Aziz Tanriseven

Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer.

This study aims at carrying out lipase-catalyzed synthesis of fatty acid methyl esters (biodiesel) from various vegetable oils using lipase immobilized onto a novel microporous polymeric matrix (MPPM) as a low-cost biocatalyst. The research is focused on three aspects of the process: (a) MPPM synthesis (monolithic, bead, and powder forms), (b) microporous polymeric biocatalyst (MPPB) preparation by immobilization of lipase onto MPPM, and (c) biodiesel production by MPPB. Experimental planning of each step of the study was separately carried out in accordance with design of experiment (DoE) based on Taguchi methodology. Microporous polymeric matrix (MPPM) containing aldehyde functional group was synthesized by polyHIPE technique using styrene, divinylbenzene, and polyglutaraldehyde. Thermomyces lanuginosus lipase was covalently attached onto MPPM with 80%, 85%, and 89% immobilization efficiencies using bead, powder, and monolithic forms, respectively. Immobilized enzymes were successfully used for the production of biodiesel using sunflower, soybean, and waste cooking oils. It was shown that immobilized enzymes retain their activities during 10 repeated batch reactions at 25 degrees C, each lasting 24h. Since the developed novel method is simple yet effective, it could have a potential to be used industrially for the production of chemicals requiring immobilized lipases.

Immobilization of invertase within calcium alginate gel capsules

Abstract Saccharomyces cerevisiae invertase was immobilized in alginate capsules. The immobilization resulted in 87% relative activity and activity for 36 days without decrease in activity. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4.3 and 60°C, respectively. Immobilized invertase was however, more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. Since the process is simple and invertase does not leak out of capsules, this method can be used for the industrial production of invert sugar.

Covalent attachment of microbial lipase onto microporous styrene–divinylbenzene copolymer by means of polyglutaraldehyde

A novel method for immobilization of Thermomyces lanuginosus lipase onto polyglutaraldehyde-activated poly(styrene-divinylbenzene) (STY-DVB), which is a hydrophobic microporous support has been successfully developed. The copolymer was prepared by the polymerization of the continuous phase of a high internal phase emulsion (polyHIPE). The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. Lipase from T. lanuginosus was immobilized covalently with 85% yield on the internal surface of the hydrophobic microporous poly(styrene-divinylbenzene) copolymer and used as a biocatalyst for the transesterification reaction. The immobilized enzyme has been fully active 30 days in storage and retained the activity during the 15 repeated batch reactions. The properties of free and immobilized lipase were studied. The effects of protein concentration, pH, temperature, and time on the immobilization, activity, and stability of the immobilized lipase were also studied. The newly synthesized microporous poly(styrene-divinylbenzene) copolymer constitutes excellent support for lipase. It given rise to high immobilization yield, retains enzymatic activity for 30 days, stable in structure and allows for the immobilization of large amount of protein (11.4mg/g support). Since immobilization is simple yet effective, the newly immobilized lipase could be used in several application including oil hydrolysis, production of modified oils, biodiesel synthesis, and removal of fatty acids from oils.

Production of isomalto-oligosaccharides using dextransucrase immobilized in alginate fibres

Abstract Isomalto-oligosaccharides were produced using Leuconostoc mesenteroides B-512 FM dextransucrase immobilized in alginate fibres. Immobilization resulted in 90% relative activity. The immobilized enzyme retained activity for ten batch reactions without a decrease in activity and was reacted with a substrate composed of sucrose (1 M) and glucose (1 M) to obtain isomalto-oligosaccharides. Immobilization of dextransucrase in alginate fibre is superior to that in alginate beads in terms of immobilization yield and repetitive use.

A novel method for the immobilization of glucoamylase to produce glucose from maltodextrin

Immobilized glucoamylase from Aspergillus niger was used to produce glucose from maltodextrin. The enzyme was immobilized using a newly developed combined method of adsorption of the enzyme to gelatinized corn starch and subsequent alginate fiber entrapment. The immobilization resulted in 22% relative activity and has been active for 21 days without decrease in activity. The optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4.3 and 60°C, respectively. The kinetic parameters were also determined for the hydrolysis of maltodextrin by free and immobilized glucoamylase.

Novel method for the production of a mixture containing fructooligosaccharides and isomaltooligosaccharides

A sugar mixture containing fructooligosaccharides and isomaltooligo-saccharides was produced. Sucrose was converted to fructooligosaccharides by a commercial enzyme preparation. The sugar mixture contained kestose (33.5%), nystose (13.3%), fructofuranosyl nystose (5.7%), glucose (20.9%), and unreacted sucrose (26.6%). The unreacted sucrose was converted to isomaltooligosaccharides by reacting the sugar mixture with Leuconostoc mesenteroides B-512FM dextransucrase. The final product comprised fructooligosaccharides (kestose, nystose, fructofuranosyl nystose), isomaltooligosaccharides (isomaltose through isomaltodecaose), glucose, and fructose.

