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

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 b-(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 5.0-6.0 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.

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

Keywords:

b-(1, 3-1 4)-glucanase, licA, Lactococcus lactis, lichenan, Orpinomyces,

PDF

___

Demirbas β-Glucan and mineral nutrient contents of cereals A. grown in Turkey. Food Chem 90: 773-777, 2005. 2. Planas A. Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochim Biophys Acta 1543: 361-382, 2000.

Annison G, Choct M. Anti-nutritive activities of cereal non-starch polysaccharides in broiler diets and strategies minimizing their eff ects. World Poultry Sci J 47: 232-242, 1991.

Wang ZR, Qiao SY, Lu WQ et al. Eff ects of enzyme supplementation on performance, nutrient digestibility, gastrointestinal morphology, and volatile fatty acid profi les in the hindgut of broilers fed wheat-based diets. Poult Sci 84: 875- 881, 2005.

Liu JR, Yu B, Liu FH et al. Expression of rumen microbial fi brolytic enzyme genes in probiotic Lactobacillus reuteri. Appl Environ Microbiol 71: 6769-6775, 2005.

Wegmann U, O’connell-Motherway M, Zomer A et al. Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363. J Bacteriol 189: 3256-3270, 2007.

Çömlekcioğlu U, Akyol İ, Kar B et al. Anaerobik rumen funguslarının izolasyonu, tanımlanması ve kültür koleksiyonunun oluşturulması. Hayvansal Üretim 49: 29-35, 2008.

Orpin CG. Studies on the rumen fl agellate Sphaeromonas communis. J Gen Microbiol 94: 270-280, 1976.

Simon D, Chopin A. Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 20: 559-566, 1988.

Leenhouts K, Buist G, Bolhuis A et al. A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet 253: 217-224, 1996.

Gasson MJ. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci aft er protoplast induced curing. J Bacteriol 154: 1-9, 1983.

Th ompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specifi c gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680, 1994.

Cantarel BL, Coutinho PM, Rancurel C et al. Th e Carbohydrate-Active EnZymesdatabase (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37 (Database issue): D233-D238, 2009.

Mandel M, Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol 53: 159-162, 1970.