Tengku Haziyamin Tengku Abdul Hamid

Molecular Modelling and Functional Studies of the Non-Stereospecific α-Haloalkanoic Acid Dehalogenase (DehE) from Rhizobium SP. RC1 and its Association with 3-Chloropropionic Acid (β-Chlorinated Aliphatic Acid)

ABSTRACT Many environmental pollutions are caused by the abundance of xenobiotic compounds in nature. For instance, halogenated compounds released from chemical industries were proven to be toxic and recalcitrant in the environment. However, haloalkanoic acid dehalogenases can catalyse the removal of halides from organic haloacids and thus have gained interest for bioremediation and synthesis of industrial chemicals. This study presents the first structural model and the key residues of the non-stereospecific haloalkanoic acid dehalogenase, DehE, from Rhizobium sp. RC1. The enzyme was built using a homology modelling technique; the structure of DehI from Pseudomonas putida PP3 was used as a template, because of its homology to DehE. The structure of DehE consists of only α-helices. Twelve conserved residues that line the active site were identified: Trp34, Ala36, Phe37, Asn114, Tyr117 Ala187, Ser188, Asp189, Tyr265, Phe268, Ile269, and Ile272. These residues are consistent with the residues found in the active site of DehI and D, L-DEX 113 from Pseudomonas sp. 113. Asp189 activates the water molecule as a nucleophile to attack the substrate chiral centre, which would result in an inversion of configuration of either D- or L-substrates. Both D- and L-substrates bind to and interact with the enzyme by hydrogen bonding with three residues, Trp34, Phe37, and Ser188. In addition, a putative tunnel was also identified that would provide a channel for the substrate to access the binding site. Based on computational analysis, DehE was proven to have the substrate affinity towards 3-chloropropionic acid (3CP)/β-chlorinated aliphatic acid, however, its dehalogenation process is far from clear. This DehE structural information will allow for rational design of non-stereospecific haloalkanoic acid dehalogenases in the future.

A review on non-stereospecific haloalkanoic acid dehalogenases

Haloalkanoic acid dehalogenases remove halides from organic haloacids and have potential as bioremediation agents. DehE from Rhizobium sp. RC1, DehI from Pseudomonas putida PP3 and D,LDEX 113 from Pseudomonas sp. 113 are non-stereospecific dehalogenases that invert the configurations of D- and L- carbons bound to a halogen. The kinetics of DehE has been partially characterized and brominated compounds have greater specificity constant values than do the corresponding chlorinated compounds. The sequence of DehE is similar to that of DehI; therefore, the two enzymes may have similar structures and functions. The three-dimensional structure of DehI is known and its reaction mechanism was inferred from its structure and a mutagenesis study of D,L-DEX 113. Aspartate residues at positions 189 and 194 in DehI and D,L-DEX 113 were predicted to be involved in catalysis. These residues activate a water molecule that directly attacks the chiral carbon. Because DehE and DehI are sequentially related, delineating the structure of DehE is important to ascertain if the catalytic residues and reaction mechanism are the same for both enzymes. A structural prediction, sequence-homology modeling and a site-directed mutagenesis study of DehE might help achieve this goal. Key words: Haloalkanoic acids, non-stereospecific dehalogenase, DehE, Rhizobium sp. RC1, enzyme kinetics, protein structure prediction, site-directed mutagenesis.

Lactic acid bacteria: bacteriocin producer: a mini review

Summary: LAB is a diverse bacterial group consisting of 11 genuses. These bacteria are Gram-positive, non-spore-forming, coccus or rods but aerotolerant, able to ferment carbohydrates into energy and lactic acid. Lactic acid bacteria produce various compounds such as organic acids, diacetyl, hydrogen peroxide, and bacteriocins or bactericidal proteins during lactic acid fermentations. Bacteriocins are peptides produced by a variety of microbes and have antimicrobial activity against closely related species. These antimicrobial agents are gaining more and more attention as an alternative therapeutics not only in pharmaceutical but also as a preservative in food industries. The main aim of this review is to highlight lactic acid bacteria and its bacteriocins.

