isopropyl-β-D-thiogalactoside Research Articles

High-level expression of Aerococcus viridans pyruvate oxidase in Escherichia coli by optimization of vectors and induction conditions.

This study demonstrates that a highly soluble pyruvate oxidase can be obtained in recombinant Escherichia coli by optimizing vectors and induction conditions. The pyruvate oxidase yield achieved is the highest reported so far, which provides a convenient and cost-saving way to produce pyruvate oxidase. This research promotes pyruvate oxidase application in the pharmaceutical and biochemical industries.

Expression, Purification and Characterization of a Novel Antimicrobial Peptide: Gloverin A2 from Bombyx mori

Worldwide, antibacterial resistance has increased dramatically in the past years. Antimicrobial peptides are a group of immune-related peptides/proteins that protect the host from microbial infections, and were identified broad activity against pathogenic microorganism and not easy to generate insecticide resistance. It appears that antimicrobial peptides possess great potential for developing into a new ideal antimicrobial drug. Bombyx mori Gloverin A2 (BMGlvA2) was an antimicrobial peptide of B. mori, however, directly extracted and purified BMGlvA2 was difficult and uneconomic. In this study, BMGlvA2 gene was successfully cloned from B. mori and heterologous expression with vector pET28a (+) by E. coli Rosetta (DE3). The selected E. coli Rosetta (DE3)-BMGlvA2 strain yielded 241 µg/ml recombinant BMGlvA2 (rBMGlvA2) protein after 9 h inducted by 1.0 mM IPTG (isopropyl-β-d-thiogalactoside) at 28 °C. rBMGlvA2 protein was identified by western blot and MALDI-TOF. MALDI-TOF analysis indicated that its amino acid sequence is consistent with naturally secreted proteins by B. mori (sequence coverage 78.71%). Importantly, inhibition zone assay shown that rBMGlvA2 efficiently inhibited the growth of several Gram-negative bacteria, including E. coli DH5α, E. coli Rosetta, E. coli K88, S. typhimurium and several Gram-positive bacteria, including S. aureus and B. subtilis. Furthermore, The MIC of rBMGlvA2 showed antimicrobial activity against E. coli DH5α. Meaningfully, rBMGlvA2 did not display hemolytic activity against pig red blood cells. In conclusions, the antimicrobial activity and hemolytic of rBMGlvA2 suggested that it could be one of potential alternatives to conventional antibiotics against Gram-negative and Gram-positive bacteria.

Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations.

We have constructed novel plasmids pANY2, pANY3, and pANY6 for flexible cloning with low false positives, efficient expression, and convenient purification of proteins. The pANY2 plasmid can be used for efficient isopropyl-β-d-thiogalactoside (IPTG) induced protein expression, while the pANY3 plasmid can be used for temperature-induced expression. The pANY6 plasmid contains a self-cleaving elastin-like protein (ELP) tag for purification of recombinant protein by simple ELP-mediated precipitation steps and removal of the ELP tag by self-cleavage. A urea-based denaturation and refolding processes for renaturation of insoluble inclusion bodies can be conveniently integrated into the ELP-mediated precipitation protocol, removing time-consuming dialysis steps. These novel vectors, together with the described strategies of gene cloning, protein expression, and purification, may have wide applications in biosciences, agricultural, food technologies, and so forth.

Efficient gamma-aminobutyric acid bioconversion by engineered Escherichia coli

ABSTRACTGamma-aminobutyric acid (GABA) can be converted into 2-pyrrolidone, an intermediate in the synthesis of nylon 4 and agrochemicals, which has great potential for application in the chemical industry. The main aim of this work was to construct a simple and efficient process for industrial production of GABA from L-glutamic acid (L-Glu) by whole-cell bioconversion. The gene encoding glutamate decarboxylase (GAD) from Lactococcus lactis FJNUGA01 was overexpressed using the most available vector in Escherichia coli, pET-28a. GABA production was enhanced by optimizing the concentration of the catalyst isopropyl-β-D-thiogalactoside (IPTG), induction temperature and induction time. After optimization, GABA production reached 98.4 g L−1 with 96 mol% conversion within 6 h was achieved by whole-cell conversion using the cells induced with 1 mmol L−1 IPTG for 12 h at 30 °C. Furthermore, when the initial concentration of L-Glu was adjusted, 204.12 g L−1 GABA with 34 g L−1 h−1 productivity was reached using 2 mol L−1 L-Glu as the substrate. During the 6 h whole-cell bioconversion process, almost all L-Glu was converted to GABA with a conversion efficiency of ∼99%. The high conversion efficiency and high productivity made the GABA purification occur downstream, and the whole process is more simple and economical for industrial application.

