Biosci. Biotechnol. Biochem., 77 (5), 961–965, 2013
Comparative Purification and Characterization of Two Distinct Extracellular Monocrotophos Hydrolases Secreted by Penicillium aculeatum and Fusarium pallidoroseum Isolated from Agricultural Fields Rachna JAIN,1 Veena G ARG,1; y Koushalya D ANGWAL,2 and Madhuri Kaushish L ILY2 1 2
Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan 304022, India Department of Biotechnology, Modern Institute of Technology, Rishikesh, Uttarakhand 249201, India
Received November 29, 2012; Accepted January 21, 2013; Online Publication, May 11, 2013 [doi:10.1271/bbb.120907]
The present study aimed at a comparative characterization of two distinct extracellular monocrotophos hydrolases, from Penicillium aculeatum ITCC 7980.10 (M3) and Fusarium pallidoroseum ITCC 7785.10 (M4), isolated from agricultural fields. The MCP hydrolases were purified by Sephadex G-100 column and DEAESepharose CL-6B ion-exchange column followed by SDS–PAGE analysis, which showed the presence of two hydrolases, of 33 and 67 kDa respectively. Both enzymes were most active at alkaline pH and were stable over a wide range of temperatures (60–70 C). Between the strains, the MCP hydrolases from M3 were 2-fold more active than that from M4. Enzyme kinetic studies showed lowest Km (33.52 mM) and highest Vmax (5.18 U/mg protein) for OPH67 of M3 in comparison to the Km and Vmax of the other hydrolases purified from M3 and M4, suggesting that M3 OPH67 was the most efficient MCP hydrolase. To the best of our knowledge, this is the first report of the purification of two distinct extracellular thermostable MCP hydrolases from fungal strains Penicillium aculeatum ITCC 7980.10 and Fusarium pallidoroseum ITCC 7785.10. Owing to its potential MCP hydrolyzing activity, M3 OPH67 can perhaps used directly or in the encapsulated form for remediation of MCP contaminated sites. Key words:
enzyme activity; hydrolase; monocrotophos (MCP); organophosphorous (OP); pesticide
reducing the toxicity of OP pesticides. The ability of OPHs to hydrolyze various OP pesticides is attributable to their molecular structure resemblance, except for substituents. OPH mediated hydrolysis of OP pesticides is potentially more efficient than chemical hydrolysis, as revealed by a previous study that found a faster rate of enzymatic hydrolysis (2,450 times) of parathion than of chemical hydrolysis.4) There have been several reports on the cloning of OPH genes from Flavobacterium sp. (ATCC27551),5) Pseudomonas sp.,6,7) Alteromonas sp.,5) Arthrobacter sp.,8) Plesiomonas sp.,9) Agrobacterium radiobacter,10) and Chromobacterium violaceum (ATCC12472).11) In addition, of two genes from Pseudomonas sp., one was found to be identical to the corresponding gene from Flavobacterium sp. (ATCC27551),5,6) while the other revealed 99.6% homology with the gene from Plesiomonas sp.7,9) In contrast to bacterial mediated biotransformation of OP pesticides, little is known about fungal OPHs.12) Fungi participate in biogeochemical cycles and the degradation of some xenobiotics in the biosphere.13,14) To gain insight into fungus mediated degradation of OP pesticides, the present study was aimed to isolate and characterize broad-spectrum fungal OPHs capable of hydrolyzing MCP containing both the P–O–C linkage and the amide bond effectively.
