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The first ITS2 sequence data set of eDNA from honey of Malaysian giant honeybees (Apis dorsata) and stingless bees (Heterotrigona itama) reveals plant species diversity

Huda N, Ullah S, Wahab RA, Lani MN, Daud NHA, Shariff AHM, et al.

BMC Res Notes, 2023 Sep 12;16(1):211.
PMID: 37700361 DOI: 10.1186/s13104-023-06495-9

OBJECTIVES: Pollen is a useful tool for identifying the provenance and complex ecosystems surrounding honey production in Malaysian forests. As native key pollinators in Malaysia, Apis dorsata and Heterotrigona itama forage on various plant/pollen species to collect honey. This study aims to generate a dataset that uncovers the presence of these plant/pollen species and their relative abundance in the honey of A. dorsata and H. itama. The information gathered from this study can be used to determine the geographical and botanical origin and authenticity of the honey produced by these two species.
RESULTS: Sequence data were obtained for both A. dorsata and H. itama. The raw sequence data for A. dorsata was 5 Mb, which was assembled into 5 contigs with a size of 6,098,728 bp, an N50 of 15,534, and a GC average of 57.42. Similarly, the raw sequence data for H. itama was 6.3 Mb, which was assembled into 11 contigs with a size of 7,642,048 bp, an N50 of 17,180, and a GC average of 55.38. In the honey sample of A. dorsata, we identified five different plant/pollen species, with only one of the five species exhibiting a relative abundance of less than 1%. For H. itama, we identified seven different plant/pollen species, with only three of the species exhibiting a relative abundance of less than 1%. All of the identified plant species were native to Peninsular Malaysia, especially the East Coast area of Terengganu.
DATA DESCRIPTION: Our data offers valuable insights into honey's geographical and botanical origin and authenticity. Metagenomic studies could help identify the plant species that honeybees forage and provide preliminary data for researchers studying the biological development of A. dorsata and H. itama. The identification of various flowers from the eDNA of honey that are known for their medicinal properties could aid in regional honey with accurate product origin labeling, which is crucial for guaranteeing product authenticity to consumers.
Fulltext Green honey of Banggi Island: A preliminary anti-diabetic study on zebrafish model

Ullah S, Huyop F, Huda N, Ab Wahab R, Hamid AAA, Mohamad MAN, et al.

Heliyon, 2024 Feb 29;10(4):e26469.
PMID: 38404777 DOI: 10.1016/j.heliyon.2024.e26469

Zebrafish is a developing vertebrate model with several advantages, including its small size, and high experimental efficiency. Malaysia exhibit one of the highest diabetes rates in the Western Pacific and incurring an annual cost of 600 million US dollars. The objective of the study is to determine the antidiabetic properties of green honey (GH) using a zebrafish model. Adult zebrafish, aged 3-4 months, were subjected to overfeeding and treated with streptozotocin (STZ) through intraperitoneal injection (IP) on days 7 and 9. The study assessed the oral sucrose tolerance test (OSTT) and the anti-diabetic effects of green honey. The evaluation was conducted at three time points: 30, 60, and 120 min after treatment and sucrose administration. The study utilised a model with a sample size of 5. The study was performed in six groups. These groups are (1) Normal control (non-diabetic, no intervention), (2) Normal control + GH (non-diabetic, supplemented with GH 3 μl), (3) DM control (diabetic, no intervention), (4) DM Gp1 (diabetic, 3 μL GH), (5) DM Gp2 (diabetic, 6 μ L GH), (6) DM Acarbose (diabetic, treated with acarbose). Fasting blood glucose levels for non-diabetic (non-DM) and diabetic (DM) groups were evaluated before and after the 10 days of diabetic induction. DM groups (excess of food and two injections of STZ) have caused a significant increment in the fasting blood glucose to 11.55 mmol/l (p
Comparative modeling and enzymatic affinity of novel haloacid dehalogenase from Bacillus megaterium strain BHS1 isolated from alkaline Blue Lake in Turkey

Wahhab BH, Oyewusi HA, Wahab RA, Mohammad Hood MH, Abdul Hamid AA, Al-Nimer MS, et al.

