Lipases [triacylglycerol acyl hydrolases; (EC 18.104.22.168)] are a class of enzymes which are able to hydrolyze ester bonds of triacylglyceride substrates at oil-water interfaces [1-3]. Microbial lipases vary widely in enzymatic properties and substrate specificities.Consequently, they are currently receiving much attention because of their potential applications in various industrial and pharmaceutical processes. For example alkaline pH-resistant lipases are currently used as additives in detergents [4-6].
Bacillus subtilis 168 lipase (EC-000964) has become an attractive enzyme for industrial applications because:
a) It could be cloned and expressed in E.coli showing a broad substrate range which includes various–chain–length p-nitro phenyl esters and triglycerides.
b) Nucleotide and deduced amino acid sequences predict a remarkably alkaline pH of 9.73 and a very small size. In fact the B. subtilis enzyme is one of the smallest lipases described so far [2, 7, 8]. Additionally, interest in lipases has been increased recently due to their recognition as important virulence factors and their biotechnological potentials . As for the bacteria, B. subtilis has long been used in industery for the production of secretory proteins. Because of its nonpathogenic nature and high secretion capacity and existence of a great deal of fermentation technology, B. subtilis has been regarded as an attractive host for the secretion of endogenous as well as heterologous proteins [2, 9]. Therefore, it is evident that finding new strains of B. subtilis and isolation of industrially favorable lipases is of extreme importance. For this purpose, the present study was undertaken for the optimization of the polymerase chain reaction (PCR) method for the detection of lipase gene from B. subtilis 168.
Figure 1. Electrophoresis of the obtained PCR products using different annealing temperatures. MgCl2 concentration was kept constant at 2 mM. Column 1: DNA size marker (1kb ladder); columns 2-8: annealing temperatures of: 63.9, 62.2, 59.9, 56.2, 53.7, 52.0, 51.0 °C, respectively. A PCR product of 371 bp was expected. The bands observed below 100 bp are nonspecific (possibly primer dimmers).
2. Materials and methods
Ethidium bromide, loading buffer, 10X PCR buffer, and dNTP were purchased from CinnaGen, Iran. Primers were obtained from FazaBiotech, Iran. Taq polymerase (5 U/ìl) was purchased from Biotools, Spain. Gen Ruler (1kb DNA Ladder 250 ìg) was from Fermentas, Poland. High Pure PCR Template preparation kit was purchased from Roche, Germany, and Thermal cycler-MyCycler was from Bio-Rad, USA.
2.2. DNA extraction
Bacterial DNA from B. subtilis was extracted by high pure PCR template preparation kit. In this method, the bacterial sample was centrifuged and phosphate buffered saline (PBS) solution was added to the pellet. The mixture was centrifuged and buffer containing lysosyme was then added. After incubation for 15 min. at 37 °C, protease solution was added and the mixture was incubated for 10 min. at 70 °C. Then isopropanol was added and this solution was transferred to a filter tube and centrifuged. After a couple of washes, separating buffer (warmed to 70 oC) was added and centrifuged with the extracted DNA collected in an eppendorf tube. This DNA was kept at –20 °C for future use.
2.3. PCR reaction
The following primers were used for the amplification of the lipase A gene (targeting conserved regions of lipase coding sequence). Primer1: Sense 5´ATGGTTCACGGTATTGGAGG- 3´ Primer2: Antisense 5´-CTGCTGTAAATGGATGTGTA-3´ PCR reactions were performed according to the following conditions: Template DNA (50 ng) was added to 10X PCR buffer (5ìM), Taq polymerase (5U/ml), dNTP (100mM), forward and reverse primers (25 ìM) and (1, 1.5, 2, 2.5 and 3 mM) MgCl2 in a final volume of 50 µl. PCR conditions were as following: One cycle of (94 °C, 5 min.); thirty five cycles of denaturation (94 °C, 1 min.), annealing (different temperatures: 51.0, 52.0, 53.7, 56.2, 59.5, 62.2 and 63.9 °C, 2 min.), and extension (72 °C, 3 min.). The PCR product was electrophoresed on a 0.7% agarose gel. Based on the sequence of lipase Agene, a band of 371 bp was expected.
Electrophoresis of PCR products in different annealing temperatures showed that the optimal annealing temperature was 63.9 °C (keeping the MgCl2 concentration constant at 2 mM for all these experiments). When annealing temperatures of 56.2 °C and below were used, the lipase gene could not be consistently amplified (Figure 1). Using different concentrations of MgCl2 (keeping the annealing temperature constant at 55 oC) demonstrated that the optimal concentration was 2.5 mM. At lower MgCl2 concentrations no DNA bands could be detected (Figure 2).
Figure 2. Electrophoresis of the obtained PCR products using different MgCl2 concentrations. The annealing temperature was kept constant at 55.0 °C. Column 1: DNA size marker (1 kb ladder); columns 2-6: MgCl2 concentrations of: 3.0, 2.5, 2.0, 1.5 and 1.0 mM, respectively. Letters a and b show the expected band (371 bp). The bands observed below 100 bp are nonspecific (possibly primer dimmers).
Detection and isolation of lipase from new strains of bacteria is industrially and commercially very important. In the present study PCR conditions for the detection of lipase A gene from B. subtilis was optimized. Since higher annealing temperatures produced more intense bands, it can be concluded that the used primers were specific for the lipase A gene.
In fact because a highly conserved region of this gene was used for the primer design, it seems that the optimized PCR conditions can be used for the detection of lipase A gene from any B. subtilis strain. Therefore, this method can be used for the screening of various resources such as soil for the detection and isolation of B. subtilis strains.
In one study, the annealing temperature of 45-50 °C was used for the detection of lipase gene . However, in that study, the template used was a mutated lipase sequence present in a plasmid. In another study, the DNA from B. pulmilis was used for the amplification of lipase gene using the annealing temperatures of 50 °C and 65 °C . However, the experimental conditions for the detection of lipase gene had not been optimized.
In conclusion, for the first time, we have optimized the experimental conditions for the detection of lipase gene in B. subtilis which can be used for finding and isolation of new strains of these bacteria as sources for the production this enzyme.