Process Biochemistry 46(2011)168–173
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Process
Biochemistry
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /p r o c b i
o
Bioconversion of d -glucose into d -glucosone by immobilized glucose 2-oxidase from Coriolus versicolor at moderate pressures
Amin Karmali ?,JoséCoelho
Chemical Engineering and Biotechnology Research Center,Department of Chemical Engineering,Instituto Superior de Engenharia de Lisboa,Rua Conselheiro Emídio Navarro,1959-007Lisboa,Portugal
a r t i c l e i n f o Article history:
Received 19January 2010
Received in revised form 29June 2010Accepted 2August 2010
Keywords:
Biocatalysis at moderate pressures Immobilized glucose 2-oxidase from Coriolus versicolor
Immobilization in polyethersulfone membranes Catalase
Bioconversion of d -glucose into d -glucosone
Apparent kinetic constants
a b s t r a c t
The immobilized glucose 2-oxidase (pyranose oxidase,pyranose:oxygen-2-oxidoreductase,EC 1.1.3.10)from Coriolus versicolor was used to convert d -glucose into d -glucosone at moderate pressures,up to 150bar,with compressed air in a modi?ed commercial batch reactor.Several parameters affecting bio-catalysis at moderate pressures were investigated as follows:pressure,different forms of immobilized biocatalysts,glucose concentration,pH,temperature and the presence of catalase.Glucose 2-oxidase (GOX2)was puri?ed by immobilized metal af?nity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II)column at pH 6.0.Puri?ed enzyme and catalase were immobilized into a polyethersulfone (PES)membrane in the presence of glutaraldehyde and gelatin.Enhancement of the bioconversion of d -glucose was done by the pressure since an increase in the pressure with compressed air increases the conversion rates.The optimum temperature and pH for bioconversion of d -glucose were found to be 62?C and pH 6.0,respectively and the activation energy (E a )was 28.01kJ mol ?1.The apparent kinetic
constants (V max ,K m ,K cat and K cat /K
m )for this bioconversion were 2.27U mg ?1protein,11.15mM,8.33s ?1and 747.38s ?1M ?1,respectively.The immobilized biomass of C.versicolor as well as crude extract con-taining GOX2activity were also useful for bioconversion of d -glucose at 65bar with a yield of 69.9±3.8%and 91.3±1.2%,respectively.The immobilized enzyme was apparently stable for several months without any signi?cant loss of enzyme activity.On the other hand,this immobilized enzyme was also stable at moderate pressures,since such pressures did not affect signi?cantly the enzyme activity.
?2010Elsevier Ltd.All rights reserved.
1.Introduction
Enzymes are ideal biocatalysts for stereo-and regioselective reactions which take place in aqueous medium under mild con-ditions [1].Fungal glucose 2-oxidase (GOX2,pyranose oxidase,pyranose:oxygen-2-oxidoreductase,EC 1.1.3.10)has been used to catalyse the oxidation of d -glucose at C-2position producing 2-keto-d -glucose (d -glucosone,d -arabino-hexos-2-ulose)which is an important precursor for biosynthesis of the antibiotic cor-talcerone [2].Moreover,this enzyme also exhibits activity over other carbohydrates such as d -galactose,l -sorbose and 1-deoxy-d -glucose producing the corresponding keto-sugars [3].Therefore,dicarbonyl sugars are widely used for synthesis of rare sugars such as antibiotics,pyrrolidine and piperidine aminosugars.However,the bioconversion of d -glucose and other carbohydrates in the pres-ence of GOX2and catalase into their corresponding keto-sugars
?Corresponding author at:Chemical Engineering and Biotechnology Research Center,Instituto Superior de Engenharia de Lisboa,Rua Conselheiro Emídio Navarro,1,1959-007Lisboa,Portugal.Tel.:+351218317052;fax:+351218317267.
E-mail address:akarmali@deq.isel.ipl.pt (A.Karmali).is very slow and time-consuming at atmospheric pressure [4,5].Moreover,GOX2activity from basidiomycete strains (i.e.Coriolus versicolor and Phanerochaete chrysosporium )is highly unstable [6,7]and there is a great need to stabilize the enzyme activity by several ways such as by protein engineering and by immobilization in a suitable support [8].
Immobilization of enzymes to solid carriers is the most widely used strategy to improve stability of biocatalysts such as storage and operational stabilities.Moreover,this strategy can increase selectivity towards other substrates compared with the corre-sponding free enzymes.The four main methods of immobilization are as follows:(a)adsorption,(b)covalent binding,(c)entrapment and (d)membrane con?nement.In general,thermal stability of bio-catalyst is due to the molecular rigidity introduced by binding to a rigid support and creation of a protected microenvironment for the biocatalyst [9,10].
Enzyme reactions can be carried out under high pressure (i.e.over 2kbar)or in supercritical ?uids which is a new and promis-ing ?eld of enzyme engineering [11].High pressure is responsible for direct conformational changes in enzymes which affect their biological activities [12,13].However,such high pressures may inactivate some enzymes since undesirable changes may occur in
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A.Karmali,J.Coelho/Process Biochemistry46(2011)168–173169
enzyme conformation which results in enzyme inactivation[13]. Moreover,pressure also in?uences the reaction rate constant which changes according to transition state theory and standard thermo-dynamics[14].There are many reports in the literature on the effect of high pressure(i.e.over2kbar)on enzyme structure and activ-ity[12,15].However,few published work have been found in the literature regarding the use of moderate pressures,in the range of 1–150bar,on enzyme structure and activity.Therefore,the present work is concerned with the use of moderate pressure up to150bar by compressed air in a pressure batch reactor in order to increase the rate of bioconversion of d-glucose since oxygen is one of the substrates of the reaction catalysed by immobilized GOX2.Several parameters affecting this bioconversion will be investigated such as pressure,different forms of biocatalysts,d-glucose concentration, pH,temperature and the presence of catalase.
2.Materials and methods
2.1.Materials
C.versicolor was isolated from old growth forest of Olympic Peninsula(Port Townsend,Washington,USA).Corn-steep liquor was kindly donated by COPAM, Portugal.O-dianisidine,iminodiacetic acid,d-glucose,d-glucosone,1,4-butanediol diglycydil ether,glutaraldehyde,catalase and peroxidase were purchased from Sigma Chemical Company(USA).Sepharose6B was obtained from GE Health Care, Sweden.
Sugar Pak column was obtained from Waters(USA)and discs of3.0cm diameter of modi?ed polyethersulfone(PES)membranes(Ultrabind,US4500.45?m)were purchased from Pall Gelmam Laboratories(USA).
2.2.Enzyme production and puri?cation
GOX2from C.versicolor was produced by submerged fermentation by using a basal culture medium containing agro-industrial wastes as carbon sources such as whey powder(2.5%)and other components as follows:corn-steep liquor(0.7%), MgSO4(0.15%)CaCl2(0.0125%)and KH2PO4(0.1%).They were all dissolved in tap water which also contained trace amounts of salts as follows:1.0?M CuSO4,1.0mM CaCO3,0.06mM MgCl2,0.9?M AlCl3,1.3?M MnCl2,0.01mM NaNO3,0.85?M NaF, 1.7?M FeCl3,1.1?M ZnCl and0.1mM NaCl.The pH of the medium was adjusted to5.5and sterilized at121?C for20min in an autoclave.Alternatively,other agro-industrial wastes could be used such as tomato pomace and rice bran.
