Km Incresase and decrease
Journal of Molecular Catalysis B:Enzymatic21(2003)
239–252
In?uence of the spacer length on the activity of enzymes immobilised on nylon/polyGMA membranes
Part1.Isothermal conditions
A.De Maio a,b,M.M.El-Masry a,1,M.Portaccio a,b,N.Diano a,S.Di Martino a,
A.Mattei b,2,U.Bencivenga a,D.G.Mita a,b,?
a Institute of Genetics and Biophysics of CNR,Via Guglielmo Marconi12,80125Naples,Italy
b Department of Experimental Medicine,Second University of Naples,Via S.M.di Costantinopoli16,80138Naples,Italy
Received29May2002;received in revised form23September2002;accepted23September2002
Abstract
?-Galactosidase was immobilized on nylon/poly(glycidyl methacrylate)membranes through spacers of different length: hexamethylenediamine,ethylenediamine or hydrazine.The effect of the spacer length on the catalytic behavior of the three membranes was studied in isothermal bioreactors.The behavior of the soluble and insoluble enzymes was compared to know the effects of the immobilization process and of the spacer length.
The enzyme derivatives in comparison with the soluble enzyme exhibited shifts of the optimum pH values towards more acidic solutions.These shifts were found decreasing with the spacer length;while an opposite trend was observed when the optimum temperature values were considered.Also the values of the apparent K m were found to decrease with the spacer length. All these results indicated that a soluble enzyme could be considered as an enzyme immobilized on a solid support through a spacer of in?nite length.
?2002Elsevier Science B.V.All rights reserved.
Keywords:?-Galactosidase;Chemical grafting;Biocatalytic membranes;Nylon membranes;Isothermal bioreactors
1.Introduction
Immobilized catalyst,enzymes or whole cells,are successfully employed in an increasing number of industrial processes[1–3].Typical examples are the optical resolution of racemic aminoacids by aminoa-cylase[4],the development of an enzymatic process ?Corresponding author.Tel.:+39-81-2395887;
fax:+39-81-2395887.
E-mail address:mita@https://www.wendangku.net/doc/677830255.html,r.it(D.G.Mita).
1Permanent address:Department of Polymers and Pigments, National Research Center,Dokki,Cairo,Egypt.
2Permanent address:Dipartimento di Scienze Chirurgiche ed Anestesiologiche,Seconda Universit`a di Napoli,Napoli,Italia.by means of immobilized bacterial cells for manufac-turing acrylamide[5],the lactose hydrolysis in milk by?-galactosidase[6],the waste water treatment by urease[7,8].
The success encountered by the employment of immobilized enzymes in biotechnological processes stimulated the interest of the researchers towards the basic research addressed to improve the performance of the biocatalytic membranes.To this aim,the study of the interactions between the enzyme and the sup-port became of fundamental relevance.
It is well known that the immobilization process af-fects the enzyme activity in respect to that of the sol-uble counterpart.The physico-chemical nature of the
1381-1177/02/$–see front matter?2002Elsevier Science B.V.All rights reserved. PII:S1381-1177(02)00229-1
240 A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic21(2003)239–252
carrier as well as the immobilization methods are the main causes of the observed differences.The carrier nature acts mainly through the“partitioning effect”
[9]which is responsible of the changes in the chem-ical composition of the microenvironment in which the immobilized enzyme is operating in respect to that of the bulk solution.The immobilization method acts through the nature of the binding forces or through the type and the position of the aminoacidic residues in-volved in the enzyme attachment.Covalent coupling is the most convenient immobilization technique since its allows high accessibility and reusability of the bound enzyme[10].Covalent attachment,in addition,offer the advantage that no relevant leaking of enzyme takes place in repeated uses since the binding forces are stronger than those occurring in the absorption or ionic binding.
When the support and the immobilization method are the same,one more subtle cause which can affect the activity of the immobilized enzyme is the length of the spacer between the catalyst and the activated support.
In this paper,we will discuss the results obtained with catalytic membranes prepared by using the same support(a nylon/polyGMA membrane),the same immobilization method(condensation by means of glutaraldehyde as bifunctional binding agent),but spacers of different https://www.wendangku.net/doc/677830255.html,parison with the be-havior of the soluble enzyme will be performed to evidenziate the effects induced by the immobilization process,with particular reference to the spacer length. All experiments were carried out under isothermal conditions.
In a separate paper[11],we have reported the be-havior in a non-isothermal bioreactor of the mem-branes characterized in this paper.The advantage of using non-isothermal bioreactors in biotechnological processes has been demonstrated[12–21].
2.Apparatus,materials and methods
2.1.The bioreactor
The bioreactor consisted of two cylindrical half-cells,2.5mm in depth and35mm in diameter,?lled with the working solution and separated by the cat-alytic membrane.Substrate solutions were recirculated in each half-cell at a rate of3.5mL min?1by means of a peristaltic pump through hydraulic circuits starting and ending in a common container.In this way,the whole solution reacted with the two faces of the cat-alytic membrane and the catalytic power of both faces was averaged.Thermocouples,placed at1.5mm from each of the membrane surfaces,were used to measure the temperatures inside each half-cell.The tempera-ture in each half-cell was programmed by means of circulation in external jackets of water coming from a thermostatic baths.A3D picture,not to scale,of the core of the bioreactor is represented in Fig.1.
