ORIGINAL PAPER
Purification and characterization of a thermostable uricase from Microbacterium sp.strain ZZJ4-1
Lei Kai ÆXiao-Hang Ma ÆXue-Lai Zhou ÆXiao-Ming Jia ÆXia Li ÆKang-Ping Guo
Received:10April 2007/Accepted:30June 2007/Published online:25July 2007ÓSpringer Science+Business Media B.V.2007
Abstract In order to study the properties of a thermo-stable uricase produced by Microbacterium sp.strain ZZJ4-1,the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange,hydro-phobic and molecular sieve chromatography.The molec-ular mass of the purified enzyme was estimated to be 34kDa by SDS-PAGE.The enzyme was stable between pH 7.0and 10.00.The optimal reaction temperature of the enzyme was 30°C at pH 8.5.The K m and K cat of the enzyme were 0.31mM and    3.01s –1,respectively.Fe 3+could enhance the enzyme activity,whereas Ag +,Hg 2+,o -phenanthroline and SDS inhibited the activity of the enzyme considerably.After purification,the enzyme was purified 19.7-fold with 31%yield.As compared with uri-cases from other microbial sources,the purified enzyme showed excellent thermostability and other unique char-acteristics.The results of this work showed that strains of Microbacterium could be candidates for the pro
duction of a thermostable uricase,which has the potential clinical application in measurement of uric acid.
Keywords Uricase ÁThermostability ÁMicrobacterium .ÁPurification ÁCharacterization ÁUric acid
Introduction
Uricases (urate oxidoreductase;EC 1.7.3.3),which catalyse the oxidative breakdown of uric acid,belong to a group of
enzymes in the purine degradation pathway found in ani-mals (Keilin 1959;Wallrath and Friedman 1991),plants (Montalbini et al.1997),fungi (Montalbini et al.1999),yeasts (Adamek et al.1990;Hongoh et al.2000;Koyama et al.1996)and bacteria (Yamamoto et al.1996).Since Bongaert et al.(Bongaert et al.1978)found that Bacillus fastidiosus could produce uricase and use uric acid as the only carbon source for growth in 1978,many reports on bacterial and yeast uricases,such as those from Arthrob-acter globiformis (Nobutoshi et al.2000),Bacillus subtilis (Hunag and Wu 2004),Candida utilis (Liu et al.1994)and Pseudomonas aeruginosa (Ishikawa et al.2004),have been published.
In the human body,uric acid is a final product of purine catabolism and is excreted out of the body by th
e kidney.When the level of uric acid in blood increases over the normal value,it can lead to a group of diseases,such as gout (Nakagawa et al.2006),idiopathic calcium urate nephrolithiasis (Masseoud et al.2005)and renal failure (Capasso et al.2005),and it was also reported that a high level of uric acid was related to leukemia in children (Larsen &Loghman-Adham 1996).Consequently,uric acid concentration is an important parameter monitored in urine and blood samples in routine clinical examinations.At present,the colorimetric method that employs uricase and peroxidase is widely accepted as a simple,sensitive and highly specific test for uric acid examination.In this enzymatic system of uric acid analysis,uricase plays an important role:
uric acid À!uricase
5Àhydroxyisourate þH 2O 2þCO 2
By determining the amount of peroxide formed in the reaction,the concentration of uric acid can be estimated.For the best result in this examination,the properties of the
L.Kai ÁX.-H.Ma (&)ÁX.-L.Zhou ÁX.-M.Jia ÁX.Li ÁK.-P.Guo
College of Life Sciences,Zhejiang University,Yuhangtang Road 388#,Hangzhou,China
e-mail:maxiaohong@zju.edu
World J Microbiol Biotechnol (2008)24:401–406DOI 10.1007/s11274-007-9489-1
uricase in this reaction system are important,especially its stability,which will determine the precision of the mea-surement.At present,most enzymes applied in the clinical test are used in solution,and most proteins,including this enzyme,are relatively unstable when dissolved in aqueous solution.Therefore,for the enzymatic examination appli-cation,research was undertaken to search for a thermostable enzyme(Guo et al.2006;Huang et al.1998;Zhou et al.2005).
As mentioned above,there were many uricases that have been isolated from microorganisms,but the thermostability of the published uricases were relatively low(Suzuki et al. 2004).The most thermostable uricase was reported by Suzuki,but it would lose its activity after a short period of treatment at60°C(Suzuki et al.2004).This low stability is a disadvantage in clinical applications.