Erleynmeyer?asks(500ml)containing100ml of sterile medium were inoc-ulated with two pieces of0.5cm×0.5cm plugs of the appropriate culture grown in petri dishes and the culture was grown at200rpm,25?C for several days.By this time it was possible to detect GOX2activity in the biomass.This inoculum was used to inoculate batch fermenter(2.5l)containing the same culture medium (2.0l)and the culture was grown at200rpm,pH5.5and25?C for several days and GOX2production was followed as a function of time.Aliquots(10ml)were removed daily from the fermenter under aseptic conditions;the biomass was recovered by ?ltration and washed with saline.The biomass was resuspended in2volumes of 50mM phosphate buffer pH6.5and sonicated at100W for2min in an ice bath.The suspension was centrifuged at10,000×g for5min and the cell-free supernatant was the source of GOX2activity.The enzyme was puri?ed by immobilized metal af?nity chromatography on epoxy-activated Sepharose6B-IDA-Cu(II)as described previously[16].
2.3.Electrophoretic analysis
Crude extract and puri?ed enzyme were analysed for purity by SDS-PAGE and native PAGE as reported previously[17,18].
2.4.Enzyme assays
For routine enzyme assays in fungal cultures and chromatographic fractions, GOX2activity at atmospheric pressure was determined by using o-dianisidine, d-glucose(0.1M)and peroxidase(1U)in50mM phosphate buffer pH6.5.The reaction mixture contained0.79ml of o-dianisidine(6mg/100ml),0.01ml peroxi-dase(1mg ml?1),0.1ml glucose(1M)and0.1ml of crude extract.After10min,the absorbance was recorded at450nm at25?C(ε=8.3×103cm?1M?1).Alternatively, enzyme activity was assayed at atmospheric pressure by using0.1ml of appropriate concentration of d-glucose in50mM phosphate buffer pH6.0and suitable amount of either crude extract or puri?ed enzyme in a?nal volume of1ml by bubbling with air.Aliquots were removed from the reaction mixture at suitable time inter-vals which were analysed by HPLC in terms of substrate and product as described below.Catalase activity was assayed in the presence of H2O2as reported previously [16].2.5.Protein assay
Protein concentration was determined by Coomassie blue dye binding method [19].
2.6.Immobilization of GOX2and catalase,crude extract and biomass of C.
versicolor
Either crude extract containing GOX2activity or puri?ed enzyme and catalase were immobilized by covalent bonding in PES membranes(discs of3.0cm diameter) by using the following reaction mixture:80U of puri?ed GOX2,10mg of catalase (1000U mg?1protein),2?l of glutaraldehyde(5%,v/v)and20?l gelatin(25%)in 50mM phosphate buffer pH6.0in a?nal volume of200?l.The mixture was imme-diately transferred to membrane discs which were dried and washed several times with50mM phosphate buffer pH6.0[20].In some experiments,GOX2was immo-bilized on PES membranes without catalase by using the methodology described above.Alternatively,crude extract(0.21U mg?1protein)was immobilized in PES membranes by using the following reaction mixture:crude enzyme having80U of activity,10mg catalase(1000U mg?1protein),10?l of glutaraldehyde(5%,v/v) and50?l gelatin(25%)as described above.Regarding biomass immobilization,1.0g (wet weight)of biomass of C.versicolor having80U of enzyme activity,was mixed with1.0ml of glutaraldehyde(5%,v/v)and1.0ml gelatin(25%)in50mM phosphate buffer pH6.0.The biomass was allowed to dry,washed with50mM phosphate buffer pH6.0and subsequently it was wrapped in a dialysis membrane(P10)for biocatalysis in a batch reactor.Alternatively,biomass of C.versicolor was wrapped in a dialysis membrane(P10)and used for biocatalysis.All immobilized forms of biocatalysts were stored at4?C in50mM phosphate buffer pH6.0.
2.7.Biocatalysis at moderate pressure
2.7.1.Apparatus set-up
The experimental set-up consists basically of one batch reactor(Micro Reactor Parr Instruments CO,4843)with25ml of capacity equipped with agitation,temper-ature and relative pressure reading devices.Two principal valves inlet and outlet connections and a rupture disk set a250bar were used to perform the enzyme reactions.In each experiment samples were withdrawn at appropriate intervals to determine product concentration by using an HPLC system(Jasco Instruments).
Biocatalysis at moderate pressure was carried out by using either immobilized crude extract and catalase(0.05U mg?1protein)in PES membranes,immobilized puri?ed enzyme and catalase in membranes(1.5U mg?1protein),biomass wrapped in a dialysis membrane or immobilized biomass from C.versicolor(1.0g)wrapped in a dialysis membrane.The reaction mixture contained appropriate concentration of d-glucose in50mM phosphate buffer pH6.5(19.0ml)and appropriate form of enzyme which was stirred at110rpm at23?C.The reaction was performed at several relative pressures at23?C by using compressed air.Aliquots(100?l)were removed from the batch reactor at suitable time intervals and the substrate and product of the reaction were analysed by HPLC as described below.
2.7.2.Calibration curve for d-glucose and d-glucosone
Several concentrations of d-glucose and its corresponding keto-sugar were pre-pared in50mM phosphate buffer pH6.5.These samples were analysed in an HPLC system(Jasco)by using a Sugar Pak column at75psi,90?C,0.1mM Ca(II)EDTA in millipore water as the solvent and at a?ow rate of0.5ml min?1.
2.8.Assay of GOX2by HPLC analysis
Samples(25?l)from reaction mixtures were injected into the HPLC system and the chromatograms were obtained in12min time since the retention time of d-glucose and d-glucosone were9.3and10.3min,respectively.The peak area was determined as a function of either d-glucose or d-glucosone concentration.
2.9.Kinetic characterization of puri?ed preparation of immobilized GOX2and catalase at moderate pressures
Immobilized GOX2activity containing co-immobilized catalase in PES mem-branes was used throughout these kinetic studies.It was assayed by using d-glucose as the substrate and one unit of GOX2activity was de?ned as the amount of enzyme required to oxidize1(mol substrate per min.under the same experimental condition.Assays in pressure batch reactor were performed at suitable moderate pressures,up to120bar,in50mM of the appropriate buffer and pH,and suitable aliquots were removed from the pressure reactor at appropriate time intervals and analysed by HPLC as described above.The optimum pH of GOX2activity was inves-tigated at110bar by using puri?ed immobilized enzyme preparation in a reaction mixture containing0.1M d-glucose in several buffers as follows:50mM citrate pH 4.6and5.6,50mM phosphate buffer pH6.0and7.0and50mM Tris pH7.0and 8.0.The optimum temperature of GOX2activity was investigated by using puri-?ed immobilized enzyme preparations at110bar in a reaction mixture containing 100mM d-glucose in50mM phosphate buffer pH6.0which was incubated at dif-ferent temperatures(i.e.25,35,45,55,62,66,70and80?C)for5min.The activation energy for immobilized GOX2reaction was determined by measuring the enzyme
170 A.Karmali,J.Coelho/Process Biochemistry46(2011)168–173
activity at different temperatures and transforming them according to the Arrhenius
equation:
Ln(v)=A?E a
R
×1
T
where v is reaction rate;T is the absolute temperature(K),E a is the activation energy and R is gas constant.
