Studys On The Breeding Of Agarase Overproduction Strain And The Conditions Of Agarase Production

An agar-decomposing strain was isolated from some kinds of red agar in the costal water in Qingdao, and presumptively classified within the genus Pseudomonas. It grows at an optimum temperature of 20~40C and a pH of 5.0~9.0. NaCl or seawater are not strictly required for growth, but 2.0~4.0% NaCl is helpful. Besides agar, Pseudomonas Strain AT-22 can also degrade carbohydrates such as alginate and starch.This original strain was treatd repeatedly with various mutagens(UV, EMS) in screening high producer of agarse, and the mutant with high and stable yield of agarase is selected after repeated natural selection. With the single factor test and relationship test, the optimal culture media for AT-22 strain producing agarase is decided as: agar 0.3%, (NH4)2SO4 0.5%, peptone 0.3 %, NaCl 3.0%, KH2PO4 0.1 %, MgCl2 0.1 %; and the optimal culture conditions such as incubating temperature, medium pH, rotation rate and amount of inoculation are 24C, 7.0, 120rpm and 1% respectively, 300mL Erlenmeyer flask containing 100mL medium. The agarase activity of fermented broth is 800U/mL when Pseudomonas sp. AT-22 is incubated in Erlenmeyer flask under the optimal conditions at 36~48h.The agarase from Pseudomonas sp. AT-22 is inducible and secretes extracellularly. Enzyme production is reduced if monosaccharide and oligosaccharide, such as glucose, galactose or acid hydrolysis products, are added to the mudium,Its optimal temperature and pH for hydrolyzing agar are 40C and pH7.0 respectively. And the agarase is stable in the pH range of 6.0-7.0, the optimal concentration of substrate is 1.0-1.2%, addition of Ca2+ will greatly promote the enzyme activity, whereas Fe3+, Mn2+, Zn2+ and Pb2+cause inhibition. The kinetic parameters Km was 0.53mg/mL.