J Biomol Struct Dyn, 2024;42(3):1429-1442.
PMID: 37038649 DOI: 10.1080/07391102.2023.2199870

This study presents the initial structural model of L-haloacid dehalogenase (DehLBHS1) from Bacillus megaterium BHS1, an alkalotolerant bacterium known for its ability to degrade halogenated environmental pollutants. The model provides insights into the structural features of DehLBHS1 and expands our understanding of the enzymatic mechanisms involved in the degradation of these hazardous pollutants. Key amino acid residues (Arg40, Phe59, Asn118, Asn176, and Trp178) in DehLBHS1 were identified to play critical roles in catalysis and molecular recognition of haloalkanoic acid, essential for efficient binding and transformation of haloalkanoic acid molecules. DehLBHS1 was modeled using I-TASSER, yielding a best TM-score of 0.986 and an RMSD of 0.53 Å. Validation of the model using PROCHECK revealed that 89.2% of the residues were located in the most favored region, providing confidence in its structural accuracy. Molecular docking simulations showed that the non-simulated DehLBHS1 preferred 2,2DCP over other substrates, forming one hydrogen bond with Arg40 and exhibiting a minimum energy of -2.5 kJ/mol. The simulated DehLBHS1 exhibited a minimum energy of -4.3 kJ/mol and formed four hydrogen bonds with Arg40, Asn176, Asp9, and Tyr11, further confirming the preference for 2,2DCP. Molecular dynamics simulations supported this preference, based on various metrics, including RMSD, RMSF, gyration, hydrogen bonding, and molecular distance. MM-PBSA calculations showed that the DehLBHS1-2,2-DCP complex had a markedly lower binding energy (-21.363 ± 1.26 kcal/mol) than the DehLBHS1-3CP complex (-14.327 ± 1.738 kcal/mol). This finding has important implications for the substrate specificity and catalytic function of DehLBHS1, particularly in the bioremediation of 2,2-DCP in contaminated alkaline environments. These results provide a detailed view of the molecular interactions between the enzyme and its substrate and may aid in the development of more efficient biocatalytic strategies for the degradation of halogenated compounds.Communicated by Ramaswamy H. Sarma.
The mechanistic role of active site residues in non-stereo haloacid dehalogenase E (DehE)

Zainal Abidin MH, Abd Halim KB, Huyop F, Tengku Abdul Hamid TH, Abdul Wahab R, Abdul Hamid AA

J Mol Graph Model, 2019 07;90:219-225.
PMID: 31103914 DOI: 10.1016/j.jmgm.2019.05.003

Dehalogenase E (DehE) is a non-stereospecific enzyme produced by the soil bacterium, Rhizobium sp. RC1. Till now, the catalytic mechanism of DehE remains unclear although several literature concerning its structure and function are available. Since DehE is non-stereospecific, the enzyme was hypothesized to follow a 'direct attack mechanism' for the catalytic breakdown of a haloacid. For a molecular insight, the DehE modelled structure was docked in silico with the substrate 2-chloropropionic acid (2CP) in the active site. The ideal position of DehE residues that allowed a direct attack mechanism was then assessed via molecular dynamics (MD) simulation. It was revealed that the essential catalytic water was hydrogen bonded to the 'water-bearer', Asn114, at a relatively constant distance of ∼2.0 Å after 50 ns. The same water molecule was also closely sited to the catalytic Asp189 at an average distance of ∼2.0 Å, signifying the imperative role of the latter to initiate proton abstraction for water activation. This reaction was crucial to promote a direct attack on the α-carbon of 2CP to eject the halide ion. The water molecule was oriented favourably towards the α-carbon of 2CP at an angle of ∼75°, mirrored by the formation of stable enzyme-substrate orientations throughout the simulation. The data therefore substantiated that the degradation of a haloacid by DehE followed a 'direct attack mechanism'. Hence, this study offers valuable information into future advancements in the engineering of haloacid dehalogenases with improved activity and selectivity, as well as functionality in solvents other than water.
Biodegradation of 3-chloropropionic acid (3-CP) by Bacillus cereus WH2 and its in silico enzyme-substrate docking analysis

Muslem WH, Edbeib MF, Aksoy HM, Kaya Y, Hamid AAA, Hood MHM, et al.