The apparent kinetic constants(V max,K m,K
cat and K cat/K m)for d-glucose were
determined by Michaelis–Menten plot by using the software from Sigma Plot2004 (version9.01).
2.10.Biocatalysis at atmospheric pressure
For comparative purposes,biocatalysis at atmospheric pressure,was also car-ried out by using either immobilized cell-free extract and catalase in PES membranes (0.05U mg?1protein),immobilized biomass of C.versicolor(1.0g wet weight)and puri?ed immobilized enzyme and catalase in PES membranes(1.5U mg?1protein). The reaction mixture contained appropriate concentration of d-glucose in50mM phosphate buffer pH6.5(19.0ml)and appropriate form of enzyme/biocatalyst which was stirred at110rpm in a magnetic stirrer.The reaction was performed by using the reaction mixture bubbled with air([O2]=8.89mg l?1).The solubility of O2in the solution is assumed as in fresh water,using the toolbox and results from [21]and the respective corrections when O2is pure against the concentration in the air.
2.11.Stability of immobilized GOX2and catalase at moderate pressures
Membrane preparations containing immobilized GOX2and catalase were stored in50mM phosphate buffer pH6.0at4?C and assayed for activity in a pressure batch reactor at100bar,110rpm at23?C in a reaction mixture containing0.1M d-glucose in50mM phosphate buffer pH6.0as described above.Such membranes were assayed at appropriate time intervals for several months in order to investi-gate its stability.Alternatively,PES membrane preparations containing immobilized GOX2were incubated50mM phosphate buffer pH6.0(20ml)in batch reactor at 70bar,100rpm at23?C for several days.Membrane preparations were removed at suitable time intervals and immobilized GOX2activity was measured at atmo-spheric pressure by HPLC.For comparative purposes,the stability of the immobilized enzymes(i.e.GOX2and catalase)in PES membranes was also investigated at atmo-spheric pressure for several days at100rpm in a magnetic stirrer at23?C.Membrane preparations were removed at suitable time intervals and immobilized GOX2activ-ity was measured by HPLC.
3.Results and discussion
3.1.Biocatalysis at moderate and atmospheric pressures
The calibration curves for d-glucose and d-glucosone were car-ried out by HPLC[22]which exhibited linear relationship between the peak area(mV)and either d-glucose or d-glucosone concentra-tion(data not shown).
The immobilization of GOX2and catalase on PES membranes revealed that the recovery of immobilized enzyme activity was 14.1±0.92%and11.5±1.01%,respectively(data not shown).The amount of GOX2immobilized on PES membranes was found to be 5.1mg which was determined by the indirect method(i.e. Coomassie blue dye binding method)because extensive washing of PES membrane was not required.This protein value is?ve-fold higher than the manufacturer data for this membrane in the absence of glutaraldehyde.However,in the present work the enzyme was immobilized in the presence of glutaraldehyde which may increase the membrane capacity.
The formation of d-glucosone and the consumption of d-glucose were followed by HPLC when the reaction was carried out at atmospheric pressure and batch reactor by using either puri?ed immobilized enzyme and catalase or puri?ed soluble enzyme and catalase(Fig.1).These data revealed that the enzyme reaction is very slow at atmospheric pressure(Fig.1),compared with the reac-tion in a batch reactor(i.e.70bar,[O2]=2960.0mg l?1)(Fig.1).This result may be explained on the basis that molecular O2is one of the substrates of this enzyme reaction and its concentration is much higher in batch reactor at70bar compared with atmospheric pres-sure([O2]=8.89mg l?1).However,the activating effect of moderate pressures on immobilized GOX2activity cannot be ruled out
since Fig.1.Progress curve of consumption of d-glucose by free and immobilized GOX2. Atmospheric pressure:immobilized GOX2and catalase( )and free GOX2(9.0U) and catalase(10mg)( );72bar:immobilized form( )and free form(?).
several reports in the literature have described this effect on sev-eral enzyme reactions[12,23].The oxidation of d-glucose in the presence of immobilized GOX2and catalase was also carried out at several working pressures at room temperature as shown in Fig.2A.These data suggest that by increasing the pressure there is an increase in the conversion of d-glucose compared with atmo-spheric pressure because[O2]is much higher in batch reactor than at atmospheric pressure.Several experiments were carried out by increasing the pressure up to160bar in the batch reactor which exhibited an increase in rate of reaction catalysed by immobilized GOX2and catalase(data not shown).Furthermore,the conversion of d-glucose into d-glucosone was investigated by using differ-ent enzyme preparations such as immobilized crude extract and immobilized biomass from C.versicolor,either in a batch reactor or at atmospheric pressure(Fig.2B).The data presented in Fig.2B revealed that crude extract exhibited a higher degree of conversion compared with puri?ed enzyme preparation by using the same amount of enzyme activity.Surprisingly,the biomass of C.versi-color containing GOX2activity was also useful for conversion of d-glucose into d-glucosone at moderate pressure.This rather low degree of bioconversion for the biomass compared with the crude extract may be due to mass transfer limitations(Fig.2B)which has been reported by several researchers[24,25].The aim of this experiment was to show that it was possible to immobilize several forms of biocatalysts and use them in batch reactor for biocatalysis either at moderate or atmospheric pressures.However,the com-parative analysis of these biocatalysts is dif?cult because:1.the amount of glutaraldehyde used for each biocatalyst was different since the total amount of protein in each case was also different;
2.the microenvironment as well as steric hindrance of each immo-bilized biocatalyst in PES membrane is different;
3.mass transfer limitations for each immobilized biocatalyst are also different and
4.the amount of immobilized enzyme may be different for each bio-catalyst.Biocatalysis at atmospheric pressure revealed a low degree of d-glucose bioconversion with different immobilized biocatalysts compared with the batch reactor(Fig.2B).Immobilized biomass of C.versicolor could be used several times for bioconversion of d-glucose and about2/3of GOX2activity was lost after the10th re-utilization of immobilized biomass in batch reactor(Fig.2C).In fact,the data in Fig.2C revealed that the initial velocity in the1st use of biomass was0.507mmol d-glucosone h?1whereas the initial velocity in the10th re-use of biomass was0.156mmol h?1.There-fore,after10th re-utilization of biomass,the initial velocity was 30.77%of the1st use of biomass.Moreover,the yield of d-glucosone from d-glucose by using either the immobilized biomass of C.versi-color or immobilized crude extract was found to be69.9±3.8%and
A.Karmali,J.Coelho/Process Biochemistry46(2011)168–173
171
Fig.2.(A)The effect of pressure on bioconversion of d-glucose by immobilized GOX2.The reaction at atmospheric pressure was carried out at23?C by bubbling the reaction mixture with air.(B)Bioconversion of d-glucose by different forms of biocatalysts by applying70bar of pressure(white bar)and without pressure(dark bar).(C)The reusability of immobilized biomass from C.versicolor on the rate of bioconversion of d-glucose at65bar at1st use of biomass(?)and10th re-utilization of biomass( ).
