Studies On Screening & Determinant Of Microbial Esterase-producing And The Enzyme Production From Bacillus Sp. Strain EB-87 And Its Properties.

In this paper, the screening and determinant of microbial esterase-producing, the fermentation conditions and properties of the Bacillus sp. Strain EB-87 were studied systematically, because the application of microbial esterase was increasing, especially in the field of bioconversion, kinetic resolution. Based on studies and comparisons among methods of screening microbial esterase, a rapid, simple and efficient Screening Model for microbial esteraseroducing was developed. More than thirty soil samples from all kinds of water ester or oil and strains from myself laboratory. An efficient and rapid plating methods for determinant of microbial esterase activity by observing the diameter of the strain and the hydrolytic color circle around on the culture was adopted. The underlying principle employed in this Model was based on color change of the indicator-Fast Blue B Salt caused by a -NA, f3 -NA from a -NP and P -NP under the proper conditions. This was preliminary screening and 174 strains obtained according to relative esterase activity more than 40%. After culturing in a triangle bottle on the shake and color halo is used to determine their esterase activity. This was secondary screening and 24 fine strains were selected and the highest esterase activity of the strain EB-87 was 38.5UIml. The mixed liquor of esterase results of the preliminary screening was a nearly linear with results of the secondary screening, but the Bromocresol Purple later didn so. Besides, studies and compared esterase activity of these strains with different substance, including the esterase roducing strain for stereospecific hydrolysis of (D,L)-Pantyl lactone and (D,L)-Ethyl lactate. Isolation aiid purification the strain EB-87, and through detection of its appearance, physiological and biochemical characteristics, growth characters, it was identified Bacillus Genus according to Bergeysmanual of Deternmiutue Bacteriology(8) and Microbiology Bacteriological Analytical Manual(FDA), and named Bacillus sp. Strain EB-87. In order to investigate the conditions and the principle of producing esterase by Bacillus sp. Strain EB-87, the methods of combination Single Factor design Plackett-Burman design and Response Surface Analysis had been employed for the fermentation conditions and the composition of the fermentation medium.The results showed that the optimum composition of the fermentation medium asfollowing: Corn steep liquor 2.4%, Glucose l.0%, Peptone 2.6%, MgSO.7H,O0.07l%, peH.),SO. 0.2%, K,HPO, 0.2%, NaH,PO. 0.05%, the optimum fermentationconditions as following: the optimum fermentation tempendre was 32oC, theoptimum fermentation shaking frequency was l50rpm, the optimum fermentation pHwas 7.2, the optimum fermentation time was 26-28h. In doing so Bacillus sp. strainEB-87 esterase activity was 84.5U/ml, enlarging 2.2 times.Finally the purification and properties including substance specificity, temperature,pH, Bacillus sp. strain EB-87 esterase were studied in detail. The results showed thatthe optimal temperature of BaciUus sp. strain EB-87 esterase reaction was 40C,pH7.2, the esterase activity was stable in the range of pH6.0-8.0. and the enzyme wasstable less than 40C, but not stable above 50C. The parameters: tI/2 was 56.4min, k 85was l l .2min at 50oC and tll2 was 2l .7min, to85 was 8.6min at 60C. The lions Fe2+.Mg'+. K+. Na+ and Ca2+ were beneficial to esterase activity, but the lions Co2+. Zn2+and Cu2+ were inhibited esterase activity. The kinetic parameters K. was0.33 lmmoI/L, V. was l .005 ll mol/ml.minThe enzyme exhibits different esterase...