Surface expression of carbonic anhydrase on E. coli as a sustainable approach for enzymatic CO2 capture.

The utilisation of carbonic anhydrase (CA) in CO2 sequestration is becoming prominent as an efficient, environment friendly and rapid catalyst for capturing CO2 from industrial emissions. However, the application of CA enzyme in soluble form is constrained due to its poor stability in operational conditions of CO2 capture and also production cost of the enzyme. Addressing these limitations, the present study focuses on the surface display of CA from Bacillus halodurans (BhCA) on E coli aiming to contribute to the cost-effectiveness of carbon capture through CA technology. This involved the fusion of the BhCA-encoding gene with the adhesion molecule involved in diffuse adherence (AIDA-I) autotransporter, resulting in the efficient display of BhCA (595 ± 60 U/gram dry cell weight). Verification of the surface display of BhCA was accomplished by conjugating with FITC labelled anti-his antibody followed by fluorescence-activated cell sorting (FACS) and cellular fractionation in conjunction with zymography. Biochemical characterisation of whole-cell biocatalyst revealed a noteworthy enhancement in thermostability, improvement in the thermostability with T1/2 of 90 ± 1.52 minutes at 50 ˚C, 36 ± 2.51 minutes at 60 ˚C and18 ± 1.52 minutes at 80˚C. Surface displayed BhCA displayed remarkable reusability retaining 100% activity even after 15 cycles. Surface displayed BhCA displayed highly alkali stable nature like free counterpart in solution. The alkali stability of the surface-displayed BhCA was comparable to its free counterpart in solution. Furthermore, the study investigated the impact of different metal ions, modulators, and detergents on the whole-cell biocatalysts. The present work represents the first report on surface display of CA utilising the AIDA-1 autotransporter.

Carbonic anhydrase as a tool to mitigate global warming.

The global average temperature breaks the record every year, and this unprecedented speed at which it is unfolding is causing serious climate change which in turn impacts the lives of humans and other living organisms. Thus, it is imperative to take immediate action to limit global warming. Increased CO2 emission from the industrial sector that relies on fossil fuels is the major culprit. Mitigating global warming is an uphill battle that involves an integration of technologies such as switching to renewable energy, increasing the carbon sink capacity, and implementing carbon capture and sequestration (CCS) on major sources of CO2 emissions. Among all these methods, CCS is globally accepted as a potential technology to address this climate change. CCS using carbonic anhydrase (CA) is gaining momentum due to its advantages over other conventional CCS technologies. CA is a metalloenzyme that catalyses a fundamental reaction for life, i.e. the interconversion of bicarbonate and protons from carbon dioxide and water. The practical application of CA requires stable CAs operating under harsh operational conditions. CAs from extremophilic microbes are the potential candidates for the sequestration of CO2 and conversion into useful by-products. The soluble free form of CA is expensive, unstable, and non-reusable in an industrial setup. Immobilization of CA on various support materials can provide a better alternative for application in the sequestration of CO2. The present review provides insight into several types of CAs, their distinctive characteristics, sources, and recent developments in CA immobilization strategies for application in CO2 sequestration.

Characterization of a novel thiol activated phospholipase TAPLB1 from Trichosporon asahii MSR 54.

Phospholipases are hydrolytic enzymes that play crucial roles in vivo and also possess immense biotechnological potential. In the present study, the phospholipase B of Trichosporon asahii MSR54 was overexpressed in E. coli and characterized. The 68-kDa enzyme was monomeric in solution and possessed phospholipase, lysophospholipase, esterase and acyltransferase activities. It was maximally active at pH 8.0 and 40 °C. The enzyme retained >50% activity between pH 3.0-8.0 and had a half-life of 30 min at 60 °C. Its activity was not metal dependent and was stable in the presence of most metal ions. Its catalytic efficiency on lysophosphatidyl choline was 1.0 × 103 mM-1 h-1. Site directed mutagenesis revealed R121 (present in the GYRAMV motif), S194 (present in the conserved GLSGG motif) and D420 (present in LVDXGE motif) to be the crucial amino acid residues for esterolytic activity. S194 and D420 were also the catalytic amino acids for lysophospholipase and phospholipase activities of the enzymes, while R121 was not involved in catalysis of phospholipid substrates. Further, it was found that cysteine residues in C61 and C354 were involved in disulphide linkages that imparted the properties of thiol activation and thermostability, respectively.

