Fabrika atıklarından izole edilen Bacillus sp.den aktif ve kararlı lipaz üretim koşullarının ve üretilen enzimin deterjan endüstrisinde kullanımının araştırılması
The investigation of active and stable lipase production conditions from Bacillus sp. isolated from factory wastewaters and of their utilization in detergent industry
Yazar: BAŞAK MARUL
Danışman: PROF.DR. SERPİL TAKAÇ
Ankara Üniversitesi / Biyoteknoloji Enstitüsü / Temel Biyoteknoloji Ana Bilim Dalı
Derece: Yüksek Lisans, 2007
Konu: Biyoteknoloji = Biotechnology ; Kimya Mühendisliği = Chemical Engineering ; Mikrobiyoloji = Microbiology
Dizin: Enzim aktivitesi = Enzyme activity ; Enzim üretimi = Enzyme production ; Lipaz = Lipase

Özet:
Lipazlar (E.C.3.1.1.3; trigliserol açilhidrolazlar) enzim siniflandirmasinda hidrolazlarana sinifinda yer alan, triaçilgliserollerin serbest yag asitlerine ve gliserole hidrolizinikatalizleyen enzimlerdir. Endüstriyel lipazlar, genellikle hücre disi lipaz üretenmikroorganizma türlerinden saglanir. Mikrobiyal lipazlar çogunlukla sivi ortamlarda üretilirve aktiviteleri karbon/azot kaynaklari tür ve derisiminden, ortam pH'sindan, sicakliktan vemetal iyonlarindan etkilenir. Bu çalismada; lipaz kaynagi olarak yagli fabrika atiklari ilekontamine olmus topraklardan izole edilen Bacillus'lar arasindan en yüksek enzim aktivitesigösteren seçilerek, lipaz üretiminde önem tasiyan ortam kosullari ile ortam bilesenleriparametre olarak incelenmistir. Karbon kaynaklari olarak ülkemizde yaygin olarak kullanilanbitkisel yaglarin (zeytin yagi, ayçiçek yagi, soya yagi, misir özü yagi, susam yagi), yagasitlerinin (triasetin, tribütirin, trioktanoat, tripalmitin, tristearin, triolein), yag asidiesterlerinin (palmitik asit, stearik asit, oleik asit, linoleik asit) ve glukozun farkliderisimlerinin; azot kaynaklari olarak ise bazi dogal kaynaklar (misir unu, soya unu vebugday unu) ile organik kaynaklarin (pepton, maya özütü, üre, jelatin) ve ayrica metaltuzlarinin (NaCl, Na2SO4, KCl, K2SO4, MgCl2, MgSO4, FeCl3, CaCl2, ZnCl2, LiCl, CuSO4)hücre disi ve hücre içi lipaz aktivitesi, hücre derisimi ve protein derisimi üzerine etkileriincelenmistir. Optimal ortam kosullarini belirlemek amaciyla, 6-9 baslangiç pH araligi ve 37-50 °C sicaklik araliginda üretimler yapilarak bu degiskenlerin lipaz aktivitesine etkileriincelenmistir. Yapilan deneyler sonucunda Bacillus sp.'nin hücre disi lipaz ürettigi, en uygunkarbon kaynaginin % 1 (h/h) derisimde susam yagi, en uygun azot kaynaginin % 0.5 (a/h)pepton + % 0.3 (a/h) maya özütü ve lipaz üretimini en fazla indükleyen metal tuzunun 1 mMK2SO4 oldugu belirlenmistir. Optimal ortam kosullari ise pH=7.5 ve T=45 °C olarakbulunmustur. Üretilen enzim ultrafiltrasyon ve anyon degistirici kromatografi ile kismisaflastirilmis ve enzime olasi deterjan katkilarinin etkisi (proteaz, Tween 20, Tween 80,Triton X-100, EDTA, SDS) belirlenerek elde edilen lipazin endüstriyel kullanim potansiyeliortaya konulmustur.Anahtar Kelimeler: Deterjan Enzimleri, Enzim Üretimi, Lipaz, Lipaz Aktivitesi

Abstract:
Lipases (E.C.3.1.1.3; trigliserol acylhydolyses), belong to the hydrolase family ofenzymes, hydrolyze triacylglycerides into fatty acids and glycerol. Industrial lipases aremainly produced from microorganisms that secrete extracellular lipase. Microbial lipases areusually produced by submerged fermentation and their activities are influenced by the typeand concentration of carbon/nitrogen sources, pH, temperature, and metal ions. In the presentstudy, a Bacillus strain exhibiting the highest lipase activity was selected amongst isolatedfrom soil contaminated by olive oil plants wastewater; and the effects of medium conditionsand components were investigated for a higher enzyme activity. Vegetable oils (olive,sunflower, soybean, corn and sesame oils), triacylgliserides (triacetin, tributyrin, trioctanoat,tripalmitin, tristearin and triolein), fatty acids (palmitic, stearic, oleic and linoleic acids), andglucose were used for their different concentrations as carbon sources. Effects of some naturalsources (corn flour, soybean flour and wheat mill bran) in addition to conventional sources(peptone, yeast extract, urea, gelatin) as nitrogen sources; and NaCl, Na2SO4, KCl, K2SO4,MgCl2, MgSO4, FeCl3, CaCl2, ZnCl2, LiCl, CuSO4 as salts of metal ions on extracellular andintracellular lipase activities were investigated. The effects of initial pH and temperature onthe lipase activity were investigated within 6-9 and 37-50 oC ranges, respectively. Sesame oil(1% v/v) and the combination of peptone (0.5% w/v) + yeast extract (0.3 % w/v) were foundto be the best carbon and nitrogen sources, respectively. 1 mM K2SO4 favored the highestlipolytic activity. The optimal temperature and initial pH for lipase activity were 45oC and7.5, respectively. The lipase produced under optimal conditions partially purified byultrafiltration and anion exchange chromatography; and its molecular mass found to be about40 kDa by SDS-PAGE. The purified enzyme was tested for its activity recovery in thepresence of protease, Tween 20, Tween 80, Triton X-100, EDTA and SDS; and its potentialutilization in industry was discussed.Key Words: Detergent Enzymes, Enzyme Production, Lipase, Lipase Activity