Expression of haloacid dehalogenase gene and its molecular protein characteriza on from Klebsiella pneumoniae ITB 1

Organohalogen compounds are widely used industrially and agriculturally, as well as in households as flame retardants and refrigerants. However, these compounds can become significant pollutants through their accidental or deliberate release into the environment in large quan es. Dehalogenase is an enzyme with the poten al to be used in the removal of organohalogen contaminants. A previous study successfully subcloned a 690 bp of haloacid dehalogenase gene (hakp1) from Klebsiella pneumoniae ITB1 into a pET-30a(+) expression system to achieve high enzyme produc vity. IPTG was used as an inducer to express a pET-hakp1 recombinant clone in Escherichia coli BL21 (DE3). The molecular mass of the haloacid dehalogenase Hakp1 protein was 30 kDa as determined by SDS-PAGE. Zymogram analysis showed that this recombinant protein has dehalogenase ac vity as shown by the forma on of AgCl white precipitate. A quan ta ve assay of haloacid dehalogenase Hakp1 gave a specific ac vity of 84.29 U/mg with the op mum temperature of 40°C at pH 9. Predicted threedimensional structure of Hakp1 showed α/β mo f folding which comprised of cap and core domain. The predicted ac ve sites of Hakp1 were Asp8, Glu10, Leu22, Phe23, Trp90, Ser125, Ser126, Lys159, and Asp184 with Asp8, Glu10, Ser126, and Lys159 act as binding residue. This recombinant haloacid dehalogenase clone provides an alterna ve agent for effec ve bioremedia on of organohalogen pollutants.

Organohalogen compounds are compounds that contain carbon-halogen bond.Large-scale synthesis and extensive uses of these organic chemicals in many areas of agriculture and industry have led to widespread distribution of harmful compounds in the environment and created pollution problems.These chemicals, which are produced industrially and introduced into the environment as novel compounds, or at a concentration that exceeds the normal amount, are called xenobiotics (Top and Springael 2003).Organohalogen compounds, which constitute of more than 75% compounds listed as "priority pollutants", have been widely used as herbicides, pesticides, fungicides, solvents, plasticizers, paints, printing ink, adhesives, hydraulic and heat transfer fluids, flame retardants, refrigerants, additives for cutting oils, textile auxiliaries, and intermediates for other fine chemical synthesis (Fetzner and Lingens 1994;Janssen et al. 1994).
These chemically synthesized organic compounds are not readily degraded in the environment, and many are accumulate in soil water, groundwater, lake, and river (Esteve-Núñez et al. 2001;Pervova et al. 2002).Besides local and regional contamination, organohalogen continues to be a global issue pollutants, partly because its transport through water and air helps these compounds to spread across the Earth (Iwata et al. 1993).The persistence of these compounds in the environment causes considerable human health problems because of their toxicity and bioaccumulation in the food chain and ground water (Brokamp et al. 1997;Dórea 2008).It is well-known that some organohalogen compounds degrade slowly and form toxic intermediates which may affect cellular metabolic processes (Slater et al. 1995;Janssen et al. 2001).
Reactions between natural organic matter and chlorine compounds produce haloacetic acids at ppt to ppb ranges in drinking water distribution systems and in ppb to ppm ranges in wastewater (Rebhun et al. 1997).The concern over the carcinogenicity of haloacetic acids led the United States Environmental Protection Agency to regulate the allowable concentration of haloacetic acids in drinking water as part of the Disinfectants and Disinfection by Products Rule promulgated in 1998.Five haloacetic acids, known as HAA, are regulated as a part of the rule.These are monochloroacetic acid (MCA), dichloroacetic acid (DCA), trichloroacetic acid (TCA),

