Optimization of haloacid dehalogenase production by recombinant E. coli BL21 (DE3)/pET-hakp1 containing haloacid dehalogenase gene from Klebsiella pneumoniae ITB1 using Response Surface Methodology (RSM).

Organohalogens, including monochloroacetic acid (MCA), are abundantly synthesized compounds for various industrial purposes. MCA is widely used as a raw material or as an intermediate compound for the production of pesticides, herbicides, fungicides, plastics, surfactants, shampoos, liquid soaps, and emulsion agents. Nonetheless, widespread and large-scale utilization of organohalogens might negatively impact life quality as these compounds are toxic to organisms and persistently present in the environment. An effort to decrease the effect of MCA pollutant is by performing bioremediation, taking advantage of microorganisms that produce haloacid dehalogenases, a class of enzymes that catalyze the breakage of carbon halogen bonds. In this sense, we have isolated Klebsiella pneumoniae ITB1 that could degrade MCA. The haloacid dehalogenase gene from this bacterium has been successfully cloned into pGEM-T vector and subcloned into pET-30a(+) expression vector to yield pET-hakp1 recombinant clone in Escherichia coli BL21 (DE) host cell. This research aimed to find an optimum condition for producing haloacid dehalogenase from this recombinant clone using Response Surface Methodology (RSM). Among the independent variables studied were the concentration of inducer, incubation temperature after the induction, and incubation period after the induction. We obtained the crude extract of the enzyme as cells' lysate after sonicating the bacterial cells. Haloacid dehalogenase activity against MCA substrate was determined by measuring the amount of chloride ions released into the medium of the enzymatic reaction using the colorimetry method, according to Bergmann and Sanik. The result indicated that the optimum condition for haloacid dehalogenase production by E. coli BL21 (DE3)/pET-hakp1 was observed when using 1.8 mM IPTG (isopropyl-ß-D-1-thiogalactopyranoside) as the inducer, followed by 4 h incubation with shaking at 37 °C, which was predicted to result in a maximum of 0.48 mM chloride ions from 0.50 mM of MCA substrate. This report provides an insight into applying RSM for optimization of enzyme production from E. coli recombinant clones.

In Silico Analysis on the Interaction of Haloacid Dehalogenase from Bacillus cereus IndB1 with 2-Chloroalkanoic Acid Substrates.

Recently, haloacid dehalogenases have gained a lot of interest because of their potential applications in bioremediation and synthesis of chemical products. The haloacid dehalogenase gene from Bacillus cereus IndB1 (bcfd1) has been isolated, expressed, and Bcfd1 enzyme activity towards monochloroacetic acid has been successfully studied. However, the structure, enantioselectivity, substrate range, and essential residues of Bcfd1 have not been elucidated. This research performed computational studies to predict the Bcfd1 protein structure and analyse the interaction of Bcfd1 towards several haloacid substrates to comprehend their enantioselectivity and substrates' range. Structure prediction revealed that Bcfd1 protein consist of two domains. The main domain consists of seven ß-sheets connected by six α-helices and four 310-helices forming a Rossmannoid fold. On the other hand, the cap domain consists of five ß-sheets connected by five α-helices. The docking simulation showed that 2-chloroalkanoic acids bind to the active site of Bcfd1 with docking energy decreases as the length of their alkyl chain increases. The docking simulation also indicated that the docking energy differences of two enantiomers of 2-chloroalkanoic acids substrates were not significant. Further analysis revealed the role of Met1, Asp2, Cys33, and Lys204 residues in orienting the carboxylic group of 2-chloroalkanoic acids in the active site of this enzyme through hydrogen bonds. This research proved that computational studies could be used to figure out the effect of substrates enantiomer and length of carbon skeleton to Bcfd1 affinity toward 2-chloroalkanoic acids.

Molecularly Imprinted Affinity Membrane: A Review.

A molecularly imprinted affinity membrane (MIAM) can perform separation with high selectivity due to its unique molecular recognition introduced from the molecular-printing technique. In this way, a MIAM is able to separate a specific or targeted molecule from a mixture. In addition, it is possible to achieve high selectivity while maintaining membrane permeability. Various methods have been developed to produce a MIAM with high selectivity and productivity, with their respective advantages and disadvantages. In this paper, the MIAM is reviewed comprehensively, from the fundamentals of the affinity membrane to its applications. First, the development of a MIAM and various preparation methods are presented. Then, applications of MIAMs in sensor, metal ion separation, and organic compound separation are discussed. The last part of the review discusses the outlook of MIAMs for future development.