91.3±1.2%,respectively(data not shown).In addition,the presence of catalase in PES membranes increased signi?cantly the degree of conversion of d-glucose(data not shown)suggesting that H2O2acts as a powerful inhibitor for this enzyme which is in agreement with published reports[6].
3.2.Kinetic characterization of immobilized GOX2from C. versicolor at moderate pressures
The bioconversion of d-glucose in the presence of immobilized GOX2and catalase was carried out at several working temperatures at moderate pressures as shown in Fig.3A.These data suggest that optimum temperature for maximum conversion of d-glucose into d-glucosone under these experimental conditions is62?C which is higher than the value of50?C reported for the free enzyme at atmospheric pressure[26].From the linear range of the Arrhenius plot,the activation energy(E a)was determined to be28.01kJ mol?1 which is lower than the value of34.6kJ mol?1reported for this sol-uble enzyme at atmospheric pressure[4].There are few reports on immobilization of GOX2for biocatalysis in the literature
[10,27–29]Fig.3.The effect of temperature and pH on the initial velocity of the reaction catalysed by immobilized GOX2and catalase at110bar.(A)Temperature and(B)
pH.
Fig.4.The effect of d-glucose concentration on the initial velocity of the reac-tion catalysed by immobilized GOX2and catalase at pH 6.0and110bar. Michaelis–Menten(A)and Lineweaver–Burk(B)plots.
172 A.Karmali,J.Coelho /Process Biochemistry 46(2011)
168–173
Fig.5.Stability of immobilized GOX2at moderate pressures.Several PES mem-branes containing GOX2and catalase (1.2U)were incubated in 50mM phosphate buffer pH 6.0either in batch reactor at 70bar (?)or at atmospheric pressure ( ).
which have described that there are no differences in optimum temperature of activity of the free and immobilized forms of this enzyme [29].The effect of pH on the bioconversion of d -glucose into d -glucosone at moderate pressure was also investigated which revealed that the optimum pH is 6.0for maximum conversion of d -glucose into d -glucosone by using immobilized GOX2and cata-lase (Fig.3B).These data are in agreement with the pH value of 6.2reported for this soluble enzyme at atmospheric pressure [3,26].
The effect of d -glucose on initial velocity of immobilized GOX2-catalysed reaction at moderate pressures of 110bar was carried out by using PES membrane containing GOX2and catalase (Fig.4).
The apparent kinetic constants (i.e.V max ,K m ,K cat and K cat /K m )
were determined by using Sigma Plot which were found to be 2.27U mg ?1protein,11.15mM,8.33s ?1and 747.38s ?1M ?1,respectively (Fig.4).These results are lower than the data reported in the literature for soluble enzyme preparations which may be due to mass transfer limitations as well as the use of different assay
con-
Fig.6.Storage stability of immobilized GOX2.A PES membrane was stored in 50mM phosphate buffer pH 6.0.(A)The membrane was assayed for activity at suitable time intervals at 110bar.(B)Initial velocity of the reaction catalysed by immobilized GOX2on the 4th day ( )and on the 75th day ( )at 110bar.
ditions for this enzyme since linked enzyme assays with peroxidase were used in such kinetic studies [24–26,29].
3.3.Storage stability of immobilized GOX2at moderate pressures The effect of moderate pressures on the stability of immobi-lized GOX2activity was studied by incubating several preparations of immobilized enzyme in batch reactor for several days.The data presented in Fig.5revealed that it did not affect signi?cantly the enzyme activity at 70bar compared with the enzyme stored at atmospheric pressure.In addition,the storage stability of immo-bilized GOX2on PES membranes containing catalase was also investigated which revealed that this enzyme preparation was apparently stable for several months without any signi?cant loss of enzyme activity (Fig.6).The operational stability of this immo-bilized enzyme was studied at moderate pressures which revealed that it was stable for at least 30days without any substantial loss of GOX2activity (data not shown).On the other hand,this is also the ?rst report on the use of either immobilized crude extract or biomass from C.versicolor for bioconversion of d -glucose at mod-erate pressures.Such membrane supports are highly suitable for immobilization of GOX2which exhibit high stability for several months.
4.Conclusions
