Electrophoretic and zymographic techniques for production monitoring of two lipase forms from Candida antarctica DSM 70725

Abstract

Yeast Candida antarctica produces two lipase forms, which are widely used as catalysts in variety of organic reactions, many of which are applied on a large scale. In this work, production of two forms of lipase from C. antarctica DSM 70725 (CAL A and CAL B) was monitored during seven days of cultivation in the optimal medium using different electrophoretic and zymographic techniques. According to electrophoresis after silver staining, C. antarctica lipase A (molecular mass 45 kDa) was produced starting from the second day of cultivation. C. antarctica lipase B (CAL B) was also produced starting from the second day, but protein was present in the fermentation broth predominantly as dimer (molecular weight 66 kDa), while presence of monomeric form of CAL B (molecular weight of 33 kDa) was observed starting from the fourth day of cultivation. Both types of zymograms (based on hydrolysis and synthesis reactions) were used for detection of lipase activity in the fermentation broth. C. antarctica lipase A showed activity only in hydrolytic zymogram, when α-naphtyl butyrate was used as substrate. In the same zymogram, with α-naphtyl acetate as substrate no CAL A activity was detected. Similarly, CAL A showed no activity in synthesis based zymograms towards oleic acid and octanol as substrates, indicating that CAL A is not active towards very short or long-chain substrates. As opposite of CAL A, both monomeric and dimeric form of CAL B were detected in the all zymograms, suggesting that CAL B is active towards wide range of substrates, regardless to the chain length. Thus, zymogram based on hydrolysis of α-naphtyl butyrate represents a simple method for monitoring the production of two forms of lipase from C. antarctica, that greatly differ in their characteristics.

Keywords

• Lipase
• Candida antarctica
• electrophoresis
• zymogram