Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
Browse
3 results
Search Results
Master Thesis Production of Commercially Suitable Pectin Methylesterase and Polyphenol Oxidase From Agro-Industrial Wastes(Izmir Institute of Technology, 2004) Şimşek, Şebnem; Yemenicioğlu, Ahmet; Yemenicioğlu, Ahmet; 03.08. Department of Food Engineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyIn this study, some simple and effective extraction and/or partial purification procedures were developed to obtain pectin methylesterase (PME) and polyphenol oxidase (PPO) enzymes from orange peels and mushroom stems, respectively. Also, some characteristics of enzymes were investigated and their stable preparations were obtained in liquid or lyophilized forms. Valencia orange peels contain considerable PME activity (300-350 mL NaOH/min/100 g) that is quite stable during season for at least 5 months. The enzyme was ionically bound to cell walls and can not be extracted by homogenization with water. However, the addition of suitable amounts of NaCl (10 g /100 g extraction mixture) to pellet, obtained by homogenization of peels several times with water, and 30 min mixing (at 200 rpm) may be effectively used to extract the enzyme. The PME in orange peels contains almost the same amount of heat stable and heat labile fractions and the enzyme can not be activated by mild heating. A slight activation (almost 20 %) may be achieved by adding 1 mM CaCl2 to enzyme extracts. However, at higher concentrations the addition of CaCl2 was inhibitory. The PME activity in extracts, stabilized by use of 0.1 % Na-benzoate and 0.1 % K-sorbate, is stable almost 5 months at + 4 oC (maintains > 90 % of its activity). Thus, the commercial preparations of enzyme may be obtained in liquid form. The extracted PME was successfully used to prepare edible films from citrus pectin For the extraction of PPO, on the other hand, mushroom stems were first processed to acetone powder. The acetone powders were then extracted with Na-phosphate buffer and partially purified with ammonium sulfate (90 % saturation) or acetone precipitation (2-fold). Following dialysis, the recoveries and purification folds obtained from the partial purification of monophenolase activity of PPO from the same acetone powder were 74-86 % and 3.4-4.3 and 55-67 % and 5.4-6.2 for ammonium sulfate and acetone precipitations, respectively. Thus, it appears that the ammonium sulfate precipitation gives a higher yield but lower purity. The monophenolase activity of partially purified PPO is heat labile and showed inactivation above 45 oC. The enzyme exhibited a pH optimum between pH 6.0 and 8.0. The pH stability of enzyme was maximal at pH 7.0 and 8.0. However, at pH 4.0 the enzyme lost most of its activity after 24 h incubation. The optimum temperature of enzyme was found as 40 C. The monophenolase activity of PPO enzyme showed no stability in acetone powders at + 4 oC. However, it showed good stability at -18 oC for two months with retention of 60-70 % of its activity. The PPO partially purified with ammonium sulfate precipitation and dialysis, and lyophilized by using dextran or saccharose as supporting materials also retained its monophenolase and diphenolase activities for three months at -18 oC. The effect of lyophilization with dextran on temperature stability of enzyme was insignificant. However, lyophilization with dextran reduced the pH stability of monophenolase activity at 4.0 moderately. In addition to its monophenolase activity on tyrosine and diphenolase activity on L-DOPA, PPO lyophilized with dextran can also use phloridzin as substrate. Thus, it appears that the enzyme may be used in different food applications including the production of antioxidants and colorants, modification of proteins, fermentation of cocoa and black tea, etc.Master Thesis Isolation and Molecular Characterization of Extracellular Lipase and Pectinase Producing Bacteria From Olive Oil Mills(Izmir Institute of Technology, 2004) Altan, Asena; Yenidünya, A.Fazil; 01. Izmir Institute of TechnologyLipases and pectinases are industrially important enzymes. These enzymes are produced by a variety of microorganisms. However there are few studies on the production of these enzymes by thermoacidophilic Bacillus species. The aim of this research was the isolation of extracellular lipase and pectinase producing thermoacidophilic Bacillus from olive oil mills and their identification by phenotypic tests, 16S-ITS rDNA RFLP and DNA sequencing. Eighty-six thermoacidophilic strains were isolated from olive, olive husk and soil contaminated with alpechin collected within different olive oil mills in Ayvalık. The strains were screened for the presence of 5 extracellular enzyme activities. These were lipase, pectinase, amylase, xylanase and cellulase. In total, 69 lipase (Tween 20 as subtrate), 32 pectinase and 68 amylase activities were detected. None of the isolates were able to produce xylanase or cellulase enzyme. All of the isolates were Gram(+) endospore forming rods, thus they were identified as Bacillus sp. Taq I was used for 16S-ITS rDNA based RFLP. The isolated strains were clustered into four groups by Taq I restriction profiles of 16S-ITS rDNA. One representative isolate among the members of each of the 16S-RFLP homology groups was chosen and used for 16S rRNA gene partial sequence analysis. Sequencing results were submitted to GenBank. So far the indicated accession numbers were obtained: AY601903 (isolate H 22 of G-3, 679 nucleotides), AY606276 (isolate S1 of G1, 330 nucleotides)Master Thesis Isolation and Identification of Lipase Producing Soil Fungus; Cloning, Sequencing and Partial Characterization of Its Lipase(Izmir Institute of Technology, 2010) Ergülen, Elvan; Arslanoğlu, Alper; Arslanoğlu, Alper; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyLipases are well known enzymes which catalyze the hydrolysis of long chain triglycerides. Contrary to many other enzymes, lipases show a wide range of substrate specificity and remarkable levels of activity and stability in non-aqueous environments. Therefore, they have a great potential in many industrial applications such as detergent industry, paper and food technology, as biocatalysts for the synthesis of organic intermediates. Lipases can be obtained from animals, plants as well as from natural and recombinant microorganisms in good yields. The aim of this thesis was isolation and identification of a lipolytic fungus and purification and characterization of its lipase enzyme. For this purpose, a lipolytic fungus was isolated from soil sample collected from Kula. Lipase activity of this fungus was detected rapidly by Rhodamin B - olive oil plate assay. The lipolytic fungus was identified by 28S rRNA gene sequence analysis and determined to be a strain of Rhizopus stolonifer. Because this lypolytic fungus was isolated from soil sample collected from Kula, it was named as R. stolonifer K45. This fungus showed best growth at 25°C and did not grow above 35°C. In the second part of the study, lipase enzyme of the fungus was partially purified but previously, optimum time and carbon source for lipase production was determined. According to this, optimum lipase production was obtained at 7th day of growth in the media including only olive oil as carbon source. Glucose when included in the growth media was observed to reduce the amount of lipase. In order to purify fungal lipase, aceton precipitation (30 %) and ultrafiltration methods were used. Lipase activity assay was performed spectrophotometrically. The chain length specificity of this lipase was detected and highest activity was observed towards p-NP laurate. The effect of different temperature and pH values on lipase activity and stability was also determined and optimum temperature and pH were found 45°C and pH 8, respectively. Furhermore, different organic solvents and metal ions were tested on lipase activity. The lypolytic enzyme was inhibited by n-hexane. However, methanol and DMSO were detected to enhance the lipase activity.