J Biomol Struct Dyn, 2020 07;38(11):3432-3441.
PMID: 31401940 DOI: 10.1080/07391102.2019.1655482
Molecular docking and molecular dynamics simulations of a mutant Acinetobacter haemolyticus alkaline-stable lipase against tributyrin

Anuar NFSK, Wahab RA, Huyop F, Amran SI, Hamid AAA, Halim KBA, et al.

J Biomol Struct Dyn, 2021 Apr;39(6):2079-2091.
PMID: 32174260 DOI: 10.1080/07391102.2020.1743364

We previously reported on a mutant lipase KV1 (Mut-LipKV1) from Acinetobacter haemolyticus which optimal pH was raised from 8.0 to 11.0 after triple substitutions of surface aspartic acid (Asp) with lysine (Lys). Herein, this study further examined the Mut-LipKV1 by molecular docking, molecular dynamics (MD) simulations and molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) calculations to explore the structural requirements that participated in the effective binding of tributyrin and its catalytic triad (Ser165, Asp259 and His289) and identify detailed changes that occurred post mutation. Mut-LipKV1 bound favorably with tributyrin (-4.1 kcal/mol) and formed a single hydrogen bond with His289, at pH 9.0. Despite the incongruent docking analysis data, results of MD simulations showed configurations of both the tributyrin-Mut-LipKV1 (RMSD 0.3 nm; RMSF 0.05 - 0.3 nm) and the tributyrin-wildtype lipase KV1 (tributyrin-LipKV1) complexes (RMSD 0.35 nm; RMSF 0.05 - 0.4 nm) being comparably stable at pH 8.0. MM-PBSA analysis indicated that van der Waals interactions made the most contribution during the molecular binding process, with the Mut-LipKV1-tributyrin complex (-44.04 kcal/mol) showing relatively lower binding energy than LipKV1-tributyrin (-43.83 kcal/mol), at pH 12.0. All tributyrin-Mut-LipKV1 complexes displayed improved binding free energies over a broader pH range from 8.0 - 12.0, as compared to LipKV1-tributyrin. Future empirical works are thus, important to validate the improved alkaline-stability of Mut-LipKV1. In a nutshell, our research offered a considerable insight for further improving the alkaline tolerance of lipases.Communicated by Ramaswamy H. Sarma.
An Overview of Crosslinked Enzyme Aggregates: Concept of Development and Trends of Applications

Bouguerra OM, Wahab RA, Huyop F, Al-Fakih AM, Mahmood WMAW, Mahat NA, et al.

Appl Biochem Biotechnol, 2024 Jan 05.
PMID: 38180645 DOI: 10.1007/s12010-023-04809-y

Enzymes are commonly used as biocatalysts for various biological and chemical processes in industrial applications. However, their limited operational stability, catalytic efficiency, poor reusability, and high-cost hamper further industrial usage. Thus, crosslinked enzyme aggregates (CLEAs) are developed as a better enzyme immobilization tool to extend the enzymes' operational stability. This immobilization method is appealing because it is simpler due to the absence of ballast and permits the collective use of crude enzyme cocktails. CLEAs, so far, have been successfully developed using a variety of enzymes, viz., hydrolases, proteases, amidases, lipases, esterases, and oxidoreductase. Recent years have seen the emergence of novel strategies for preparing better CLEAs, which include the combi- and multi-CLEAs, magnetics CLEAs, and porous CLEAs for various industrial applications, viz., laundry detergents, organic synthesis, food industries, pharmaceutical applications, oils, and biodiesel production. To better understand the different strategies for CLEAs' development, this review explores these strategies and highlights the relevant concerns in designing innovative CLEAs. This article also details the challenges faced during CLEAs preparation and solutions for overcoming them. Finally, the trending strategies to improve the preparation of CLEAs alongside their industrial application trends are also discussed.
Fulltext Baseline amplicon sequencing data for the ITS2 region in the green honey of Banggi Island, Sabah

