Anti-His tag antibody, clone I9

Bacterium Bacillus sp. SS105, isolated from Free Air CO2 Enriched (FACE) soil was previously screened for carbonic anhydrase activity and CO2 sequestration. In this study, strain was selected to amplify carbonic anhydrase encoding genes. The CA genes from Bacillus sp. SS105 were found to be homologous with beta-carbonic anhydrase (beta-CA) and gamma-carbonic anhydrase (gamma-CA). Both types of CA genes was cloned in pET30b (+) and expressed in E coliBL21 (DE3) with His-tag at the N-terminus. The recombinant proteins were purified by Ni-NTA affinity chromatography. The molecular size of beta-CA and gamma-CA were approximately 27 kDa and 25 kDa respectively. The optimum pH and temperature were found to be 8.0 and 37 degrees C respectively. The Zn+ was enhancing the CAs enzyme activity. Anions and modulators showed inhibitory effect on CAs at specific concentration. Functional domain analysis of both CA proteins showed conserved region of respective proteins. Recombinant enzymes were used for bio-mineralization based conversion of atmospheric CO2 into valuable calcite. Calcite formation was evaluated with or without use of enzymes and confirmed by SEM and XRD analysis. SEM result confirmed the conversion of flower-shaped unstable form of vaterite to hexagonal cubic stable form of calcite in presence of enzymes. (C) 2019 Elsevier B.V. All rights reserved.

Thraustochytrids are heterotrophic marine protists that are considered as important decomposers in the marine ecosystem; however, how they digest and uptake lipid nutrients from the environment is largely unknown. Genomic clustering analysis using thraustochytrid draft genome databases revealed that novel proteins with a Lipase_3 domain are commonly present in thraustochytrids, including Aurantiochytrium limacinum. After heterologous expression and His tag-based purification, protein ID: 145138 was identified as lipase/phospholipase capable of hydrolyzing triacylglycerol (TG) and phosphatidylcholine (PC). 145138 was secreted into the medium, and deletion of the 145138 gene in A. limacinum reduced the degradation of extracellular lipids. Fatty acids generated by 145138 were reused for the biosynthesis of PC and TG, and 145138 allowed A. limacinum to survive in the medium containing TG as a sole carbon source. 145138 hydrolyzed all the acyl-ester linkages of TG; however, the enzyme showed strict positional specificity toward phospholipids, generating 2-acyl lysophospholipids. The 2-acyl lysophospholipids showed stronger antimicrobial activity compared with 1-acyl lysophospholipids. These results suggested that 145138 is a bifunctional enzyme that contributes to the acquisition of lipid nutrients from the environment, as well as to generate antimicrobial lysophospholipids that are beneficial for competition with bacteria over lipid nutrients in the marine environment.