organism among different strains can be a valuable starting point for this study. Various isolates from various potantial microorganisms which differ significantly from each other, may exhibit different catalytic effectiveness. The microbial isolates for this study are maintained by monthly transfers on MGYP agar slants and stored at + 5◦ C. For the production stage, microorgansims are transferred to MGYP preculture medium. After overnight incubation, this culture is inoculated to production medium including different types of oil. The culture is incubated in appropriate conditions. Samples are taken at different time intervals to determine the growth and lipase activity. The enzyme activity is shown by p-nitrophenyl palmitate (pNPP) as substrate. P-nitrophenol is obtained from pNPP and absorbance is measured spectrophotometrically against an enzyme free sample. One unit of lipase activity (U) is defined as the amount of enzyme which liberates 1 mol p-nitrophenol/min under assay conditions. Protein is measured by the Coomassie Blue G-250 binding method using bovine serum albümin as standard. http://dx.doi.org/10.1016/j.nbt.2014.05.1821
PA-36 A cutinase from Fusarium oxysporum with potential for PET surface modification Evangelos Topakas ∗ , Efstratios Nikolaivits, Maria Kanelli, Paul Christakopoulos National Technical University of Athens
Cutinases are small extracellular serine hydrolases whose natural function is the hydrolysis of the polyester cutin. Their ability to hydrolyze a wide range of substrates (from low molecular weight esters to high molecular weight polymers) makes cutinases very useful biocatalysts for various applications. In the present study, a cutinase (FoCut5a) from the ascomycete fungus Fusarium oxysporum was functionally overexpressed in Escherichia coli BL21, harboring pET22b(+)-cut16606. In order to improve enzyme sta-
S90
www.elsevier.com/locate/nbt
bility, the folding of the expressed protein took place also in the periplasm in addition to cytosolic protein production by cloning cut16606 gene downstream of the pelB signal peptide. The heterologous expression was induced with IPTG in 16о C for 20 h of incubation. The recombinant cutinase was purified from the cytosol or culture supernatant using immobilized-metal affinity chromatography (IMAC), resulting in a monomeric protein of ca. 23 kDa that is optimally active at 40 ◦ C. FoCut5a cutinase was tested for its potential use in surface modification of PET fabrics, with the intention to increase their hydrophilicity and to improve their properties. Towards this direction, the enzymatic hydrolysis of two model substrates bis(benzoyloxyethyl)terephthalate (1) and commercial bis(2-hydroxyethyl)terephthalate (2) was attempted, in order to evaluate the capability of the recombinant cutinase in PET modification. FoCut5a succeeded in hydrolyzing both models, releasing bis(2-hydroxyethyl)terephthalate and benzoic acid from substrate 1 and a bis(2-hydroxyethyl)terephthalate derivative in case of substrate 2. Experiments are in progress for the surface modification and functionalization of PET fibers. http://dx.doi.org/10.1016/j.nbt.2014.05.1822
PA-37 Synthesis of biological active compounds using carbohydrate esterases as biocatalysts Paul Christakopoulos 1,∗ , Io Antonopoulou 1 , Evangelos Topakas 2 1
2
Luleå University of Technology National Technical University of Athens
Various fungal and bacterial carbohydrate esterases represent appealing biocatalysts that have the ability not only to deconstruct plant biomass but also to modify compounds with a potential use in food, cosmetic and pharmaceutical industries. Feruloyl esterases (FAEs, E.C. 3.1.1.73) have been proved promising candidates for the enzymatic synthesis of antioxidants allowing more flexible process configurations. Among the advantages they provide are use of lower temperatures (50-60 ◦ C) comparing to the counterpart chemical process (150о C), one step production of one product instead of mixtures and no need of by-product and catalyst residues removal in order to produce clean and high quality substances. Glucuronoyl esterase (GE) synthetic ability needs to be explored towards the production of alkyl branched glucuronic acid derivatives which are non-ionic surfactants and have good surface properties, including biodegradability. In addition, due to their tastelessness, non skin-irritation and non toxicity, these bioactive compounds find diverse uses in the cosmetic and pharmaceutical industries. Aim of this work is the development of competitive and eco-friendly bioconversions based on transesterification reactions catalyzed by FAEs and GEs, for the production of molecules with antioxidant activity, such as phenolic fatty and sugar esters. The synthesis of four biological active compounds (prenyl ferulate, prenyl caffeate, 5-O-(trans-feruloyl)-arabinofuranose, and glyceryl ferulate) was evaluated using recombinant FAEs from Myceliopthora thermophila and Fusarium oxysporum, while the