Ullah S, Huda N, Wahab RA, Hamid AAA, Nasir MHM, Mohamad MAN, et al.

Data Brief, 2024 Feb;52:110044.
PMID: 38328502 DOI: 10.1016/j.dib.2024.110044

Green honey, was discovered on Banggi Island, Sabah, showing high in essential amino acids and chlorophyll derivatives. Despite its lucrative market potential owing to its distinctive color, uncertainties persist regarding its nature. This study leverages amplicon sequencing by targeting micro- and macro-organisms present in honey environmental DNA (eDNA) using Internal Transcribed Spacer 2 (ITS2) region, enabling the identification of floral and microorganism sources that represent the honey's composition. The investigation into green honey from Banggi Island concerns the prevalence of honey adulteration and authenticity for economic gain. Adulteration methods, such as the addition of sugar syrups, compromise honey purity. Using a sequencing approach would help in determining the geographic origin and verifying the authenticity of the honey. The study aims to identify plant species or microorganisms in honey's eDNA. To authenticate honey, we utilized ITS2 with Illumina sequencing, exploring the diversity of green honey samples. Raw sequence reads obtained for the green honey sample revealed 1,438,627 raw reads, with a GC average of 49.22 %. A total of 44 amplicon sequence variances (ASVs) were identified, including three genera: Zygosaccharomyces with two species, Fraxinus with three species, and the genus Ficaria with only one species. Their respective relative abundances were 98.55%, 0.94%, and 0.51%. Zygosaccharomyces rouxii and Zygosaccharomyces mellis were identified as the pre-dominant yeast species in honey, while the Fraxinus and Ficaria genus represent common plant species in Sabah, particularly in Banggi Island. The dominance of Zygosaccharomyces species aligns with their known prevalence in honey, affirming the reliability of our findings. The presence of Fraxinus and Ficaria in the honey sample correlates with its abundance in the local environment. This amplicon sequencing approach not only contributes to our understanding of green honey composition but also serves as a valuable resource for authenticating honey origin in Malaysia, particularly for green honey from Banggi Island, Sabah. Our study pioneers the application of ITS2 amplicon sequencing for green honey amplicon sequencing, providing valuable insights into its composition and origin. This methodology, with a focus on eDNA, contributes to the authentication and quality determination of honey in Malaysia, addressing the pressing concerns of adulteration and variability in production practices.
Fulltext Physicochemical and antioxidant properties of Apis cerana honey from Lombok and Bali Islands

Huyop F, Ullah S, Abdul Wahab R, Huda N, Sujana IGA, Saloko S, et al.