Box-Wilson Design for Optimization of in vitro Levan Production and Levan Application as Antioxidant and Antibacterial Agents.

Background: Levan or fructan, a polysaccharide of fructose, is widely used in various commercial industries. Levan could be produced by many organisms, including plants and bacteria. The cloning of the gene from Bacillus licheniformis, which expressed levansucrase in Escherichia coli host, was carried out successfully. In the present study, we performed the in vitro production of levan and analyzed its potential application as antibacterial and antioxidant agents. Methods: In vitro levan production catalyzed by heterologous-expressed levansucrase Lsbl-bk1 and Lsbl-bk2 was optimized with Box-Wilson design. The antibacterial activity of the produced levan was carried out using agar well diffusion method, while its antioxidant activity was tested by free radical scavenging assays. Results: The optimum conditions for levan production were observed at 36 °C and pH 7 in 12% (w/v) sucrose for levansucrase Lsbl-bk1, while the optimum catalysis of levansucrase Lsbl-bk2 was obtained at 32 oC and pH 8 in the same sucrose concentration. The in vitro synthesized levan showed an antibacterial activity within a concentration range of 10-20% (w/v) against Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa. The same levan was also able to inhibit the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with the antioxidant strength of 75% compared to ascorbic acid inhibition. Conclusion: Our study, therefore, shows that the optimized heterologous expression of levansucrases encoded by Lsbl-bk1 and Lsbl-bk2 could open the way for industrial levan production as an antibacterial and antioxidant agent.

Enhancement of antioxidant activity of levan through the formation of nanoparticle systems with metal ions.

Levan, a natural polymer, is widely used in biomedical applications, such as antioxidants, anti-inflammatory, and anti-tumor. The present study aimed to enhance the antioxidant activity of levan by combining it with various metal ions in the nanoparticle (NP) system. Levansucrase encoding gene from Bacillus licheniformis BK1 has been inserted into an expression vector and the obtained recombinant was labeled as Lsbl-bk1 (accession number MF774877.1). That enzyme was used for in vitro levan synthesis in 12% (w/v) sucrose as a substrate and about 4.28 mg/mL of levan was obtained. Levan-based metal ion NPs were synthesized using the coprecipitation method. In the production of NPs, levan acts as a reducing and stabilizing agent. Four types of levan-based metal ion NPs were synthesized, namely, levan-Fe2+ NPs, levan-Cu+ NPs, levan-Co2+ NPs, and levan-Zn2+ NPs. The transmission electron microscopy (TEM) technique was applied to visualize the size and shape of the synthesized levan-metal NPs. All levan-based metal ion NPs have a particle size of less than 100 nm, and even levan-Cu+ and levan-Zn2+ have particle sizes less than 50 nm. Levan-Fe2+ NPs and levan-Cu+ NPs exhibited prominent antioxidant activity with an inhibition level of up to 88% and 95%, respectively. And the inhibition level of two metal ion NPs had about 33%-40% higher antioxidant activity compared with the inhibition level of levan only. The two levan-metal ion NPs, therefore, have future prospects to be developed as the new formulation for the antioxidant drugs.

Production of hydrophobic amino acids from biobased resources: wheat gluten and rubber seed proteins.

Protein hydrolysis enables production of peptides and free amino acids that are suitable for usage in food and feed or can be used as precursors for bulk chemicals. Several essential amino acids for food and feed have hydrophobic side chains; this property may also be exploited for subsequent separation. Here, we present methods for selective production of hydrophobic amino acids from proteins. Selectivity can be achieved by selection of starting material, selection of hydrolysis conditions, and separation of achieved hydrolysate. Several protease combinations were applied for hydrolysis of rubber seed protein concentrate, wheat gluten, and bovine serum albumin (BSA). High degree of hydrolysis (>50 %) could be achieved. Hydrophobic selectivity was influenced by the combination of proteases and by the extent of hydrolysis. Combination of Pronase and Peptidase R showed the highest selectivity towards hydrophobic amino acids, roughly doubling the content of hydrophobic amino acids in the products compared to the original substrates. Hydrophobic selectivity of 0.6 mol-hydrophobic/mol-total free amino acids was observed after 6 h hydrolysis of wheat gluten and 24 h hydrolysis of rubber seed proteins and BSA. The results of experiments with rubber seed proteins and wheat gluten suggest that this process can be applied to agro-industrial residues.

Screening, gene sequencing and characterising of lipase for methanolysis of crude palm oil.

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/ß hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K(M) of 0.75 mM and a V(max) of 0.33 mMmin(-1) on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.