Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs

Abstract

Background: Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc.

Results: In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins.

Conclusions: The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Keywords: Esterase; GDSL family lipase; Geothermal springs; Lipase; Thermophilic bacilli.

Fig. 1

Location of study sites. Maps of Armenia and Nagorno Karabakh showing the locations of studied geothermal springs with red marks. Close up photograph of 1. Akhourik geothermal spring. 2. Tatev geothermal spring and 3. Karvachar geothermal spring. The source of the map: http://map-caucasus.com/

Fig. 2

Phylogenetic tree obtained by distance matrix analysis showing the position of the strains Akhurik 107, Tatev 4 and Karvachar QB2. The tree was generated by the Neighbour-Joining method using MEGA 6.06 software. Bootstrap values per 100 bootstrap analysis are presented on the tree. Scale bar represents 0.005 substitutions per site

Fig. 3

Multiple sequence alignment for A. flavithermus QB2 a B. licheniformis Akhurik 107 b Geobacillus sp. Tatev 4 c strains and closely related lipases/esterases. Protein sequences were aligned with CLUSTALX (2.0.11) and ESPrint (3.0). Identical residues were marked with red background and the highly-conserved residues were showed as red font. The catalytic triads are indicated with arrow. Тhe triangle is showed aspartic residues involving in the Ca²⁺-binding site

Fig. 4

Phylogenetic analysis of lipase sequences (partial sequence, 200 amino acids). The data are illustrated as a neighbour-joining tree based on 12 lipase sequences from the NCBI Database