In a previous study,we isolated a bacterium Micro-bacterium sp.strain ZZJ4-1that produced a thermostable uricase.The enzyme was stable at65°C and its solution retained its original activity even after storage at37°C for 40days(Zhou et al.2005).Considering that this enzyme has a potential value in practical application,the present study was undertaken to purify and study the properties of this new en
zyme.
Materials and methods
Materials and chemicals
The culture of Microbaterium sp.(strain ZZJ4-1)was maintained in our laboratory(Zhou et al.2005).
The protein standards for SDS-PAGE were purchased from Invitrogen(Shanghai,China).All other chemicals used were of reagent or molecular biology grade and pur-chased from Hangzhou Huadong Medicine Group Co.,Ltd (Hangzhou,China).
Cultivation conditions
The fermentation medium consisted of3.0g of uric acid, 10.0g of maize milk,0.5g of MgSO4Á7H2O,0.5g of KH2PO4,2.0g of K2HPO4Á3H2O,0.1g of NaCl,1.0l of tap water and the pH was adjusted to7.5(Zhou et al. 2005).To cultivate the strain for production of the uricase, a loop of bacteria from a slant was inoculated into a500ml flask containing100ml liquid medium and incubated at 30°C for30h with a rotary shaker at120rev/min.
Enzyme assay and protein measurement
The principle of enzyme measurement was as follows: uricase can catalyse the oxidation of uric acid to form 5-hydroxyisourate and H2O2,which is then measured using a reaction system containing4-aminoantipyrine,phenol and peroxidase as chromogens.In practical analysis, 0.10ml enzyme solution was incubated with a mixture of 0.6ml0.1M sodium borate buffer(pH8.5)containing 2mM uric acid,0.15ml of30mM4-aminoantipyrine, 0.1ml of1.5%phenol and0.05ml peroxidase(15U/ml) at37°C for20min(Masaru1981).The reaction was stopped by addition of1.0ml ethanol and the absorbance at540nm was read against the blank in a spectropho-tometer.One unit of enzyme was defined as the amount of enzyme that produces1.0l mole of H2O2per minute under the standard assay conditions.
The protein was measured by the Folin-phenol method (Lowry et al.1951).
Enzyme purification
Unless otherwise stated,all of the enzyme purification processes were performed with0.1M phosphate buffer at pH7.0(buffer A)at4°C.
Cells from10.0l of media were harvested by centrifu-gation and washed twice with50mM phosphate buffer(pH 7.5),re-suspended in buffer A and disrupted by ultrasonic oscillation(120W oscillating for3s with6s intervals, repeated100times).After the cell debris had been sepa-rated by centrifugation,solid a
mmonium sulfate was added to the enzyme solution and the precipitate of the fractions from55%to80%saturation was collected by centrifuga-tion(8000rpm,40min,4°C).The enzyme was then dis-solved in a small amount of buffer(2:1,volume of the buffer/weight of the precipitate),dialyzed against0.01M phosphate buffer until the ammonium sulfate was removed.
The dialyzed enzyme solution was put onto a DEAE-Cellulose column(5.5·50cm)previously equilibrated with buffer A and it was then eluted with2.0l of buffer A with a linear gradient of0–1.5M KCl(flow rate:2ml/ min).The fractions containing enzyme activity were col-lected.After the ammonium sulfate had been added to65% saturation,the enzyme solution was loaded onto a Toyo-pearl HW-65column(5.5·85cm)equilibrated previ-ously with buffer A containing65%ammonium sulfate. The enzyme was eluted with2.0l of the same buffer with a linear gradient of ammonium sulfate from65%to0%(flow rate:2ml/min).When the fractions containing enzyme activity had been collected,the ammonium sulfate was added to get85%saturation.The precipitate of the enzyme was then collected by centrifugation,dissolved in a small amount of buffer A,dialysed with the same buffer con-taining0.1M KCl and applied to a Sephadex G-75column (5.5·100cm)equilibrated with the same buffer.The enzyme was then eluted with2.0l of the same buffer(flow rate:2ml/min)and the fractions containing the highest specific activity were collected for further study.
Characterization of enzyme
To study the effect of pH on the activity of uricase,the enzyme was assayed at different pHs in the range from4.0 to11.0with intervals of0.5.The following buffers were used:100mM citrate for pH4.0–6.0,100mM phosphate for pH6.0–8.5and100mM borate for pH8.5–11.The pH stability was studied by incubating the purified enzyme solution in the corresponding buffers in the range from4.0 to11.0at25°C for18h and measuring the residual activity.
To study the effect of temperature on the uricase activity,the standard enzyme reaction solution was pre-incubated at temperatures of20–60°C with5°C intervals for5min and the enzyme solution was then added and incubated for20min at the same temperature to measure its activity.For thermostability testing,the purified uricase solution was incubated at temperatures in the range from 20°C to80°C for30min and the remaining activity was then measured.