2.2.Materials
As solid support to be grafted nylon Hydrolon mem-branes,a gift of Pall Italia(Pall Italia srl,Milano,Italy) were used.These membranes,150?m in thickness, are hydrophobic and have a nominal pore diameter of 0.2?m.Pore size is the measure of the diameter of the smallest particles that the membrane retains,since the membrane has no“classical pores”,but irregu-lar and interconnected cavities crossing the membrane thickness.Every kind of transmembrane matter trans-port,such as water or solute?uxes driven by concen-tration,pressure,temperature or electrical gradients, takes place through these cavities.
All chemicals,including the enzyme,were purcha-sed from Sigma(Sigma-Aldrich srl,Milano,Italy) and used without further puri?cation.Glycidyl metha-crylate(GMA)was used as monomer to be grafted. Hexamethylenediamine,ethylenediamine or hydra-zine were separately employed as spacer between the grafted membrane and the enzyme.A2.5%glu-taraldehyde aqueous solution was also used as bifunc-tional coupling agent for covalently binding the enzyme to the activated membranes.
The enzyme employed was a?-galactosidase(EC 3.2.1.23)from Aspergillus oryzae.?-Galactosidase has been used in view of the employment of these membranes in lactose hydrolysis in milk or in the treat-ment of the waste waters coming from dairy industry.
2.3.Methods
2.3.1.Preparation of the catalytic membranes
The preparation of the catalytic membranes was car-ried out in two steps:(a)grafting copolymerization, and(b)enzyme immobilization.
A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic21(2003)239–252
241
Fig.1.A3D picture,not to scale,of the core of the bioreactor.The hydraulic circuits,through which the substrate solutions are recirculated and the common cylinder have been omitted.
2.3.1.1.Grafting copolymerization.Grafting copoly-
merization was carried out by using as initiating
system K2S2O8/Na2S2O3in the ratio1:1.The mem-
branes were immersed,for1h at40?C,in a reaction
vessel?lled with a1:1water/ethanol solution contain-
ing0.3M GMA,0.008M K2S2O8and Na2S2O3and
in the presence of0.004%(w/v)copper https://www.wendangku.net/doc/677830255.html,ter
on,the membranes were treated with methylethyl ke-
tone to remove the produced homopolymer,then dried
at40?C until a constant weight was measured.At this
point,a nylon/polyGMA membrane was obtained,
The grafting percentage(X,%)was determined by
the difference between membrane masses before,G B,
and after,G A,the grafting by means of the formula:
X(%)=G A?G B
G B
×100(1)
The GMA grafting occurred also into the membrane
pores.
2.3.1.2.Enzyme immobilization.Hexamethylenedi-
amine,ethylenediamine or hydrazine were used as
spacers of different length.To this aim the membranes
were treated,for45min at room temperature,with a
1%(v/v)spacer solution in0.1M sodium carbonate
buffer,pH9.After washing with running tap water
to remove the unreacted amines,the membranes were
treated for90min at room temperature in a2.5%(v/v)
glutaraldehyde aqueous solution.At the end of this
treatment each membrane type was at?rst washed at
room temperature with double distilled water,after
with0.1M phosphate buffer solution,pH6.5,?nally
treated for16h at4?C with the same buffer solution
A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic21(2003)239–252243
containing?-galactosidase at a concentration of 3mg/mL.When this step was over,the membranes were washed with the0.1M phosphate buffer so-lution,pH 6.5,to remove the unbound enzymes. These conditions were found to be optimal in prelim-inary experiments aimed to obtain membrane types with comparable grafting degree,hydrophobicity and amount of immobilized enzyme.
The steps for the preparation of the catalytic mem-branes are reported in Fig.2,were the spacer has been indicated with NH2–(R)i–NH2,R being the CH2 group.When i is equal to6,hexamethylenediamine is the spacer and the corresponding catalytic membrane henceford will be named M6;when i is4,ethylene-diamine is the spacer and the corresponding catalytic membrane will be named M4,when i is0the spacer is hydrazine and the corresponding catalytic membrane will be named M0.
2.3.2.Determination of membrane activity and stability
?-Galactosidase hydrolyses lactose to glucose and galactose.Enzyme activity was determined by sam-pling at regular time intervals in the common container the solution in contact with the two catalytic surfaces of the membrane and by measuring the glucose con-centration by the GOD-Perid test.The test uses a cou-pled enzyme reaction by which a colored solution is obtained.The glucose concentration,proportional to the color solution intensity,is spectrophotometrically determined at570nm.
Membrane activity,expressed as?moles min?1, is given by the angular coef?cient of the straight line interpolating the experimental point of the glu-cose production(?moles)as a function of time (min).
