Farklı pH ve sıcaklık değerlerinin Pichia pastoris'de rekombinant ekspanzin üretimi üzerine etkisi

Bu çalışma kapsamında rekombinant Ekspanzin üretimi amacıyla domatesten izole edilen LeExp1 geni Pichia pastoris mayalarına pPIC9K vektörü vasıtasıyla aktarılmıştır. Ekspresyon koşulları 20, 25, 30°C ve pH 4.0, 5.0, 6.0 ve 7.0 değerleri için ayrı ayrı sağlanmıştır. Fermentasyon özütlerinin Biomass analizi ve Bradford assay deneyleri sonucunda toplam rekombinant protein miktarları ve toplam hücre konsantrasyonları belirlenmiştir. Farklı pH ve sıcaklık değerlerinin rekombinant protein üretim miktarına ve toplam hücre konsantrasyonuna olan etkileri incelenmiştir. Deneysel sonuçlara göre maksimum rekombinant ekspanzin üretimi için P. pastoris mayalarının optimum fermantasyon koşulları 25oC sıcaklık ve pH 7.0 olarak tespit edilmiştir.

Effect of different pH and temperature on the production of recombinant expansin in Pichia pastoris

In this study LeExp1 gene isolated from tomato genome was transferred to Pichia pastoris by using pPIC9K vector for production of recombinant Expansin protein. Recombinant protein expression was carried out at 20, 25, 30°C temperature and 4.0, 5.0, 6.0 and 7.0 pH levels. Total protein and dry cell weight amounts was determined in fermentation medium. The effects of different fermentation temperature and pH level in the medium on protein expression and total cell concentration were investigated. The optimum fermentation conditions for P. pastoris were determined as 25oC and pH 7.0 according to experimental results. 

___

Ahmad, M., Hirz, M., Pichler, H., Schwab, H., 2014. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Applied Microbiology and Biotechnology., 98: 5301-5317.

Bayraktar E., Effects of pH on human growth hormone production by pichia pastoris considering the expression levels of regulatory genes, Master Thesis, Supervised, Pınar ÇALIK, 2009.

Bradford, M.M., 1976. Rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding, Analytical biochemistry, 7: 248 - 254.

Calık P, Bayraktar E, Inankur B, Soyaslan ES, Sahin M, Taspınar H, Acık E, Yılmaz R ve Ozdamar TH, 2010. Influence of pH on recombinant human growth hormone production by Pichia pastoris; Journal of Chemical Technology and Biotechnology, 85: 1628–1635.

Cereghino, J.L., Cregg, J.M., 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews,. 24: 45 - 66.

Cereghino, G.P.L., Sunga, A.J., Cereghino, J.L., Cregg, J.M., 2001. Expression of foreign genes in the yeast Pichia pastoris, Genetic Enginering, 23: 157 – 169.

Cosgrove, D.J., 2000. Loosening of plant cell walls by expansins. Nature 407:321–326.

Chang S,W, Shieh C,J, Lee G,C, Akoh C, C, Shaw J, -F, 2006. Optimized Growth Kinetics of Pichia pastoris and Recombinant Candida rugosa LIP1 Production by RSM. Journal of Molecular Microbiology and Biotechnology, 11:28-40.

Cosgrove, D. J., 2005. Growth of the plant cell wall. Nature Review Molecular & Cell Biology, 6: 850-861.

Cregg, J.M., Vedvick, T.S., Raschke ,W.C, 1993. Recent advances in the expression of foreign genes in Pichia pastoris, BioTechnology 11: 905 – 910.

Cregg, J.M., Cereghino, J.L., Shi, J., Higgins, DR., 2000. Recombinant protein expression in Pichia pastoris. Molecular Biotechnology,. 16:23-52.

Dehnavi, E., ve ark., 2016. Cloning and high-level expression of β-xylosidase from Selenomonas ruminantium in Pichia pastoris by optimizing of pH, methanol concentration and temperature conditions. Protein Expression and Purification, 124: 55–61.

Dale F, C. Robert, W. Tracy, 2010. Optimization of the expression of recombinant human Activin A in the yeast Pichia pastoris;; Biotechnology Progress, 26: 372-383.

Files D, Ogawa M, Scaman CH, Baldwin SA, 2001. A Pichia pastoris fermentation process for producing high-levels of recombinant human cystatin-C. Enzyma and Microbiol Technology, 29: 335-340.

Goodrick, J.C., Kurtzman, C., 2009. Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. Journal of Industrial Microbiology and Biotechnology, 36:1435–1438.

Higgins, D.R., Cregg, J.M., 1998. Introduction to Pichia pastoris. Methods in Molecular Biology,.103:1-15. Hu M., 2014. An Alkaline pH Control Strategy for Methionine Adenosyltransferase Production in Pichia pastoris Fermentation. Biotechnology and Bioprocess Engineering, 19: 900-907.

Inan, M., Chiruvolu, V., Eskridge, K.M., Vlasuk, G.P., Dickerson, K., Brown, S., Meagher, M.M., 1999. Optimization of temperature-glycerol-pH conditions for a fedbatch fermentation process for recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production by Pichia pastoris. Enzyme and Microbial Technology, 24: 438-445.