Materials and Methods Monocrotophos [dimethyl-(e)-1-methyl-2-(methylcarbamoyl) vinyl phosphate] (MCP), an organophosphorous (OP) pesticide is used globally to control common mites, ticks, and spiders. Due to its hydrophilic nature, it is rapidly absorbed into plant tissues.1) Intense agricultural use of MCP results in the occurrence of residues in food, water, and the environment, threatening human life owing to its elevated toxicity to acetyl cholinesterase (AChE), a key enzyme involved in nerveimpulse transmission. OP hydrolases are known to hydrolyze various OP pesticides.2,3) Flavobacterium sp. (ATCC27551), isolated from OP-treated soil has been reported to be capable of hydrolyzing OPs such as diazinon and parathion.2) Further research findings point to the ability of many microorganisms to produce OP hydrolase (OPH, EC 3.1.8.1) or phospho-triesterase, which hydrolyze the phosphoester bonds of OPs, y
Chemicals. MCP of analytical grade (99.5% purity) was procured from Sigma (St. Louis, MO, USA) and its stock solution of 1 mg/mL was prepared in ethanol. All other chemicals employed were of analytical grade, and were purchased from Himedia (Mumbai, India) and Rankem (Mumbai, India). Medium. Modified Czapek-Dox medium (CZM) was used as growth medium for the fungal strains to produce MCP hydrolase, which contained 30 g sucrose, 2 g NaNO3 , 0.5 g KCl, 0.5 g MgSO4 7H2 O, 10 g glucose, 10 mg FeCl3 , 0.2 g BaCl2 , 0.05 g CaCl2 , in 1 L distilled water and was supplemented with MCP (150 mg/mL) and KH2 PO4 (0.5 g/L) as an addition source of phosphorus.
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Soil sampling. Soil samples were collected from various agricultural farms within a 10 km range of the Banasthali Campus, Rajasthan, India. The collected samples were mixed, air dried, and sieved through, a 2-mm mesh prior to use. They were stored at 4 C until use.
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R. JAIN et al.
Isolation of MCP hydrolyzing fungal strains. Isolation of MCP hydrolyzing fungal strains was done by standard enrichment culture techniques.15) One g of soil sample was suspended in 10 mL of 0.85% saline and serially diluted up to 104 . Each dilution (100 mL) was spreaded onto a potato dextrose agar (PDA) plate containing penicillin and streptomycin at the optimum concentrations (30 mg/L). Screening of MCP hydrolyzing fungi was done using a gradient of MCP concentrations (50, 100, 150, 200, 250, and 300 mg/mL). The plates were incubated at 28 2 C for 5 d and observed for the presence of fungal colonies. Pure cultures of fungi were isolated by streaking the isolated fungal colonies on PDA medium. They were further identified based on colony morphology and staining properties. MCP hydrolase assay. The MCP hydrolase activity of fungal strains was estimated by the method described by Jia et al., with some modifications.16) The assay system included 900 mL of 50 mM Tris–HCl (pH 8.0) assay buffer containing 50 mM of MCP as substrate, to which 100 mL of fungal culture suspension or purified enzyme fraction was added, followed by incubation at 37 C for 10 min. The reaction was terminated by adding 1 mL of 10% trichloroacetic acid. Residual MCP was estimated by measurement of the absorbance at 254 nm.16) All experiments were performed in triplicate, and control experiments lacking the enzyme were used. Enzyme activities were expressed as units (mM of MCP hydrolyzed per min) per mL. After each purification step, the enzyme activities and the total protein contents of the crude and purified fractions were determined in order to estimate specific activity and fold purification. Protein content was determined by the method of Lowry et al.17) Enzyme purification. The experiments described below were carried out at 0 and 4 C unless otherwise specified. MCP hydrolyzing fungal strains M3 and M4 were grown for 10 d in 500-mL Erlenmeyer flasks containing 150 mL of modified Czapekdox medium supplemented with MCP (150 mg/mL) on a rotary shaker at 100 rpm and incubated