LA QUITINA DESACETILASA DE Bacillus thuringiensis CEPA Bt-132

Abstract

Deacetylase produced by the strain of B. thuringiensis Bt-132 is a periplasmic non-specific enzyme hydrolying substances different such as glycol chitin, EDTA, uranyl acetate, p-nitro phenyl acetate and 2,4-dichlorophenoxyacetic acid. Therefore, it may also enzyme could degrade acetylated xenobiotic compounds. This enzyme is constitutive. In order to purify and characterize the enzyme with activity of chitin deacetylase (CDA) present in the strain Bt-132, both glycol chitin and colloidal chitin previously demineralized and deproteinized were used as its substrates. The purification of the CDA was carried out, by a process starting with the collection of bacterial cells grown in nutrient broth, until de middle zone of the logarithmic phase of growth. Such cells were broken by using different abrasive materials, combined with vigorous shaking, friction, high-pressure and sonication treatments. However, the best results to release the enzyme, were obtained when the bacteria were autolyzed after an incubation for 96 h, under shaking (280 rpm) at 28 °C. The autolyzate was then clarified by centrifugation, and sterilized by filtration. The cell-free crude enzymatic extract was concentrated by lyophilization, and then dissolved in a minimal volume of one appropriate buffer. The sample obtained was at once fractionated in a hydrophobic column of sepharose CL-4B, eluting with a descending discontinuous gradient of ammonium sulphate. The enzyme eluted fastly, showing its low hydrophobicity. The collected fractions with the highest activity of CDA were pooled and lyophilized. The new sample obtained was again fractionated in a column of Sephadex G-25, in order to eliminate the ammonium sulphate derived from the previous chromatography, and also to obtain a still more purified sample of the CDA. The molecular homogeinity of the resulting CDA was electrophoretically verified by PAGE-SDS, staining the protein bands by means of the silver nitrate staining technique. The enzyme obtained was purified 33 times with a recovery of 11 %, wich are satisfactory data, with respect to published previous reports. The pure enzyme showed a molecular weight of 60.2 kDa, and it was inactivated with EDTA, being its activity restored by addition of salts as ZnCl2, CaCl2, MgCl2 and CoCl2; therefore, it may be considered as a metalloenzyme. Besides, the CDA presented its greatest stability and activity at pH 4.5. On the other hand, although its thermostability is high, the more favorable temperature for its reaction was 40 °C, a lower value compared with the temperature required (50-60°C) by other reported CDAs. This study is the first report related to the purification and characterization of the CDA from B. thuringiensis.