Jahic, M., Wallberg, F., Bollok, M., Garcia, P., Enfors, S.O., 2003. Temperature limited fedbatch technique for control of proteolysis in Pichia pastoris bioreactor cultures, Microbial Cell Factories, 2: 6 - 17.

Kobayashi, K., Kuwae, S., Ohya, T., Ohda, T., Ohyama, M., Tomomitsu, K, 2000. High level secretion of recombinant human serum albumin by fedbatch fermentation of the methylotrophic yeast, Pichia pastoris, with minimal protease production and activation; Journal of Bioscience and Bioengineering, 89: 55-61.

Ling LY, Ithoi I ve Yik FM, 2010. Optimization for high-level expression in Pichia pastoris and purification of truncated and full length recombinant sag2 of toxoplasma gondii for diagnostic use. Southeast Asian J Trop Med Public Health, 41:507 - 513.

Sampedro J and Cosgrove, D.J., 2005. The expansin superfamily. Genome Biology 6: 242-253.

Sinha J, Plantz BA, Zhang W, et al., 2003. Improved production of recombinant ovine interferon-T by Mut+ strain of Pichia pastoris using an optimised methanol feed profile. Biotechnology Progress, 19: 794 - 802.

Spohner SC, Müller H, Quitmann H, Czermak P., 2015. Expression of enzymes for the usage in food and feed industry with Pichia pastoris. Journal of Biotechnology., 202:118-134.

Soyaslan, E.S., Calık, P., 2011. Enhanced recombinant human erythropoietin production by Pichia pastoris in methanol fed-batch/sorbitol batch fermentation through pH optimization; Biochemical Engineering Journal 55: 59–65.

Wang, Z., ve ark., 2010. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation, Bioresource Technology, 101: 1318-1323.

Wanderley et al., Influence of pH on cellular growth of Pichia pastoris KM71H by fed‐ batch process, 2009. Microbial Physiology, Molecular Biology and Functional Genomics, ISBN 978-972-97810-6-3. p. 29.

___

APA Aytekin Akbaba, M. , Karaaslan, A. , Alihanoğlu, S. , Vardin, H. & Karaaslan, M. (2018). Farklı pH ve sıcaklık değerlerinin Pichia pastoris'de rekombinant ekspanzin üretimi üzerine etkisi . Harran Tarım ve Gıda Bilimleri Dergisi , 22 (2) , 207-214 . DOI: 10.29050/harranziraat.315532
Harran Tarım ve Gıda Bilimleri Dergisi
  • Yayın Aralığı: Yılda 4 Sayı
  • Yayıncı: Harran Üniversitesi

6.7b290

Sayıdaki Diğer Makaleler

Besi Sığırı Rasyonlarında Tamamlayıcı Yem Olarak Farklı Şekillerde İşlenmiş Arpa ve Mısır Kullanımının Performans, Bazı Biyokimyasal Parametreler ile Serum Laktat ve Bikarbonat Düzeyi Üzerine Etkileri

Neşe Nuray TOPRAK, İsmail YAVAŞ, Canberk BİLGEL

Denizli İli Koşullarında Organik Yetiştirilen İzmir Kekiği (Origanum onites L.) Bitkisinin Beslenme Durumları ve Bazı Kalite Öğeleri Arasındaki İlişkiler

Seda ERDOĞAN BAYRAM

Fitopatolojide Nanoteknoloji

AYDIN ATAKAN, Hülya ÖZGÖNEN ÖZKAYA

Metabolik Gıda İntoleransları

Aylin Akoğlu, Murat Oruç

Şanlıurfa Tek Tek Dağları’nda Farklı Yöneylerdeki Meraların Bitki Kompozisyonları ile Ot Verimi ve Kalitelerinin Belirlenmesi

Tahir POLAT, Şehnaz BÜYÜKHATİPOĞLU, Gökhan AKKAYA

Biocontrol of Alternaria alternata Causing Leaf Spot Disease on Faba Bean (Vicia faba L.) Using Some Trichoderma harzianum Isolates Under In vitro Condition

Mukaddes KAYIM, Amin Mohammed YONES, Ali ENDES

Kırmızı Mercimekte Zararlı Dolycoris baccarum L. ve Piezodorus lituratus (F.)’ a Karşı Deltamethrin’in Biyolojik Etkinliği

Çetin MUTLU, Vedat KARACA, Ayhan ÖĞRETEN, Musa BÜYÜK, Yunus BAYRAM

Pamuk (Gossypium hirsutum L.) Genotiplerinin Çoklu Dizi (line x tester) Melezlerinde Bazı Lif Özelliklerinin Kalıtımı

Nazife Özkan, Osman ÇOPUR

Keçiboynuzu Pekmezi İlave Edilerek Üretilen Meyveli Yoğurdun Bazı Özellikleri

Şerafettin ÇELİK, Naciye ÜNVER, Betül GÜÇ, Pınar CEYLAN

Diyarbakır İli Pamuk Ekim Alanlarında Bulunan Yeşilkurt, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)’un Popülasyon Dalgalanmasının Belirlenmesi

Merve AKYILDIZ, Erol BAYHAN