BIOTECHNOLOGY AND BIOENGINEERING Chinese Journal of Chemical Engineering, 19(4) 644648 (2011)

Purification and Characterization of a Nonylphenol (NP)-degrading Enzyme from Bacillus cereus. Frankland*
YANG Ge ()1,2,**, ZHANG Ying ()1 and BAI Yanfen ()2
1 2

College of Textiles Tianjin Polytechnic University, Tianjin 300160, China Key Lab of Biogeology and Environmental Geology of Ministry of Education, China University of Geosciences, Wuhan 430074, China

Abstract An extracellular NP-degrading enzyme secreted by Bacillus cereus. Frankland was purified to homogeneity by a combination of ammonium sulfate precipitation, Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography. On SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis analysis, the purified enzyme showed a relative molecular mass of 58.3 kDa. The depolymerzation of subunits was accompanied with the loss of NP-degrading enzyme activity, and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity. The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60 C, and was the most active at pH 6.0. The enzymatic activity was stable at pH 4-8 and inhibited by Cu2+. Ten N-terminal amino acids were determined to be ASVNSIKIGY, demonstrating that the purified enzyme was a novel one. The hydrolysis pattern of the purified enzyme indicated that the NP-degrading enzyme was an endo NP-degrading enzyme. The extraordinary thermo-stability provided the enzyme with a good prospect to be used as a new tool for clean-production process for textile industry. Keywords nonylphenol (NP), Bacillus cereus. Frankland, NP-degrading enzyme, purification, characterization

INTRODUCTION

Alkylphenol polyethoxylates (APEOs) were a group of non-ionic surfactants and the hard-degradable polymer that was found widespread use as detergents, emulsifiers, wetting agents, stabilisers, defoaming agents and intermediates in the synthesis of anionic surfactants. It had been reported that these substances were degraded into more toxic products, mainly as NP [1-3]. In particular, concerns had been expressed regarding the possible endocrine disrupting effects of these hormone mimicking degradation products [4-9]. In recent years, the development of cleanproduction process for textile industry attracted great interest and therefore, the biodegradation of NP at the desizing stage, which could greatly reduce discharge of NP waste water and minimize damage of cotton fiber in the desizing process, became one of the key points in textile biotechnology [10, 11].Till now, the research about biodegradation of NP was mainly focused on the screening of NP-degrading microorganisms and the characteristics of PVA (polyvinyl alcohol)degrading enzymes from obtained strains. PVAdegrading enzymes were still not applicable in real industry process due to their low activity and the producing strains of NP-degrading enzyme were limited and they often grew very slowly. Here the recent research of NP biodegradation, including purification and characterization of the NP-degrading enzyme was reported [12-16]. Bacillus cereus. Frankland No. BCF83 was a newly isolated strain from soil in Shandong of China for its high NP-degrading enzyme activity secreted in

culture medium. In this study the extracellular NPdegrading enzyme was purified to homogeneity from the fermented broth of Bacillus cereus. Frankland No. BCF83 to investigate its physico-chemical properties. With the determination of partial N-terminal amino acid sequence and its characteristics, it was demonstrated that the purified enzyme was a novel endo NP-degrading enzyme. 2 2.1 MATERIALS AND METHODS Bacterial strain and culture condition

Bacillus cereus. Frankland No. BCF83 was isolated from soil in Shandong of China. Cultures were maintained on nutrient agar slants and incubated at 37 C for 24 h. The cells were then inoculated into a 500-ml Erlenmeyer flask containing 200ml liquid medium, cultured at 37 C for 24-36 h on a shaker. The medium adjusted to pH 7.0 was composed of (gL1): 1 NP, 1 NH4NO3, 1 yeast extract, 0.5 KH2PO4, 0.2 MgSO47H2O, 0.02 FeSO47H2O, and 0.1 CaCl2. 2.2 Chemicals

Phenyl-Sepharose CL-4B, DEAE-Sepharose Fast Flow and Sephadex G-150 were purchased from Pharmacia LKB (Uppsala Sweden). SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis) protein markers were purchased from Sigma (Santa Clara, USA). Other chemicals were of analytical grade.