Thermo-alkali-stable α-carbonic anhydrase of Bacillus halodurans: heterologous expression in Pichia pastoris and applicability in carbon sequestration.

Recombinant α-carbonic anhydrase of the polyextremophilic bacterium Bacillus halodurans TSLV1 (rBhCA) has been produced extracellularly in active form in Pichia pastoris under methanol inducible (AOX1) as well as constitutive (GAP) promoters. A marked improvement in rBhCA production was achieved by developing a P. pastoris recombinant that produces rBhCA constitutively as compared to that under inducible promoter. The purified rBhCA from P. pastoris is a glycosylated protein that displays a higher molecular mass (79.5 kDa) than that produced from E. coli recombinant (75 kDa); the former has a Tm of 75 °C, which is slightly higher than that of the latter (72 °C). The former rBhCA exhibits higher thermostability than the latter. The former sequestered CO2 efficiently similar to that of the native BhCA and the latter. This is the first report on the production of recombinant carbonic anhydrase extracellularly in P. pastoris.

Characteristics of recombinant α-carbonic anhydrase of polyextremophilic bacterium Bacillus halodurans TSLV1.

Carbonic anhydrase (CA) is a biocatalyst that catalyzes the hydration of CO2 to bicarbonate and protons, thus useful in mitigating green house effect by sequestering CO2 from various point sources. An alkalistable and moderately thermostable α- carbonic anhydrase encoding gene (BhCA) from Bacillus halodurans TSLV1 has been cloned and expressed in Escherichia coli. A 31.4-fold enhancement in CA production was achieved due to cloning and expression in E. coli. About 50% of the CA produced was secreted when recombinant E. coli with BhCA-pET22b was cultivated in a medium with EDTA and lysozyme because of the efficient pelB leader sequence. rBhCA is a ∼75kDa homodimeric protein with a Tm of 72°C and T1/2 values of 66 and 24min at 50 and 60°C, respectively. SDM analysis revealed that H137, H139, H156 and H110 present in the active site play an important role in catalysis. Mineralization of CO2 using rBhCA led to the accelerated precipitation of CaCO3 in calcite form. rBhCA also functions as an efficient virtual peroxidase when Zn(2+) is substituted with Mn(2+).

Novel alkalistable α-carbonic anhydrase from the polyextremophilic bacterium Bacillus halodurans: characteristics and applicability in flue gas CO2 sequestration.

The emissions of CO2 into the atmosphere have been constantly rising due to anthropogenic activities, which have led to global warming and climate change. Among various methods proposed for mitigating CO2 levels in the atmosphere, carbonic anhydrase (CA)-mediated carbon sequestration represents a greener and safer approach to capture and convert it into stable mineral carbonates. Despite the fact that CA is an extremely efficient metalloenzyme that catalyzes the hydration of CO2 (CO2 + H2O ↔ HCO3 (-) + H(+)) with a kcat of ∼10(6) s(-1), a thermostable, and alkalistable CA is desirable for the process to take place efficiently. The purified CA from alkaliphilic, moderately thermophilic, and halotolerant Bacillus halodurans TSLV1 (BhCA) is a homodimeric enzyme with a subunit molecular mass of ~37 kDa with stability in a broad pH range between 6.0 and 11.0. It has a moderate thermostability with a T1/2 of 24.0 ± 1.0 min at 60 °C. Based on the sensitivity of CA to specific inhibitors, BhCA is an α-CA; this has been confirmed by nucleotide/amino acid sequence analysis. This has a unique property of stimulation by SO4 (2-), and it remains unaffected by SO3 (2-), NOx, and most other components present in the flue gas. BhCA is highly efficient in accelerating the mineralization of CO2 as compared to commercial bovine carbonic anhydrase (BCA) and is also efficient in the sequestration of CO2 from the exhaust of petrol driven car, thus, a useful biocatalyst for sequestering CO2 from flue gas.