The Role of Lid in Protein-Solvent Interaction of the Simulated Solvent Stable Thermostable Lipase from Bacillus Strain 42 in Water-Solvent Mixtures

ABSTRACT Lip 42 lipase, isolated from Bacillus sp. strain 42 was previously shown to be stable in polar organic solvent such as dimethyl sulfoxide (DMSO). Stabilities in different solvent compositions were studied based on 40°C pre-incubation in solvent, and the purified lipase was shown to retain at least 100% residual activity in up to 45% v/v DMSO. In 60% v/v DMSO, 68% of residual activity was retained, however, this dramatically reduced to 6.5% at 65% v/v DMSO. Activity enhancements were recorded at lower solvent compositions (less than 45% v/v solvent), whereby, at 30% v/v DMSO, enhancement was as much as 35%. Based on these solvent stability profile, molecular dynamic simulations were then carried out in the presence ofwater, 60% v/v DMSO + 40% v/v water and 100% v/v DMSO, by using a structure predictedfrom a highly homologous (97%) lipase (PDB:1JI3). Results showed that the flexibility changes in the helix-loop-helix motif covering catalytic triad were found to be associated with a hydrophobic cluster region. The presence of 60% v/v DMSO resulted in the disorganization of the cluster, accompanied with nonnative H-bonds formations. The cluster was retained in 100% v/v DMSO which resembled to that of water simulation. Mutant form of Lip 42, V171S contained residue substitution in the cluster and within helix-loop-helix motif. At 50°C pre-incubation, the mutant lost as much of the high temperature enhancements observed in low DMSO compositions. This indicated the potential role of hydrophobic residues in helix-loop-helix motif and the cluster in interfacial activation.

An S188V Mutation Alters Substrate Specificity of Non-Stereospecific α- Haloalkanoic Acid Dehalogenase E (DehE)

The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3-chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by Km. This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.

Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1

The non‐stereospecific α‐haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α‐haloalkanoic acid D,L‐stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D‐stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active‐site amino acids by site‐specific mutagenesis. Ten active‐site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D‐ and L‐2‐chloropropionic acid was assessed. Interestingly, the mutants W34 → A, F37 → A, and S188 → A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189 → N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non‐stereospecific α‐haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties.

Isolation of coagulase negative Enterococcous sp. strains from non-broiler chicken producing bacteriocin active against Staphylococcus aureus

Abstract Lactic acid bacteria (LAB) were isolated from the bile, gizzard, intestine and caecum of the Malaysian domestic non-broiler chicken. Of the 56 isolates grown on MRS agar plates, 7 - namely, B3L3, B4L4, G5L5, B5L6, B10L7, I1L8, and C4L10 - showed inhibition against the methicillin resistant Staphylococcus aureus (MRSA). These isolates were all Gram positive cocci, non-spore forming, and catalase negative. The purified fraction from the three phase partitioning (TPP) method, employing t-butanol and ammonium sulfate, produced inhibitory zones of 8 to 18 mm in diameter on the indicator organism, Staphylococcus aureus (MRSA). The TPP purified fraction contained heat stable proteins of about 10 kDa in size and was inactivated by trypsin. Based on the partial rDNA sequences (>89% similarity), isolates B4L4 and G5L5 belonged to Enterococcus hirae; isolate B3L3 belonged to Enterococcus faecium; isolates B5L6, B10L7, I1L8 to Enterococcus faecalis; and isolate C4L10 to Enterococcus mundtii. These probiotic strains, exclusively isolated from the Malaysian non-broiler chicken, produced an antimicrobial protein or bacteriocin which was active against a pathogenic strain of methicillin resistant Staphylococcus aureus (MRSA).