Time stability of the biocatalytic membranes was assessed by analysing every day their activity un-der the same experimental conditions,i.e.0.2M lactose in0.1M phosphate buffer solution,pH6.5 and T=25?C.After3/4days,during which the membranes lost some activity,a stable condi-tion was reached,remaining unchanged for over 2months.Only these stabilized membranes were used in the comparative experiments reported in the following.When not in use the membranes were stored at4?C in0.1M buffer phosphate solution, pH6.5.2.3.3.Experimental data treatment
Every experimental point reported in the?gures represents the average value of?ve experiments per-formed under the same conditions.Each experiment lasted30min,but only the initial reaction rates were accounted for in the construction of the?gures.The duration of each experiment,the composition of our solutions,and the hydrophobic nature of the mem-brane excluded the occurrence of membrane fouling. In any case,to avoid fouling due to membrane reuse, a cleaning0.1M phosphate buffer solution was circu-lated for60min through the bioreactor and the mem-brane between two subsequent experiments.Effects due to concentration polarization,even if present, have not been taken in account for considering the hydrophobic nature of the membrane.
3.Results
To compare the behavior of membranes M0,M4 and M6,it is important to know the amount of enzyme immobilized on each membrane.This has been done by measuring the enzyme activity in the initial solution used for the immobilization,the residual activity of this solution after the immobilization process and the activity in all the solutions used to wash the membrane. Since at constant substrate concentration the activity is proportional to the enzyme concentration,it is easy to evaluate the amount of immobilized enzyme through a calibration curve of the catalytic activity of the free enzyme as a function of its concentration.
To this aim the equation:a=b?c?
n
i=1
d i, has been used.“a”is th
e amount o
f immobilized en-zyme,“b”and“c”are the amount of enzyme in the initial and?nal solution used for immobilization,re-spectively,and d i the amount of enzyme found in the i th washing,n bein
g the number of washings.The washing ends when d n becomes zero.The result of this procedure gives the amount of enzyme immobi-lized on eac
h membrane.In Table1,the amount of immobilized enzyme as well as the absolute and rela-tive catalytic activities for each of the three membrane types are reported.Absolute membrane activity has been de?ned as the activity for the total membrane surface(two surfaces of35cm2each one),while the speci?c activity as the activity for milligram of immo-bilized protein.In Table1,the grafting percentages,
244 A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic21(2003)239–252 Table1
Physical and biochemical properties of the catalytic membranes
Membrane type Grafting
degree(%)
Immobilized
enzyme(mg)
Absolute activity
(?moles min?1)
Speci?c activity
(?moles min?1mg?1)
Activity retention
(%)
M013.96 2.80.120.043 2.5
M414.03 3.10.410.1327.2
M613.86 2.9 1.990.68638.0
The activity retention has been calculated considering that1mg of soluble enzyme in a0.2M lactose concentration in0.1M phosphate buffer,pH6.5and T=25?C,gives a catalytic activity of1.82?moles min?1.
measured according to Eq.(1),are also listed for each of the three membranes.
Inspection of the data in Table1shows the strong dependence of the activity of the immobilized enzyme on the spacer length since the physical parameters, such as grafting degree and amount of immobilized enzyme,are practically the same for all the three mem-brane types.The high value of the activity retention of membrane M6,three times in respect to that of mem-brane M4and14times higher in comparison to mem-brane M0,induces to consider a soluble enzyme as an enzyme immobilized on a support through a spacer of in?nite length.Under these conditions the inter-actions between the enzyme and the support,indeed, vanish.
3.1.Kinetic parameters
When a biocatalyst is immobilized,the kinetic pa-rameters K m and V max undergo variations in compar-ison with the corresponding parameters of the soluble enzyme.To indicate that the kinetic parameters are changed they are indicated as K m,app.and V max,app.. These variations are attributed to several factors such as:(i)the changes in the protein conformation in-duced by the interactions between the support and the enzyme;(ii)the immobilization methods which, in the case of covalent attachment,can involve dif-ferent aminoacidic residues;(iii)the steric hindrances and the diffusional effects introduced by the grafted monomers or by the spacer.These factors may oper-ate simultaneously or separately.Consequently the ap-parent K m,app.value may increase[22,23]or decrease [24,25]in comparison with that of the soluble enzyme.
A decrease of the K m,app.value leads to a faster reac-tion rate,whereas an increase of the K m,app.implies the use of a higher substrate concentration in order to get the same reaction rate observed for the free en-zyme.The K m,app.increases if,for example,the elec-tric charges on support and substrate are of the same sign.The contrary occurs if the support and the sub-strate have opposite electric charges.
Also the V max values are affected by the immobi-lization process.In general similar values of V max have been found for the free and the immobilized form of the enzyme,even if increases or decreases have also been reported.Nevertheless it is dif?cult to compare the values of the V max,app.with the ones of the soluble enzymes,as the reaction rates are proportional to the amount of enzymes,and in the case of immobilization, even if we know the amount of immobilized enzymes, we don’t know the percentage of active enzymes.
