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. 2015 Summer;14(3):901-6.

Isolation and Characterization of a New Thermoalkalophilic Lipase from Soil Bacteria

Mohammad Rabbani  1 , Fatemeh Shafiee  1 , Zahra Shayegh  1 , Hamid MirMohammadSadeghi  1 , Ziaedin Samsam Shariat  2 , Zahra Etemadifar  3 , Fatemeh Moazen  1
Affiliations

Isolation and Characterization of a New Thermoalkalophilic Lipase from Soil Bacteria

Mohammad Rabbani et al. Iran J Pharm Res. 2015 Summer.

Abstract

Lipases are diversified enzymes in their properties and substrate specificity, which make them attractive tools for various industrial applications. In this study, an alkalinethermostable lipase producing bacteria were isolated from soil of different regions of Isfahan province (Iran) and its lipase was purified by ammonium sulfate precipitation and ion exchange chromatography. To select a thermoalkalophil lipase producing bacterium, Rhodamine B and Horikoshi media were used and the strain that can grow at 45° Cwas selected. The isolated strain was identified using microbial and biochemical tests. One strain showed an orange colored zone on plate and grew on Horikoshi plate. Microbial and biochemical tests showed that the isolated strain was Bacillus subtilis, a Gram positive rod. In PCR, an expected band was obtained with about 371 bp. The activity of the purified lipase was 10.2 folds that of the standard enzyme using ammonium sulfate precipitation and ion exchange chromatography. The molecular weight of lipase determined by SDS-PAGE electrophoresis, was 21 and 35 KDa. Existence of two bands in SDS-PAGE electrophoresis and low amount of obtained purified enzyme highlights the necessity of optimization of purification and concentrating process.

Keywords: Bacillus subtilis; Lipase; Soil; Thermoalkalophile.

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Figures

Figure 1
Figure 1
large colonies from lecitinase medium were transferred to Rhodamine B medium. Colonies with a clear halo in 302 nm wavelength were selected in this medium
Figure 2
Figure 2
PCR amplification of the B. subtilis lipase A gene. In this experiment the DNA templates used were as follows: lane 1: leader, lane 2 to 9: temperature gradients. (2: 45, 3:46.3, 4: 48.6, 5: 52, 6:56.6, 7: 60.2, 8: 62.5 and 9: 64).
Figure 3
Figure 3
SDS-PAGE analysis of purified lipase in a 12% polyacrylamide gel. In this experiment the protein templates used were as follows Lane 1: the standard molecular weight marker (kDa), lane 2: purified sample with 31(kDa).
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