Hydroxylapatite chromatography, isolation

Three extracellular P-mannanases (M-1, M-II and M-III) were purifled by anunonium sulfate precipitation (80% saturation) followed by chromatography on a DEAE-Toyopearl 650 M column (4.6 x 35 cm) equilibrated and eluted with 0.01 M phosphate buffer (pH 7.0 ) and by a hydroxylapatite column (1.6 x 25 cm). As shown in Fig. 1, two active fractions (Fraction 1 and 2) were detected after hydroxylapatite chromatography. Each fraction 1 and 2 was applied onto a Sephacryl S-200 column (2.6 x 90 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 0.1 M NaCl and eluted with the same buffer. Mannanase-I and -II were isolated from fraction 1 and mannanase-III was from fraction 2. 

Once cell wall breakage or enzymatic dissolution has occurred, a variety of procedures can be used for isolation and purification of the DNA. These generally rely on an initial deproteinization using phenol or a mixture of perchlorate, sarcosine, and chloroform-isoamyl alcohol.6 9 The crude DNA is further purified by enzyme treatments, alcohol precipitation and spooling, hydroxylapatite chromatography, cesium chloride gradient centrifugation, or any combination of these methods. 

Clostripain [9028-00-6] Mr -55,000 [EC 3.4.22.8]. Clostripain is isolated from Clostridium histolyticum callogenase by extraction in pH 6.7 buffer, followed by hydroxylapatite chromatography with a 0.1-0.2 M phosphate gradient, then Sephadex G-75 gel filtration with 0.05M phosphate pH 6.7, dialysis and a second... 

The purified hydrolases were stimulated greater than 10-fold by Mg2+ and dithiothreitol (9). Optimal Mg2+ concentration was approximately 5 to 10 mM (9). The dithiothreitol sensitivity of the hydrolase was dependent on the purification procedure. Following hydroxylapatite chromatography, die impure enzyme was relatively insensitive to dithiothreitol activation under the standard assay procedure required approximately 10 mM thiol (9). The enzyme isolated by procedure A exhibited a similar sensitivity (9). In contrast, the hydrolase purified by procedure B was dramatically more sensitive to dithiothreitol approximately 50 pM produced considerable activation. Both hydrolases were partially resistant to sulfhydryl reagents such as N-ethylmaleimide (MEM) unless subjected to prior incubation with thiol as expected, higher concentrations of tiiiol were required to convert hydrolase A to an NEM-sensitive state than was the case with hydrolase B. 

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