High cell‐density cultivation of phenolic acid decarboxylase‐expressing Escherichia coli and 4‐vinylguaiacol bioproduction from ferulic acid by whole‐cell catalysis

AbstractBACKGROUND4‐vinyl guaiacol (4‐VG) is a high value‐added product widely used in the cosmetic, pharmaceutical, and chemical industries. The practical bioproduction of 4‐VG using phenolic acid decarboxylases has been limited by its relatively high biocatalyst cost and low yield and product concentration.RESULTSIn the present study, high‐cell density cultivation was employed to improve the activity and production of phenolic acid decarboxylase from Bacillus licheniformis (BLPAD) in recombinant Escherichia coli. The factors influencing enzyme production in E. coli such as the induction point and temperature for induction and feeding strategies were optimized. The highest BLPAD activity (531 U mL‐1) and productivity (20.4 U mL‐1 h‐1), respectively, were achieved in a 5 L bioreactor using a glucose exponential feeding strategy with isopropyl β‐D‐thiogalactoside (IPTG) as inducer. Furthermore, a high BLPAD production level (512 U mL‐1) was achieved using lactose as an inducer and continuous lactose feeding. Using a biphasic emulsion system with equal volumes (1 L) of cyclohexane as a organic solvent and a substrate fed‐batch strategy, the concentration and conversion yield of 4‐VG reached 129.9 g L‐1 (85.6%) in a 5 L bioreactor by whole‐cell biocatalysis, which is the highest reported to date.CONCLUSIONThis study describes a strategy for large‐scale 4‐VG bioproduction using the biocatalytic method. © 2018 Society of Chemical Industry

Enhanced a novel β-agarase production in recombinant Escherichia coli BL21 (DE3) through induction mode optimization and glycerol feeding strategy

Agarases are hydrolytic enzymes that act on the hydrolysis of agar and have a broad range of applications in food, cosmetics and pharmaceutical industries. In this study, a glycerol feeding strategy based on induction mode optimization for high cell density and β-agarase production was established, which could effectively control acetate yield. First, exponential feeding strategy of glycerol with different overall specific growth rates (μ) was applied in the pre-induction phase. The results showed that the low μ (μ=0.2) was suggested to be the optimal for cell growth and β-agarase production. Second, the effects of induction temperature and the inducer concentration on cell growth and β-agarase production were investigated in the post-induction phase. When induced by isopropyl-β-d-thiogalactoside (IPTG), the strategy of 0.8 mmol/L IPTG induction at 20°C was found to be optimal for β-agarase production. When cultivation was induced by continuous lactose feeding strategy of 1.0 g/(L·h), the β-agarase activity reached 112.5 U/mL, which represented the highest β-agarase production to date. Furthermore, the β-agarase was capable of degrading G. lemaneiformis powder directly to produce neoagarooligosaccharide, and the hydrolysates were neoagarotetraose (NA4) and neoagarohexaose (NA6). The overall research may be useful for the industrial production and application of β-agarase.

Expression of Mastoparan B, a Venom Peptide, Via Escherichia coli C43 (DE3) Coupled with an Artificial Oil Body-Cyanogen Bromide Technology Platform.