A new lipase as a pharmaceutical target for battling infections caused by Staphylococcus aureus: Gene cloning and biochemical characterization

Staphylococcus aureus lipases along with other cell‐wall‐associated proteins and enzymes (i.e., catalase, coagulase, protease, hyaluronidase, and β‐lactamase) play important roles in the pathogenesis of S. aureus and are important subject of drug targeting. The appearance of antibiotic‐resistant types of pathogenic S. aureus (e.g., methicillin‐resistant S. aureus, MRSA) is a worldwide medical problem. In the present work, a novel lipase from a newly isolated MRSA strain from a cow with subclinical mastitis was cloned and biochemically characterized. The mature part of the lipase was expressed in Escherichia coli and purified by nickel affinity chromatography. It displays a high lipase activity at pH 8.0 and 25 °C using p‐nitrophenyl palmitate and has a preference for medium–long‐chain substrates of p‐nitrophenyl esters (pNPC10–C16). Furthermore, in search of inhibitors, the effect of farnesol on the growth of S. aureus and the lipase activity was also studied. Farnesol inhibits the growth of S. aureus and is a mixed‐type inhibitor with Ki and Ki′ values of 0.2 and 1.2 mmol L−1, respectively. A lipase with known properties could not only serve as a template for developing inhibitors for S. aureus but also a valuable addition to enzyme toolbox of biocatalysis. The discovery of this lipase can be potentially important and could provide a new target for pharmaceutical intervention against S. aureus infection.

Mutation of AN39-1 for production and characterization of constitutive, thermostable and pH-resistant dextransucrase / Konstitutif, sıcaklığa ve pH’ya dayanıklı dekstransukrazların üretimi ve karakterizasyonu için AN39-1’in mutasyonu

Abstract Objective: Leuconostoc mesenteroides AN39-1 has recently been isolated from Crataegus orientalis var. Orientalis. It produces inducible extracellular dextransucrase (EC 2.4.1.5) forming dextran from sucrose. The aim of this study was (1) to obtain constitutive, pH-resistant and thermostable dextransucrase, (2) to characterization of these dextransucrase. Methods: Mutagenesis was carried out on the parent strain (AN39-1) using UV, ethyl methane sulfonate, and N- methyl- N´-nitro-N-nitrosoguanidine. Dextransucrases from wild type (AN39-1) and the mutant strain (A26-2/11) were purified by polyethylene glycol (PEG) precipitation and characterized. Results: Mutants (A26, A26-2, and A26-2/11) hyper producing and constitutive for dextransucrase were isolated. The mutants (A26, A26-2, A26-2/11) produced 7.2, 8.1, and 2.0 times more dextransucrase activity as compared to parent strain on sucrose medium, respectively. In addition, the mutants produced dextransucrase on glucose medium with higher activities (3.0-5.8 times) than what the parental strain produced on sucrose medium. The mutant enzyme (A26-2/11) was much more thermostable than the native enzyme and resistant to pH more than dextransucrase of AN39-1. The dextransucrase from mutant strain was stable up to 35°C and pH of 7.5 for 3 hr. Conclusion: The structures of dextrans produced by wild type and mutant enzymes were similar to commercially produced B-512 F dextran. Thus, the newly dextransucrases produced by mutant strain could find industrial applications at higher temperature and pH. Özet Amaç: Leuconostoc mesenteroides AN39-1, Crataegus orientalis var. Orientalis.’den izole edilmiştir. Bu izolat sukrozdan dekstran sentezini gercekleştirebilen ve induklenebilen hucre dışı dekstransukraz (EC 2.4.1.5) uretmektedir. Bu calışmada, konstitutif uretilebilen ve sıcaklık ile pH’ya direncli dekstransukraz sentezleyebilen mutantların elde edilmesi amaclanmıştır. Metod: Ana soy (AN39-1) UV, etil metan sulfonat ve Nmetil- N´-nitro-N-nitrozoguanidin ile mutasyona uğratılıp konstitutif dekstransukraz uretebilen mutantlar elde edilmeye calışılmıştır. Ana soyun (AN39-1) ve mutantının dekstransukrazı polietilen glikol (PEG) ile cokturulerek saflaştırılıp karakterize edilmişlerdir. Bulgular: Mutasyon calışmaları sonrasında, dekstransukrazı konstitutif ve yuksek miktarlarda ureten mutantlar (A26, A26-2, and A26-2/11) elde edilmiştir. Bu mutantlar (A26, A26-2, and A26-2/11) sukroz medyumda ana soya oranla sırasıyla 7.2, 8.1 ve 2.0 kat daha fazla dextransukraz aktivitesine sahiplertirler. Ayrıca, bu mutantlar glukoz medyumda, ana soyun sukroz medyumdaki aktivitesinden 3.0-5.8 kat daha fazla dekstransukraz aktivitesine sahiptirler. Mutant suşa ait enzimin ana soyun sentezlediği enzime oranla pH’ya ve ısıya daha direncli olduğu belirlenmiştir. Mutant suşun sentezlediği enzim pH 7.5’de 35°C’ye kadar 3 saat stabildir. Sonuç: Ana soyun ve mutantının sentezlediği enzimlerin urunu olan dekstranlar ticari boyutta uretilen B-512F dekstran ile benzer yapıdadır. Bu nedenlerle mutant suşa ait yeni uretilen dekstransukrazlar daha yuksek pH ve sıcaklıklarda daha fazla urun elde edilmesi amacı ile endustriyel uygulamalarda kullanılabilirler.