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.

The use of Mycobacterium tuberculosis H37Ra-infected immunocompetent mice as an in vivo model of persisters.

Persistence of Mycobacterium tuberculosis (Mtb) is one of the challenges to successful treatment of tuberculosis (TB). In vitro models of non-replicating Mtb are used to test the efficacy of new molecules against Mtb persisters. The H37Ra strain is attenuated for growth in macrophages and mice. We validated H37Ra-infected immunocompetent mice for testing anti-TB molecules against slow/non-replicating Mtb in vivo. Swiss mice were infected intravenously with H37Ra and monitored for CFU burden and histopathology for a period of 12 weeks. The bacteria multiplied at a slow pace reaching a maximum load of ∼106 in 8-12 weeks depending on the infection dose, accompanied by time and dose-dependent histopathological changes in the lungs. Surprisingly, four-weeks of treatment with isoniazid-rifampicin-ethambutol-pyrazinamide combination caused only 0.4 log10 and 1 log10 reduction in CFUs in lungs and spleen respectively. The results show that ∼40 % of the H37Ra bacilli in lungs are persisters after 4 weeks of anti-TB therapy. Isoniazid/rifampicin monotherapy also showed similar results. A combination of bedaquiline and isoniazid reduced the CFU counts to <200 (limit of detection), compared to ∼5000 CFUs by isoniazid alone. The study demonstrates an in vivo model of Mtb persisters for testing new leads using a BSL-2 strain.

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.

Bio-imprinted magnetic cross-linked polyphenol oxidase aggregates for enhanced synthesis of L-dopa, a neurodegenerative therapeutic drug.

Bio-imprinted magnetic cross-linked enzyme aggregates (i-m-CLEAs) of polyphenol oxidase (PPO) obtained from potato peels were prepared using amino-functionalized magnetic nanoparticles. Bio-imprinting is being used to improve the catalytic efficiency and conformational stability of enzymes. For bio-imprinting, PPO was incubated with different imprint/template molecules (catechol, 4-methyl catechol and l-3,4-dihydroxy phenylalanine) before cross-linking with glutaraldehyde. CLEAs imprinted with 4-methyl catechol showed maximum activity as compared with non-bio-imprinted magnetic CLEAs (m-CLEAs). They were further characterized by scanning electron microscopy and confocal microscopy. In bio-imprinted m-CLEAs, half-life (t1/2) of PPO significantly improved (364.74 min) as compared to free PPO (43.58 min) and non-bio-imprinted m-CLEAs (266.54 min). Bio-imprinted m-CLEAs showed excellent thermal and storage stability as well as reusability. The CLEAs preparation were used for the synthesis of l-3,4-dihydroxyphenylalanine (L-dopa, a therapeutic drug to treat neurodegenerative disorder) and a remarkable increase in L-dopa yield (23.5-fold) was obtained as compared to free enzyme. A cost effective and reusable method has been described for the production of L-dopa.