PLoS One, 2024;19(4):e0301213.
PMID: 38578814 DOI: 10.1371/journal.pone.0301213

Limited honey production worldwide leads to higher market prices, thus making it prone to adulteration. Therefore, regular physicochemical analysis is imperative for ensuring authenticity and safety. This study describes the physicochemical and antioxidant properties of Apis cerana honey sourced from the islands of Lombok and Bali, showing their unique regional traits. A comparative analysis was conducted on honey samples from Lombok and Bali as well as honey variety from Malaysia. Moisture content was found slightly above 20% in raw honey samples from Lombok and Bali, adhering to the national standard (SNI 8664:2018) of not exceeding 22%. Both honey types displayed pH values within the acceptable range (3.40-6.10), ensuring favorable conditions for long-term storage. However, Lombok honey exhibited higher free acidity (78.5±2.14 meq/kg) than Bali honey (76.0±1.14 meq/kg), surpassing Codex Alimentarius recommendations (≤50 meq/kg). The ash content, reflective of inorganic mineral composition, was notably lower in Lombok (0.21±0.02 g/100) and Bali honey (0.14±0.01 g/100) compared to Tualang honey (1.3±0.02 g/100). Electric conductivity, indicative of mineral content, revealed Lombok and Bali honey with lower but comparable values than Tualang honey. Hydroxymethylfurfural (HMF) concentrations in Lombok (14.4±0.11 mg/kg) and Bali (17.6±0.25 mg/kg) were slightly elevated compared to Tualang honey (6.4±0.11 mg/kg), suggesting potential processing-related changes. Sugar analysis revealed Lombok honey with the highest sucrose content (2.39±0.01g/100g) and Bali honey with the highest total sugar content (75.21±0.11 g/100g). Both honeys exhibited lower glucose than fructose content, aligning with Codex Alimentarius guidelines. The phenolic content, flavonoids, and antioxidant activity were significantly higher in Lombok and Bali honey compared to Tualang honey, suggesting potential health benefits. Further analysis by LC-MS/MS-QTOF targeted analysis identified various flavonoids/flavanols and polyphenolic/phenolic acid compounds in Lombok and Bali honey. The study marks the importance of characterizing the unique composition of honey from different regions, ensuring quality and authenticity in the honey industry.
Synthesis of geranyl propionate in a solvent-free medium using Rhizomucor miehei lipase covalently immobilized on chitosan-graphene oxide beads

Isah AA, Mahat NA, Jamalis J, Attan N, Zakaria II, Huyop F, et al.

Prep Biochem Biotechnol, 2017 Feb 07;47(2):199-210.
PMID: 27341522 DOI: 10.1080/10826068.2016.1201681

The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan-graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box-Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76 ± 0.02% of recovered protein had yielded 7.2 ± 0.04 mg g(-1) and 211 ± 0.3% U g(-1) of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20 hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70 rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.
Insights into the stereospecificity of the d-specific dehalogenase from Rhizobium sp. RC1 toward d- and l-2-chloropropionate

Sudi IY, Hamid AA, Shamsir MS, Jamaluddin H, Wahab RA, Huyop F

Biotechnology, biotechnological equipment, 2014 Jul 04;28(4):608-615.
PMID: 26740767

Halogenated compounds are recalcitrant environmental pollutants prevalent in agricultural fields, waste waters and industrial by-products, but they can be degraded by dehalogenase-containing microbes. Notably, 2-haloalkanoic acid dehalogenases are employed to resolve optically active chloropropionates, as exemplified by the d-specific dehalogenase from Rhizobium sp. RCI (DehD), which acts on d-2-chloropropionate but not on its l-enantiomer. The catalytic residues of this dehalogenase responsible for its affinity toward d-2-chloropropionate have not been experimentally determined, although its three-dimensional crystal structure has been solved. For this study, we performed in silico docking and molecular dynamic simulations of complexes formed by this dehalogenase and d- or l-2-chloropropionate. Arg134 of the enzyme plays the key role in the stereospecific binding and Arg16 is in a position that would allow it to activate a water molecule for hydrolytic attack on the d-2-chloropropionate chiral carbon for release of the halide ion to yield l-2-hydroxypropionate. We propose that within the DehD active site, the NH group of Arg134 can form a hydrogen bond with the carboxylate of d-2-chloropropionate with a strength of ∼4 kcal/mol that may act as an acid-base catalyst, whereas, when l-2-chloropropionate is present, this bond cannot be formed. The significance of the present work is vital for rational design of this dehalogenase in order to confirm the involvement of Arg16 and Arg134 residues implicated in hydrolysis and binding of d-2-chloropropionate in the active site of d-specific dehalogenase from Rhizobium sp. RC1.
Fulltext Structure prediction, molecular dynamics simulation and docking studies of D-specific dehalogenase from Rhizobium sp. RC1