Anggoro and Ratnaningsih
Indonesian Journal of Biotechnology 22(1), 2017, 1-5 bromoacetic acid (BAA), and dibromoacetic acid (DBA).HAA, which is expressed as the sum of the concentrations of these acids, is currently limited there to 60 ppb (Xie and Zhou 2002).The current concentration of HAA in the Sidoarjo (Rosyidi 2010) and Sukabumi (Indraningsih et al. 2006) water supply sampled was approximately 50 ppb.Because of their widespread occurrence, toxicity to plants and aquatic organisms, and most importantly their suspected human carcinogenicity, there is a great need to find treatment methods for haloacetic acids.
In nature, some microorganisms are known as able to degrade organohalogen compounds and play a major role in biodegradation and decontamination (Weightman and Slater 1980;de Lorenzo 2008).Microbes degrade organohalogen compounds in order either to exploit them for growth as a carbon source and/or as a means of protection against its toxicity (Müller and Lingens 1986).There are several key requirements for a microorganism to degrade xenobiotics: (a) the ability to transport the compound into the cell where enzyme action can occur; (b) degradative catabolic genes must be expressed producing functional enzymes; (c) and the product of enzyme must be able to enter metabolism pathways (i.e. to be a growth substrate) (Weightman and Slater 1980;Weightman et al. 1985).Dehalogenases are key enzymes in the degradation of organohalogen compounds (Fetzner and Lingens 1994;Janssen et al. 1994).
A problem in using a wild-type bacterial dehalogenase for industrial biotransformation is rare because of their basal level availability and its low efficiency in degrading organohalogen pollutants.The advance of molecular biotechnology provides a solution to increase dehalogenases production by microbes through cloning and subcloning of the gene encoding dehalogenase into expression system for high enzyme productivity.Previous studies had been successfully cloned the haloacid dehalogenase gene from Klebsiella pneumoniae ITB1 (hakp1) into pET-30a(+) expression vector, named as pET-hakp1 (Anggoro and Ratnaningsih 2017).This system would make good expression because it contains a strong regulatable promoter for gene expression.In order to analyze the function of hakp1 gene, the pET-hakp1 should be transformed into Escherichia coli BL21 (DE3) and expressed by IPTG induction, which is performed in this research.

Transforma on and clone selec on
The pET-hakp1 recombinant clone isolated from E. coli TOP10 was transformed into competent E. coli BL21(DE3) cells using heat shock method (Sambrook and Russell 2001).The obtained transformants were plated on LB medium supplemented with 50 µg kanamycin for antibiotic screening.The E. coli BL21 (DE3) colonies harboring pET-hakp1 recombinant clone would survived on this medium due to the presence of kanamycin resistance gene in pET-30a(+).

Expression and extrac on of haloacid dehalogenase
Single colony of E. coli BL21(DE3) harboring pET-hakp1 was grown at 37°C on 100 mL liquid LB medium supplemented with 50 µg kanamycin.Induction was performed by adding 1 mM IPTG when the 450 nm optical density of this culture reached around 0.6.These induced cells were then harvested by centrifugation at 5,000 rpm for 10 min after 4-h induction, washed and resuspended in 10 mL of 50 mM Tris-acetate buffer (pH 7.5).Cells were disrupted by sonication for 30 min at 4°C.A crude extract was obtained after the cell debris had been removed by centrifugation.

Es ma on of haloacid dehalogenase molecular mass
The molecular mass of denatured protein from the crude extract was estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) utilizing protein ladder standard.Gel was visualized using staining Commassie Brilliant Blue solution.

Zymogram assay
Qualitative assay of haloacid dehalogenase activity in the crude extract was determined by zymogram.Crude extracts were electrophorized using Native-PAGE, and the resulted gel was incubated with 50 mM monochloroacetic acid (MCA) at 40°C for 10 min, and then soaked and incubated at room temperature for 1 h in 0.1 M AgNO 3 solution.Haloacid dehalogenase activity was showed by the formation of AgCl white precipitate.

Enzyma c assay
Haloacid dehalogenase activities were routinely measured with MCA as substrate.A suitable enzyme solution was incubated in 1 mL of 5 mM MCA with various pH and temperature for 10 min.Liberation of chloride ion was followed spectrophotometrically as described by Bergmann and Sanik (1957).One unit of dehalogenase activity was defined as the amount of enzyme that catalyzed the forma- tion of 1 µmol of chloride ion per min.Protein was determined using Bradford reagent with bovine serum albumin as standard (Bradford 1976).