To determine the kinetic parameters for?-galacto-sidase immobilized on M0,M4and M6,the activity of the catalytic membranes was studied as a function of substrate concentration.The pH and temperature of the solutions were6.5and25?C,respectively.The results, reported in Fig.3a,show that:(i)each of the three membrane types exhibited a Michaelis–Menten be-havior;(ii)at each lactose concentration investigated the reaction rate of membrane M0was lower than that of membrane M2which,in turn,had a reaction rate lower than that of membrane M6.The latter obser-vation con?rms the conclusion derived from Table1: more far the enzyme is from the support,more high is its reaction rate.This means that the enzyme af?n-ity towards the substrate increases with to the spacer length,by assuming that greater apparent af?nities to-ward the substrate must correspond at higher reaction rate,under equal substrate concentrations.
To verify this hypothesis,in Fig.3b,the experi-mental points of Fig.3a have been reported in form of Hanes plots.The K m,app.and V max,app.values,calcu-lated from Fig.3b,are listed in Table2,together with
A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic21(2003)239–252
245
Fig.3.(a)Catalytic membrane activity as a function of substrate concentration.(b)Hanes plots of the experimental points in Fig.3a.(?) Membrane M6;(?)membrane M4;(?)membrane M0.
the ones relative to the free enzyme[21].Different values of K m,app.and V max,app.have been found.The af?nity of the enzyme towards the substrate is smaller for the immobilized enzymes in comparison with that found for the soluble one.Moreover,the af?nity towards the substrate for the?-galactosidase immo-bilized on membrane M6is higher than that of the enzyme immobilized on M2,the latter being,in turn, higher than that of the enzyme immobilized on M0. Practically,the apparent af?nity towards the substrate
246 A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic 21(2003)
239–252
Fig.4.(a)K m ,app .as a function of the number of CH 2groups in the spacer chain.(b)Normalized maximum reaction rates (?,left scale)and activity retention (?,right scale)as a function of the number of CH 2groups in the spacer chain.
linearly increases with the increase of the spacer length,as it can be seen in Fig.4a ,where the K m ,app .values are reported as a function of the number of CH 2groups in the spacer chain.The analytical equa-tion representing the experimental data of Fig.4a is:y =110.57?4.07x ,where y is the value of the K m ,app .and x the number of (CH 2)groups in the spacer chain.The coef?cient R is equal to 0.99.
A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic21(2003)239–252247
Table2
Kinetic parameters
Enzyme status K m,app.(mM)V max(?moles min?1) Soluble21.40 3.20
Immobilized on M081.00.18
Immobilized on M492.00.59
Immobilized on M6112.0 2.67
The values of the kinetics parameters relative to the soluble enzyme have been taken from[21].
The V max,app.values increase exponentially with the increase of the spacer length(Fig.4b).A similar be-havior was found in the case of the activity retention, how it appears in the same Fig.4b.The increase of the reaction rate with the number of CH2groups in the spacer chain agrees with the increase of the enzyme af?nity with this parameter.
All these results indicate that the presence of the spacer in somewhat reduces the modi?cation on the enzyme structure induced by the interactions between the electric charges present on the nylon membranes and the surface charges exposed by the enzyme.This protective effect increases with the spacer length ac-cording to the Coulomb’s law.
3.2.Effect of pH
The pH-activity pro?le of an immobilized en-zyme is characteristic of the enzyme,immobilization method and carrier used.The support,indeed,can change the pH value around the catalytic site,thus determining appreciable differences in the catalytic behavior of the soluble and insoluble form of the catalyst.This effect,known as partitioning effect,is directly related to the nature of support(and grafted monomers)which induce electrostatic or hydrophobic interactions between the matrix and the low molecular weight species present in bulk solution.Partition-ing effect,indeed,causes in the microenvironment in which the immobilized enzymes are operating concentration changes of the charged species(e.g. hydrogen and hydroxyl ions)in respect to the bulk solution.
To know the effect of the spacer length on the parti-tioning effect we have investigated the activity of the ?-galactosidase,in the free and immobilized forms, as a function of pH in the range between2.5and
6.5.Fig.5.Relative activity as a function of pH for membrane M0(a), membrane M4(b)and membrane M6(c).(?)Soluble enzyme; (?)membrane M6;(?)membrane M4;(?)membrane M0. We used0.1M NaCl–HCl buffer solution for pH2.5;
0.1M citrate buffer solution in the pH range from3 to5,and0.1M phosphate buffer solution in the pH range from5to6.5.The results of this investigation are reported in Fig.5a–c,where the relative activi-ties of membranes M0,M4and M6are,respectively, reported as a function of pH.In the same?gures the relative activity of the soluble?-galactosidase is also reported,to allow comparison.Inspection of the
248 A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic 21(2003)239–252
?gures shows different positions of the optimum pH between the free and insoluble forms of ?-galac-tosidase,with a shift of the optimum position to-wards more acidic solutions in the case of the enzyme derivatives.Indeed,the position of the optimum pH for the free enzyme occurs at pH 4.6;while this posi-tion occurs at pH 4.0for the enzyme immobilized
on Fig.6.Optimum pH (a)and optimum temperature (b)as a function of the number of CH 2groups in the spacer chain.