Sudi IY, Wong EL, Joyce-Tan KH, Shamsir MS, Jamaluddin H, Huyop F

Int J Mol Sci, 2012;13(12):15724-54.
PMID: 23443090 DOI: 10.3390/ijms131215724

Currently, there is no three-dimensional structure of D-specific dehalogenase (DehD) in the protein database. We modeled DehD using ab initio technique, performed molecular dynamics (MD) simulation and docking of D-2-chloropropionate (D-2CP), D-2-bromopropionate (D-2BP), monochloroacetate (MCA), monobromoacetate (MBA), 2,2-dichloropropionate (2,2-DCP), d,l-2,3-dichloropropionate (d,l-2,3-DCP), and 3-chloropropionate (3-CP) into the DehD active site. The sequences of DehD and D-2-haloacid dehalogenase (HadD) from Pseudomonas putida AJ1 have 15% sequence similarity. The model had 80% of the amino acid residues in the most favored region when compared to the crystal structure of DehI from Pseudomonas putida PP3. Docking analysis revealed that Arg107, Arg134 and Tyr135 interacted with D-2CP, and Glu20 activated the water molecule for hydrolytic dehalogenation. Single residue substitutions at 25-30 °C showed that polar residues of DehD were stable when substituted with nonpolar residues and showed a decrease in activity within the same temperature range. The molecular dynamics simulation of DehD and its variants showed that in R134A variant, Arg107 interacted with D-2CP, while in Y135A, Gln221 and Arg231 interacted with D-2CP. It is our emphatic belief that the new model will be useful for the rational design of DehDs with enhanced potentials.
An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA

Biotechnology, biotechnological equipment, 2015 Mar 04;29(2):205-220.
PMID: 26019635

The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies.
Interactions of non-natural halogenated substrates with D-specific dehalogenase (DehD) mutants using in silico studies

Sudi IY, Shamsir MS, Jamaluddin H, Wahab RA, Huyop F

Biotechnology, biotechnological equipment, 2014 Sep 03;28(5):949-957.
PMID: 26019583

The D-2-haloacid dehalogenase of D-specific dehalogenase (DehD) from Rhizobium sp. RC1 catalyses the hydrolytic dehalogenation of D-haloalkanoic acids, inverting the substrate-product configuration and thereby forming the corresponding L-hydroxyalkanoic acids. Our investigations were focused on DehD mutants: R134A and Y135A. We examined the possible interactions between these mutants with haloalkanoic acids and characterized the key catalytic residues in the wild-type dehalogenase, to design dehalogenase enzyme(s) with improved potential for dehalogenation of a wider range of substrates. Three natural substrates of wild-type DehD, specifically, monochloroacetate, monobromoacetate and D,L-2,3-dichloropropionate, and eight other non-natural haloalkanoic acids substrates of DehD, namely, L-2-chloropropionate; L-2-bromopropionate; 2,2-dichloropropionate; dichloroacetate; dibromoacetate; trichloroacetate; tribromoacetate; and 3-chloropropionate, were docked into the active site of the DehD mutants R134A and Y135A, which produced altered catalytic functions. The mutants interacted strongly with substrates that wild-type DehD does not interact with or degrade. The interaction was particularly enhanced with 3-chloropropionate, in addition to monobromoacetate, monochloroacetate and D,L-2,3-dichloropropionate. In summary, DehD variants R134A and Y135A demonstrated increased propensity for binding haloalkanoic acid and were non-stereospecific towards halogenated substrates. The improved characteristics in these mutants suggest that their functionality could be further exploited and harnessed in bioremediations and biotechnological applications.
Candida rugosa Lipase Immobilized onto Acid-Functionalized Multi-walled Carbon Nanotubes for Sustainable Production of Methyl Oleate