Bioinforma c analysis
The three-dimensional structure of deduced Hakp1 protein was predicted using Swiss Model and I-TASSER program.The active site of this protein was analyzed using Sequences Annotated by Structure (SAS).Molecular docking was done using Autodock Vina to determine the binding site, ligand conformation, and affinity energy.

Expression of recombinant hakp1 gene on E. coli BL21(DE3) biofilm qualita ve analysis by SEM
Expression of hakp1 gene in pET-hakp1 recombinant clone was studied by induction of IPTG.The SDS-PAGE analysis on the cell pellet protein showed that the overexpression was occurred after 4-h induction (Figure 1a).On this gel, haloacid dehalogenase was seen more concentrated in the cell lysate compare to that in the cell debris.This fact indicated that the haloacid dehalogenase was present as soluble protein.Molecular mass estimation of this haloacid dehalogenase recombinant was obtained at around 30 kDa (Figure 1b).This size was larger than 25.5 kDa in silico analysis of Hakp1 protein, which was possibility due to the addition of fusion 6x His-Tag, S-Tag, and enterokinase site on its N-terminus.

Dehalogenase ac vity of recombinant haloacid dehalogenase
The zymogram assay showed that the haloacid dehalogenase produced by pET-hakp1 has detectable activity on MCA.This result was proved by the formation of AgCl white precipitate observable on the gel (Figure 2).The size of this active halaocid dehalogenase was approximately 30 kDa.
Further assay of recombinant haloacid dehalogenase activity were determined by identified chloride ion re- leased upon incubation with 5 mM MCA, by varying pH (Figure 3a) and temperature (Figure 3b) to obtain optimum condition of enzymatic reaction.The result suggests that the maximum activity of the recombinant haloacid dehalogenase was 84.29 U/mg at 40°C and pH 9. At this condition, the total chloride ion detected was 684 µM representing 13.68% of MCA degradation.This activity was three times higher compare to haloacid dehalogenase activity from the wild-type K. pneumoniae ITB1 (Tahya and Ratnaningsih 2015).The specific activity was not as high as expected because the haloacid dehalogenase crude used was still impure.

Conclusions
The pET-hakp1 system have been successfully express a 30 kDa recombinant haloacid dehalogenase.This recombinant dehalogenase has 84.29 U/mg optimum activity at 40°C and pH 9. Dehalogenase activity assay showed that the enzymes can degrade 13.68% chloride ion from 5 mM monochloroacetic acid substrate.Predicted threedimensional Hakp1 structure showed α/β folding motif with Asp8, Glu10, Ser126, and Lys159 as binding residues.These result suggested that the recombinant haloacid dehalogenase clone has potentially used as bioremediation agent for organohalogen pollutants in the environment.Further studies with enzyme purification and gene mutation needed to be done to improve the purity and activity of the recombinant haloacid dehalogenase.

FIGURE 1
FIGURE 1 SDS-PAGE electropherogram of the pET-hakp1 gene expression in E. coli BL21(DE3).(a) Overexpression analysis; M: Promega Broad Range Protein Ladder; 0: Total cell protein before IPTG induc on; 1-4: Total cell protein a er IPTG induc on from 1 to 4 h; Deb: Total protein in cell debris; Eks: Protein content in the medium (represen ng extracellular protein); Lis: Total protein in the cell lysate.(b) Molecular mass analysis of Hakp1.M: Promega Broad Range Protein Ladder; 1-2: Cell lysate protein from recombinant E. coli BL21 (DE3); C : Cell lysate protein from non-recombinant E. coli BL21 (DE3).

FIGURE 3 FIGURE 4
FIGURE 3 Ac vity assay of recombinant haloacid dehalogenase (a) Effect of pH on enzyme ac vity.(b) Effect of temperature on enzyme ac vity