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Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80-pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)-1-phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R-S)-1-phenylethanol with vinylacetate.

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Interfacial activation via surfactant (Tween 80, Triton X-100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross-linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non-pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899-904, 2016.

Porcine pancreatic lipase (EC 3.1.1.3) was covalently immobilized onto 2,4,6-trichloro-s-triazine (cyanuric chloride) activated polyvinyl alcohol (PVA). The influence of activating agent and enzyme concentration on the immobilization process were evaluated. Hydrolytic activities of free and immobilized enzyme were determined and the immobilization yield was estimated by measuring the quantity of protein, both in free enzyme solution and in washing solutions after immobilization. After the optimization of immobilization process, the physical and chemical characterization of immobilized enzyme was performed. Additionally, the thermal, pH, storage, and operational stability of the immobilized and free enzymes were tested. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme.

Porcine pancreatic lipase was covalently immobilized on polyvinyl alcohol using adipoyl dichloride as a cross-linker. The effect of pre-treatment of lipase previously with various types of oils on immobilization efficiency was investigated. The increment in immobilization efficiency was observed after pre-treatment of lipases with oils. The highest immobilization efficiency obtained was 20% (v/v) for olive oil pre-treated lipase, which was 8 times higher than that of non-pretreated immobilized lipase. Immobilized lipase had better stability and had some advantages in comparison with free enzyme.

Production and partial purification of extracellular lipase produced by a novel yeast strain Candida odintsovae TY42[Yeni bir maya suşu olan Candida odintsovae TY42 tarafından üretilen hücre dışı lipazın üretimi ve kısmi saflaştırılması] ABSTRACT Objective: Nowadays, in most of industrial technology, especially biotechnological processes, use of enzymes is increasingly widespread. With respect to discovery of new enzymes, one of the most successful methods is isolation of microorganisms from natural habitats. Therefore, in this research, we aimed to isolate and characterize lipase producing yeasts and purify the extracellular lipase produced by a novel yeast strain Candida odintsovae TY42. Methods:The yeasts used in the study were isolated from olive oils and identified based on sequence analyses of ITS1-5.8S rRNA-ITS2 and D1/D2 domain of 26S rRNA. The optimal nutritional and physicochemical conditions were determined by investigating the effects of initial pH and temperature and different carbon sources on enzyme production in basal medium. The lipase was purified by ammonium sulfate and acetone precipitation and anion exchange chromatography. Results: Three lipase producing yeast isolates were obtained from olive oils produced in Aegean region. The sequencing of rRNA regions revealed that novel strains, TY17, TY42 and TY54 identified as Candida odintsovae. C. odintsovae TY42 showed higher lipase activity and therefore, the yeast was selected for further investigations. The maximum lipase production was achieved in a medium containing fish oil after 144 h of incubation. The lipase was purified 12.8 fold. The molecular weight of the purified lipase was estimated to be about 39 kDa. Conclusion: This paper is the first study representing lipase production by C. odintsovae. Key Words: Candida odintsovae, enzyme purification, extracellular lipase, lipase production. Conflict of Interest:The authors declare no conflict of interest. ÖZETAmaç: Günümüzde, endüstriyel teknolojilerin çoğunda, özellikle de biyoteknolojik işlemlerde enzimlerin kullanımı giderek yaygınlaşmaktadır. Yeni enzimlerin keşfi ile ilgili olarak, en başa-rılı metotlardan bir tanesi, doğal habitatlardan mikroorganizmaların izolasyonudur. Bu yüzden bu araştırmada, lipaz üretici mayaların izolasyonu ve karakterizasyonu ve yeni bir maya suşu olan Candida odintsovae TY42 tarafından üretilen hücre dışı lipazın saflaştırılması amaçlan-mıştır. Metod: Bu çalışmada kullanılan mayalar, zeytinyağlarından izole edilmiş ve ITS1-5.8S rRNA-ITS2 ve 26S rRNA D1/D2 domaininin dizi analizine göre tanımlanmıştır. Optimal besinsel ve fizikokimyasal koşullar, başlangıç pH, sıcaklık ve farklı karbon kaynaklarının bazal ortamda enzim üretimi üzerine etkileri incelenerek saptanmıştır. Lipaz, amonyum sülfat ve aseton çök-türmesi ve anyon değişim kromatografisi ile saflaştırılmıştır. Bulgular: Lipaz üretici maya izolatları, Ege bölgesinde üretilen zeytinyağlarından elde edilmiştir. rRNA bölgelerinin dizi analizi, bu yeni suşların, TY17, TY42 ve TY54, Candida odintsovae'ye dahil...