Protein Expression And Purification And Analysis Of Enzymatic Properties Of Esterase EstB

Soil microorganisms represent a vast untapped source of novel enzymes. However,many soil microorganisms can not be cultured in laboratory by conventional techniques.Over the last decade, there has been increasing success in isolating novel microbialenzymes directly from various habitats using a number of new culture-independentmethods. Among those methods, the metagenomics is one of the most promising tools, as itallows novel functional genes to be identified directly from an environmental sample.Recently, metagenomic libraries from soil communities were used successfully in cloninggenes encoding lipolytic enzymes, proteases, amylases and oxidoreductases etc.This work described the screening of lipolytic enzymes from soil metagenomic libary.One positive clone possessing the activity of lipolytic enzyme was obtained in the LB platecontaining 0.5% tributyrin. Sequencing and bioinformatic analyses suggested a novel genethat was designated as estB. This gene consisted of 1185 bp encoding 394 amino acids witha theoretical molecular weight of 40900 Da. Sequence alignment analysis revealed that thededuced amino acid sequence exhibited only moderate identity (38%) to known lipolyticenzyme and, the protein maybelonged to the GDSLhydrolase superfamily.It was predicted that EstB contained a signal peptide by online software. In order toavoid negative-influence of the signal peptide on soluble protein expression, a pair ofprimer which lack the signal peptide coding domain was designed. The PCR products werecloned into the expression vector pET28a and then over-expressed in Escherichia coliBL21(DE3). The cells were homogenized by ultrasonic, both the supernatant and theprecipitate were subjected to SDS-PAGE (Sodium Dodecyl Sulfate-PolyacrylamideElectrophoresis Gel) analysis. The result indicated that most of the EstB formed insolubleaggregates in the precipitate. The soluble protein of EstB was gained by adding 0.5%sodium deoxycholate (DOC) into the lysis buffer.The soluble protein was purified by Ni-NTA Agarose and the enzymatic properties ofEstB were further explored. The detail results were shown as follows:(1) We examined seven substrates constituting 2 through 16 carbon elements, andfound that 10 carbon substrate exhibited the highest enzyme activity under the givenconditions, suggesting that the EstB is likelybelonged to Esterase family;(2) The optimal temperature of EstB was measured to be 47.5℃under the givenconditions;(3)The optimal pH was found at 10.0 under the given conditions;(4) We anlysised the effects of metal ions and commonly used detergents on theactivity of EstB and observed that the enzyme appeared to require Cu2+, Mn2+, Mg2+. Itwas also noticed that addition of DOC (1%) and Tween-20 (0.1%) promoted not only thesolubility of EstB but also the enzyme activity. Other metal ions and detergent test such asCa2+, Fe3+, Ni2+, Zn2+and SDS (0.1%) exhibited little influence on the enzyme activity.