The apparent K m of the uricase was estimated by the double reciprocal plot method.At different concentrations of uric acid,the enzyme activity was assayed and the Km was calculated by the Lineweaver-Burk plotting according to the Michaelis-Menten equation.
To study the effects of chemicals on the uricase activity, the enzyme solution was pre-incubated with th
e chemicals for30min at room temperature in phosphate buffer and the remaining uricase activity was assayed with the standard reaction system containing the corresponding chemical.
The relative molecular mass of the enzyme was deter-mined by SDS-PAGE with10%polyacrylamide gels (Laemmli1970).The purity was determined by the specific activity of the enzyme and PAGE with10%polyacryl-amide gels.The protein was stained with0.1%Coomassie brilliant blue R250in4:1:5methanol/acetic acid/water (vol/vol/vol)solution and destained in the same solution without dye.
Results
Purification of the enzyme
In thefirst step of the purification,ammonium sulfate precipitation was applied.The amount of protein and the enzyme activity of each fraction were measured.The fractions with ammonium sulfate concentrations from65% to80%had the highest enzyme specific activity(0.43U/ mg),while fractions from35–55,55–65to80–85had the specific activity of0.02U/mg,0.09U/mg and0.07U/mg, respectively.The fractions with concentrations from65% to80%were collected,dialyzed and loaded onto a DEAE-cellulose column.The enzyme was eluted with the same buffer containing a linear concentrati
on gradient of KCl from0M to1.5M.Enzyme activity was found in fractions from75to130and fractions from80to105were pooled, solid ammonium sulfate was added to65%saturation and the solution was loaded onto a Toyopearl HW65-C col-umn.The enzyme was then eluted with the same buffer with a decreasing linear concentration of ammonium sul-fate from65%to0%.The enzyme activity was found in the fractions from100to160and fractions from110to135 were pooled,concentrated and applied to the Sephadex G-75column.After being washed with buffer A containing 0.1M NaCl,the enzyme activity was observed in the fractions from30to80.The fractions from50to60were pooled as purified enzyme for further study.
The purity of the enzyme after each purification step was examined by SDS-PAGE and is shown in Figure1. The results of the purification process are summarized in Table1.During the purification,the enzyme was purified 19.7-fold with a recovery of31%and the purified enzyme had a specific activity of5.32U mg-1.
Molecular mass determination
The purified enzyme showed a single protein band in SDS-PAGE and its molecular mass was estimated to be34kDa (Fig.2).
Optimum reaction temperature and thermostability of
the purified enzyme
The purified enzyme was stable at a relative high temper-ature.As shown in Fig.3,it was stable at65°C and it retained64%of its original activity even after being
treated Fig.1SDS-PAGE pattern of uricase samples from different steps of purification.(A)crude extract;(B)ammonium sulfate precipitation;
(C)DEAE-cellulose chromatography;(D)Toyopearl HW-65chro-matography;(E)Sephadex G-75chromatography
at70°C for30min.By comparison between the optimal reaction and stable temperature,it was shown that although the enzyme was stable at65°C,its optimum temperature was30°C,which was relatively low,and it only showed 21%relative activity at60°C.
Optimum pH and the stability of the enzyme
at different pHs
The activity of the enzyme was measured in different buffers with a pH range from4.0to11.0.The results showed that the enzyme had low activity at pH below5.5 or over10.5and had relatively high activity in the range from7.0to10.0,with the optimal reaction pH at8.5 (Fig.4).As shown in Fig.4,after being incubated in dif-ferent buffers at25°C for18h,the uricase was stable in the pH range from5.5to9.5.
Kinetics and effect of chemicals on the activity
of the enzyme
The K m of the purified enzyme for uric acid was estimated to be0.31mM,which was calculated from the slopes and intercepts of the regression lines of the Lineweaver-Burk plot by determining the enzyme activity at37°C and the K cat of the enzyme was estimated to be3.01s–1.
The effects of different chemicals on the activity of the enzyme are summarized in Table2.It was shown that among the metal ions,Li+,Ag+and Hg+greatly inhibited
Table1Summary of the uricase purification process Purification steps Activity(U)Total protein
(mg)
Specific activity
(U mg–1)
Purification
(fold)
Yeild(%)
Crude enzyme6012220400.271100 Ammonium
sulfate
488595190.51  1.9081 DEAE-cellulose338540840.83  3.0756 Toyopearl HW-6528451530  1.86  6.8947 Sephdax G-751866351  5.3219.70
31 Fig.2SDS-PAGE electrophoregram of uricase.M:Standard Protein
Marker;U:Uricase
the enzyme activity.The strongest suppression was ob-served in the case of Hg+,which suppressed almost all of the activity of this enzyme.The chelating reagents had different effects;EDTA had no inhibitory effect,whereas o-phenanthroline(OPT)could inhibit the activity of uricase considerably.