Received 2010-04-07, accepted 2011-04-18. * Supported by the Fund of Open Subject of Key Lab of Biogeology and Environmental Geology of Ministry of Education (BGEG1006). ** To whom correspondence should be addressed. E-mail: yangge100@126.com

Chin. J. Chem. Eng., Vol. 19, No. 4, August 2011

645

2.3

Purification of NP-degrading enzyme

The fermented broth of Bacillus cereus. Frankland No. BCF83 was collected by centrifugation at 8000 g for 10 min, and the proteins fractionated with 50% saturation (NH4)2SO4 were collected by centrifugation at 8000 g for 20 min. The protein precipitate was dissolved in 0.8 molL1 (NH4)2SO4 solution and the insoluble materials were removed by centrifugation at 15000 g for 30 min. The derived supernatant was applied onto a Phenyl-Sepharose CL-4B column (1.2 cm 10 cm) preequilibrated with 1 molL1 (NH4)2SO4. The column was washed with 1.5 bed volumes of 1 molL1 (NH4)2SO4, 2 bed volumes of 0.1 molL1 (NH4)2SO4, and eluted with distilled water. The flow rate was maintained at 0.5 mlmin1. The fractions with NP-degrading enzyme activity were pooled and dialyzed overnight at 4 C against 10 mmolL1 tris-HCI buffer with pH 6.2. The dialysate was collected and immediately applied on a DEAE-Sepharose Fast Flow column (1.2 cm4 cm) pre-equilibrated with 10 mmolL1 tris-HCI buffer with pH 6.2. The flow rate was maintained at 0.25 mlmin1. The fractions with NP-degrading enzyme activity were pooled, 10 times concentrated and kept at 20 C until use. For N-terminal amino acid sequencing, the enzyme fraction was further purified by reverse-phase HPLC (High Performance Liquid Chromatography). NP-degrading enzyme fractions derived from DEAE-Sepharose Fast Flow were loaded on a Zorbas 300SBCN column (du Pont, 250 mm4.6 mm I. D.), and the column was developed with acetonitrile gradient (0-10% for 20 min supplemented with 0.1% trifluoroacetic acid (TFA). The elution pattern was monitored by absorbance at 220 nm and 280 nm. The major peak was collected and lyophilized for automatic amino acid sequencing. 2.4 Enzymatic activity assay

Coomassie brilliant blue R-225. For isoelectric focusing (IEF) experiment, about 5 g of sample proteins in 20 l solution were loaded to a precasted capillary gel with 0.75% Ampholine (pH range 3.5-10), and run under 200 V for 5 h. Amyloglucosidase (pl 3.6), trypsin inhibitor (pI 4.6), -lactoglubin A (pI 5.1), conalbumin (pI 6.0), myoglobin (pI 6.8, 7.2), lentil lectin (pI 8.2, 8.6, 8.8), and trypsinogen (pI 9.3) were used as markers. 2.6 Zymogram

To identify the protein of NP-degrading enzyme, zymographic approach was applied on samples derived from DEAE Fast Flow chromatography. Samples were separated on 10% polyacrylamide gel electrophoresis at pH 8.3. As soon as the electrophoresis was finished, the gel was immediately placed on an agarose slab gel containing 10 gL1 nonylphenol. After incubation for 1.5 h at 37 C, a transparent band could be seen on the agarose slab. The sections of the polyacrylamide gel overlapping with the transparent band were carefully cut out and pestled with the transparent band were carefully cut out and pestled with sample buffer in an Eppendorf tube. The derived paste was analyzed on 10% SDS-PAGE. 2.7 Gel filtration chromatography

Two methods for enzymatic activity assay were used in this work. For rapidly tracing NP-degrading enzyme activity during the chromatographic separation processes, 100 l of each fraction was mixed with 2 ml 10 gL1 nonylphenol (NP) in 50 mmolL1 acetate buffer (pH 7.0) and incubated at 50 C for 10 min. The enzyme activity was calculated from a standard curve obtained with known concentration of nonylphenol (NP). One unit of NP-degrading enzyme activity was defined as the amount of enzyme that liberated 1 mol NP-degrading per min at pH 7.0 and 50 C. Negative control tubes contained all components except substrate, and blanks contained all components except the enzyme. 2.5 Electrophoresis

Gel filtration chromatography was used for determination of molecular weight of molecular weight of NP-degrading enzyme. Sephadex G-150 was packaged in a 1.2 cm60 cm column and equilibrated with 10 mmolL1 tris-HCI buffer (pH 6.2) at a flow rate of 0.15 mlmin1. About 0.4 ml concentrated enzyme obtained from the DEAE Fast Flow column was applied to the column and eluted with the same buffer. Protein profile was monitored at 280 nm. The molecular weight was estimated from a standard curve obtained from the proteins with their relative molecular mass known [17]. 2.8 N-Terminal amino acid sequencing

