Wild-type CYP102A1 as a biocatalyst: turnover of drugs usually metabolised by human liver enzymes

This work provides functional data showing that the bacterial CYP102A1 recognises compounds metabolised by human CYP3A4, CYP2E1 and CYP1A2 and is able to catalyse different reactions. Wild-type cytochrome CYP102A1 from Bacillus megaterium is a catalytically self-sufficient enzyme, containing an NADPH-dependent reductase and a P450 haem domain fused in a single polypeptidie chain. An NADPH-dependent method (Tsotsou et al. in Biosens. Bioelectron. 17:119–131, 2002) together with spectroscopic assays were applied to investigate the catalytic activity of CYP102A1 towards 19 xenobiotics, including 17 commercial drugs. These molecules were chosen to represent typical substrates of the five main families of drug-metabolising human cytochromes P450. Liquid chromatography–mass spectrometry analysis showed that CYP102A1 catalyses the hydroxylation of chlorzoxazone, aniline and p-nitrophenol, as well as the N-dealkylation of propranolol and the dehydrogenation of nifedipine. These drugs are typical substrates of human CYP2E1 and CYP3A4. The K _M values calculated for these compounds were in the millimolar range: 1.21 ± 0.07 mM for chlorzoxazone, 2.52 ± 0.08 mM for aniline, 0.81 ± 0.04 mM for propranolol. The values of v _max for chlorzoxazone and propranolol were 46.0 ± 9.0 and 7.6 ± 3.4 nmol min⁻¹ nmol⁻¹, respectively. These values are higher then those measured for the human enzymes. The v _max value for aniline was 9.4 ± 1.3 nmol min⁻¹ nmol⁻¹, comparable to that calculated for human cytochromes P450. The functional data were found to be in line with the sequence alignments, showing that the identity percentage of CYP102A1 with CYP3A4 and CYP2E1 is higher than that found for CYP1A2, CYP2C9 and CYP2D6 families.     

Screening of bioemulsifier-producing micro-organisms isolated from oil-contaminated sites

Eighty-eight micro-organisms were isolated from oil-contaminated soils and checked for their extracellular bioemulsifier producing potential. The micro-organisms were screened on the basis of oil spread, drop collapse and emulsification index. Most efficient strains were characterized as Lysinibacillus sp. SP1025 and Bacillus cereus SP1035. In Lysinibacillus sp. SP1025, the E-24 index, surface tension and production of crude bioemulsifier were found to be 83.3 % with diesel, 34.20?±?0.03 mN/m and 3.07?±?0.62 g/L, respectively, whereas in the case of Bacillus cereus SP1035, the E-24 index, surface tension and production of crude bioemulsifier recorded were 76.5 % with diesel, 43.42?±?0.03 mN/m and 3.90?±?0.3 g/L. Crude biomemulsifier produced by selected micro-organisms was stable, withstanding a wide temperature and pH range with an E-24 Index value greater than 50 %. All emulsions formed were oil-in-water type. Emulsions formed with tested aliphatic and aromatic hydrocarbons, except those formed with ester based oils, were 100 % stable with the entire organic layer converted into emulsion. To the best of our knowledge this is the first report for bioemulsifier production from the genus Lysinibacillus.     

Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation

In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790±690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed K_m and V_max values in the CYP2A6 reconstituted system were 1.48±0.37 μM and 3360±180 pmol product/min/nmol CYP, respectively.     

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b₅ enhanced the k_cat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min⁻¹) with little effect on K_M (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.     

Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus

CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized. We report here the cloning and expression of CYP102A5, a new member of this family that is very closely related to CYP102A4 from Bacillus anthracis. Characterization of the substrate specificity of CYP102A5 shows that it, like the other CYP102s, will metabolize saturated and unsaturated fatty acids as well as N-acylamino acids. CYP102A5 catalyzes very fast substrate oxidation, showing one of the highest turnover rates for any P450 monooxygenase studied so far. It does so with more specificity than other CYP102s, yielding primarily ω-1 and ω-2 hydroxylated products. Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in CYP102A5 than in CYP102A1, providing a likely explanation for the increased monooxygenation rate. The availability of this new, very fast fusion P450 will provide a great tool for comparative structure-function studies between CYP102A5 and the other characterized CYP102s.     