Discussion
At present,the uricases from many microorganisms have been studied and some gene sequences of uricases have been cloned and studied.It was shown that uricases belong to a group of enzymes that have the same catalytic char-acter,but a great diversity of molecular structures.Uricases from different
sources may have different molecular mas-ses and amino acid sequences.In this study,the molecular mass of the uricase from strain ZZJ4-1was estimated to be 34kDa by SDS-PAGE,whereas uricases produced by Candida utilis(Koyama et al.1996)and Pseudomonas aeruginosa(Ishikawa et al.2004)had the molecular mas-ses of34and54kDa,respectively.The apparent K m value of this uricase was0.31mM,while uricases from Ar-throbacter,Bacillus sp.and Candida sp.had K m values of 75,75,46l M,respectively(Suzuki et al.2004).
Some relatively thermostable uricases,such as those produced by Arthrobacter globiformis FERM BP-360, Bacillus sp.TB-90and Candida utili s,have been studied and these were stable at55or60°C(Suzuki et al.2004). In this work,the uricase produced by strain ZZJ4-1showed good thermostability.After incubation at65°C for30min, the enzyme from strain ZZJ4-1still retained98.9%of its original activity,and even after incubation at70°C for 30min,the remaining activity was64%.Additionally,the pH stability of the enzyme from strain ZZJ4-1was a little broader than that of the uricases from the above three species(Suzuki et al.2004).
The effects of metal ions on the enzyme activity from strain ZZJ4-1were also compared with the above three enzymes(Suzuki et al.2004).Considering that some uri-cases require metal ion cofactors for activity(Wu et al. 1989;Chu et al.1996)and whether or not an enzyme is inhibited by certain metals is a
n important characteristic, experiments were carried out to examine the effect of metals on the activity of this enzyme.It was shown that the enzyme from strain ZZJ4-1was not inhibited by Cu2+,Fe3+ or Zn2+,while uricases from the other three strains were strongly inhibited by these ions.Ag+is a strong inhibitor of the uricases from strain ZZJ4-1,Candida utilis and Bacillus sp.TB-90,while it had no such effect on the uricase from Arthrobacter globiformis FERM BP-360. When EDTA,a chelating reagent,was added to the enzyme solution at afinal concentration of20mM,the activity of the uricase was only slightly inhibited.When the stronger metal-ion-chelating reagent o-phenanthroline was added to the enzyme solution,the activity of the enzyme was strongly inhibited.This phenomenon indicates that some yet unidentified metal ion is strongly bound in the enzyme and forms part of the uricase structure,which is very important to keep its catalytic activity.This property is also different from the uricase of Arthrobacter globiformis FERM BP-360(Suzuki et al.2004).
It was shown that although the uricase from the strain ZZJ4-1was stable at65°C,the enzyme was at its optimal activity at30°C and it only showed21%of its maximal activity at60°C.This implied that the molecular structure of enzyme had a reversible change at a temperature be-tween30°and60°C,which had a negative effect on its catalysis activity.But considering that most clinical enzy-matic examinations are undertaken at25to37°C,this property will not affect its application in clinical exami-nation.
References
Adamek V,Suchova M,Demnerova K et al(1990)Fermentation of Candida utilis for uricase production.J Indu Microbiol6:85–90 Bongaerts GP,Uitzetter J,Brouns R et al(1978)Uricase of Bacillus fastidiosus.Properties and regulation of synthesis.Biochim Biophys Acta527:348–358
Capasso G,Jaeger Ph,Robertson WG et al(2005)Uric acid and the kidney:urate transport,S tone disease and progressive renal failure.Curr Pharm Des11:4153–4159
Table2Effects of chemicals on uricase activity
Chemicals Final Concentration Residual activity(%)
Blank–100
Fe3+1mM118.5
Ca2+1mM107.7
Ba2+1mM103.1
Mn2+1mM101.5
Zn2+1mM100.7
reaction between pvp and aminoCu2+1mM98.9
Li+1mM81.5
Ag+1mM7.5
Hg2+1mM  1.8
NaN320mM101.2
EDTA20mM99.8
o-Phenanthroline2mM  1.2
Tween200.10%(w/v)99.7
Tween800.10%(w/v)80.5
Triton X-1000.10%(w/v)99.7
SDS0.50%(w/v)51.3

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