Samples obtained from reverse-phase HPLC were lyophilized and subjected to N-terminal amino acid sequencing on an automatic protein sequencer ( Model 473A, Applied Biosystems Inc., USA). 3 3.1 RESULTS AND DISCUSSION Purification of NP-degrading enzyme

SDS-polyacrylamide gel electrophoresis (SDSPAGE) was performed. The proteins were stained with

A NP-degrading enzyme secreted by this new strain of Bacillus cereus. Frankland No. BCF83 was purified for further study. Proteins in the fermented broth were recovered with (NH4)2SO4 precipitation at 50% saturation. The protein precipitates were dissolved

646

Chin. J. Chem. Eng., Vol. 19, No. 4, August 2011

in 0.8 molL1 (NH4)2SO4 solution and separated by Phenyl-Sepharose CL-4B hydrophobic interaction chromatography. In a typical separation, 30 ml of the sample solution containing 68.4 mg of crude proteins was applied to a 1.2 cm10 cm column, and developed as described in Section 2. Four protein peaks were detected at 280 nm as shown in Figs. 1 and 2. NP-degrading enzyme activity was only found in the last peak eluted with distilled H2O.

shown in Fig. 2. When this peak was analyzed on 10% SDS-PAGE, a protein band with relative molecular mass of 58.3 kDa (Fig. 3) was shown. Meanwhile, zymographic approach was applied to identify the protein with NP-degrading enzyme activity. Table 1 is the summary of purification.

Figure 1 Elution profile of NP-degrading enzyme on PhenylSepharose CL-4B column The protein solution was eluted stepwise with 0.1 molL1 (NH4)2SO4 and distilled water. Absorbance at 280 nm () and relative enzymatic activity () were determined.

Figure 3 SDS-PAGE analysis of NP-degrading enzyme under various conditions Lanes 1 and 2 are from DEAE-Sepharose. Lanes 3,4,5 and 6 are from Phenyl-Sepharose.

3.2

N-terminal amino acid sequence

For N-terminal sequencing, the enzyme fractions from DEAE-Sepharose Fast Flow were further purified by reverse-phrase HPLC. As shown in Fig. 4, only one protein peak was detected. The protein peak was collected, lyophilized and subjected to amino acid sequencing. The first 10 amino acids in the N-terminal sequence were determined to be ASVNSIKIGY. The sequence was blasted against GenBank, however, no

Figure 2 Elution profile of NP-degrading enzyme on DEAESepharose Fast Flow column chromatography 0.04 molL1 NaCl to 0.14 molL1 NaCI (); relative enzymatic activity ()

The fractions with NP-degrading enzyme activity were pooled and dialyzed against 10 mmolL1 tris-HCl buffer (pH 6.2) overnight. The dialysate containing 23.46 mg proteins was then applied onto a DEAESepharose Fast Flow column for anion-exchange chromatography described as Section 2. The NP-degrading enzyme activity was detected in the third peak as
Table 1
Purification step crude enzyme 50% ammonium sulfate precipitation Pheny-Sepharose CL-4B column DEAE-Sepharose Fast column

Figure 4 Elution profile of NP-degrading enzyme on reverse-phase HPLC

Purification of NP-degrading enzyme from Bacillus cereus. Frankland No. BCF83

Total protein/mg 617.7 75.3 27.12 3.21 Total activity/ 51.3 10.1 6.94 4.19 Specific activity/mg1 0.08 0.17 0.28 1.51 Purification factor 1.00 1.83 3.67 16.00 Yield/% 100 24.21 14.52 8.46