Effect of subcritical water hydrolysate in the brown seaweed Saccharina japonica as a potential antibacterial agent on food-borne pathogens

Seaweeds are rich in bioactive compounds which have well-documented antioxidant properties. They also have antimicrobial activities against food pathogenic microorganisms. This study uses an extract of the brown seaweed, Saccharina (Laminaria) japonica, produced by subcritical water hydrolysis (SWH) for investigating its potential to inhibit bacteria. De-oiled S. japonica was obtained by supercritical carbon dioxide extraction. The reaction temperatures for hydrolysis of raw and de-oiled S. japonica were maintained from 200 to 280 °C. The experiment was done with condition 1.3–6.0 MPa for the reaction pressure and 1:10 (w/v) for the ratio of material to water. The antibacterial activities of raw and de-oiled S. japonica produced by SWH were determined by using the agar diffusion method. Antibacterial activity was tested against two Gram-negative (Escherichia coli and Salmonella typhimurium) and two Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus). The antibacterial activities of hydrolysate water with catalyst at 240 °C showed better bacterial inhibition than the others. Strong antibacterial activity was found using de-oiled material with acetic acid added, with a zone of inhibition of S. typhimurium (14.33?±?0.06 mm) and E. coli (13.00?±?0.09 mm). On the other hand, the weakest antibacterial inhibition was found for S. aureus (12.83?±?0.10 mm) and B. cereus (12.50?±?0.09 mm).     

Demonstration of human platelet β-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol

The radioiodinated pindolol analogs ¹²⁵I-labeled cyanopindolol ([¹²⁵I]CYP) and ¹²⁵I-labeled hydroxybenzylpindolol ([¹²⁵I]HBP) have been used to study binding to human platelet β-adrenergic receptors. [¹²⁵I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (K_d) of 14±3 pM and maximal binding capacity (B_max) of 18±4 fmol/mg protein. Binding of [¹²⁵I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8·10⁷ s^?1·M^?1 and 3.8·10^?4 s^?1, respectively. [¹²⁵I]HBP binds to a saturable class of platelet membrane sites with a K_d of 50±10 pM and B_max of 32±6 fmol/mg protein. [¹²⁵I]HBP also binds to a saturable class of sites on intact platelets with a K_d of 58±14 pM and B_max of 24±4 molecules per platelet. Binding of [¹²⁵I]CYP and [¹²⁵I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (?) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol ? isoproterenol > epinephrine > practolol > norepinephrine > phenylephrine. These observations indicate that [¹²⁵I]CYP and [¹²⁵I]HBP bind to platelet sites which have the pharmacological characteristics of β-adrenergic receptors but which are not typical of either the β₁ or β₂ sub-type.     

Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents

The P450 monooxygenases CYP102A1 from Bacillus megaterium and CYP102A3 from Bacillus subtilis are fusion flavocytochromes comprising of a P450 heme domain and a FAD/FMN reductase domain. This protein organization is responsible for the extraordinary catalytic activities making both monooxygenases promising enzymes for biocatalysis. CYP102A1 and CYP102A3 are fatty acid hydroxylases that share 65% identity, and their mutants are able to oxidize a wide range of substrates. In an attempt to increase the process stability of CYP102A1, we exchanged the more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3. Stability of the chimeric fusion protein was determined spectrophotometrically as well as by measuring the hydroxylation activity towards 12-para-nitrophenoxydodecanoic acid (12-pNCA) after incubation at elevated temperatures. In the reaction with 12-pNCA, the new chimeric protein exhibited 88 and 38% of the activity of CYP102A3 and CYP102A1, respectively, but was able to hydroxylate substrates within a wider temperature range compared with the parental enzymes. Maximum activity was obtained at 51°C, and the half-life at 50°C was with 100 min more than ten times longer than that of CYP102A1 (8 min).     