at 30 C. The culture was centrifuged at 12;000 3 g for 20 min and the supernatant collected was filtered through Whatman filter paper no. 1 (GE healthcare UK limited, UK) and stored for further processing. To prepare fungal mycelial extract, the mycelial pellet was washed twice with cold 50 mM Tris–HCl buffer (pH 8.0). The mycelia were crushed in liquid nitrogen, followed by the addition of 5 mL of protein isolation buffer (PIB) (10 mM Tris–HCl pH 8.0, 1 mM EDTA, 2% PVPP) per 2 g of mycelium and centrifuged at 8,000 rpm for 30 min. The supernatant was collected and frozen overnight with an equal amount of acetone. The mixture was centrifuged again at 8,000 rpm, and the pellet was dissolved in a minimum amount of 50 mM Tris–HCl buffer (pH-8). The stored supernatant and the mycelial extract were considered to be a crude enzyme source, and were checked for the presence of extracellular and intracellular hydrolase activity. For purification of extracellular hydrolases, the culture filtrate was precipitated overnight with 80% ammonium sulfate. The precipitate was collected by centrifugation at 12;000 3 g for 20 min, and dissolved in the smallest possible volume of 50 mM Tris–HCl buffer (pH 8.0). Then it was dialyzed against the same buffer and concentrated by means of PEG (polyethylene glycol). The concentrated enzyme solution was loaded onto a Sephadex G-100 column (1.8 by 100 cm) pre-equilibrated with 50 mM Tris–HCl buffer (pH 8.0). The column was washed at a flow rate of 24 mL/h with 400 mL of the same buffer, and 5-mL fractions were collected. Fractions possessing high specific activity (3 mL) were pooled and concentrated for further purification. The concentrated active fractions were loaded on a DEAE-Sepharose CL-6B ionexchange column (1.2 by 30 cm) pre-equilibrated with 50 mM Tris–HCl buffer (pH 8.0). The column was washed at a flow rate of 20 mL/h with 500 mL of the same buffer, and proteins were eluted with a linear gradient of NaCl from 0 to 1.0 M. Five-mL fractions were collected and enzyme activity was calculated. Enzyme characterization was done by SDS–PAGE by the method of Laemmli.18) The molecular weight of the native enzyme was determined by the method of Andrew using blue dextran and molecular weight markers from Sigma as standards.19) Optimization of MCP hydrolase activity. Optimum temperature and pH for the purified MCP hydrolases from M3 and M4 were determined by evaluating hydrolase activity at various temperatures ranging 30 C to 100 C at pH 1–10 (at a gap of one unit) after 10 min of incubation. Results were expressed as % relative activity.
MCP hydrolase stability. The stability of the purified MCP hydrolases with respect to temperature and pH was determined by pre-incubating them at various pH values (1–10) and temperatures (30 C–100 C) for 18 h. Hydrolase activity was estimated by the standard method described above. A blank with the reaction mixture lacking substrate or enzyme was set up. Kinetics analysis of MCP hydrolases. The Km and Vmax of the purified MCP hydrolases from M3 and M4 were calculated in 50 mM Tris–HCl buffer (pH-8) at 37 C by various concentrations of MCP as substrate using the standard protocol. The values of the kinetic constants were calculated by Lineweaver and Burk plot.20) Effects of physical and chemical agents on MCP hydrolase activity. The effects of physical agents including UV and sunlight as well as chemicals (SDS, NaN2 , EDTA, potassium ferricyanide, HgCl2 , mercaptoethanol, CoCl2 , tartarate, NaF, ZnCl2 , CuCl2 , FeCl3 , and MnCl2 ; 1 mM) on the activity of the purified MCP hydrolases from M3 and M4 was assessed by pre-incubating the enzymes with the respective agents for 30 min followed by hydrolase assay. Statistical analysis. All experiments were carried out in triplicate and the results were expressed as means standard deviation. Statistical analysis was done using the SPSS program (Statistical Package for the Sciences System). The variables were subjected to t-test and ANOVA. Significance was set at p 0:05.
