Nowadays there is increased industrial demand for enzymes produced by microorganisms (Gupta et al., 2004; Jaeger et al., 1999). Among these are lipases, which are glycerol ester hydrolyses, such as true lipases and esterases that break long or short acylglycerols to fatty acids and glycerol, respectively (Brockerhoff and Jensen, 1974). The Bacillus subtilis lipase is one of the smallest lipases reported and because of its unique characteristics has commercial and research applications for example, the B. subtilis lipase A gene can be expressed in its active form in Escherichia coli without the need to co-express any specific chaperones. (Droge et al., 2003). Several investigators have focused their attention on the isolation, cloning and mutation of this enzyme (Reetz and Carballeira 2007; Acharya et al., 2004). Screening for microorganisms in soil from areas, which have extreme conditions such as very hot climates may lead to the isolation of highly useful enzymes (Masayama et al., 2007). In the present study, the first step towards obtaining this goal was investigated, which involved the setting up of a fast and reliable technique for screening collected soil samples for lipase-containing Bacillus strains.
Soil samples from different areas of Isfahan in Iran that included river banks, gardens and agricultural lands were collected and dried at room temperature for 48 h. Subsequently, different dilutions of these samples were plated onto nutrient agar plates (incubated for 24 hours at 37ºC) and bacterial colonies were obtained. From these colonies, the ones displaying the colony morphology of B. subtilis were separated, streaked onto nutrient agar plates and incubated at 37ºC for 24-48 h. For the biochemical and molecular experiments, B. subtilis PTCC 1254 and Bacillus licheniformis (PTCC) 1331 obtained from the Persian Type Culture Collection were used as positive and negative controls, respectively.
Biochemical tests for the detection of B. subtilis strains were performed according to the procedures reported by Mc Fadden (2000) which included tests for catalase, lecithinase, nitrate, Voges-Proskauer (VP), citrate and maltose. DNA extraction of the chosen colonies was performed using the “high pure template preparation kit” (Roche, Germany). Primers were designed according to the two conserved regions of the B. subtilis lipase A gene (Forward: 5´-ATGGTTCACGGTATTGGAGG-3´ and Reverse: 5´-CTGCTGTAAATGGATGTGTA-3´). The polymerase chain reaction (PCR) was performed according to a previous procol (Mir Mohammad Sadeghi et al., 2008) and the obtained products were electrophoresed on 0.7% (w/v) agarose gel. Based on the B. subtilis lipase A gene sequence, the amplified PCR product should be approximately 371 bp. Single strand conformational polymorphism (SSCP) of the amplified PCR products was conducted using procedures described by Sambrook and Russell (2001).
The results of the biochemical tests performed on 15 colonies are shown in Table 1. Based on these tests, four samples were identified as being putative B. subtilis (colony numbers 3, 6, 8 and 13).
PCR products obtained after amplification of the lipase A gene are illustrated in Figure 1. Only in the positive control, a weak band of approximately 370 bp was observed. Regarding the samples isolated from Iranian soil, four samples which were identified as putative B. subtilis also showed a band of approximately 370 bp. Since the intensity of the obtained bands between these samples was different, optimization of the experimental conditions was performed by altering MgCl2 concentration (1-, 1.5-, 2-, 2.5- and 3 mM) and the annealing temperature (55-, 58.3- and 64ºC). As can be seen in Figure 2, the MgCl2 concentration of 2.5 mM gave the most intense band. Regarding the annealing temperature, the intensity of the bands decreased when higher annealing temperatures (64ºC) was applied (Fig. 3).
Form the results obtained, the optimum PCR conditions for the detection of the lipase A gene in putative B. subtilis strains included an annealing temperature of 55ºC and MgCl2 concentration of 2.5 mM. Thus, the samples were amplified under these optimized conditions and then electrophoresed (Fig. 4). Which showed that the amount of amplified DNA for all samples increased in comparison to previous experiments (Fig. 1). However the differences observed in these samples regarding the intensity of the obtained DNA bands were still present.
The SSCP results are shown in Figure 5. The patterns of the obtained bands for samples 3 and 8 were similar to each other. Sample 13 and the control showed the same pattern (lanes 4 and 5) but were different from samples 3, 6 and 8 (lanes 1-3, respectively).
The results of this study indicate that the designed primers were suitable for use in molecular identification of the B. subtilis lipase A gene. Additionally, when B. licheniformis DNA (a bacterium closely related to B. subtilis) was used as template, no PCR bands were obtained demonstrating the specificity of these primers.
The putative B. subtilis colonies showing a faster response to the citrate test also had less intense PCR bands. This correlation can offer a possible tool for identifying differences in B. subtilis strains using the intensity of the PCR bands as an indicator. The SSCP experiments also revealed that the differences in the intensity of the PCR bands could have been due to the polymorphism of this gene.
Pinchuk et al. (2002) have studied the genetic diversity and production of isocoumarin production by various B. subtilis strains isolated from different habitats however, they did not detect the lipase gene. In another investigation, Ruiz et al. (2005) collected samples from three different areas in Argentina and after obtaining colonies, PCR amplification of the lipase gene from the high enzyme producing strains was carried out. The difference in that study was the use of enzyme activity for the screening method rather than the obtained PCR bands. It seems that a combination of this method and those that have been devised in our study can create a successful and easy procedure for isolating B. subtilis strains having the desired lipolytic activity.
In conclusion, in this study, a fast and reliable method for the detection of the B. subtilis lipase A gene has been developed. Morever, the bacterial strains containing this gene have been identified in the Iranian soil samples. This will help in further screening of soil samples for lipase producing B. subtilis strains.