Immunogenicity studies of recombinant RBD SARS-CoV-2 as a COVID-19 vaccine candidate produced in Escherichia coli.

The severe acute respiratory syndrome coronavirus 2 -related global COVID-19 pandemic has been impacting millions of people since its outbreak in 2020. COVID-19 vaccination has proven highly efficient in reducing illness severity and preventing infection-related fatalities. The World Health Organization has granted emergency use approval to multiple, including protein subunit technology-based, COVID-19 vaccines. Foreseeably, additional COVID-19 subunit vaccine development would be essential to meet the accessible and growing demand for effective vaccines, especially for Low-Middle-Income Countries (LMIC). The SARS-CoV-2 spike protein receptor binding domain (RBD), as the primary target for neutralizing antibodies, holds significant potential for future COVID-19 subunit vaccine development. In this study, we developed a recombinant Escherichia coli-expressed RBD (rRBD) as a vaccine candidate and evaluated its immunogenicity and preliminary toxicity in BALB/c mice. The rRBD induced humoral immune response from day 7 post-vaccination and, following the booster doses, the IgG levels increased dramatically in mice. Interestingly, our vaccine candidate also significantly induced cellular immune response, indicated by the incrased IFN-É£-producing cell numbers. We observed no adverse effect or local reactogenicity either in control or treated mice. Taken together, our discoveries could potentially support efficient and cost-effective vaccine antigen production, from which LMICs could particularly benefit.

Isolation, expression, and characterization of raw starch degrading α-amylase from a marine lake Bacillus megaterium NL3.

A land-locked marine lake Kakaban with its significant ecological paramaters provides a unique habitat for bacteria with novel biotechnology potential that uses a diverse array of catalytic agents, including α-amylase. Aiming at the isolation of raw starch degrading α-amylase from marine biodiversity, a gene encoding BmaN2 from a sea anemone associated bacterium Bacillus megaterium NL3 was cloned and expressed in Escherichia coli ArcticExpress (DE3). It comprises an open reading frame of 1,563 nucleotides encoding BmaN2 of 520 amino acids and belongs to the glycoside hydrolase family 13 subfamily 36 (GH13_36). This α-amylase has a maximum activity at pH 6.0 and 60 °C with a specific activity of 28.7 U mg-1. The BmaN2 activity is enhanced strongly by Ca2+ but inhibited by EDTA. BmaN2 also exhibits high catalytic efficiency on soluble starch with k cat /K M value of 14.1 mL mg-1 s-1. Despite no additional starch-binding domain, BmaN2 is able to hydrolyze various raw starches, such as wheat, corn, cassava, potato, rice, sago, and canna, in which granular wheat is the preferred substrate for BmaN2. These characteristics indicate that BmaN2 is a promising raw starch degrading enzyme within the subfamily GH13_36.

Alkyl hydroperoxide reductase from Bacillus aquimaris MKSC 6.2 protects Esherichia coli from oxidative stress.

Alkyl hydroperoxide reductase genes (ahpCF) from the soft coral associated Bacillus aquimaris MKSC6.2 have been isolated. The cloned 546 bp ahpC gene encodes a 181 amino acid residues polypeptide. The AhpC belongs to typical 2-Cys peroxiredoxin (Prx) containing conserved peroxidatic cysteine residue (C46 ) required for hydroperoxide reduction and conserved resolving cysteine (C166 ). The isolated 1530 bp ahpF gene encodes a polypeptide of 509 amino acid residues with two conserved C128 HNC131 and C337 PHC340 catalytic residues required for reduction of oxidized-AhpC during catalytic turnover. A survival study with Escherichia coli showed that overexpression of AhpC and AhpF resulted in a total protection against 0.16 mM t-butyl hydroperoxide.

Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase.

Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 µM, while that of Sfamy01 was 31 ± 3.9 µM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.