Evaluation and comparison of microbial cells disruption methods for extraction of pyruvate decarboxylase

An enzymatic biotransformation process based on extracted pyruvate decarboxylase (PDC) overcomes the problem of by-product benzyl alcohol production. Seven methods of cells disruption which included application of glass beads, freezing/thawing sequence, silver nanoparticles, ultrasonication, freezing/thawing sequence with glass beads, freezing/thawing sequence with silver nanoparticles, and freezing/thawing sequence with ultrasonication were investigated to choose the best method for partial isolation of PDC from Candida tropicalis TISTR 5350. Ultrasonication was an effective method for cells disruption with the corresponding specific PDC activity of 0.36 ± 0.01 U/mg of protein. Ultrasonication method should thus be selected as the method of choice to preserve enzyme activity. The PDC from C. tropicalis TISTR 5350 released from ultrasonic cells disruption method was prepared for partial purification in comparison with 40-60% (w/v) ammonium sulphate and 50% (v/v) acetone precipitation techniques. Specific PDC activity, purification and percentage recovery (yield) of precipitated PDC enzyme based on 50% (v/v) acetone at 4ºC were higher (0.53 ± 0.02 U/mg protein, 1.24 ± 0.10, and 94.41 ± 2.12 %, respectively) than the precipitation obtained using the 40 to 60% (w/v) ammonium sulphate saturation (0.49 ± 0.01 U/mg protein, 1.13 ± 0.07, and 92.87 ± 2.50%, respectively). The result indicated that the precipitation of PDC using the 50% (v/v) acetone was the most effective strategy for partially purifying PDC and suitable for application in a commercial process which had a relatively low cost and simple process.