Che Marzuki NH, Mahat NA, Huyop F, Buang NA, Wahab RA

Appl Biochem Biotechnol, 2015 Oct;177(4):967-84.
PMID: 26267406 DOI: 10.1007/s12010-015-1791-z

The chemical production of methyl oleate using chemically synthesized fatty acid alcohols and other toxic chemicals may lead to significant environmental hazards to mankind. Being a highly valuable fatty acid replacement raw material in oleochemical industry, the mass production of methyl oleate via environmentally favorable processes is of concern. In this context, an alternative technique utilizing Candida rugosa lipase (CRL) physically adsorbed on multi-walled carbon nanotubes (MWCNTs) has been suggested. In this study, the acid-functionalized MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) was used as support for immobilizing CRL onto MWCNTs (CRL-MWCNTs) as biocatalysts. Enzymatic esterification was performed and the efficiency of CRL-MWCNTs was evaluated against the free CRL under varying conditions, viz. temperature, molar ratio of acid/alcohol, solvent log P, and enzyme loading. The CRL-MWCNTs resulted in 30-110 % improvement in the production of methyl oleate over the free CRL. The CRL-MWCNTs attained its highest yield (84.17 %) at 50 °C, molar ratio of acid/alcohol of 1:3, 3 mg/mL of enzyme loading, and iso-octane (log P 4.5) as solvent. Consequently, physical adsorption of CRL onto acid-functionalized MWCNTs has improved the activity and stability of CRL and hence provides an environmentally friendly means for the production of methyl oleate.
Fulltext An S188V mutation alters substrate specificity of non-stereospecific α-haloalkanoic acid dehalogenase E (DehE)

Hamid AA, Hamid TH, Wahab RA, Omar MS, Huyop F

PLoS One, 2015;10(3):e0121687.
PMID: 25816329 DOI: 10.1371/journal.pone.0121687

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.
Haloadaptation: insights from comparative modeling studies between halotolerant and non-halotolerant dehalogenases

Edbeib MF, Aksoy HM, Kaya Y, Wahab RA, Huyop F

J Biomol Struct Dyn, 2020 Aug;38(12):3452-3461.
PMID: 31422756 DOI: 10.1080/07391102.2019.1657498

Halophiles are extremophilic microorganisms that grow optimally at high salt concentrations by producing a myriad of equally halotolerant enzymes. Structural haloadaptation of these enzymes adept to thriving under high-salt environments, though are not fully understood. Herein, the study attempts an in silico investigation to identify and comprehend the evolutionary structural adaptation of a halotolerant dehalogenase, DehHX (GenBank accession number: KR297065) of the halotolerant Pseudomonas halophila, over its non-halotolerant counterpart, DehMX1 (GenBank accession number KY129692) produced by Pseudomonas aeruginosa. GC content of the halotolerant DehHX DNA sequence was distinctively higher (58.9%) than the non-halotolerant dehalogenases (55% average GC). Its acidic residues, Asp and Glu were 8.27% and 12.06%, respectively, compared to an average 5.5% Asp and 7% Glu, in the latter; but lower contents of basic and hydrophobic residues in the DehHX. The secondary structure of DehHX interestingly revealed a lower incidence of α-helix forming regions (29%) and a higher percentage of coils (57%), compared to 49% and 29% in the non-halotolerant homologues, respectively. Simulation models showed the DehHX is stable under a highly saline environment (25% w/v) by adopting a highly negative-charged surface with a concomitant weakly interacting hydrophobic core. The study thus, established that a halotolerant dehalogenase undergoes notable evolutionary structural changes related to GC content over its non-halotolerant counterpart, in order to adapt and thrive under highly saline environments.Communicated by Ramaswamy H. Sarma.
In silico mutation on a mutant lipase from Acinetobacter haemolyticus towards enhancing alkaline stability

Anuar NFSK, Wahab RA, Huyop F, Halim KBA, Hamid AAA

J Biomol Struct Dyn, 2020 Sep;38(15):4493-4507.
PMID: 31630644 DOI: 10.1080/07391102.2019.1683074