The data presented in this work have revealed that at moder-ate pressure,there are several parameters that affect the degree of conversion of d -glucose into d -glucosone namely,temperature,pH,different forms of immobilized biocatalysts and the presence of catalase.The optimum pH and temperature for this reaction at moderate pressures were slightly different compared with the data published for this enzyme at atmospheric pressure.It is important to stress that this is the ?rst report on biocatalysis at moderate pres-sures regarding immobilized GOX2for bioconversion of d -glucose into d -glucosone by using compressed air.Acknowledgements
The authors would like to thank Instituto Politécnico de Lisboa (Project No.25/2003)and Fundac ??o para a Ciência e a Tecnologia (POCTI/EQU/48812/2002)for research grants.References
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50个可接双宾语的高考高频动词 (一) 双宾语易位时需借助介词to的常用动词 award sb. sth. = award sth. to sb. 颁奖给某人 bring sb. sth. = bring sth. to sb. 把某物带给某人hand sb. sth. =hand sth. to sb. 把某物递给某人 lend sb. sth. = lend sth. to sb. 把某物借给某人 mail sb. sth. = mail sth. to sb. 把某物寄给某人 offer sb. sth. = offer sth. to sb. 将某物给某人 owe sb. sth. = owe sth. to sb. 欠某人某物 pass sb. sth. = pass sth. to sb. 把某物递给某人 pay sb. sth. = pay sth. to sb. 付给某人某物（钱） post sb. sth. = post sth. to sb. 把某物寄给某人 read sb. sth. = read sth. to sb. 把某物读给某人听return sb.sth. = return sth. to sb. 把某物还给某人send sb. sth. = send sth. to sb. 把某物送给某人 sell sb. sth. = sell sth. to sb. 把某物卖给某人 serve sb. sth. = serve sth. to sb. 拿某物招待某人show sb. sth. = show sth. to sb. 拿某物给某人看 take sb. sth. = take sth. to sb. 把某物拿给某人 teach sb. sth. = teach sth. to sb. 教某人某物 tell sb. sth. = tell sth. to sb. 告诉某人某情况 throw sb. sth. = throw sth. to sb. 把某物扔给某人 write sb. sth. = write sth. to sb. 给某人写信 (二) 双宾语易位时需借助介词for的常用动词 book sb. sth. = book sth. for sb. 为某人预定某物 buy sb. sth. = buy sth. for sb. 为某人买某物 choose sb. sth. = choose sth. for sb. 为某人选某物cook sb. sth. = cook sth. for sb. 为某人煮某物 draw sb. sth. = draw sth. for sb. 为某人画某物 fetch sb. sth. = fetch sth. for sb. 为某人去取某物 find sb. sth. = find sth. for sb. 为某人找到某物 fix sb. sth. = fix sth. for sb. 为某人准备某物 get sb. sth. = get sth. for sb. 为某人拿来某物 make sb. sth. = make sth. for sb. 为某人做某物 order sb. sth. = order sth. for sb. 为某人订购某物pick sb. sth. = pick sth. for sb. 为某人采摘某物prepare sb. sth. = prepare sth. for sb. 为某人准备某物save sb. sth. = save sth. for sb. 为某人留某物 sing sb. sth. = sing sth. for sb. 为某人唱某物（歌）
、名词 表示人、事物、地方、现象或抽象概念等的名称的词都叫做名词。名词可分为专有名词和普通名词。 名词在句子中可担任除谓语外的任何成分，即主语、标语、宾语（动词宾语和介词宾语）、状语、宾补、定语等。 【举例】桌子，凳子，椅子，沙发，桌子 2、冠词 冠词是虚词，本身无词义，也不能单独使用，它用在名词前，帮助指明名词的含义。冠词只有三个，分别是定冠词(the)和不定冠词(a、an) 【举例】因无词义，所以无法举例 3、数词 表示数目多少的词叫做数词。数词可分为基数词和序数词。 数词在句子中可作主语、宾语、定语、表语和同位语（同位语仅限基数词）【举例】一、二、三、第一、第二、第三 4、代词 代词是代替名词的一种词类。大多数代词具有名词和形容词的功能。英语中的代词，按起意义、特征及在句中的作用分为：人称代词、物主代词、指示代词、自身代词、相互代词、疑问代词、关系代词和不定代词八种 【举例】我、你、他的、我自己 5、形容词 形容词修饰名词，说明事物/人的性质或特征。通常，可将形容词分为性质形容词和叙述形容词两类，其位置不一定在名词 形容词可在句中可作表语、定语、宾补、状语，需要注意的是作定语修饰名词时，要放在名词的前面。但是如果形容词以-thing（如something）为字尾的词语时，要放在这些词语之后 【举例】小的，大的（翻译时词尾一定是“的”） 6、副词 副词和形容此一样，也具有修饰的功能。形容词是修饰名词，而副词是修饰动词，此外副词还可以修饰形容词和其它的副词。副词在句子中修饰动词、形容词、副词或整个句子。用来表示时间、场所、状态、程度等。 副词在句中可作定语、状语、表语和宾补 【举例】小心地，仔细地（翻译时词尾一定是“地”） 7、介词 介词是一种虚词，它不能单独担任句子成分，必须和名词或代词或相当于名词的其他词类、短语或从句构成介词短语，才能担任句子成分 【举例】在……里，在……上
50个可接双宾语的高频动词 (一) 双宾语易位时需借助介词to的常用动词 award sb. sth. = award sth. to sb. 颁奖给某人bring sb. sth. = bring sth. to sb. 把某物带给某人hand sb. sth. =hand sth. to sb. 把某物递给某人lend sb. sth. = lend sth. to sb. 把某物借给某人mail sb. sth. = mail sth. to sb. 把某物寄给某人 offer sb. sth. = offer sth. to sb. 将某物给某人owe sb. sth. = owe sth. to sb. 欠某人某物 pass sb. sth. = pass sth. to sb. 把某物递给某人pay sb. sth. = pay sth. to sb. 付给某人某物（钱）post sb. sth. = post sth. to sb. 把某物寄给某人read sb. sth. = read sth. to sb. 把某物读给某人听return sb.sth. = return sth. to sb. 把某物还给某人send sb. sth. = send sth. to sb. 把某物送给某人sell sb. sth. = sell sth. to sb. 把某物卖给某人serve sb. sth. = serve sth. to sb. 拿某物招待某人 show sb. sth. = show sth. to sb. 拿某物给某人看take sb. sth. = take sth. to sb. 把某物拿给某人teach sb. sth. = teach sth. to sb. 教某人某物 tell sb. sth. = tell sth. to sb. 告诉某人某情况throw sb. sth. = throw sth. to sb. 把某物扔给某人write sb. sth. = write sth. to sb. 给某人写信 (二)双宾语易位时需借助介词for的常用动词book sb. sth. = book sth. for sb. 为某人预定某物buy sb. sth. = buy sth. for sb. 为某人买某物choose sb. sth. = choose sth. for sb. 为某人选某物cook sb. sth. = cook sth. for sb. 为某人煮某物draw sb. sth. = draw sth. for sb. 为某人画某物 fetch sb. sth. = fetch sth. for sb. 为某人去取某物find sb. sth. = find sth. for sb. 为某人找到某物 fix sb. sth. = fix sth. for sb. 为某人准备某物 get sb. sth. = get sth. for sb. 为某人拿来某物
动词16个时态 —、一般现在时 1. 概念：表示经常发生的情况；有规律出现的情况；总是发生的；和事实真理。 2. 时间状语：Always, usually, often, sometimes, every week (day, year, month once a …)，week(day, year, month …)onS un days ( on Mon days …)， 3. 基本结构：动词原形(如主语为第三人称单数，动词上要改为第三人称单数形式) 4. 否定形式：主语+ am/is/are + no t + 其他；此时态的谓语动词若为行为动词，则在其前加don't , 如主语为第三人称单数，则用does n't ，同时还原行为动词。 5?—般疑问句：把be动词放于句首；用助动词do提问，如主语为第三人称单数，则用does，同时，还原行为动词。 6.例句：It seldom sn ows here. 这里很少下雪。 He is always ready to help others. 他总是乐于帮助别人。 Action speaks louder tha n words. 事实胜于雄辩。 二、一般过去时 1. 概念：过去某个时间里发生的动作或状态；过去习惯性、经常性的动作、行为。 2. 时间状语：ago, yesterday, the day before yesterday, last week, last(year, night, month ), in 1989,just now, at the age of 5,one day, long long ago, once upon a time, etc. 3. 基本结构：主语+动词的过去式或be的过去式+名词 4. 否定形式：主语+ was/were + not + 其他；在行为动词前加did n't ，同时还原行为动词。 5. 一般疑问句：was或were放于句首；用助动词do的过去式did 提问，同时还原行为动词。 6. 例句：She ofte n came to help us in those days. 那些天她经常来帮助我们。
