Identification and in-silico analysis of a novel restriction enzyme coding gene from Pseudomonas anguilliseptica

Abstract

Type II restriction enzymes (REs) which can cleave double stranded DNA in a sequence specific manner have many applications in recombinant DNA technology and are considered the workhorses of molecular biology. This study was carried out to screen bacterial communities for restriction enzymes. Soil and water samples were screened for isolation of bacteria, harboring restriction enzymes. Cell lysates of isolated bacteria were incubated with λ DNA, followed by analysis by agarose gel electrophoresis. The presence of distinct banding patterns indicated the presence of REs. Nine putative isolates harboring REs were morphologically and molecularly characterized using 16S rRNA analysis and belonged to four different genera (Acinetobacter, Lysinibacillus, Pseudomonas, and Brevibacillus). A HindIII like restriction digestion profile was observed in a lysate of a soil bacterium belonging to genus Pseudomonas. Based on 16S rRNA analysis, the bacterial species was identified as P. angulliseptica. The enzyme was partially purified and optimum conditions for enzyme activity and its recognition sequence were determined. The enzyme showed optimum activity at 40 °C and was stable at 40 °C for 20 min without the DNA substrate. The recognition sequence of the enzyme was determined and found to be 5’AAGCTT 3′ indicating it to be an isoschizomer of HindIII. The whole genome of the Pseudomonas species was sequenced and the coding sequence of the gene for the putative HindIII isoschizomer was identified together with other genes encoding putative REs. The gene coding for the HindIII isoschizomer was analyzed in-silico and its homology and evolutionary relationship to other known isoschizomers of HindIII were determined. The enzyme was tentatively designated as PanI.Our study clearly demonstrated that PanI is an isoschizomer of HindIII with very little sequence similarity to the latter. It can be used as an alternative to HindIII in molecular biological applications.