Results Isolation and screening of MCP hydrolyzing fungal strains Ten fungal strains (M1–M10) possessing MCP tolerance were isolated from soil samples collected from different agricultural fields by the enrichment culture method. These strains were subjected to various concentrations of MCP to isolate the most efficient MCP hydrolyzing strain. Among them, two strains, having the highest MCP hydrolyzing activity, were designated M3 and M4. These were identified on a morphological basis. The fungal isolates were identified and authenticated by the Indian Agricultural Research Institute (IARI) (New Delhi, India) as Penicillium aculeatum and Fusarium pallidoroseum (nos. ITCC 7980.10 and ITCC 7785.10 respectively). They were tested for the production of hydrolase in the presence and the absence of MCP. It was observed that the MCP hydrolase was expressed even in the absence of MCP, suggesting that the activity of the constitutively expressing hydrolase was found to be higher in M3 and M4 than that in the other isolates. These two strains were further selected for characterization of the hydrolases responsible for MCP hydrolysis. MCP hydrolase purification In order to characterize the MCP hydrolases from M3 and M4, these strains were grown in modified Czapekdox medium for 7 d, followed by estimation of MCP hydrolase activity in the culture filtrate and mycelial extracts. Our results indicated the presence of MCP hydrolase in the filtrate as well as the mycelial extracts, but the filtrate had higher hydrolase activity (7.65 U and 2.43 U/mg protein for M3 and 5.44 U and 1.98 U/mg protein for M4) than the mycelial extracts (0.98 U and 1.76 U/mg for M3 and 1.34 U and 1.34 U/mg for M4). Hence the culture filtrate was processed for further purification as a source of extracellular hydrolases. The purification strategy involved ammonium sulphate pre-
Comparative Characterization of Two Distinct Extracellular Monocrotophos Hydrolases
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Table 1. Comparative Purification Profile of Extracellular Enzyme from Penicillium aculeatum (M3) and Fusarium pallidoroseum (M4) Total activity (U)
Total protein (mg)
Sp. Activity (U/mg)
Purification fold
Yield %
M3
1;309:9 2:61
93:15 1
14:06 0:18
1
100
M4
1;227:93 2:48
51:3 198
23:93 0:134
1
100
(S)
M3
128:17 2:83
35:49 0:9
3:61 0:17
0:26 0:011
9:78 0:2
M4
114:76 56
34:26 1:5
1:43 0:05
9:34 0:04
Ppt
M3
1;248:68 5:73
30:28 0:78
41:24 1:18
2:93 0:005
95:32 0:369
M4
1;030:58 1:39
15:24 0:33
67:62 1:46
2.82
83:93 0:18
Crude
AmSO4
G-100
3:35 0:145
OPH33
M3
516:71 5:75
3:78 0:098
136:6 3:8
16:94 0:45
68:81 1:21
kDa
M4
448:69 2:84
1:39 0:08
321:66 18:5
17:64 1:02
47:98 0:17
OPH67
M3
1;088:98 4:85
3:78 0:098
287:9 8:68
20:47 51
83:13 0:32
963:19 2:4
kDa
M4
DEAE
OPH33
M3
CL6B
kDa
M4
442:9 2:91 367:58 6:8
1:39 0:08
690:75 45:89
28:85 1:79
78:44 0:04
1:62 0:11
272:86 18:76
33:849 2:36
58:98 0:33
0:68 0:02
537:35 10:12
29:48 0:49
39:31 0:62
OPH67
M3
980:34 3:05
3:77 0:09
259:48 6:79
18:45 0:37
74:84 0:084
kDa
M4
759:25 1:02
0:88 0:05
857:2 57:8
35:8 2:27
61:8 0:04
an anomaly for M3 OPH67, which showed a decrease of 2 units at higher purification levels whereas M4 OPH67 had the highest value at 36-fold purification.
Fig. 1. SDS–PAGE of the Purified Extracellular Enzymes from Penicillium aculeatum (M3) and Fusarium pallidoroseum (M4). Lane 1, marker proteins (from top to bottom): phosphorylases b (Mr, 97,400), bovine serum albumin (Mr, 67,000), ovalbumin (Mr, 43,000), carbonic anhydrase (Mr, 30,000), soyabean trypsin inhibitor (Mr, 20,100) and lysozyme (Mr, 14,300); Lanes 2–4, M3 and lanes 5–7 M4; lane 2, purified enzyme G100; lane 3, high molecular weight protein DEAE CL6B; lane 4, low molecular weight protein DEAE CL6B; lane 5, purified enzyme G100; lane 6, high molecular weight protein DEAE CL6B; lane 7, low molecular weight protein DEAE CL6B. The gel was stained for protein with Coomassie Brilliant Blue R-250, and destained in methanol-acetic acid-water (7:2:1).