可接双宾语的个动词 IMB standardization office【IMB 5AB- IMBK 08- IMB 2C】
英语中可接双宾语的38个动词 （1）双宾语易位时需借助介词to的常用动词 award sb. sth. = award sth. to sb. 颁奖给某人 bring sb. sth. = bring sth. to sb. 把某物带给某人 hand sb. sth. =hand sth. to sb. 把某物递给某人 lend sb. sth. = lend sth. to sb. 把某物借给某人 mail sb. sth. = mail sth. to sb. 把某物寄给某人 offer sb. sth. = offer sth. to sb. 将某物给某人 owe sb. sth. = owe sth. to sb. 欠某人某物 pass sb. sth. = pass sth. to sb. 把某物递给某人 pay sb. sth. = pay sth. to sb. 付给某人某物(钱) post sb. sth. = post sth. to sb. 把某物寄给某人 read sb. sth. = read sth. to sb. 把某物读给某人听 return sb. sth. = return sth. to sb. 把某物还给某人 send sb. sth. = send sth. to sb. 把某物送给某人 sell sb. sth. = sell sth. to sb. 把某物卖给某人 serve sb. sth. = serve sth. to sb. 拿某物招待某人 show sb. sth. = show sth. to sb. 拿某物给某人看 take sb. sth. = take sth. to sb. 把某物拿给某人 teach sb. sth. = teach sth. to sb. 教某人某物 tell sb. sth. = tell sth. to sb. 告诉某人某情况 throw sb. sth. = throw sth. to sb. 把某物扔给某人 write sb. sth. = write sth. to sb. 给某人写信 (2) 双宾语易位时需借助介词for的常用动词 book sb. sth. = book sth. for sb. 为某人预定某物 buy sb. sth. = buy sth. for sb. 为某人买某物 choose sb. sth. = choose sth. for sb. 为某人选某物 cook sb. sth. = cook sth. for sb. 为某人煮某物 draw sb. sth. = draw sth. for sb. 为某人画某物 fetch sb. sth. = fetch sth. for sb. 为某人去取某物 find sb. sth. = find sth. for sb. 为某人找到某物 fix sb. sth. = fix sth. for sb. 为某人准备某物 get sb. sth. = get sth. for sb. 为某人拿来某物 make sb. sth. = make sth. for sb. 为某人做某物 order sb. sth. = order sth. for sb. 为某人订购某物 pick sb. sth. = pick sth. for sb. 为某人采摘某物 prepare sb. sth. = prepare sth. for sb. 为某人准备某物 save sb. sth. = save sth. for sb. 为某人留某物 sing sb. sth. = sing sth. for sb. 为某人唱某物(歌)
award sb. sth. = award sth. to sb. 颁奖给某人bring sb. sth. = bring sth. to sb. 把某物带给某人hand sb. sth. =hand sth. to sb. 把某物递给某人lend sb. sth. = lend sth. to sb. 把某物借给某人mail sb. sth. = mail sth. to sb. 把某物寄给某人offer sb. sth. = offer sth. to sb. 将某物给某人 owe sb. sth. = owe sth. to sb. 欠某人某物pass sb. sth. = pass sth. to sb. 把某物递给某人 pay sb. sth. = pay sth. to sb. 付给某人某物（钱）post sb. sth. = post sth. to sb. 把某物寄给某人read sb. sth. = read sth. to sb. 把某物读给某人听return sb. sth. = return sth. to sb. 把某物还给某人send sb. sth. = send sth. to sb. 把某物送给某人sell sb. sth. = sell sth. to sb. 把某物卖给某人serve sb. sth. = serve sth. to sb. 拿某物招待某人show sb. sth. = show sth. to sb. 拿某物给某人看take sb. sth. = take sth. to sb. 把某物拿给某人teach sb. sth. = teach sth. to sb. 教某人某物 tell sb. sth. = tell sth. to sb. 告诉某人某情况throw sb. sth. = throw sth. to sb. 把某物扔给某人write sb. sth. = write sth. to sb. 给某人写信 (2) 双宾语易位时需借助介词for的常用动词 book sb. sth. = book sth. for sb. 为某人预定某物buy sb. sth. = buy sth. for sb. 为某人买某物choose sb. sth. = choose sth. for sb. 为某人选某物cook sb. sth. = cook sth. for sb. 为某人煮某物draw sb. sth. = draw sth. for sb. 为某人画某物fetch sb. sth. = fetch sth. for sb. 为某人去取某物find sb. sth. = find sth. for sb. 为某人找到某物fix sb. sth. = fix sth. for sb. 为某人准备某物 get sb. sth. = get sth. for sb. 为某人拿来某物make sb. sth. = make sth. for sb. 为某人做某物order sb. sth. = order sth. for sb. 为某人订购某物pick sb. sth. = pick sth. for sb. 为某人采摘某物prepare sb. sth. = prepare sth. for sb. 为某人准备某物save sb. sth. = save sth. for sb. 为某人留某物sing sb. sth. = sing sth. for sb. 为某人唱某物（歌）spare sb. sth. = spare sth. for sb. 为某人让出某物steal sb. sth. = steal sth. for sb. 为某人偷某物
一、延续性动词和终止性动词的概念 英语中，动词按其动作发生的方式、动作发生过程的长短，可分为延续性动词和终止性 动词。 延续性动词表示能够延续的动作，这种动作可以延续下去或产生持久影响。如：learn, work, stand, lie, know, walk, keep, have, wait, watch, sing, read, sleep, live, stay等。 终止性动词也称非延续性动词、瞬间动词或短暂性动词，表示不能延续的动作，这种动作发生后立即结束。如open, close, finish, begin, come, go, arrive, reach, get to, leave, move, borrow, buy等。 二、延续性动词的用法特征 1.延续性动词可以用于现在进行时和现在完成进行时；延续性动词还可以用于现在完成时与表示"段时间"的状语连用。表示"段时间"的短语有：for two years, during the past three years, since last year, how long等。如： I have learned English since I came here.自从我来到这儿就学英语了。 2.延续性动词不能与表示短暂时间的"点时间"状语连用。如：It rained at eight yesterday morning.(误) rain为延续性动词，而at eight表示"点时间"，前后显然矛盾。如果用延续性动词表示一瞬间的动作，可以借助come, begin, get等终止性动词来表示。 上句可改为：It began to rain at eight yesterday morning.(正) 又如：-When did you get to know Jack? -Two years ago. -Then you've known each other for more than two years. -That's right. 三、终止性动词的用法特征 1.终止性动词可用来表示某一动作完成，因此可用于现在完成时。如： The train has arrived.火车到了。 Have you joined the computer group? 你加入电脑小组了吗？ 2.终止性动词表示的动作极其短暂，不能持续。因此，不可与表示一段时间的状语连用 (只限肯定式)。如：
