(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Firmicutes: NE > Bacilli: NE > Bacillales: NE > Bacillaceae: NE > Bacillus: NE > Bacillus pumilus: NE
Warning: This entry is a compilation of different species or line or strain with more than 90% amino acide identity. You can retrieve all strain data
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) Bacillus pumilus ATCC 7061: N, E.
Bacillus pumilus SAFR-032: N, E.
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MKVICFKKKSLQILVALALVIGSMAFIQPKEIKAAEHNPVVMVHGMGGAS YNFASIKSYLVTQGWDRNQLFAIDFIDKTGNNRNNGPRLSRFVKDVLGKT GAKKVDIVAHSMGGANTLYYIKNLDGGDKIENVVTLGGANGLVSLRALPG TDPNQKILYTSVYSSADMIVVNSLSRLIGARNVLIHGVGHISLLASSQVK GYIKEGLNGGGQNTN
The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic-psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus, which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and steady-state kinetics confirms the psychrophilic nature of the B. pumilus enzyme. These results provide a useful benchmark for examining the structural basis of cold-adaptation and should now make it possible to disentangle the effects of the 34 mutations between the two enzymes on catalytic properties and thermal stability.
Title: ThermoSlope: A Software for Determining Thermodynamic Parameters from Single Steady-State Experiments Lund BA, Brandsdal BO Ref: Molecules, 26:, 2021 : PubMed
The determination of the temperature dependence of enzyme catalysis has traditionally been a labourious undertaking. We have developed a new approach to the classical Arrhenius parameter estimation by fitting the change in velocity under a gradual change in temperature. The evaluation with a simulated dataset shows that the approach is valid. The approach is demonstrated as a useful tool by characterizing the Bacillus pumilus LipA enzyme. Our results for the lipase show that the enzyme is psychrotolerant, with an activation energy of 15.3 kcal/mol for the chromogenic substrate para-nitrophenyl butyrate. Our results demonstrate that this can produce equivalent curves to the traditional approach while requiring significantly less sample, labour and time. Our method is further validated by characterizing three alpha-amylases from different species and habitats. The experiments with the alpha-amylases show that the approach works over a wide range of temperatures and clearly differentiates between psychrophilic, mesophilic and thermophilic enzymes. The methodology is released as an open-source implementation in Python, available online or used locally. This method of determining the activation parameters can make studies of the temperature dependence of enzyme catalysis more widely adapted to understand how enzymes have evolved to function in extreme environments. Moreover, the thermodynamic parameters that are estimated serve as functional validations of the empirical valence bond calculations of enzyme catalysis.
Title: Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium, Bacillus pumilus Wi AR, Jeon SJ, Kim S, Park HJ, Kim D, Han SJ, Yim JH, Kim HW Ref: Biotechnol Lett, 36:1295, 2014 : PubMed
A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 degrees C, respectively. The enzyme retained 85 % of its activity at 5 degrees C. There was a significant difference between temperatures for maximal activity (20 degrees C) and for protein denaturation (approx. 45 degrees C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.
Bacillus pumilus (Bacillus mesentericus) Lipase
