| Summary: | Bu çalışmada, enzimatik halka açılımı polimerizasyon yöntemi kullanılarak polilaktik asit sentezi gerçekleştirilmiş ve elde edilen polimerin proteaz enzimi ile biyobozundurulması incelenmiştir. Candida cylindracea lipaz katalizörlüğünde laktitten halka açılımı reaksiyonunun optimize edilmesi amacıyla, reaksiyon şartları değiştirilerek farklı sıcaklıklarda gerçekleştirilen polimerizasyon işlemlerinde, reaksiyona giren Candida cylindracea lipaz konsantrasyonuna bağlı olarak oluşan polilaktik asitin molekül ağırlığı, polidispersitesi ve dönüşüm verimi değerleri tespit edilmiştir. Elde edilen polilaktik asit, proteaz DSM enzimi ile biyobozundurma işlemine tabii tutulmuş ve polimerin yapısındaki ester grupları ile biyobozundurma mekanizması takip edilmiştir. Biyobozunma süresince molekül ağırlığı kaybı izlenmiş ve orjinal polimerin morfolojik ve termal geçiş özelliklerinin değişimi incelenmiştir. Candida cylindracea lipaz kullanılarak laktitin halka açılımı polimerizasyonunda, yüksek molekül ağırlıklı polimer sentezinde, en uygun reaksiyon şartı, sıcaklığın 80ºC ve lipaz konsantrasyonunun ağırlıkça %4 olduğu durum olarak belirlenmiştir. Bu sıcaklık değerinde, sabit enzim konsantrasyonunda, sürenin artmasıyla birlikte molekül ağırlığı ve monomer dönüşümü belirgin bir şekilde artış göstermiş, polidispersite değerleri yaklaşık 1.5 civarında olan polimerler sentezlenmiştir. Proteaz DSM ile yapılan biyobozundurma işlemi sonucunda ise, ilk 15 günlük süre içersinde, polimerin bozunmasına ilişkin molekül ağırlığı kaybı son derece düşük seviyede olduğu, bu süreden sonra molekül ağırlığı kaybında belirgin bir artış gözlenerek 90 günlük süre sonunda ’lük bir kayıp meydana geldiği saptanmıştır. Biyobozundurma işlemine tabii tutulan polimerin çeşitli özellikleri incelenerek orijinal polimer ile arasındaki farklar tespit edilmiştir.Anahtar Kelimeler: Lipaz, halka açılımı polimerizasyonu, biyobozunma. Because of its biocompatibility and degradability to non-toxic products, polylactic acid (PLA) based polymers and copolymers have been employed in novel applications, such as absorbable bone plates, artificial skin, tissue scaffolds, and carriers of drugs for controlled release systems. Enzymatic polymerization using lipase has been receiving much attention as one of the new methodologies that provide biodegradable polymer synthesis without toxic catalysts. Lipases are the most versatile biocatalysts because they can be applied to the synthesis of a wide range of substrates with a high stereospecificity and enantioselectivity. In general lipases used in polyester synthesis are of mammalian (Porcine pancreatic lipase (PPL)), fungal (Candida antarctica lipase B (CAL)), or bacterial origin (Pseudomonas cepacia (PCL)). The hydrolytic degradation of PLA polymers has been extensively investigated. It is now well know that degradation of macroscale PLA devices is heterogeneous: it is faster inside than at the surface. This phenomenon has been assigned to an internal autocatalytic effect of carboxyl end groups. Biodegradation of PLA polymers in soil under natural conditions has also been examined. It was reported that degradation by-products resulting from the hydrolytic degradation can be totally assimilated by microorganisms such as fungi, bacteria, or earthworms, thus indicating that PLA polymers are environmentally benign materials. Hydrolysis reactions may be catalyzed by enzymes known as hydrolases, which include proteases, esterases, glycosidases, and phosphatases, among others. This class of enzymes comprises cell-derived proteins that are responsible for the catalysis of several reactions in the human body. The degradation of PLA in the presence of enzymes has been investigated by many groups. Early in 1981, it was found that proteases showed strong effects on PLLA degradation. In this work, polylactic acid was prepared by lipase catalyzed ring opening polymerization of lactide. Enzymatic polymerization was performed with Candida cylindracea lipase, enzyme and the effect of enzyme concentration, temperature and time parameters on polymerization mechanisms were investigated in detail. A typical procedure for the polymerization of lactide using Candida cylindracea lipase is described as follow. To a mixture of lactide (2.50 mmol) and toluene (0.72 mL) at 80 and 100oC was added Candida cylindracea (2 and 4 wt.-%) under nitrogen atmosphere at different time (1-7 days). After the reaction, the enzyme was removed by filtration and the polymer was isolated by precipitation in choloform. Then degradation mechanisms were examined. PLLA films were prepared using a solvent casting method. One gram of PLLA was dissolved in 20 mL of chloroform while mixing vigorously at room temperature. The dissolved solution was poured onto a leveled Teflon film coated glass plate, spread evenly with a bent glass rod and then allowed to dry for about 24 h at room temperature. The resultant film was peeled from the casting surface. Each of polymeric films were placed in test tubes and dipped in 5 mL of DSM protease solution (50 mM Tris-HCl buffer, pH 8.6). The test tube was sealed and kept constant at 37°C for a predetermined period and enzyme solutions were added every 48 h so that enzyme activity remained at a desired level throughout the experiment. The time course of the weight loss and enzymatic degradation were evaluated and the appearance of the samples was examined. It was found that the L-lactide was polymerized in bulk by Candida cylindracea lipase between 3 and 7 days, in a temperature range 80-100oC to yield the polylactide with an Mw of up to 69800 g.mol-1. Both the conversion and Mw of the resultant polymeric fraction increased with increasing reaction time 3 days to 7 days respectively at constant lipase concentrations. The chemical structures of the degraded polymer samples were characterized using DSC, FTIR and XRD. The molecular weight of the polymer samples after enzymatic degradation process with protease DSM, decreased 25% after 2 months degradation period. PLA polymers investigated in this study showed degradation as illustrated by the decrease in molecular weight, disappearance of amorphous phase and formation of micro fractures and acicular structures. After detailed evaluation of biocompatibility and toxicity of PLAs, it is expected that they will take the petroleum-based plastics' place in future. Keywords: Lipase, ring opening polymerization, biodegradation.
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