接双宾语常用动词 The Standardization Office was revised on the afternoon of December 13, 2020
（1）双宾语易位时需借助介词to的常用动词 bring sb. sth. = bring sth. to sb. 把某物带给某人 hand sb. sth. =hand sth. to sb. 把某物递给某人 lend sb. sth. = lend sth. to sb. 把某物借给某人 mail sb. sth. = mail sth. to sb. 把某物寄给某人 offer sb. sth. = offer sth. to sb. 将某物给某人 pass sb. sth. = pass sth. to sb. 把某物递给某人 pay sb. sth. = pay sth. to sb. 付给某人某物（钱）read sb. sth. = read sth. to sb. 把某物读给某人听return sb. sth. = return sth. to sb. 把某物还给某人 sell sb. sth. = sell sth. to sb. 把某物卖给某人 send sb. sth. = send sth. to sb. 把某物送给某人show sb. sth. = show sth. to sb. 拿某物给某人看 take sb. sth. = take sth. to sb. 把某物拿给某人 teach sb. sth. = teach sth. to sb. 教某人某物 tell sb. sth. = tell sth. to sb. 告诉某人某情况 throw sb. sth. = throw sth. to sb. 把某物扔给某人write sb. sth. = write sth. to sb. 给某人写信 (2) 双宾语易位时需借助介词for的常用动词 book sb. sth. = book sth. for sb. 为某人预定某物 buy sb. sth. = buy sth. for sb. 为某人买某物 choose sb. sth. = choose sth. for sb. 为某人选某物cook sb. sth. = cook sth. for sb. 为某人煮某物 draw sb. sth. = draw sth. for sb. 为某人画某物 find sb. sth. = find sth. for sb. 为某人找到某物 get sb. sth. = get sth. for sb. 为某人拿来某物 make sb. sth. = make sth. for sb. 为某人做某物 order sb. sth. = order sth. for sb. 为某人订购某物pick sb. sth. = pick sth. for sb. 为某人采摘某物prepare sb. sth. = prepare sth. for sb. 为某人准备某物
常用的能接双宾语的及物动词 有：give, teach, buy, lend, find, hand, leave, sell, show, read, pay, make, offer, build, pass, bring, cook, refuse等。 I’ll fetch you a chair. 我给你拿一只椅子。 （a chair是直接宾语，you是间接宾语） 间接宾语可以用一个由to( 表示动作方向）或for (表示动作目标）引起的短语来表示。这时，间接宾语置于直接宾语之后。我们可以把上面例句改写为：I'll fetch a chair for you. 一般用法如下:for example: give sth to sb = give sb sth. 一、双宾语 英语中，有些及物动词可接两个宾语（双宾语），即指人的间接宾语和指物的直接宾语。常用句型为：主语+及物动词+间接宾语+直接宾语。如：She gave me a cup of tea. 她给了我一杯茶. 有时，间接宾语也可改为由介词to或for引起的短语，放在直接宾语的后面。如： She passed him the salt. = She passed the salt to him. 她把盐递给了他。
Her uncle bought her an English-Chinese dictionary yesterday. = Her uncle bought an English-Chinese dictionary for her yesterday. 昨天，她叔叔给她买了一本英汉词典。 双宾语的特殊情况 下面几种情况，通常要用介词to或for引起的短语： 1. 当直接宾语是人称代词（it/them），间接宾语是名词时：或两个宾语都是人称代词时。如： The watch is Li Lei's. Please give it to him. 这块手表是李雷的。请把它给他。 2. 当强调间接宾语时。如： Mother cooks breakfast for us every day. 母亲每天都为我们做早饭。 3. 当间接宾语比直接宾语长时。如： On the bus, he often gives his seat to an old person. 在公共汽车上，他经常把座位让给老人。 注：由to连接间接宾语的动词有：pass, give, show, tell, lend, take等；由for 连接间接宾语的动词有：buy, cook, get, sing, make等。
动词短语的概念： 动词常和某些其他词类用在一起，构成固定词组，形成所谓短语动词(phrasalverb)。和动词一样，短语动词也可分为及物和不及物两种。短语动词可以作为一个整体看待，同一般动词一样使用。 动词短语的搭配类型： 1）动词+介词： 这类短语动词用作及物动词，后面须跟宾语。 如：The small boy insisted on going with his parents. 那男孩坚持要跟父母一起去。 Do you often listen to broadcasts in English? 你常听英语广播吗? Look at the children. Aren't they lovely? 看着这些孩子们。他们多么可爱呀! We stand for self-reliance. 我们是主张自力更生的。 这一类的短语动词还有很多，如depend on(upon)(依靠)，wait on(服侍)，look for(寻找)，deal with(对待)，look after(照料)，wait for(等待)等。 2）动词+副词： 这类短语动词有的用作及物动词，有的用作不及物动词。 如：I always get up as soon as the bell rings. 我总是一打铃就起床。(不及物) Look out, there's a car coming! 当心，来汽车了!(不及物) Have you handed in your exercises already? 你已经交练习了吗?(及物) Please don't forget to put on your coat, it's cold outside. 请不要忘记穿外衣，外面很冷。(及物) 这一类的短语动词还有很多，及物如put out(扑灭)，eat up(吃光)，put down(放下)；不及物如set off(出发)，come up(走近)，go on(继续)。 注："动词+副词"这类短语动词和上面第一类"动词+介词"的不同之处在于："动词+介词"用作及物动词，后面须跟宾语。"动词+副词"则有的及物，有的不及物；用作及物动词而宾语为人称代词或自身代词时，副词往往放在宾语之后。
创作编号： GB8878185555334563BT9125XW 创作者：凤呜大王* 50个可接双宾语的高考高频动词 (一) 双宾语易位时需借助介词to的常用动词 award sb. sth. = award sth. to sb. 颁奖给某人 bring sb. sth. = bring sth. to sb. 把某物带给某人 hand sb. sth. =hand sth. to sb. 把某物递给某人 lend sb. sth. = lend sth. to sb. 把某物借给某人 mail sb. sth. = mail sth. to sb. 把某物寄给某人 offer sb. sth. = offer sth. to sb. 将某物给某人 owe sb. sth. = owe sth. to sb. 欠某人某物 pass sb. sth. = pass sth. to sb. 把某物递给某人 pay sb. sth. = pay sth. to sb. 付给某人某物（钱） post sb. sth. = post sth. to sb. 把某物寄给某人 read sb. sth. = read sth. to sb. 把某物读给某人听 return sb.sth. = return sth. to sb. 把某物还给某人 send sb. sth. = send sth. to sb. 把某物送给某人 sell sb. sth. = sell sth. to sb. 把某物卖给某人 serve sb. sth. = serve sth. to sb. 拿某物招待某人 show sb. sth. = show sth. to sb. 拿某物给某人看 take sb. sth. = take sth. to sb. 把某物拿给某人 teach sb. sth. = teach sth. to sb. 教某人某物 tell sb. sth. = tell sth. to sb. 告诉某人某情况 throw sb. sth. = throw sth. to sb. 把某物扔给某人 write sb. sth. = write sth. to sb. 给某人写信 (二) 双宾语易位时需借助介词for的常用动词 book sb. sth. = book sth. for sb. 为某人预定某物 buy sb. sth. = buy sth. for sb. 为某人买某物 choose sb. sth. = choose sth. for sb. 为某人选某物 cook sb. sth. = cook sth. for sb. 为某人煮某物 draw sb. sth. = draw sth. for sb. 为某人画某物 fetch sb. sth. = fetch sth. for sb. 为某人去取某物