M 0,at pH 4.2for the enzyme immobilized on M 2and at pH 4.4for the enzyme immobilized on M 6.It is interesting to observe (Fig.6a )that the values of the optimum pH positions for the enzyme derivatives lin-early increase with the increase of the spacer length.The analytical expression interpolating the experi-mental points of Fig.6a is y =3.92+0.08x ,where
A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic21(2003)239–252249
y is the value of the position of the optimum pH and x the number of(CH2)groups in the spacer chain. The coef?cient R is equal to0.99.Once again,the behavior represented in Fig.6a suggests that the free enzyme can be considered as an enzyme immobilized on a solid support through a spacer of in?nite length, in a position in which the partitioning effects induced by the carrier are zero.On the contrary,the more short is the spacer,the more effective is the partition-ing effect.In other words,the hexamethylenediamine, being enough long and keeping the enzyme enough far from the electric?eld of the nylon membrane,cre-ates the conditions by which the microenvironment around the catalytic site of the immobilized enzyme is quite similar to that around the free form.
By de?ning“optimum pH range”the range in which the relative enzyme activity is higher than90%from Fig.5a-c,it is possible to see that this range occurs between4.20and5.08for the free enzyme;between 3.69and4.44for the enzyme immobilized on M0, between3.80and4.61for the enzyme immobilized on M4,and between3.87and4.84for the enzyme immobilized on M6.When the optimum pH range is considered,it is possible to conclude that the extent of the optimum pH range is little affected by the spacer length.
3.3.Temperature dependence
The isothermal characterization of membrane ac-tivity is one of the principal parameters required to know how the immobilization procedure and the spacer length affect the enzyme activity.Generally, enzymatic derivatives show optimum temperatures shifted towards higher temperatures than those of the soluble counterpart.
In Fig.7,the relative activities of the three mem-brane types as a function of temperature are reported, together with that of the free enzyme,for comparison. Fig.7a refers to membrane M0,Fig.7b to membrane M4,and Fig.7c to membrane M6.All membranes exhibit a shift of the optimum activity towards higher temperatures in comparison to the position of the sol-uble enzyme,evidencing in this way that the immobi-lization procedure protects the enzyme structure.The optimum temperature for the free enzyme occurs at about48?C,while for membrane M0at about65?C, for membrane M4at about62?C,and for
membrane Fig.7.Relative activity as a function of temperature for membrane M0(a),membrane M4(b)and membrane M6(c).(?)Soluble enzyme;(?)membrane M6;(?)membrane M4;(?)membrane M0.
M6at about60?C.In Fig.6b,the value of the opti-mum temperature of the three membrane types as a function of the spacer length are reported.Results in Fig.6b show that the values of the optimum temper-ature of the immobilized enzymes linearly decrease with the increase of the spacer length.Once again, the more far is the enzyme from the carrier,i.e.the more long is the spacer,the more the optimum tem-perature of the immobilized enzyme approaches that
250 A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic 21(2003)239–252
of the soluble counterpart.This means that the immo-bilization process,besides strengthening the enzyme structure,gives to the macromolecule a protective effect against the heat denaturation.This protective effect decrease with the increase of the spacer
length.Fig.8.(a)Thermal inactivation of the membrane:relative activity as a function of the incubation time at 60?C.(?)Membrane M 6;(?)membrane M 4;(?)membrane M 0.(b)Thermal inactivation constants as a function of the number of CH 2groups in the spacer chain.
The analytical expression representing the linear re-lationship between the optimum temperature value (y )and the number of CH 2groups (x )in the spacer chain is given by:y =65.07?0.82x ,with R =0.99.
A.De Maio et al./Journal of Molecular Catalysis B:Enzymatic21(2003)239–252251
Calling“optimum temperature range”the range
in which the relative enzyme activity is higher than
90%,it is possible to see that this range is between
41.5and54.9?C for the free enzyme;between56.3
and68.8?C for the enzyme immobilized on M0;be-
tween52.1and66.9?C for the enzyme immobilized
on M4,between53.6and66.3?C for the enzyme
immobilized on M6.The simultaneous existence of a
large optimum temperature range and of the shift of
the optimum temperature position suggest the use of
our membranes in processes requiring high working
temperatures.
3.4.Thermal stability
In view of industrial applications it is important to
know the thermal stability of a catalytic membrane.To
measure the thermal stability of the three membrane
types we have adopted the following procedure.Cat-
alytic membranes of large surface were prepared ac-
cording to the methodology described in Section2.3.1.
Then the membranes were cut in several pieces.The
catalytic activity of each piece was then measured
under standard conditions,i.e.0.2M lactose in0.1M
phosphate buffer solution,pH6.5and T=25?C,be-
fore the heat treatment and after incubation at60?C for
the required time.Calling A0the initial membrane ac-
tivity and A the activity measured at the end of the ther-
mal treatment,the ratio A/A0gives directly the thermal
deactivation of the membrane.In Fig.8a,the A/A0
ratio is reported as a function of time for each of the
three membrane types used in this experimentation.