cipitation (80%), followed by gel filtration chromatography (Sephadex G-100) and ion exchange chromatography (DEAE-Sepharose CL-6B). The data for the purification of MCP hydrolase from M3 and M4 are summarized in Table 1. DEAE-Sepharose CL-6B analysis revealed the presence of two peaks demonstrating the presence of two hydrolases differing in molecular weight. The data were confirmed by SDS PAGE of the purified fractions (Fig. 1). Based on molecular weights, the two hydrolases were designated OPH33 and OPH67. The specific activity of the enzymes varied over a range of 259 to 857 U/mg, with the highest for M4 OPH67 and the lowest for M3 OPH67. Enzyme yield followed the reverse pattern, with a variation from 39 to 75%. M3 showed the highest yield at 59% for OPH33 and 75% for OPH67. However, the purification fold showed
Optimization of MCP hydrolase activity The optimum pH values (1–10) and temperatures (30– 100 C) for the purified OPH33 and OPH67 of both M3 and M4 were determined. The pH activity profile of both the enzymes is represented in Fig. 2 A. All the enzymes were found to be alkaline, with pH optima ranging from 7 to 9. OPH33 of both M3 and M4 demonstrated highest relative activity at pH 8, retaining 60 and 40% activity respectively. In contrast, M3 OPH67 retained 100% activity at pH 7, whereas M4 OPH67 retained 50% activity at pH 9. Thus it is clear that in both M3 and M4, OPH67 had higher activity than OPH33. The temperature profile shown in Fig. 2 B follows the traditional pattern of a bell shaped curve. OPH33 of M3 and M4 showed highest activity, of 50% and 30% at 70 C respectively, while M3 and M4 OPH67 showed highest activity of 100% and 50%, at 60 C, respectively. Thus OPH33 showed almost half the activity of OPH67. MCP hydrolase stability The MCP hydrolase stability data against pH and temperature are summarized in Fig. 3 A and B. The results showed that M3 OPH33 was stable up to pH 7, retaining 60% activity, while M3 OPH67 retained 100% activity at pH 8, and thereafter activity decreased rapidly, whereas M4 OPH33 retained 50% activity at pH-8 and M4 OPH67 was found to be stable at pH 9 with 80% activity (Fig. 3 A). This clearly indicates that OPH33 stability was just half that of OPH67. The temperature stability profiles showed that M3 OPH33 and OPH67 were stable, maintaining 60% and 100% activity at 80 C and 70 C respectively, whereas M4 OPH33 and OPH67 were 30% and 70% stable at 70 C and 60 C respectively (Fig. 3 B). Thus of all the hydrolases, M3 OPH67 possessed the most stability.
B
B
Relative activity (%)
A
Relative activity (%)
A
Relative activity (%)
R. JAIN et al.
Relative activity (%)
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Fig. 3. Effects of pH (A) and Temperature (B) on Stability of Hydrolases after 18 h for OPH33 and OPH67 of M3 and M4. Fig. 2. Effects of pH (A) and Temperature (B) on the Hydrolase Activity of OPH33 and OPH67 of M3 and M4.
Table 2. Comparative Kinetics of M3 and M4 M3
Effects of physical and chemical agents on MCP hydrolase activity The effects of various reagents, metal ions, sunlight, and UV were determined by pre-incubating OPH33 and OPH67 of M3 and M4 separately for 30 min under these various conditions, and the residual activity of each was estimated. As shown in Fig. 4, inhibitory effects of 2mercaptoethanol, HgCl2 , FeCl3 , CoCl2 , and UV light were observed for all the enzymes, whereas sodium potassium tartarate and CuCl2 activated all the enzymes. Negligible or little effect on enzyme activity was found for EDTA, NaF, NaN2 , ZnCl2 , potassium ferricyanide, MnCl2 , and sunlight.