Mastoparan B (MPB) is a venom peptide isolated from Vespa basalis (black-bellied hornet), one of the dangerous vespine wasps found in Taiwan. MPB is a tetradecapeptide (LKLKSIVSWAKKVL), amphiphilic venom peptide, with a molecular mass of 1.6 kDa. MPB belongs to an evolutionarily conserved component of the innate immune response against microbes. In this study, we attempted to modify a reliable oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB. The aim of this study was to develop an artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce the bioactive form of mastoparan B (MPB), which in a manner identical to that of its native counterpart. The plasmid pET30-His6-rOle(127M→L)-MPB was constructed, and then four different E. coli strains- BL21(DE3), BL21(DE3)pLysS, C41(DE3), and C43(DE3) were tested to identify the most suitable host for the pET30-His6-rOle(127M→L)-MPB fusion protein expression. We optimized the expression conditions by testing different growth temperatures, isopropyl-β-D-thiogalactoside (IPTG) concentrations, and post-induction collection times. Afterwards, the His6-rOle(127M→L)-MPB protein was purified by one-step nickel-chelated affinity chromatography (Ni2+-NTA) under denaturing conditions. The purified His6-rOle(127M→L)-MPB was selectively cleaved by thrombin protease to remove the His6-tag and the leader peptide from the N-terminus. Subsequently, rOle(127M→L)-MPB protein was constituted into AOB and incubated with CNBr for a cleavage reaction, which resulted in the release of the MPB from rOle(127M→L)-MPB protein via AOB. The purified MPB was identified by MALDI-MS and HPLC analysis, and its bioactivity was examined by antimicrobial testing. After a 2-h induction period, the E. coli C43(DE3) was found to be superior to BL21(DE3) and the other protease-deficient strains as an expression host. And, the optimal His6-rOle(127M→L)-MPB expression at 37°C for 2 h after induction with 5 µM IPTG. The purified MPB showed that a single major peak was detected by HPLC/UV detection with a retention time of 22.5 minutes, which was approximately 90% pure. The putative MPB, and over two-third of the peptide sequence was verified by the MALDI-MS analysis. Finally, the purified MPB was examined by a broth dilution-antimicrobial susceptibility test. These results indicated that the purified MPB was bioactive and very effective in anti-bacterial (E. coli J96) activity. Here, we successfully used the oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB peptide which, in a manner identical to that of its native counterpart. In this study, the recombinant oleosin based fusion strategy coupled with AOB-CNBr purification platform open a new avenue for the production of active MPB and facilitate the studies and applications of the peptide in the future for medicinal applications such as hypotension and antibacterial effect.

Functional expression, purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

ABSTRACTIn this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni2+ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.

Cloning, Expression Analysis and Enzyme Activity Assays of the α-Carbonic Anhydrase Gene from Chlamydomonas sp. ICE-L.

The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.8mM. A single band with a molecular weight of approximate 40kDa expressed in the recombinant E. coli strain harboring the α-CA vector was observed in SDS-PAGE analysis. The carbon dioxide hydration activity and esterase activity of α-CA expressed by the recombinant strain were 0.404U/mg and 0.319U, respectively. In addition, three conditions, temperature, salinity and UVB radiation exposure, were selected to analyze α-CA transcription levels by qRT-PCR. The results suggested UVB exposure increased the expression of relative mRNA; meanwhile, the α-CA mRNA expression was rapidly induced by temperature and salinity stress, indicating that Chlamydomonas sp. ICE-L might modulate the α-CA mRNA expression to adapt to the extreme environments.

Cloning and prokaryotic expression of the globular head domain of hemagglutinin antigen (HA1) of influenza A (H3N2) virus in Escherichia coli and Bacillus subtilis

Background: The influenza virus hemagglutinin is the major surface protein of the influenza A virus which is composed of HA1 and HA2 subunits. HA1 has an important role in binding of virus to cells and designing neutralizing antibodies. Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) both are known as the most useful prokaryotic hosts to express recombinant proteins. The aim of this study was to clone and express recombinant HA1protein in E. coli and B. subtilis bacteria.Materials and Methods: HA1 gene was cloned into pET-28a vector and pHT43 shuttle vector and then, both transformed to E. coli. The recombinant plasmids were extracted and then transformed into the BL21 and WB600 as expressing hosts. After induction with isopropyl-β-d-thiogalactoside (IPTG), expressed recombinant protein was analyzed by SDS-PAGE. Finally, the expressed protein was confirmed by the Western blot.Results: HA1 gene was cloned into pET-28a vector and pHT43 shuttle vector and then, both transformed to E. coli.The recombinant plasmids were extracted and then transformed into the BL21 and WB600 as expressing hosts. After induction with isopropyl-β-d-thiogalactoside (IPTG), expressed recombinant protein was analyzed by SDS-PAGE. Finally, the expressed protein was confirmed by the Western blot.Conclusion: This study demonstrated a strategy for production and purification of recombinant protein in large scale to test as vaccine candidate against influenza and it’s potentially immunogenicity be assessed in animal models.

Cloning and expression of rice glutamate decarboxylase (GAD) in Escherichia coli.