Through the expression log A=log A0?K i t it is pos-sible to calculate directly the thermoinactivation con-
stant K i(min?1)according to the indications reported
in[26].The values of this constant,reported in Fig.8b
as a function of the spacer length,show a decrease
with the increase of the spacer length.These results,
together with those of Fig.7,con?rm that membrane
M6is the most indicated for processes requiring high
temperatures.
4.Conclusions
All the results above reported have shown the
in?uence of the spacer length on the isothermal per-
formance of a catalytic membrane,when the same enzyme is attached to the same support through spac-ers of different length.
The optimum pH values of the insoluble enzymes in comparison with that of the soluble counterpart ex-hibit shifts proportional to the spacer length,whereas an opposite trend is observed when the values of the optimum temperature are considered.In both cases, increasing the spacer length the values relative to the immobilized enzymes approach to that of the free en-zyme,indicating in this way that a soluble enzyme can be considered an enzyme immobilized on a solid support through a spacer of in?nite length.This con-clusion agrees also with the behavior of the K m,app. values,which linearly decrease with the increase of the spacer length,approaching to the value of the free enzyme.
If attention is paid to the applied aspects,mem-brane M6,i.e.the membrane obtained with the longest spacer used in this research,appears the most resis-tant to high temperatures,acidic solutions,and thermal inactivation.
Acknowledgements
This work was partially supported by the Target Project“Biotechnology”of CNR,by MURST(ex40% founds),by MURST/CNR L.27/12/1997no.449,and by“Regione Campania”.
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make短语搭配
make make sth. 做，制造 make sb. do sth. 使得．．． make sb. (sth.) done make sb. (sth.) + adj. make sb. (sth.) + n. make it + adj.(n.) + that．．． make it + adj.(n.) + to do sth. make it + adj.(n.) + doing sth. make a dash for 赶往．．．，冲向．．． make a deal with 达成协议，做成交易 make a decision 作出规定 make a face = make faces 做鬼脸，做苦相 make a good effort 作很大的努力 make a record 录制唱片 make a plan for 为…作计划 make a note of 注意，记下来 make an impression on 给．．留下（某种）印象，引人注目 make fun of 取笑，和．．．开玩笑，嘲笑 make ．．．into．．．把．．．做成．．．；使成为，使变成 be made into．．． be made from．．．由．．．做的（化学变化） be made of．．．由．．．做的（物理变化）
make it 按时到达某处，办事成功；约定时间；及时赶上（火车，轮船等） make one’s living 维持生活 make progress 取得进步 make out看清楚，看出，辨识；理解，明白；开（账单，收据等）进展；假装，装出，把．．．说成是 make room (for) 让地方，让位置 make sense 有道理，好懂，有清楚的意思 make sense of 理解 make sure that．．．弄肯定，一定要做到；弄确切，弄清make sure of make sure to do sth. 一定要做．．． make ... to one's own measure 依照某人的尺寸做。。。 make up 创造，编造；弥补，把．．．补上；化妆，打扮 make up for 弥补 be made up of 有．．．组成（构成） make up one’s mind to do sth. 打定主意，决定，决心make use of 利用