Discussion Here we report the purification and characterization of two hydrolases, OPH33 and OPH67, from two soil
Km (mM) Vmax (U/mg protein)
M4
OPH33
OPH67
OPH33
OPH67
95.3 1.75
33.52 5.181347
200.02 4.69
44.49 4.37
Relative activity (%)
Kinetic analysis of MCP hydrolases MCP was used as substrate in the kinetic analysis of the MCP hydrolases. As shown in Table 2, the Km values for M3 and M4 OPH33 were found to be higher than those of M3 and M4 OPH67, revealing its lesser affinity towards the substrate. The rate of reaction was also higher for OPH67, showing its higher efficiency in converting substrate into product (Table 2).
Metal ions (1mM)
Fig. 4. Effects of Different Physical and Chemical Agents on OPH33 and OPH67 of M3 and M4. SDS, Sodium dodecyl sulphate; KF, Potassium ferricyanide; 2ME, 2-Mercaptoethanol; SU, Sunlight; Tartarate; Sodium potassium tartarate.
Comparative Characterization of Two Distinct Extracellular Monocrotophos Hydrolases
isolates, Penicillium aculeatum ITCC 7980.10 and Fusarium pallidoroseum 7785.10, that hydrolyze MCP. In contrast to our findings demonstrating the presence of two extracellular alkaline hydrolases capable of hydrolyzing MCP, previous reports have indicated the presence of a single enzyme to hydrolyze different OP pesticides. These hydrolases generally differ in their molecular weights. Parathion hydrolases have been found to have a molecular mass of 35 kDa (monomer) in Flavobacterium sp. strain ATCC 27551, 43 kDa (monomer) in strain B-1, and 67 kDa in strain SC (four identical subunits).5,21) Our findings are consistent with previous studies with respect to the molecular weights of the enzymes, 35, 43, and 67 kDa as stated above, but vary in the presence of two distinct proteins within the same strain. On the other hand, it is likely that 33 kDa was a proteolytic product of OPH67, which might have been generated at some stage in the purification process. A previous study identified the occurrence of diverse enzymes, including phosphatase, esterase, and phosphotriesterase, during the degradation process of MCP.22) Our characterization of the MCP hydrolases revealed them to be alkaline hydrolases with highest activity in a pH range from 7 to 9. The same pattern of activity was found when the enzymes were subjected to various temperatures. All the hydrolases were stable at 80 C. It has also been reported that OP pesticide hydrolases are mostly alkaline, due to the fact that these pesticides degrade more at alkaline pH. These hydrolases had a wide temperature tolerance range, as reported previously.12,23,24) The effects of potential inhibitors and activators on the hydrolases of M3 and M4 were investigated and were found to be almost same for both. M3 and M4 hydrolases were significantly inhibited by Hg2þ , Co2þ , and Fe3þ , indicating the role of thiol groups in the catalytic site. Complete inhibition of the hydrolases was observed with sulfhydryl reagent 2-mercaptoethanol, a well-known thiol group inhibitor suggesting that sulfhydryl groups are involved in the catalytic center of the enzyme.23,25) No inhibition was observed with EDTA, representing the lack of the requirement of divalent cations for hydrolase activation, but activation of the hydrolases was demonstrated with Cu2þ , similarly to that of parathion hydrolase of strain SC.5) In addition, a significant increase in the activity was found with SDS, possibly due to enhancement in the substrate accessibility to the hydrolase. We conclude that the OPH33 and OPH67 from Penicillium aculeatum ITCC 7980.10 and Fusarium pallidoroseum 7785.10 are extracellular MCP hydro-
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lases that are thermostable and pH tolerant. These properties make these, especially Penicillium aculeatum ITCC 7980.10 OPH67, as excellent candidates for the bioremediation of OP pesticide contaminated sites.
Acknowledgment This work was supported financially by the Department of Biosciences and Biotechnology of Banasthali University, Rajasthan, India, and this is gratefully acknowledged.
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