Glutamate decarboxylase (GAD) converts L-glutamate to g-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms with some activities including improvement of neurve and cytoskeltal functions. Therefore, GAD is considered as a key molecule to use in molecular therapy of genetical human diseases. Accordingly, cloning of GADs from various plants is an important aim of researchers. The aim of this study was to clone rice (Oryza sativa L.) GADs in Escherichia coli (E.coli) MC 1061 bacterium.In this study, rice GADs was cloned in E.coli in both 37°C and 28°C. Two concentrations of Isopropyl-β-D-thiogalactoside (IPTG) (0.5mM and 1mM) were investigated in TB medium. Purification was done with Ni2+-nitrilotriacetic acid (NTA) resin in various concentration of imidasol. According to SDS-PAGE analysis, rice GADs was cloned and expressed successfully in E.coli MC 1061 bacterium and the most expression was done in 37°C and 0.5mM IPTG and the best concentration of imidasol was 100mM for purification step. Based on the results, rice GADs can be expressed in E.coli MC 1061 bacterium and, hence, it is a suitable way to produce the plant enzyme in industrial scales.

Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

<p class="Els-Abstract-text">The <em>xyn</em>B gene of <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the <em>xyn</em>B gene from <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the <em>xyn</em>B gene in <em>Escherichia coli </em>Origami as host cells. The <em>x</em><em>yn</em>B gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The <em>xyn</em>B gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for <em>xyn</em>B gene and for the vector, then continued by restriction analyses. The result showed that transformants clone 9 and 10 bear the recombinant pMMB-<em>xyn</em>B plasmid. The xylanase activity of <em>xyn</em>B gene in <em>Escherichia coli</em> Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-<em>xyn</em>B recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the <em>xyn</em>B gene of <em>Bacillus subtilis</em> subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the <em>Escherichia coli</em> Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.</p>

Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing.

Objective(s):Vascular endothelial growth factor (VEGF) is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs) differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli (E. coli) system and then biological activity of this protein was evaluated in animal wound healing.Materials and Methods:E. coli BL21 (DE3) competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w) was used for external wound (25×15mm thickness) healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment.Results:The recombinant protein with molecular weight of 45 kilodaltons (kDa) and concentration of 0.8 mg/ml was produced. Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing.Conclusion:Recombinant VEGF-A produced by pET32a in E. coli, possesses acceptable structure and has wound healing capability.

Expression and Activity Analysis of Fructosyltransferase from Aspergillus oryzae.

The fructosyltransferase gene was isolated and cloned from Aspergillus oryzae. The gene was 1368bp, which encoded a protein of 455 amino acids. To analyze the activity of the expressed fructosyltransferase, the pET32a-fructosyltransferase recombined plasmid was transformed into Escherichia coli BL21. The fructosyltransferase gene was successfully expressed by Isopropyl-β-d-thiogalactoside (IPTG) induction. The molecular weight of the expression protein was about 45kDa. The optimal conditions of protein expression were 25 °C, 0.1mM IPTG, and 8h of inducing time. The optimal concentration of urea dealing with inclusion body was 2.5M. The expressed protein exhibited a strong fructosyl transfer activity. These results showed that the expressed fructosyltransferas owned transferase activity, and could catalyze the synthesis of sucrose-6-acetate.

PicW orthologs from spruce with differential freezing tolerance expressed in Escherichia coli

Spruce can grow at an extra low temperature (LT), and is inferred with important antifreezing gene resources. The research here identified 4 different spruce varieties, named as PicW1, PicW2, PicM and PicK. Sequence alignment showed base-substitution and deficiency mutations among them with sequence identity between 97.61% and 99.25%. Each gene was transferred into E. coli, where protein was induced by IPTG (isopropyl-β-d-thiogalactoside). Strains cultured at −5°C showed the lethal dose 50% (LD-50) between 53h and 57h for the transgenic strains, but 35h for the control. Strains cultivated at −20°C showed the LD-50 between 38h and 44h for the transgenic strains, but 25h for the control. Further, the soluble gene proteins were extracted and purified for Differential Scanning Calorimeter (DSC) test, which showed characteristic thermal hysteresis (TH) value of 0.77°C (PicW1), 0.78°C (PicW2), 0.72°C (PicM), and 0.86°C (PicK) respectively, significantly higher than the value of 0.05°C of the control (BSA). Summarily, four homologous proteins showed good antifreeze property with the range from high to low as PicK>PicW2>PicW1>PicM. It suggested that they can be used as resources for genetic engineering of plant cold tolerance.