Preliminary crystallographic data for beta-lactamase I from Bacillus cereus 569.

Crystals of β-lactamase I from Bacillus cereus 569 are monoclinic, space group C2 with unit cell dimensions $\text{a = 143·0 (± 0·5), b = 35·8 (± 0·1), c = 52·7 (± 0·2)}\text{A}\text{?}\text{, β = 97·0 (± 0·1) °}$, and one molecule of molecular weight about 28,000 per asymmetric unit.     

Oxidation of human cytochrome P450 1A2 substrates by Bacillus megaterium cytochrome P450 BM3

Cytochrome P450 enzymes (P450s or CYPs) are good candidates for biocatalysis in the production of fine chemicals, including pharmaceuticals. Despite the potential use of mammalian P450s in various fields of biotechnology, these enzymes are not suitable as biocatalysts due to their low stability, low catalytic activity, and limited availability. Recently, wild-type and mutant forms of bacterial P450 BM3 (CYP102A1) from Bacillus megaterium have been found to metabolize various. It has therefore been suggested that CYP102A1 may be used to generate the metabolites of drugs and drug candidates. In this report, we show that the oxidation reactions of typical human CYP1A2 substrates (phenacetin, ethoxyresorufin, and methoxyresorufin) are catalyzed by both wild-type and mutant forms of CYP102A1. In the case of phenacetin, CYP102A1 enzymes show only O-deethylation product, even though two major products are produced as a result of O-deethylation and 3-hydroxylation reactions by human CYP1A2. Formation of the metabolites was confirmed by HPLC analysis and LC–MS to compare the metabolites with the actual biological metabolites produced by human CYP1A2. The results demonstrate that CYP102A1 mutants can be used for cost-effective and scalable production of human CYP1A2 drug metabolites. Our computational findings suggest that a conformational change in the cavity size of the active sites of the mutants is dependent on activity change. The modeling results further suggest that the activity change results from the movement of several specific residues in the active sites of the mutants.     

Regioselective C-H hydroxylation of omeprazole sulfide by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> CYP102A1 to produce a human metabolite

Objectives

To find a simple enzymatic strategy for the efficient synthesis of the expensive 5′-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate.

Results

The practical synthetic strategy for the 5′-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5′-OH omeprazole sulfide with a high conversion yield (85–90 %).

Conclusions

A highly efficient synthesis of 5′-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

     

Photosynthetic response to genome methylation affects the growth of Chinese white poplar

Genome methylation plays a key role in regulating gene expression, but limited knowledge exists concerning the link between DNA methylation and economic traits in forest trees. We measured photosynthetic characteristics and growth traits in 130 intraspecific hybrids of Chinese white poplar (Populus tomentosa Carr.) and detected their genome methylation. The phenotypic data were normally distributed, and each trait had a significant difference among the hybrids. The net photosynthetic rate (Pn, 14.83?±?3.76???mol?m^?2?s^?1), stomatal conductance (Gs, 0.29?±?0.09?mol?m^?2?s^?1), and intercellular CO₂ concentration (Ci, 264.50?±?30.94???mol?mol^?1) showed similar trends. Positive correlations were found between Pn and height (H, 133.59?±?50.44?cm) and basal diameter (D, 16.29?±?5.20?mm), respectively. Using methylation-sensitive amplification polymorphism (MSAP) analysis, 32 primer-pair combinations generated 715 polymorphic markers. Positive correlations between photosynthetic characteristics, such as Pn and Gs, and total relative methylation level and relative hemimethylation (CNG methylation) level were investigated. Eighty-one candidate markers were associated with Pn, Gs, or Ci, 13 of which were also associated with growth traits using single MSAP molecular marker association. Sequencing and BLAST analysis showed that candidate markers were linked to genes encoding protochlorophyllide reductase and proteins of cytochrome P450 CYP4/CYP19/CYP26 subfamilies, and linked to genes taking part in, e.g., photosystem II. Therefore, the regions defined by the MSAP candidate markers are linked to genes that are essential for photosynthetic characteristics that respond to DNA methylation and subsequently affect growth traits.     

Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2

Salicylate is an important intermediate in the bacterial degradation of polycyclic aromatic hydrocarbons and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. Unlike the well-characterized salicylate 1-hydroxylases, the rarely occurred salicylate 5-hydroxylase (S5H) has not been characterized in detail. In this study, the three-component Fe-S protein complex (NagAaGHAb) of S5H from Ralstonia sp. strain U2 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component NagGH exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. NagAa is the ferredoxin-NADP⁺ reductase component containing flavin and plant type [2Fe–2S] cluster. The ferredoxin component NagAb was characterized as a [2Fe-2S] dimer which remains remarkably stable in denaturing gel electrophoresis after being heated at 100 °C for 1 h. Purified NagAa and NagAb, NagGH catalyzed the hydroxylation of salicylate to gentisate with a specific activity of 107.12?±?14.38 U/g and showed an apparent K _m for salicylate of 102.79?±?27.20 μM and a similar K _m value for both NADH and NADPH (59.76?±?7.81 μM versus 56.41?±?12.76 μM). The hydroxylase exhibited different affinities for two hydroxysalicylates (2,4-dihydroxybenzoate K _m of 93.54?±?18.50 μM versus 2,6-dihydroxybenzoate K _m of 939.80?±?199.46 μM). Interestingly, this S5H also showed catalytic activity to the pollutant 2-nitrophenol and exhibited steady-state kinetic data of the same order of magnitude as those for salicylate. This study will allow further comparative studies of structure–function relationships of the ring hydroxylating mono- and di-oxygenase systems.     

Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases

At low temperatures, Bacillus cereus synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions Δ5 and Δ10, as well as Δ5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative Δ5 desaturase and a fatty acid acyl-lipid desaturase in the B. cereus ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in Bacillus subtilis confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have Δ5 and Δ10 desaturase activities. Thus, these ORFs were correspondingly named desA (Δ5 desaturase) and desB (Δ10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when B. subtilis expressing B. cereus desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from B. cereus.     

Engineering class I cytochrome P450 by gene fusion with NADPH-dependent reductase and S. avermitilis host development for daidzein biotransformation

Daidzein C6 hydroxylase (6-DH, nfa12130), which is a class I type of cytochrome P450 enzyme, catalyzes a hydroxylation reaction at the C6-position of the daidzein A-ring and requires auxiliary electron transfer proteins. Current utilization of cytochrome P450 (CYP) enzymes is limited by low coupling efficiency, which necessitates extramolecular electron transfers, and low driving forces, which derive electron flows from tightly regulated NADPH redox balances into the heterogeneous CYP catalytic cycle. To overcome such limitations, the heme domain of the 6-DH enzyme was genetically fused with the NADPH-reductase domain of self-sufficient CYP102D1 to enhance electron transfer efficiencies through intramolecular electron transfer and switching cofactor preference from NADH into NADPH. 6-DH-reductase fusion enzyme displayed distinct spectral properties of both flavoprotein and heme proteins and catalyzed daidzein hydroxylation more efficiently with a k _cat/K _m value of 120.3?±?11.5 [10³ M^?1 s^?1], which was about three times higher than that of the 6-DH-FdxC-FdrA reconstituted system. Moreover, to obtain a higher redox driving force, a Streptomyces avermitilis host system was developed for heterologous expression of fusion 6-DH enzyme and whole cell biotransformation of daidzein. The whole cell reaction using the final recombinant strain, S. avermitilisΔcyp105D7::fusion 6-DH (nfa12130), resulted in 8.3?±?1.4 % of 6-OHD yield from 25.4 mg/L of daidzein.     

