Enzimatik polimerizasyon yöntemi ile polivalerolakton sentezi ve karakterizasyonu
Synthesis and characterization of polyvalerolacton via enzymatic polymerization
Yazar: ZEYNEP GÖK
Danışman: PROF. DR. FATOŞ YÜKSEL GÜVENİLİR
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Mühendisliği Ana Bilim Dalı / Kimya Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2018
Konu: Kimya Mühendisliği = Chemical Engineering
Dizin: Biyopolimerler = Biopolymers ; İmmobilize enzimler = Immobilized enzyme

Özet:
Polimer teknolojisinde biyopolimer eldesi son yıllarda giderek gelişmektedir. Bu gelişme biyopolimerlerin üstün özelliklerine dayanmaktadır, toksik olmamak, mekanik olarak yüksek dayanıma sahip olmak, kimyasal açıdan kararlı olmak, çevreye zararlı olmamak öne çıkan gelişme sebepleridir. Bunun yanında polimer teknolojisinde enzimatik polimerizasyon yöntemi de önem kazanan bir alan olarak öne çıkmaktadır. Enzimatik polimerizasyon diğer kataliz yöntemlerine göre daha düşük sıcaklık ve basınçlarda reaksiyon ortamı sunmaktadır. Bu sayede enerji tasarrufuna imkanı vermekte ve katalizlerden kaynaklanan toksik etkinin önüne geçmektedir.
Enzimatik polimerizasyonda polyesterler önemli bir yer tutmaktadır. Siklik ester olan laktonlar halka açılması polimerizasyonu ile polimerleşmektedirler. En çok bilinen ve üzerinde çalışılan lakton ɛ-kaprolakton olmakla birlikte δ-valerolakton ile de yüksek molekül ağırlıklı polimerler üretilebileceği bilinmektedir.
Bu çalışmada enzimatik halka açılması polimerizasyonu yöntemi ile polivalerolakton homopolimeri ve polikaprolakton-polivalerolakton kopolimeri sentezlenmiştir.
Çalışmanın ilk aşamasında polivalerolakton sentez reaksiyonuna ortam koşullarının etkisi araştırılmış bu amaçla farklı sıcaklık, reaksiyon ortamı ve enzim türü ile denemeler yapılmıştır. δ-valerolakton monomeri Novozyme 435 ticari enzimi ve pirinç kabuğu külüne fiziksel adsorpsiyon ile immobilize CALB L enzimi ile farklı sıcaklıklarda sabit monomer/enzim oranında, 120 rpm karıştırıcı hızında ve inert azot ortamında reaksiyona sokularak polivalerolakton sentezlenmiştir. Polivalerolakton için iki enzim türünde de en iyi sıcaklık koşulu belirlenmiş ilerleyen sentezlere belirlenen sıcaklıklarda devam edilmiştir. Reaksiyon ortamının polivalerolakton sentezindeki etkisinin belirlenmesi amacıyla farklı reaksiyon ortamlarında denemeler yapılmış ve sentez için en uygun ortamı belirlemek hedeflenmiştir.
Çalışmanın ikinci aşamasında ise kopolimer sentezine geçilmiştir. Kopolimer sentezi homopolimer sentezinde iki enzim için de ayrı ayrı belirlenmiş olan en iyi sıcaklıkta gerçekleştirilmiş. Monomer oranının kopolimer reaksiyonundaki etkisinin belirlenmesi hedeflenmiştir, farklı oranlarda monomer kullanarak denemeler yapılmıştır.
Elde edilen homopolimer ve kopolimerlerin molekül ağırlıkları dağılımı jel geçirgenlık kromatografisi (GPC) ile belirlenmiştir. Termal özellikleri için diferansiyel taramalı kalorimetri (DSC) ve termal gravimetrik analiz (TGA) analizleri kullanılmıştır. Kimyasal yapı için Fourier dönüşümlü kızılötesi spektrometrisi (FT-IR) ve proton nükleer manyetik rezonans (1H-NMR) analizleri uygulanmış ve elde edilen tüm sonuçlar karşılaştırılmıştır.

Abstract:
Prior to polymer production on the commercial scale, people used glass, wood, stone, brick, cement, steel, natural fibers such as wool, linen, cotton in their clothing and weaving needs in the making of goods and materials they used in daily life. As a result of the developments in polymer production since the 1930's, a marked increase in the diversity of human-produced materials has begun to be observed. These materials have made life easier for people.
Most of the synthetic polymers used today are petroleum based. However, alternatives have begun to be sought because of basic reasons such as increased oil prices, especially environmental concerns, and exhaustion associated with the massive use of fossil fuels. This is the basis for accelerating and improving the work for bio-based polymer production.
Biopolymer manufacturing in polymer technology has been developing in recent years. This development is based on the superior properties of biopolymers such as nontoxic, has high mechanical strength, chemically stable, not harmful to the environment. In addition, the enzymatic polymerization method in polymer technology is also an important area. Enzymatic polymerization offers reaction media at lower temperatures and lower pressures than other catalysis methods. This provides energy savings and avoids the toxic effects of catalysis.
Polyesters have an important place in the enzymatic polymerization. Lactones, which are cyclic esters, polymerize by ring opening polymerization. It is known that the most well-known and studied lactone is ɛ-caprolactone on the other hand δ-valerolactone can also produce high molecular weight polymers.
