Pichia pastoris mayasi ile rekombinant ksilanaz enzim üretiminin optimizasyonu

In recent years, xylanases are important ingredient of animal feed formulations in addition to other industrial applications. The most important issue for use of enzymes for industrial applications is reduction of production cost and the cost of goods. Studies are ongoing to determine the best production host and cheap media compositions for production of enzymes including xylanases, alpha amylase, cellulase, pectinase, proteases and lipaes etc. The other problems related to the enzyme production process relatively solved. Xylanase are produced in bacteria, yeast and by various microorganisms. This study consists of three parts. In the first part, four secretion signal sequences were evaluated for extracellular production of Aspergillusniger xylanase (XylB) in Pichia pastoris. These sequences were namely; Saccharomyces cerevisiae α -matingfactor (α -MF), Human Serum Albumin (HSA), P. pastoris Acid Phosphatase (PHO1) and P. pastoris Protein with Internal Repeats (PIR1) secretion signals. Xylanase production was achieved with all constructs. In the second part of the study, the production efficiency of xylanse was compared with four leader sequences and best signal for secretion was determined. Xylanase enzyme activities for single-copy clones with α-MF, HSA, PHO1 and PIR1 as secretion sequence were 4247, 4147, 4252 and 5366 U/mL, respectively. The best production signal was determined to be PIR. The last part of the study consist of purification of the enzyme product and determination of the optimum temperature and pH. Consequently, xylanase production in Pichia pastoris was achieved and PIR secretion signal was determined to be the best secretion signal among the tested signals. Xylanase produced in Pichia pastoris can be utilized in animal feed and bakery products after removing the His-tag sequence. KEYWORDS: Pichia pastoris, xylanase, HSA(Human serum albumin), PIR(Protein with Internal Repeats), PHO(acid phosphatase), α-mating factor