Ferit Can YAZDIÇ, Mehmet Sait EKİNCİ, Emin ÖZKÖSE, İsmail AKYOL, Uğur ÇÖMLEKÇİOĞLU

Expression of β-(1,3-1,4)-glucanase gene of Orpinomyces sp. GMLF18 in Escherichia coli EC1000 and Lactococcus lactis subsp. cremoris MG1363

β-(1,3-1,4)-Glukanaz (licA) kodlayan bir gen Orpinomyces sp. GMLF18’den izole edilmiş ve Escherichia coli’de klonlanmıştır. licA’nın DNA dizilimi, 707 bç uzunluğunda bir gen olduğunu ve 26 kDa moleküler büyüklüğünde olan glikozil hidrolaz 16 ailesine ait bir protein kodladığını göstermiştir. licA içeren transformant E. coli’de LicA enzim aktivitesinin büyük bir kısmının hücresel olduğu bulunmuş ve LicA’nın pH 5,0-6,0 ve 40-50 °C arasında en yüksek aktiviteye ulaştığı gözlenmiştir. Enzim 40 °C’de stabilitesini korurken, 50 °C’de 20 dk içerisinde aktivitesinin % 12’sini kaybetmiştir. licA stabil bir rekombinant plazmit olan pIL253 yardımıyla fakültatif anaerob bakteri olan Lactococcus lactis subsp. cremoris MG1363’e aktarılmıştır. Enzim aktivitesi E. coli’ye göre düşük olsa da anaerobik fungus kaynaklı likenaz kodlayan bir gen L. lactis’de başarılı bir şekilde ifade edilmiştir

Orpinomyces sp. GMLF18’e ait β-(1,3-1,4)-glukanaz geninin Escherichia coli EC1000 ve Lactococcus lactis subsp. cremoris MG1363’de ekspresyonu

A gene encoding β-(1,3-1,4)-glucanase (licA) was amplifi ed from Orpinomyces sp. GMLF18 and expressed in Escherichia coli. Th e DNA sequence of licA showed that the gene was 707 bp and encoded a protein with a molecular mass of 26 kDa that belongs to family glycosyl hydrolase 16. Th e main LicA activity was observed to be cell-associated for the licA containing transformant E. coli, and the enzyme expressed in E. coli showed the highest activity at pH 5.0-6.0 and at temperatures of 40-50 °C. Th e enzyme was found to be stable at 40 °C; however, 12% of LicA activity was lost at 50 °C in 20 min. Th e licA was then introduced into the facultative anaerobic bacterium Lactococcus lactis subsp. cremoris MG1363 by a stable recombinant plasmid, pIL253. Although the enzymatic activity was lower than that in E. coli, the gene encoding the fungal originated lichenase was successfully expressed in L. lactis.

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