Partial Purification and Characterization of the Recombinant Benzaldehyde Dehydrogenase from Rhodococcus ruber UKMP-5M.

Background Benzaldehyde dehydrogenase (BZDH) is encoded by the xylC that catalyzes the conversion of benzaldehyde into benzoate in many pathways such as toluene degradation. Objectives In this study, the xylC gene from Rhodococcus ruber UKMP-5M was expressed in Escherichia coli, purified, and characterized. Materials and Methods The xylC was amplified and cloned in E. coli. The recombinant plasmid pGEMT-xylC was digested by NdeI and HindIII to construct plasmid pET28b-xylC and transformed in E. coli BL21 (DE3). Expression of the recombinant protein was induced by 1 mM isopropyl β-D-thiogalactoside (IPTG) at 37°C. The BZDH was purified by ion exchange chromatography, in which the product was an NAD-dependent enzyme using benzaldehyde as a substrate for enzyme characterization. The end metabolite was identified via gas chromatography mass spectrometry (GC-MS). Results The recombinant BZDH is 27 kDa, purified by ion exchange chromatography. The activity of BZDH was 9.4 U.μL-1 The optimum pH and temperature were 8.5 and 25ºC, respectively. The Michaelis constant (Km) and maximum velocity (Vmax) were 4.2 mM and 19.7 U.mL-1, respectively. The metabolite of BZDH was benzene carboxylic acid as determined by GC-MS analysis. Conclusions BZDH has the ability to degrade benzaldehyde to less toxic compounds. The BZDH is a critical enzyme for the degradation of aromatic hydrocarbons in Rhodococcus sp. The BZDH from R. ruber UKMP-5M is showed similar function with other aldehyde dehydrogenases.

A self-inducible heterologous protein expression system in Escherichia coli.

Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter’s transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale.

Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M.

In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).

Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: A prospective recombinant enzyme for leukaemia chemotherapy

l-Asparaginase is an antineoplastic agent that selectively reduces the level of l-asparagine in blood and diminishes the proliferation of cancerous cells. Studies were carried out on the cloning and heterologous expression of l-asparaginase biosynthesis gene (ansA) from Nocardiopsis alba NIOT-VKMA08 to achieve the stable inducible system that overproduces the glutaminase-free recombinant l-asparaginase. Overexpression of recombinant l-asparaginase was achieved with an optimized final concentration of 1.5mM of isopropyl-β-d-thiogalactoside (IPTG) and the enzyme was expressed as a soluble protein. The recombinant enzyme was purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography and the purified enzyme disclosed an elevated level of asparaginase activity (158.1IU/mL). Optimum pH and temperature of the purified l-asparaginase for the hydrolysis of l-asparagine were 8.0 and 37°C and it was very specific for its natural substrate, l-asparagine. Detailed studies were carried out on the kinetics of enzyme reaction, catalytic activity, temperature and ionic strength and the thermostability of the l-asparaginase enzyme. The functional characterisation of the recombinant l-asparaginase was studied through Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), in silico sequence analysis and protein structural modelling. Glutaminase activity was not detected in the recombinant l-asparaginase, which could reduce the probable side effects during leukaemia therapy.

Development of an Indirect Competitive ELISA Kit for the Detection of Soybean Allergenic Protein Gly m Bd 28K

The complementary DNA (cDNA) sequence of Gly m Bd 28K was expressed in Escherichia coli BL21 (DE3) as an inclusion body under the induction of 1.0 mmol/L of isopropyl β-D-thiogalactoside (IPTG). The inclusion bodies were solubilized in 6 M guanidine hydrochloride and refolded by rapid dilution. After renaturation, the purity of the recovered recombinant protein was up to 90 %, and its molecular weight was 29.1 kDa. A monoclonal antibody that was specific for recombinant Gly m Bd 28K protein was produced and characterized. Based on the mAb, an indirect competitive enzyme-linked immunosorbent kit (icELISA kit) for the specific detection of recombinant Gly m Bd 28K protein was developed, which showed that the IC50 was 3.19 μg/L and the sensitivity was 0.235 μg/L. The average relative standard deviation (RSD) was 10.28 %, and the average recovery was 96.73 %, which indicated that the kit was highly sensitive and accurate. The icELISA kit remained usable for more than 6 months when stored at 4 °C. Therefore, the icELISA is a valuable tool for the rapid and sensitive detection of Gly m Bd 28K in food and feed products from soybeans.