Chin. J. Chem. Eng., Vol. 19, No. 4, August 2011

647

NP-degrading enzyme known showed significant similarity with this sequence. 3.3 Characteristics of the purified NP-degrading enzyme The difference between relative molecular mass of proteins in DEAE-Sepharose Fast Flow fractions and HPLC fractions implied a dimer structure of NP-degrading enzyme. A series of experiments or further characterization of this protein was performed. The purified NP-degrading enzyme was subjected to isoelectric focusing analysis and the pI of the NP-degrading enzyme was found to be 5.5. On the result of RPC (Reversed Phase Chromatography), the molecular weight of NP-degrading enzyme was determined to be around 56 kDa (Fig. 4), and other two peaks were not an enzyme activity. The relative molecular mass of NP-degrading enzyme protein on SDS-PAGE differed depending on conditions. If the NP-degrading enzyme in DEAE fractions was heated in boiling sample buffer before SDS-PAGE analysis, the protein band on SDS-PAGE was at the position of 58.3 kDa. After treatment of the enzyme with 4% 2-mercaptoethanol, the relative molecular mass of the purified enzyme was still 58.3 kDa on SDS-PAGE, implying that disulfide bond was not involved in the formation of NP-degrading enzyme with 8 molL1 urea at 50 C for 30 min and could cause a total loss of enzymatic activity and a shift of the protein band position from 58.3 kDa to 28.5 kDa on SDS-PAGE (Fig. 3). After dialysis of the enzymes depolymerized by 8 molL1 urea, heating at 100 C or 0.1% TFA treatment against 10 mmolL1 tris-HCl buffer (pH6.2), the enzymatic activity recovered by 79%, 75% and 87%, respectively. The dimer was found to be the major component revealed by SDS-PAGE (data not shown). These results strongly suggest that the NP-degrading enzyme had a homodimer structure based on hydrophobic interaction. After incubation the NP-degrading enzyme with 8 molL1 urea and then dialyzing it against 40% alcohol, the free NP-degrading enzyme subunits were obtained, which utterly lost the activity (the NP-degrading enzyme in 40% alcohol still exhibited hydrolytic activity). It was thus concluded that the compact structure of the dimer is necessary for NP-degrading enzyme activity. Up to now, no NP-degrading enzyme with dimer structure from bacteria had been reported. In other species only an insect inhibitor/endo NP-degrading enzyme from plant origin was shown to have a structure of dimer. By this fact, as well as the result of the N-terminal amino acid sequence, it was concluded that the NP-degrading enzyme produced by Bacillus cereus. Frankland No. BCF83 was a novel NP-degrading enzyme with an unusual structure. The optimal conditions for enzymatic reaction were studied systemically. 6g purified NP-degrading enzyme was used to determine its characteristic. The optimal pH of the NP-degrading enzyme was 6.0 (Fig. 5),

Figure 5 Effect of pH () and temperature () on enzymatic activity of the purified NP-degrading enzyme

Figure 6 Effect of temperature on stability of the purified NP-degrading enzyme

Figure 7 Effect of Cu2+ ion on enzymatic activity of the purified NP-degrading enzyme

and the NP-degrading enzyme was stable and could hydrolyze collidal nonylphenol at a wide pH range (from pH 4.0 to pH 8.0). The NP-degrading enzyme exhibited the highest activity at 60 C and retained high activity even over 80 C (Fig. 5). However, in the absence of substrate the NP-degrading enzyme lost its activity markedly above 60 C (Fig. 6), inferring that the substrate could protect the active center of the NP-degrading enzyme from denaturation. The Bacillus cereus. Frankland No. BCF83 NP-degrading enzyme could be inactivated by Cu2+ ion (Fig. 7). After incubation with 0.5 mmolL1 Cu2+ at pH 6.0 and 30 C for 30 min, only 60% of the enzyme activity remained.

648

Chin. J. Chem. Eng., Vol. 19, No. 4, August 2011

CONCLUSIONS
8 9

The Bacillus cereus. Frankland No. BCF83 NP-degrading enzyme was highly stable (retaining higher than 80% activity) in a wide range of pH (pH 6.0 to 10.0) and temperature (from 35 C to 72 C). In comparison, the NP-degrading enzyme from Bacillus cereus. Frankland No. BCF83 and other strains [18-21] exhibit their enzymatic activities in a more narrow temperature range and are less stable. Furthermore, the purified NP-degrading enzyme from Bacillus cereus. Frankland No. BCF83 was strongly resistant to the hydrolysis by trypsin. A common condition was not sufficient for trypsin digestion of the NP-degrading enzyme. The NP-degrading enzyme in fermented broth could be kept at 4 C for at least two months without loss of enzymatic activity. The crude fermented broth of the Bacillus cereus. Frankland No. BCF83 NP-degrading enzyme could be widely applied as a new tool for clean-production process of textile. REFERENCES
1 2 3 Ferguson, P.L., Brownawell, B.J., Degradation of nonylphenol ethoxylates in estuarine sediment under aerobic and anaerobic conditions, Environ. Toxicol. Chem., 22, 1189-1199 (2003). Xiao, C.B., Ning, J., Yan, H., Sun, X.D., Hu, J.Y., Biodegradation of aniline by a newly isolated Delftia sp. XYJ6, Chin. J. Chem. Eng., 17 (3), 500-5052009. Mnsson, N., Srme L., Wahlberg, C., Bergbck, B., Sources of alkylphenols and alkylphenol ethoxylates in wastewater a substance flow analysis in Stockholm, Sweden, Water, Air, & Soil Pollut: Focus, 8, 445-456 (2008). Ohtsubo, Y., Kudo, T., Tsuda, M., Nagata, Y., Strategies for bioremediation of polychlorinated biphenyls, Appl. Microbiol. Biotechnol., 65, 250-258 (2004). Wackett, L.P., Sadosky, M.J., Martinez, B., Shapir, N., Biodegradation of atrazine and related striazine compounds from enzymes to field studies, Appl. Microbiol. Biotechnol., 58, 39-45 (2002). Cravotto, G., Carlo, S.D., Binello, A., Mantegna, S., Girlanda, M., Lazzari, A., Integrated sonochemical and microbial treatment for decontamination of nonylphenol-polluted water, Wate, Air, & Soil Pollution, 187 (1-4), 353-359 (2008). Jontofsohn, M., Pfister, G., Severin, G., Schramm, K.W., Hartmann, A., Schloter, M., Bacterial community structure in lake sediments