Acetyl-coenzyme A: arylamine N-acetyltransferases in microorganisms: screening and isolation of an enzyme from Bacillus cereus

The raw extracts of a series of microorganisms were screened for the presence of acetyl-coenzyme A: arylamine N-acetyltransferase (AAAT) using a radioactive assay with ³H-acetyl-coenzyme A and aniline as substrates. Enzyme activities were primarily detected in the soluble fractions of Bacillus and Nocardia species, and in some further soil organisms. Only strains of Bacillus cereus were able to acetylate 4-nitroaniline and 3,5-dimethyl-4-nitroaniline. The fermentation conditions for the production of the enzyme were optimized. The AAAT from one strain of Bacillus cereus was purified 24-fold and characterized.Abbreviations AAAT acetyl-coenzyme A: arylamine N-acetyltransferase - AcP acetylphosphate - CoA coenzyme A - EDTA ethylenediaminetetra-acetic acid - PTA phosphotransacetylase     

Two-helper-strain co-culture system: a novel method for enhancement of 2-keto-l-gulonic acid production

A novel two-helper-strain co-culture system (TSCS) was developed to enhance 2-keto-l-gulonic acid (2-KLG) productivity for vitamin C production. Bacillus megaterium and B. cereus (with a seeding culture ratio of 1:3, v/v), used as helper strains, increased the 2-KLG yield using Ketogulonigenium vulgare compared to the conventional one-helper-strain (either B. cereus or B. megaterium) co-culture system (OSCS). After 45 h cultivation, 2-KLG concentration in the TSCS (69 g l^?1) increased by 8.9 and 7 % over that of the OSCS (B. cereus: 63.4 g l^?1; B. megaterium: 64.5 g l^?1). The fermentation period of TSCS was 4 h shorter than that of OSCS (B. cereus). The increased cell numbers of K. vulgare stimulated by the two helper strains possibly explain the enhanced 2-KLG yield. The results imply that TSCS is a viable method for enhancing industrial production of 2-KLG.     

Exploring the Energy Profile of Human IgG/Rat Anti-human IgG Interactions by Dynamic Force Spectroscopy

Interactions between antibody and antigen molecules play essential roles in biological recognition processes as well as medical diagnosis. Therefore, an understanding of the underlying mechanism of antibody?Cantigen interactions at the single molecular level would be beneficial. In the present study, human immunoglobulin (IgG) tethered cantilevers and rat anti-human IgG functionalized gold surfaces were fabricated by using self-assembled monolayers method. Dynamic force spectroscopy was employed to characterize the interactions between human (IgG) and rat anti-human IgG at the single-molecule level. The unbinding forces were determined to be 44.6?±?0.8, 65.8?±?3.0, 108.1?±?4.1, 131.1?±?11.2, 149.5?±?4.7, 239.5?±?3.1 and 294.7?±?7.7?pN with ramping loading rates of 514, 1,127, 3,058, 7,215, 15,286, 31,974 and 50,468?pN?s^-1, respectively. In addition, the unbinding forces were found to be increasing with the logarithm of apparent loading rates in a linear way. Fitting data group resulted in two distinct linear parts, suggesting there are two energy barriers. The corresponding distances in the bound and transition states (x _?? ) and the dissociation rates (K _off ) were calculated to be 0.129?±?0.006?nm, 3.986?±?0.162?s^?1 for the outer barrier and 0.034?±?0.001?nm, 36.754?±?0.084?s^?1 for the inner barrier. Such findings hold promise of screening novel drugs and discerning different unbinding modes of biological molecules.     

Functional Response of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) to Sugarcane Whitefly Aleurolobus barodensis (Maskell) in Laboratory Conditions

A functional response study of Chrysoperla carnea (Stephens) larvae to different densities of sugar cane whitefly Aleurolobus barodensis (Maskell) was conducted in test tubes at 26?±?2 °C, 65?±?5 % RH. Chrysoperla carnea showed two different types of functional response in larval instars. First instar exhibits type II. However, second and third larval instars revealed type III functional response. Based on modified Holling’s disk equation, the highest searching rates (a) of 0.82?±?0.0247 h^?1 was found for first instar larva. For second and third larval instars, the attack coefficient (b) were 0.002?±?0.030 and 0.0025?±?0.0424 respectively. The shortest handling time (T_h) per prey was observed at third instar stage (1.574?±?0.0568 h) followed by second and first instar with 1.72?±?0.0411 h and 1.919?±?0.0568 h respectively.