Lactococcus Lactis Strain A5 Producing Nisin-like Bacteriocin Active against Gram Positive and Negative Bacteria

In this study, a Lactic acid bacteria (LAB) strain was isolated on MRS medium from gastro-intestinal tissues of Broadhead catfish (Clarias macrocephalus). Out of 50 isolates, 25 isolates were found to be positive on lactose utilisation test and were identified to be gram positive cocci. Using disc diffusion methods, one out of 22 isolates, i.e., a strain A5 demonstrated inhibitions against three indicator organisms; Bacillus cereus, Staphylococcus aureus and Salmonella thyphimurium. Partial 16S rDNA sequencing identified isolate A5 as a member of Lactococcus lactis, with 100% DNA homology. Cell free supernatant fluid from Lactococcus lactis A5 showed inhibitory activities against both gram positive pathogens (Bacillus cereus and Staphylococcus aureus) and gram negative pathogens (Salmonella thyphimurium). Chloroform precipitated bacteriocin retained antagonistic activities in the presence of catalase and lysozyme; and was completely inactivated by Proteinase K treatment. The bacteriocin has a molecular weight of 3.4 kDa, based on SDS-PAGE analysis and the extract was heat stable at 37°C and 65°C, for 15 minutes. The antibacterial activity was suppressed with the addition of EDTA but was significantly increased with the addition of SDS, Triton X-100, Tween 20 and Tween 80. This bacteriocin belongs to class 1 bacteriocin, which was shown to have a nisin-like properties. This strain can be used as potential probiotics in animal or aquaculture feeding; and the bacteriocin it produces will be useful in food preservative.

Molecular characterization of monochloroacetate-degrading arthrobacter sp. Strain d2 isolated from universiti teknologi malaysia agricultural area

ABSTRACT Arthrobacter sp. strains D2 and D3 and Labrys sp. strain D1 capable of degrading 20 mM monochloroacetic acid (MCA) were isolated from soil contaminated with herbicides and pesticides. All three isolates were able to grow on MCA as the sole source of carbon and energy with concomitant chloride ion release in the growth medium (19 mM). Strains D2 and D3 (cells doubling time 7 ± 0.3 h) grew four times faster than D1 (26 ± 0.1 h). Strain D2 was then further investigated and could also grow in 10 mM of monobromoacetic acid (MBA), 2,2-dichloropropionic acid (2,2DCP), d,l-2-chloropropionic acid (D,L2CP), l-2-chloropropionic acid (L-2CP), d-2-chloropropionic acid (D-2CP), and glycolate as the sole sources of carbon and energy. Dehalogenase gene amplification using group I primers revealed a 410-bp polymerase chain reaction (PCR) product, but there was none using group II primers. The partial amino acid sequence analysis of group I DehD2 dehalogenase showed at least 32% identity to the corresponding regions of DehE, DhlIV, DehI, and D,L-DEX, with key amino acid residues Ser188, Ala187, and Asp189. These amino acid residues were involved in substrate binding and catalysis and were conserved in the partial amino acid sequence.

Effect of 2014 massive flood on well water qualities: A case study on Kelantan River basin, Malaysia

Abstract The effect of physical and biological qualities of wells after submergence was assessed following December 2014 flood in Kelantan. Studies were carried out on a total of 65 wells from 13 stations around Kelantan River basin in which the wells’ water were sampled for pH, total dissolved solid (TDS), turbidity and microbial contamination. About 95% of the well showed to be contaminated, 7 out of 65 samples (11.1%) showed TDS values >400 μS·cm−1; and 19 samples (29.2%) recorded turbidity beyond 7.0 NTU. Statistical non-parametric tests carried out on independent groups showed that the status of well contamination was neither determined by both degree of submergence nor by the geographical location. Also the physico-chemical parameters are independent of flood inundation. However, TDS and turbidity values changed based on geographical location, at p < 0.05. Well from estuary recorded higher TDS (241.2 μS·cm−1 ±159.5 SD) and turbidity (8.04 NTU ± 6.53 SD) compared to those from inner basin (TDS at 156.3 μS·cm−1± 88.9 SD; turbidity at 2.90 NTU ± 2.46 SD), respectively. The flood water had played significant role in the transmission of existing contaminant, and most of the wells were unsafe for drinking. We concluded that the degree of flood submergence does not necessarily determine the severity of the well contamination in Kelantan, but the existing contamination may exacerbate further the potential risk during post flood period.