Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system.

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.

Expression of Codon-Optimized Gene Encoding Murine Moloney Leukemia Virus Reverse Transcriptase in Escherichia coli.

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L-1 to 0.175 g L-1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.

Expression and characterization of thermostable D-allulose 3-epimerase from Arthrobacter psychrolactophilus (Ap DAEase) with potential catalytic activity for bioconversion of D-allulose from d-fructose.

A novel D-allulose 3-epimerase (DAEase) from Arthrobacter psychrolactophilus (Ap DAEase) was first characterized in this study. The enzyme catalyzes the epimerization of d-fructose into a functional rare sugar, D-allulose. Ap DAEase was the first record of DAEase identified as a homotrimer with the molecular weight of its subunit at approximately 34 kDa. It had an optimum activity at pH 8.5 and 70 °C in the presence of 1 mM Mg2+. Ap DAEase was found to be an excellent thermostable enzyme. The half-life value at 70 °C was 128.4 min. The kcat and catalytic efficiency of the enzyme toward d-fructose were 2920.00 s-1 and 3.953 mM-1 s-1, respectively. To the best of our knowledge, Ap DAEase possesses the highest kcat among the previously reported DAEases. The conversion ratio of 500 and 100 mg L-1d-fructose to D-allulose was approximately 27 % in 15 and 90 min, respectively. These research findings suggest that Ap DAEase is a promising candidate for the industrial production of D-allulose.