Alkaline-stable lipases are highly valuable biocatalysts that catalyze reactions under highly basic conditions. Herein, computational predictions of lipase from Acinetobacter haemolyticus and its mutant, Mut-LipKV1 was performed to identify functionally relevant mutations that enhance pH performance under increasing basicity. Mut-LipKV1 was constructed by in silico site directed mutagenesis of several outer loop acidic residues, aspartic acid (Asp) into basic ones, lysine (Lys) at positions 51, 122 and 247, followed by simulation under extreme pH conditions (pH 8.0-pH 12.0). The energy minimized Mut-LipKV1 model exhibited good quality as shown by PROCHECK, ERRAT and Verify3D data that corresponded to 79.2, 88.82 and 89.42% in comparison to 75.2, 86.15, and 95.19% in the wild-type. Electrostatic surface potentials and charge distributions of the Mut-LipKV1 model was more stable and better adapted to conditions of elevated pHs (pH 8.0 - 10.0). Mut-LipKV1 exhibited a mixture of neutral and positive surface charge distribution compared to the predominantly negative charge in the wild-type lipase at pH 8.0. Data of molecular dynamics simulations also supported the increased alkaline-stability of Mut-LipKV1, wherein the lipase was more stable at a higher pH 9.0 (RMSD = ∼0.3 nm, RMSF = ∼0.05-0.2 nm), over the optimal pH 8.0 of the wild-type lipase (RMSD = 0.3 nm, RMSF = 0.05-0.20 nm). Thus, the adaptive strategy of replacing surface aspartic acid to lysine in lipase was successful in yielding a more alkaline-stable Mut-LipKV1 under elevated basic conditions.Communicated by Ramaswamy H. Sarma.
Theoretical analyses on enantiospecificity of L-2-haloacid dehalogenase (DehL) from Rhizobium sp. RC1 towards 2-chloropropionic acid

Adamu A, Abdul Wahab R, Aliyu F, Abdul Razak FI, Mienda BS, Shamsir MS, et al.

J Mol Graph Model, 2019 11;92:131-139.
PMID: 31352207 DOI: 10.1016/j.jmgm.2019.07.012

Dehalogenases continue to garner interest of the scientific community due to their potential applications in bioremediation of halogen-contaminated environment and in synthesis of various industrially relevant products. Example of such enzymes is DehL, an L-2-haloacid dehalogenase (EC 3.8.1.2) from Rhizobium sp. RC1 that catalyses the specific cleavage of halide ion from L-2-halocarboxylic acids to produce the corresponding D-2-hydroxycarboxylic acids. Recently, the catalytic residues of DehL have been identified and its catalytic mechanism has been fully elucidated. However, the enantiospecificity determinants of the enzyme remain unclear. This information alongside a well-defined catalytic mechanism are required for rational engineering of DehL for substrate enantiospecificity. Therefore, using quantum mechanics/molecular mechanics and molecular mechanics Poisson-Boltzmann surface area calculations, the current study theoretically investigated the molecular basis of DehL enantiospecificity. The study found that R51L mutation cancelled out the dehalogenation activity of DehL towards it natural substrate, L-2-chloropropionate. The M48R mutation, however introduced a new activity towards D-2-chloropropionate, conveying the possibility of inverting the enantiospecificity of DehL from L-to d-enantiomer with a minimum of two simultaneous mutations. The findings presented here will play important role in the rational design of DehL dehalogenase for improving substrate utility.
A facile enzymatic synthesis of geranyl propionate by physically adsorbed Candida rugosa lipase onto multi-walled carbon nanotubes

Mohamad NR, Buang NA, Mahat NA, Lok YY, Huyop F, Aboul-Enein HY, et al.

Enzyme Microb Technol, 2015 May;72:49-55.
PMID: 25837507 DOI: 10.1016/j.enzmictec.2015.02.007

In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6h at 55°C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate.

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