——可接双宾语的常用动词 (1) 双宾语易位时需借助介词to的常用动词 award sb. sth. = award sth. to sb. 颁奖给某人 bring sb. sth. = bring sth. to sb. 把某物带给某人 hand sb. sth. =hand sth. to sb. 把某物递给某人 lend sb. sth. = lend sth. to sb. 把某物借给某人 mail sb. sth. = mail sth. to sb. 把某物寄给某人 offer sb. sth. = offer sth. to sb. 将某物给某人 owe sb. sth. = owe sth. to sb. 欠某人某物 pass sb. sth. = pass sth. to sb. 把某物递给某人 pay sb. sth. = pay sth. to sb. 付给某人某物(钱) post sb. sth. = post sth. to sb. 把某物寄给某人 read sb. sth. = read sth. to sb. 把某物读给某人听 return sb. sth. = return sth. to sb. 把某物还给某人 send sb. sth. = send sth. to sb. 把某物送给某人 sell sb. sth. = sell sth. to sb. 把某物卖给某人 serve sb. sth. = serve sth. to sb. 拿某物招待某人 show sb. sth. = show sth. to sb. 拿某物给某人看 take sb. sth. = take sth. to sb. 把某物拿给某人 teach sb. sth. = teach sth. to sb. 教某人某物 tell sb. sth. = tell sth. to sb. 告诉某人某情况 throw sb. sth. = throw sth. to sb. 把某物扔给某人 write sb. sth. = write sth. to sb. 给某人写信 (2) 双宾语易位时需借助介词for的 常用动词 book sb. sth. = book sth. for sb. 为某人预定某物 buy sb. sth. = buy sth. for sb. 为某人买某物 choose sb. sth. = choose sth. for sb. 为 某人选某物 cook sb. sth. = cook sth. for sb. 为某人煮某物 draw sb. sth. = draw sth. for sb. 为某人画某物 fetch sb. sth. = fetch sth. for sb. 为 某人去取某物 find sb. sth. = find sth. for sb. 为某人找到某物 fix sb. sth. = fix sth. for sb. 为某人准备某物 get sb. sth. = get sth. for sb. 为某人拿来某物 make sb. sth. = make sth. for sb. 为某人做某物 order sb. sth. = order sth. for sb. 为 某人订购某物 pick sb. sth. = pick sth. for sb. 为某人采摘某物 prepare sb. sth. = prepare sth. for sb. 为某人准备某物 save sth. = save sth. for sb. 为某人留某物 sing sb. sth. = sing sth. for sb. 为某人唱某物(歌) spare sb. sth. = spare sth. for sb. 为 某人让出某物 steal sb. sth. = steal sth. for sb. 为 某人偷某物
1.keep+形容词 2.keep+sth/sb+形容词 3.keep+doing一直做某事 4.keep...from doing...阻止做某事. 5.keep a pet饲养一个宠物 6.How long may I keep this book keep指借. keep you updated正确,这里的Upated作形容词用 英语里面没有keep sb do的用法 1、英语中接双宾语的动词award sb.sth.=award sth.to sb.颁奖给某人bring sb.sth.=bring sth.to sb.把某物带给某人hand sb.sth.=hand sth.to sb.把某物递给某人lend sb. sth.=lend sth.to sb.把某物借给某人mail sb.sth.=mail sth.to sb.把某物寄给某人offer sb.sth.=offer sth.to sb.将某物给某人owe sb.sth.=owe sth.to sb.欠某人某物pass sb.sth.=pass sth.to sb.把某物递给某人pay sb.sth. =pay sth.to sb.付给某人某物（钱）post sb.sth.=post sth.to sb.把某物寄给某人read sb.sth.=read sth.to sb.把某物读给某人听return sb.sth.=return sth.to sb.把某物还
给某人send sb.sth.=send sth.to sb.把某物送给某人sell sb.sth.=sell sth.to sb.把某物卖给某人serve sb.sth.= serve sth.to sb.拿某物招待某人show sb.sth.=show sth.to sb.拿某物给某人看take sb.sth.=take sth.to sb.把某物拿给某人teach sb.sth.=teach sth.to sb.教某人某物tell sb.sth.=tell sth.to sb.告诉某人某情况throw sb. sth.=throw sth.to sb.把某物扔给某人write sb.sth.= write sth.to sb.给某人写信2、双宾语易位时需借助介词for的常用动词book sb.sth.=book sth.for sb.为某人预定某物buy sb.sth.=buy sth.for sb.为某人买某物choose sb.sth.=choose sth.for sb.为某人选某物cook sb.sth. =cook sth.for sb.为某人煮某物draw sb.sth.=draw sth.for sb.为某人画某物fetch sb.sth.=fetch sth.for sb.为某人去取某物find sb.sth.=find sth.for sb.为某人找到某物fix sb.sth.=fix sth.for sb.为某人准备某物get sb.sth.=get sth.for sb.为某人拿来某物make sb.sth.=make sth.for sb.为某人做某物order sb.sth.=order sth.for sb.为某人订购某物pick sb. sth.=pick sth.for sb.为某人采摘某物prepare sb.sth. =prepare sth.for sb.为某人准备某物save sb.sth.=save sth. for sb.为某人留某物sing sb.sth.=sing sth.for sb.为某人唱某物（歌）spare sb.sth.=spare sth.for sb.为某
动词 一．动词的概念。 动词表示主语的动作或状态的词。如： I read Englih every day. (表示动作） It is autumn. (表示状态） The old man is carryinng a heavy box. (表示动作） 二．动词的种类。 ①be动词的用法 我是am,你是are,is跟着他，她和它，复数人称都用are. 如： I am a student. You are a teacher. My father and mother are workers. ②连系动词的用法 （1）连系动词一般不用于现在进行时。如： 误：The cloth is feeling soft. 正：The cloth feels soft. (2)连系动词不用副词来修饰，其后跟形容词做表语。如： 误：You should keep quietly in the hospital. 正：You should keep quiet in the hospital. ③情态动词的用法
④实义动词的用法 实义动词一般在句子中做谓语，有人称，时态的变化。实义动词可以单独用作谓语。如： I like English. The child is playing the piano. ⑤动词的基本形式 英语动词有五种基本形式，即动词原形，第三人称单数，过去式，现在分词和过去分词。动词原形用于固定用法和一般现在时中主语非三单，第三人称单数用于一般现在时中主语为三单，过去式用于一般过去时，现在分词用于进行时，过去分词用于完成时和被动语态。
（1）动词原形变第三人称单数的规则，与名词变复数的规则大致一样： (2)现在分词 (3)动词的过去式（规则变化）
50个可接双宾语的高考高频动词.txt-两个人同时犯了错，站出来承担的那一方叫宽容，另一方欠下的债，早晚都要还。-不爱就不爱,别他妈的说我们合不来。50个可接双宾语的高考高频动词 (一) 双宾语易位时需借助介词to的常用动词 1.award sb. sth. = award sth. to sb. 颁奖给某人 2.bring sb. sth. = bring sth. to sb. 把某物带给某人 3.hand sb. sth. =hand sth. to sb. 把某物递给某人 4.lend sb. sth. = lend sth. to sb. 把某物借给某人 5.mail sb. sth. = mail sth. to sb. 把某物寄给某人 6.offer sb. sth. = offer sth. to sb. 将某物给某人 7.owe sb. sth. = owe sth. to sb. 欠某人某物 8.pass sb. sth. = pass sth. to sb. 把某物递给某人 9.pay sb. sth. = pay sth. to sb. 付给某人某物（钱） 10.post sb. sth. = post sth. to sb. 把某物寄给某人 11.read sb. sth. = read sth. to sb. 把某物读给某人听 12.return sb.sth. = return sth. to sb. 把某物还给某人 13.send sb. sth. = send sth. to sb. 把某物送给某人 14.sell sb. sth. = sell sth. to sb. 把某物卖给某人 15.serve sb. sth. = serve sth. to sb. 拿某物招待某人 16.show sb. sth. = show sth. to sb. 拿某物给某人看 17.take sb. sth. = take sth. to sb. 把某物拿给某人