10 11 12

13 14

15 16

17 18 19

4 5 6

20 21

of microcosms contaminated with nonylphenol, Journal of Soils and Sediments, 2 (4), 211-215 (2002). Mai, H., EI-Dakdoky, Mona, A.M., HelaI, Reproductive toxicity of male mice after exposure to nonylphenol. Bulletin of Environmental Contamination and Toxicology, 79 (2), 188-191 (2007). Beklioglu, M., Banu Akkas, S., Elif Ozcan, H., Bezirci, G., Togan, I., Effects of 4-nonylphenol, fish predation and food availability on survival and life history traits of Daphnia magna straus, Ecotoxicology, 19 (5), 901-910 (2010). Hermuth, K., Leuthner, B., Heider, J., Operon structure and expression of the genes for benzylsuccinate synthase in Thauera aromatica strain K172, Arch. Microbiol., 177, 132-138 (2002). Krieger, J., Roseboom, W., Albracht, S.P., Spormann, A.M., A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T, J. Biol. Chem., 276, 12924-12927 (2001). Song, B., Palleroni, N.J., Haggblom, M.M., Isolation and characterization of diverse halobenzoate-degrading denitrifying bacteria from soils and sediments, Appl. Environ. Microbiol., 66, 3446-3453 (2000). Lu, J., He, Y.L., Wu, J., Jin, Q., Aerobic and anaerobic biodegradation of nonylphenol ethoxylates in estuary sediment of Yangtze River, China, Environmental Geology, 57 (1), 1-8 (2009). Liu, X., Tani, A., Kimbara, K., Kawai, F., Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria, Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90, Applied Microbiology and Biotechnology, 72 (3), 552-559 (2006). Latorre, A., Lacorte, A., Barcel, D., Presence of nonylphenol, octyphenol and bisphenol a in two aquifers close to agricultural, industrial and urban areas, Chromatographia, 57 (1-2), 111-116 (2003). Park, S.Y., Choi, J., Genotoxic effects of nonylphenol and bisphenol a exposure in aquatic biomonitoring species: freshwater crustacean, daphnia magna, and aquatic midge chironomus riparius, Bulletin of Environmental Contamination and Toxicology, 83 (4), 463-468 (2009). Chen, M., Yao, S.J., Zhang, H., Liang, X.L., Purification and characterization of a versatile peroxidase from edible mushroom Pleurotus eryngii, Chin. J. Chem. Eng, 18 (5), 824-829 (2010). Morgan, P., Watkinson, R.J., Microbiological methods for the clean up of soil and groundwater contaminated with halogenated organic compounds, FEMS Microbiol. Rev. 63, 277-300 (1989). Takasu, T., Iles, A., Hasebe, K., Determination of alkylphenols and alkylphenol polyethoxylates by reversed-phase high-performance liquid chromatography and solid-phase extraction, Anal. Bioanal. Chem., 372, 554-561 (2002). Zhang, X., Young, L.Y., Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia, Appl. Environ. Microbiol., 63, 4759-4764 (1997). Zhang, X., Sullivan, E.R., Young, L.Y., Evidence for aromaticring reduction in the biodegradation pathway of carboxylated naphthalene by a sulfate-reducing consortium, Biodegradation, 11, 117-124 (2002b).