Gene identificaiotn, cloning, expression, purification and industrial application of marine bacteria producing agarases

Abstract

Agar is an important gelatinous substance present in the cell wall of some marine red algae (Rhodophyceaea). It is mainly composed of agarose and agaropectin. Agarases are the natural enzymes of certain agarolytic organisms found mostly in marine habitats that hydrolyze the agarose. Also, agarase is the first enzyme in the agar catabolic pathway. Availability of agarases basically in the marine environment, which is consistent with the fact that agar, being a product of marine algae is available to utilize some marine organisms as a convenient carbon and energy source. Agarases are classified into two groups named as α-agarase and β-agarase based on their cleavage of α-β linkages of agarose, breaking them into oligosaccharides. α-Agarase hydrolyzes α-(1→3) linkages of agarose, producing agaro-oligosaccharises such as agarobiose, while β-agarase hydrolyses β-(1→4) linkages, resulting neoagaro-oligosaccharides. Neoagarobiose was reported to process moisturizing effect on skin and and whitening effects on melanoma cells. We screened agarase producing marine bacteria strains isolated from different marine sources at Jeju Island costal environment. Different types of selection plates were used to identify the different agarase producing bacteria. The strains include Agarivorans sp., Aquimarina sp., Cellulophaga sp., Cytophaga sp., Flammeovirga sp., Glaciecola sp., Microbulbifer sp., Pseudoalteromonas sp., Reinekia sp. and Shewanella sp. etc. that identified by 16s rRNA nucleotide sequences. The coding sequences of three different agarases were cloned from Agarivorans sp., Pseudoalteromonas sp. bacteria and purified their recombinant agarases named as AG4, AG17 and AG52, respectively. Also, purified recombinant agarase enzymes were analyzed for biochemical properties such as specific activities and optimum reaction conditions. Finally, bioactivity studies of neoagaro-oligosaccharide forms derived from agar hydrolyzes by purified agarase have been conducted f