δ-valerolactone is a renewable monomer having a melting point of -13°C, a boiling point of 226 to 229°C, and a flash point of 112°C. It has low toxicity and high solubility in water, which facilitates biological degradation. Polivalerolactone is an important hydrophobic and semi-crystalline aliphatic polyester because it is biodegradable and biocompatible and has permeability properties. They are structurally similar to polycaprolactone, another aliphatic polyester. Polivalerolactone is very unlikely to be toxic to the cell in which it is used. It also has the ability to be easily mixed with other polymer materials
These polymers, which have biodegradability, biocompatibility and superior mechanical properties, are enzymatically synthesized and therefore do not contain toxic chemical catalyst residues. This allows for use in biomedical applications such as drug delivery systems, tissue engineering and medical devices (implant, prosthesis, surgical thread, etc.).
Biocatalysts are biologically derived enzymes with chemical enzyme properties used in chemical reactions. Biocatalysts have many advantages over conventional catalysts, and their advantages are based on activation energy (Ea). The primary determinant of reaction rate value for any catalysis is Ea . Biocatalysis decreases Ea value.
Lipases have been increasing their place and importance in the enzyme market in recent years thanks to their distinct advantages compared to classical enzymes. The advantages they have are that they provide moderate environmental conditions in reactions, do not create side reactions, facilitate the control of reactions, and so on.
Candida antartica lipase B is the most well known and used biocatalyst and is widely used in hydrolysis, esterification, transesterification, aminolysis applications. The molecular weight of CALB is 33 kD at 317 amino acid residues, isoelectric point 6.0. The active site is the Ser105-His224-Asp187 catalytic triad.
Immobilization is needed to improve the use of enzymes in unnatural environments. The immobilized CALB enzyme is highly resistant to heat, even at high concentrations, maintaining its activity for a long time, especially in anhydrous environments. However, lipase shows relatively rapid denaturation in aqueous solutions even at temperatures as low as 40°C.
The commercially available CALB enzyme (Novozyme 435) was immobilized on macroporous acrylic polymer resin with an unknowned protocol. Although Novozyme 435 is an effective catalysis in enzymatic polymerisation applications, high costs and diffusion limitations restrict the use.
In this work, polyvalerolactone homopolymer and poly caprolactone-poly valerolactone copolymer were synthesized by enzymatic ring-opening polymerization method. Two different enzymes were used as catalysis. These enzymes are commercial enzyme, novozyme 435, and home-made enzyme, Candida antartica lipase (CALB L), modified by physical adsorption of immobilized silica-based material rise hush ash.
The homemade enzyme was produced by physical adsorption method on rice hush ash. Begin with, the rice hush was burned for 6 hours at 600-650℃ in an furnace. Subsequently, surface modification of RHA was provided using a silanation agent 3-aminopropyl triethoxysilane (3-APTES) and functional amine (-NH2) groups were added to the surface. Immobilization of the lipase was accomplished by physical adsorption.
In the first step of the study, polyvalerolactone homopolymer was synthesized using different temperature and time for two different enzymes. Syntheses were made at four different temperature values, which are 30, 40, 60 and 80℃, five different times values, which are 6, 24, 48, 72 and 120 hours. The optimum temperature and time values for both enzyme types were determined at the end of the syntheses.
The polymer reaction was carried out by the enzymatic ring opening method and the enzyme monomer concentration was kept constant for all reactions. The toluene medium was first selected as the reaction medium.
Since the δ-valerolactone monomer is stored at -20°C, it will be kept in the room temperature for a while before passing to the liquid form. After the enzyme, monomer and toluene are reacted at the specified ratios, the reaction will be carried out in the inert nitrogen atmosphere so that the monomer does not react with air. To terminate the reaction, the chloroform will be subjected to filtration to separate the enzyme from the medium to be used. The polymer obtained by evaporating chloroform in the filtrate containing the polymer and chloroform is precipitated using methanol and the precipitate is dried to obtain the required analysis of the polymer samples.
The highest values were obtained at 40ºC at the end of 24 h for commercial enzyme, novozyme 435, which was 9010 g/mol. At 80ºC, a molecular weight of 8040 g/mol was obtained by using modified by physical adsorption of immobilized silica-based material rise hush ash which is the best result of this enzyme. Since the best result was obtained at 80℃, additional experiments were made at 90℃.
After the optimum temperature was determined, the reaction medium was changed and the effect of the reaction medium on the polymerization was investigated. The polymerization reaction was carried out using nonsolvent and n-hexane to determine the effect of the reaction medium.
At the end of the polymer reactions, optimum temperature and reaction media for the enzymes were determined. And copolymerization reactions were carried out under these conditions.
In the second part of the study, the copolymer was synthesized by the enzymatic ring opening polymerization at the determined optimum temperature value for two different enzymes. The highest value was obtained at 40℃ at the end of 6 h for %75 δ-valerolactone with commercial enzyme which was 15100 g/mol. At 80ºC, a molecular weight of 15100 g/mol was obtained by using modified by physical adsorption of immobilized silica-based material rise hush ash which is the best result of this enzyme.
Molecular weight distribution of the obtained homopolymers and copolymers was determined by gel permeation chromatography (GPC). Differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) analyzes were used for thermal properties. Fourier transform infrared spectrometry (FT-IR) and proton nuclear magnetic resonance (1H-NMR) analyzes were performed for the chemical structure and all the results obtained were compared.