麦德龙储配方案设计
麦德龙储配方案设计 一、企业简介 麦德龙于1964年在德国以万平方米的仓储式商店开始了企业的历程。麦德龙以"现购自运"（现金交易，自选自运）营销新理念在市场上引人注目。经过近40年的发展，现在麦德龙已经成为欧洲最大的商业连锁企业之一，并自1999年开始排名世界零售百强第三位。目前，麦德龙在全球20多个国家和地区建立了3000多家分店，拥有越20万员工，年销售额超过1000亿德国马克。麦德龙于1995年来到中国并与中国着名的锦江集团合作，建立了锦江麦德龙现购自运有限公司，是第一家获得中国中央政府批准在中国多个主要城市建立连锁商场的合资企业。麦德龙提出"我们是顾客的仓库"的概念，意味着每个商店不另设仓库，同时商店本身就是仓库。通常，麦德龙标准店的规格为140米长*（90米+28米）宽，其中90米为商店宽度，28米为商店自身仓储空间的宽度。麦德龙习惯于独立运营的商业空间，单层建筑，独立的停车场，很少将店开在大型购物中心里面。2002年，麦德龙在中国北部、东部、南部和中部建立了四个销售区域。麦德龙通过其全国性分销系统将当地产品投入国内市场的同时吸引着各地顾客。同时麦德龙国际分销系统将中国商品推向国际市场。 二、企业基本特点——超市与仓储合二为一 麦德龙仓储式超市是将超市和仓储合而为一的零售业态。它省掉了传统零售企业独立的仓库和配送中心，经营中实现了快速补货，保证了超市低成本高效率的运作。仓储式超市与普通超市整体策划设计方面有明显不同。 （一）营业场所选址上 麦德龙超市通常设在大城市城乡结合部的高速公路或主干道附近。这样既避免了市中心及市区的交通拥挤，又因土地价格相对便宜，减少了投资风险。同时，选址还适应了城乡一体化的发展趋势，提前占据区位优势。它商圈的辐射半径通常为50公里。 （二）超市建筑设计 麦德龙仓储式超市从外观看就象一个现代化的大仓库，其营业面积一般为15，000～20，000平方米。外部设有与营业面积几乎相等的停车场，内部结构比较简单，通常采用高米的工业用大型货架。货架下半部分用于商品的陈列展示，与普通超市无异：而其上半部分则用于相应商品的存放，起到了仓库的作用，从而使销售和仓储合为一体。货架间距较大，便于存取货物的叉车通过，完成迅速补货的工作。 （三）商品定位 商品内容丰富，品种齐全，通常在20，000种以上，可满足客户“一站式购物”的需求。如麦德龙商品种类中食品占40%，非食品占60%。食品类商品以时令果蔬、鲜肉、鲜鱼、奶制品、冷冻品、罐头、粮食制品、饮料、甜点为主，品种相对稳定。非食品领域的商品则按季节和顾客需要定期调整，涉及范围较
make短语
ake 短语 make[mek]意思：做；制造；生产；制定；成为。 make+名词(代词)+名词、代词，介词短语，形容词或形容词短语，省去to动词不定式短语，过去分词短语。 动词＋名词： makeadifferenee?有影响，起(重要)作用makeadecision做决定makeaface?/faceS故鬼脸 makeafilm?拍电影makeafire?生火makealiving?谋生makeamistakeUE错误makefriends?交朋友makefriendswith?与…交朋友makefunof?取笑,拿…开玩笑makemoney?赚钱makeone'/thebed?整理床铺makesure弄明白，设法确保makesureof?确保；确信makesurethat查明，确信，把…弄清楚makeanoise/makemuchnoises出噪音makeupone'smind下决心，打定注意makeit到达；成功；确定时间make one' swa前进makese nten ces组句子makeuseof?利用makethemost/bestof尽量利用，充分使用makeup one' smincF决心；拿定主意make(both)e ndsmee量入为出，使收支相抵makeo neselfathome?不要拘束；别客气makesense?讲得通，有道理makesenseof?理解 makeprogress in?在…方面取得进步makepreparationsfor为.. 做准备 makeroomfor…给...腾出地方makecoffee/tea煮咖啡/沏茶makepeace议和 makeanapology道歉makeajoke开玩笑makeanappointmen约会makeasuggestior提出建议makeanexcuse 找借口makeenemies树敌 makeapromise许诺makeatrip 去旅行makebelieve假装;假扮makeclothes做衣服make on esday 整天都开心makeready 准备makehistory做出值得载入史册的事情make notes记笔记makereal兑现makepublic 公布于众makeclear打扫makebread做面包makepaper造纸 动介短语： makefor?向.. 前进；有助于 makeup组成；编造；弥补；化妆，打扮；和解；整理makeout假称，假装；听出，看出；辨认出；理解；填写 makeoff 离开 makeupof 组成 makeupfor 弥补 makeupto讨好，奉承 make(it)upwith 与... 和好 makedowith以 .. 勉强过日子；以有... 为满足 与“制成”有关：bemadeof/from/out/in/with/into
麦德龙采购模式
麦德龙采购模式 距苏州高新区商场开业仅一周时间，2006年1月，锦江麦德龙现购自运有限公司(下称“麦德龙")在其华南区总部广州的首家分店也随即开张。“麦德龙2005年计划开店6～10家，去年我们开出4家新店，现在加上苏州、广州两家门店，基本完成任务。"麦德龙总裁杜哲思在广州出席新店开幕仪式时表示。据了解，麦德龙2006年计划在中国再开新店6～8家。 首店再获五张大单 到目前为止，麦德龙在中国共有29家商场，其中华南6家，主要分布在福州、厦门、昆明、东莞、深圳、广州。“2003年，德国前总理施罗德与麦德龙全球总裁访问广州时，该项目开始立项。随后的2004年9月，广州市市长张广宁到德国慕尼黑开推介会，又将该项目作为中德合作重点。正是由于多方的支持，广州麦德龙才可以在短时间内落成。"杜哲思透露。 据了解，广州麦德龙占地面积1.1万平方米，食品和非食品区域各占一半。此外，麦德龙进军广州仍采取租赁模式。其广州合作方为著名日化企业美晨集团，它同时也是广州百安居的合作伙伴，并且正与宜家、迪卡侬等500强企业商洽合作事宜。 麦德龙广州店目前已发展了约13万家专业客户会员。近日,麦德龙再获五大专业客户，分别与中国大酒店、亚洲国际大酒店、广州酒家集团、本田(中国)汽车有限公司、箭牌糖果(中国)有限公司签署采购协议。但当事人拒绝透露采购数量。
