His-etiketli pro-Mikrobiyal Transglutaminaz Enziminin Pichia pastoris’te Üretim Şartlarının Optimizasyonu

Yıl 2019, Cilt: 9 Sayı: 2, 666 - 673, 01.06.2019

Aysun Türkanoğlu Özçelik

https://doi.org/10.21597/jist.468323

Öz

Transglutaminazlar doğada birçok organizmada bulunan ve çeşitli biyolojik işlemlerde rol alan enzimlerdir. Glutamin ve lizin aminoasitleri arasında kovalent bağ oluşumunu katalizleyerek çapraz bağ oluştururlar. Bu özelliklerinden dolayı gıda ürünlerinin fiziksel ve kimyasal özelliklerini geliştirmek amacıyla kullanılırlar. Bu çalışmada, gıda endüstrisinde kullanılan mikrobiyal transglutaminaz (MTGaz) enziminin, Pichia pastoris (P. pastoris) mayasında yapısal GAP promotoru altında üretim şartlarının optimizasyonu yapılmıştır. Bu amaçla, üç farklı sıcaklık (20°C, 25°C, 28°C) ve üç farklı pH’dan (pH 3, 5.75, 7.44) oluşan dokuz farklı koşul altında pro-MTGaz enziminin üretimi çalkalamalı erlenmayerlerde yapılmıştır. 40 saatlik protein üretimi sonrasında elde edilen sonuçlar, pro-MTGaz üretimi için 25°C sıcaklık ve 7.44 pH’nın optimum koşullar olduğunu göstermiştir.

Anahtar Kelimeler

mikrobiyal transglutaminaz, rekombinant protein üretimi, Pichia pastoris

Destekleyen Kurum

Akdeniz Üniversitesi Bilimsel AraĢtırma Projeleri Koordinasyon Birimi

Proje Numarası

FBA-2017-2474

Teşekkür

Bu çalışma Akdeniz Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi tarafından FBA-2017-2474 proje numarasıyla desteklenmiştir.

Optimization of Production Conditions of His-taqqed pro-Microbial Transglutaminase Enzyme in Pichia pastoris

Öz

Transglutaminases are enzymes involved in various biological processes and found in many organisms in nature. They form a cross-link by catalyzing the formation of covalent bonds between the amino acids glutamine and lysine. Due to these properties, they are used to improve the physical and chemical properties of food products. In this study, the production conditions of microbial transglutaminase (MTGase) enzyme, used in the food industry, in Pichia pastoris (P. pastoris) yeast under the structural GAP promoter were optimized. For this purpose, the production of the pro-MTGase enzyme under nine different conditions consisting of three different temperatures (20°C, 25°C, 28°C) and three different pHs (pH 3, 5.75, 7.44) was carried out by shake flask experiments. The results obtained after 40 hours of protein production showed that; the optimum conditions were 25°C temperature and pH 7.44 for pro-MTGase production.

Anahtar Kelimeler

microbial transglutaminase, recombinant protein production, Pichia pastoris

Proje Numarası

FBA-2017-2474

Kaynakça

• Beninati S, Bergamini CM, Piacentini S, 2009. An overview of the first 50 years of transglutaminase research. Amino Acids, 36(4): 591-598.
• Gauche C, Tomazi T, Barreto PLM, Ogliari PJ, 2009. Physical properties of yoghurt manufactured with milk whey and transglutaminase. LWT - Food Science and Technology, 42(1): 239-243.
• Gündüz B, 2012. Recombinant transglutaminase production by metabolically engineered Pichia pastoris. MSc Thesis, Middle East Technical University, Ankara, Turkey.
• Icekson I, Apelbaum A, 1987. Evidence for transglutaminase activity in plant tissue. Plant Physiology, 84: 972-974.
• Li H, Zhang L, Cui Y, Luo X, Xue C, Wang S, 2013. Expression of soluble recombinant transglutaminase from Zea mays in Pichia pastoris. World Journal of Microbiology and Biotechnology, 29:939-947.
• Moreno HM, Carballo J, Borderias AJ, 2010. Use of microbial transglutaminase and sodium alginate in the preparation of restructured fish models using cold gelation: Effect of frozen storage. Innovative Food Science and Emerging Technologies, 11: 394-400.
• Romero de A, Ordonez JA, Hoz L, Herrero AM, Cambero MI, 2010. Microbial transglutaminase for cold-set binding of unsalted/salted pork models and restructured dry ham. Meat Science, 84: 747-754.
• Rossa NP, Burin VM, Bordignon-Luiz MT, 2012. Effect of microbial transgutaminase on functional and rheological properties od ice cream with different fat contents. LWT - Food Science and Technology, 48: 224-230.
• Seravalli EAG, Iguti AM, Santana IA, Filho FF, 2011. Effects of application of transglutaminase in wheat proteins during the production of bread. Procedia Food Science, 1: 935-942.
• Sommer C, Hertel TC, Schmelzer CEH, Pietzsch M, 2012. Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-taq. Amino acids, 42: 997-1006.
• Washizu K, Ando K, Koikeda S, Hirose S, Matsuura A, Takagi H, Motoki M, Takeuchi K, 1994. Molecular cloning of the gene for microbial transglutaminase from Streptoverticillium and its expression in Streptomyces lividans. Bioscience Biotechnology and Biochemistry, 58(1): 82–87.
• Yerlikaya P, Gokoglu N, Ucak İ, Yatmaz HA, Benjakul S, 2015. Supression of the formation of biogenic amines in mackerel mince by microbial transglutaminase. Journal of the Science of Food and Agriculture, 95: 2215-2221.
• Yerlikaya P, Yatmaz HA, Gokoglu N, Ucak İ, 2017. The quality alterations of rainbow trout mince treated with transglutaminase. LWT-Food Science and Technology, 84: 815-820.
• Yokoyama K, Nio N, Kikuchi Y, 2004. Properties and applications of microbial transglutaminase. Applied Microbiology and Biotechnology, 64(4): 447-454.
• Yurimoto H, Yamane M, Kikuchi Y, Matsui H, Kato N, Sakai Y, 2004. The Pro-peptide of Streptomyces mobaraensis Transglutaminase Functions in cis and in trans to Mediate Efficient Secretion of Active Enzyme from Methylotrophic Yeasts. Bioscience, Biotechnology and Biochemistry, 68(10): 2058-2069.