Bakteriyel Kaynaklı Lipaz Geninin Pichia pastoris’te Klonlanması, Ekspresyonu ve Rekombinant Enzimin Saflaştırılması

Öz

Gıda endüstrisinde enzimler her türlü işleme ve üretim proseslerinde uygulama alanı bulan biyolojik katalizörlerdir. Süt işlemeden meyve suyu üretimine, et işlemeden ekmek üretimine kadar gıda sanayinin çeşitli alanlarında yaygın olarak ihtiyaç duyulan enzimlerden biri lipazlardır. Bu çalışmada, kodon optimize edilmiş Geobacillus stearothermophilus lipaz enzimini kodlayan genin, Pichia pastoris ekspresyon sisteminde güçlü ve indüklenebilir bir promotor olan AOX1 promotoru içeren pPICZA ekspresyon vektörü kullanılarak ekspresyonu yapılmış ve en yüksek lipaz enzimi üretimi gösteren klon belirlenmiştir. Belirlenen klon ile 400 mL indüksiyon besiyerinde 72 saat boyunca üretim gerçekleştirilmiş ve toplanan süpernatant örneğinde enzim aktivitesi, toplam protein ve SDS-PAGE analizi gerçekleştirilmiştir. Rekombinant lipaz enzimi Ni-NTA afinite kromatografisi ile saflaştırılmıştır. Saflaştırma işleminin her aşamasından alınan örneklerde SDS-PAGE analizi yapılmış ve her aşamada elde edilen örneklerde saflaştırma verimi hesaplanmıştır. Saflaştırma işleminden sonra analiz edilen örnekte, en yüksek üretim gösteren klonun 25.02 mg L-1 lipaz üretim seviyesine ulaştığı belirlenmiştir.

Anahtar Kelimeler

• Pichia pastoris
• Gıda işleme enzimi
• lipaz
• protein saflaştırma

Cloning and Expression of the Bacterial Lipase Gene in Pichia pastoris and Purification of the Recombinant Enzyme

Öz

In the food industry, enzymes are biological catalysts that find application area in all kinds of processing and production processes. Lipases are one of the enzymes that are widely needed in various fields of the food industry, from milk processing to fruit juice production, from meat processing to bread production. In this study, the gene encoding codon optimized Geobacillus stearothermophilus lipase was expressed using the pPICZA expression vector containing the AOX1 promoter, which is a strong and inducible promoter in the Pichia pastoris expression system, and the clone exhibiting the highest lipase enzyme production was determined. Production was carried out with the determined clone in 400 mL induction medium for 72 hours and the enzyme activity, total protein and SDS-PAGE analyzes were performed in the collected supernatant sample. The recombinant lipase enzyme was purified by Ni-NTA affinity chromatography. SDS-PAGE analysis was performed on the samples taken from each step of the purification process and the purification efficiency was calculated in the samples obtained at each step. In the sample analyzed after purification, it was determined that the clone with the highest production reached the lipase production level of 25.02 mg L-1.

Anahtar Kelimeler

• Food processing enzyme
• lipase
• protein purification
• Pichia pastoris

Kaynakça

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