Arabidopsis thaliana lipases: cloning and expression in pichia pastoris

Abstract

Lipases are a group of enzymes that hydrolyze the carboxyl ester bonds in acylglycerols releasing organic acids and glycerol. There is growing interest in identifying plant lipases because literature reviews show that plant lipases can be highly substrate specific and their substrate specificities can be useful in industrial applications. Accordingly, the aim of this study is to screen Arabidopsis thaliana putative lipases. Arabidopsis thaliana is chosen since it is a model plant organism with its genome sequenced. The strategy followed begins by cloning open reading frames that have sequences similar to known lipases, obtained from Arabidopsis Biological Research Center into Pichia pastoris expression vectors. Pichia pastoris is the host of expression because it is a host that is suitable for high yield expression without the requirement of time-consuming purification steps. The sequence verified constructs being transformed into Pichia pastoris are expressed in small scale in order to screen their lipase activities through a robust fluorogenic activity assay using 4-methylumbelliferryl derived substrates. The screening performed in this study investigated twenty seven putative Arabidopsis lipase activities and resulted in seven proteins of particular interest that can be further investigated through large scale expressions. The open reading frames screened in this study were successfully cloned into Pichia pastoris expression vectors; hence the constructs for large scale expressions are available. These constructs can be directly used for further investigation that may result in the annotation of new Arabidopsis thaliana lipases of commercial value in industrial applications.