Influence of Cryogenic Grinding on Release of Protein and DNA from Saccharomyces cerevisiae

Abstract

Yeast is an important organism for expression of recombinant protein and DNA in biotechnology research. Protein and DNA released by mechanical disruption are amenable to degradation due to high shear and temperature rise in conventional processes. In the present work cryogenic grinding has been employed for the first time to disrupt yeast cell by embrittlement at -196 degC, which results in less denaturation of intracellular protein and DNA. The protein release process is found to follow first-order protein release kinetics, and is dependent on the following parameters: volume of suspension, weight of impactor and rate of grinding. It was possible to release nearly 100% of soluble protein with protein denaturation of 18%. Our results suggest that cryogenic grinding enhances protein release rate to almost 1000 fold as compared to ambient condition grinding process. Although DNA release rate was found to be relatively low as compared to that of protein, the release was very high, approximately 1 mg DNA per 100 mg of yeast. High molecular weight DNA strands of 45 kbp were released with minimum shearing, which is generally difficult to obtain with other mechanical disruption processes. The results suggest that cryogenic grinding is a promising technology for release of protein and DNA.