tailieunhanh - Expression of β-(1,3-1,4)-glucanase gene of Orpinomyces sp. GMLF18 in Escherichia coli EC1000 and Lactococcus lactis subsp. cremoris MG1363
A gene encoding β-(1,3-1,4)-glucanase (licA) was amplified from Orpinomyces sp. GMLF18 and expressed in Escherichia coli. The 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. | U. ÇÖMLEKCİOĞLU, E. ÖZKÖSE, İ. AKYOL, F. C. YAZDIÇ, M. S. EKİNCİ Turk J Biol 35 (2011) 405-414 © TÜBİTAK doi: Expression of β-(1,3-1,4)-glucanase gene of Orpinomyces sp. GMLF18 in Escherichia coli EC1000 and Lactococcus lactis subsp. cremoris MG1363 Uğur ÇÖMLEKCİOĞLU, Emin ÖZKÖSE, İsmail AKYOL, Ferit Can YAZDIÇ, Mehmet Sait EKİNCİ Kahramanmaraş Sütçü İmam University, Agriculture Faculty, Animal Science Department, Biotechnology and Gene Engineering Laboratory, Kahramanmaraş - TURKEY Received: Abstract: A gene encoding β-(1,3-1,4)-glucanase (licA) was amplified from Orpinomyces sp. GMLF18 and expressed in Escherichia coli. The 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. The 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 and at temperatures of 40-50 °C. The enzyme was found to be stable at 40 °C; however, 12% of LicA activity was lost at 50 °C in 20 min. The 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. Key words: Orpinomyces, β-(1,3-1,4)-glucanase, licA, Lactococcus lactis, lichenan Orpinomyces sp. GMLF18’e ait β-(1,3-1,4)-glukanaz geninin Escherichia coli EC1000 ve Lactococcus lactis subsp. cremoris MG1363’de ekspresyonu Özet: 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.
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