采购新政策初见成效 去年7月，麦德龙中国区突然颁布采购新政，打破了以前全部商品都由上海总部说了算的传统，总部采购部门与区域采购部门联合采购。业内人士认为，这一举措旨在向采购腐败开刀。 “中国幅员辽阔，人口众多，对麦德龙未来的发展而言，意义非凡。"杜哲思首次回应此事时表示，2002年，麦德龙在中国成立了华东、华南、华北、华中四大销售区域，并以上海、广州、北京、武汉为中心城市，主要目的是进行地域协调。 据杜哲思透露，这一措施收到了很大成效，区域办公室更接近客户，了解消费者需求。据称，2002年区域采购在麦德龙体系中只占10％，而随着近期的发展，这一比例提高到了25％～30％。“除此之外，我们还设立了部分商品原产地采购，比如在昆明，我们有专人驻扎，负责直接联系采购，从而保证商品的新鲜。" 根据麦德龙集团2005年第三季度财务报告显示，去年1～9月,该集团销售增长4.2％,达到413亿欧元，预计全年销售增长可达到4％。其中现购自运商场业务增长7.3％。 证券分析人士表示，2005年1～9月，麦德龙的海外市场增长强劲，达到51.4％。在亚洲，集团拓展的焦点仍然是中国市场。根据法兰克福DAX指数显示，从2005年11月17日至今，麦德龙在证券市场一直呈上涨趋势，其股价从36欧元已经上涨到42欧元。
以make开头的短语和词组15页word
以make开头的短语和词组： make a Federal case out of sth. 过分强调某事, 过分夸大某事。 make a House (英下院)使出席议员达法定人数。 make a Virginia fence 摇摇晃晃地走。 make a balk of good ground 失去好机会。 make a balls of 把...搞糟。 make a bargain 成交。达成协议。 make a basket (篮球)投中一球。 make a beast of oneself 行同禽兽, 行为卑鄙。 make a bed 整理床铺。 make a beeline for [口]一直奔向...。抄近路向...走去。 make a bet 打赌。 make a bid for 出价买, 企图获得。 make a big deal out of 小题大作。 make a big fanfare 大吹大擂。 make a bitch of [口]弄坏, 弄糟。 make a blot for it 赶快逃走。 make a bolt for 急忙向...逃去。 make a bolt of it [俗]急逃, 奔去。 make a bow 鞠躬。 make a break for it [口](趁人不注意时)偷跑, 逃跑。 make a break of 连续得分。 make a brilliant figure 崭露头角。放异采, 引人注目, 超群出众。make a buck 挣钱。 make a butt of 嘲弄[取笑]某人。 make a call 访问。到。 make a card 以一牌赢一墩。 make a career 向上爬, 谋求发迹。 make a caricature of 使滑稽化。 make a charge against 指控, 非难。 make a choice 做一选择。 make a chump out of 使...丢脸, 侮辱。 make a circuit 迂回, 绕道而行。 make a claim for 对（赔偿等）提出要求...。 make a claim to 认为...是属于自己的。 make a clean breast 坦白。 make a clean breast of 完全承认。全盘托出。 make a clean sweep of 彻底扫除。 make a collection for 为... 募捐。 make a companion of 与...作伴，与...为友。 make a complaint 抱怨。 make a complaint against 提出不满意见，对...提出控告。 make a compliment to 恭维，夸奖，颂扬。 make a concession to 对...让步。 make a confession 招供, 坦白。 make a conspicuous figure 崭露头角。放异采, 引人注目, 超群出众。make a contract with 与...签定合同。 make a contribution to 捐赠；作出贡献。 make a contribution towards 捐赠；作出贡献。 make a convenience of sb. 利用某人。 make a countenance 假装，用脸色暗示。
麦德龙的国际市场营销分析报告
麦德龙的国际市场营销分析报告 一、基本简介 1964 年，在商业领域中，一个崭新理念和管理方式在德国诞生了，这就是麦德龙股份公司（Metro AG），由奥托·拜斯海姆创建，目前总部位于杜塞尔多夫。今天，集团已在全球33个国家拥有超过2100家门店。在10万名员工的共同努力下，公司2010年营业额达到了310亿欧元，是德国最大、欧洲第二、世界第三的零售批发超市集团。麦德龙在中国的37个城市共有56家分店。 麦德龙集团公司拥有六个独立销售区域及不同品牌。所谓的跨区域服务公司即为集团内所有销售区域提供服务。现购自运是麦德龙集团旗下最具销售能力和最国际化的分支机构。（“现购自运”是指专业顾客在仓储式商场内自选商品，以现金支付并取走商品。） 二、基本特点 （一）营业场所选址上 麦德龙超市通常设在大城市城乡结合部的高速公路或主干道附近。这样既避免了市中心及市区的交通拥挤，又因土地价格相对便宜，减少了投资风险。同时，选址还适应了城乡一体化的发展趋势，提前占据区位优势。 （二）超市建筑设计 麦德龙仓储式超市从外观看就象一个现代化的大仓库，其营业面积一般为15，000～20，000平方米。外部设有与营业面积几乎相等的停车场，内部结构比较简单，通常采用高4.5米的工业用大型货架。货架下半部分用于商品的陈列展示，与普通超市无异：而其上半部分则用于相应商品的存放，起到了仓库的作用，从而使销售和仓储合为一体。货架间距较大，便于存取货物的叉车通过，完成迅速补货的工作。 （三）商品定位 商品内容丰富，品种齐全，通常在20，000种以上，可满足客户“一站式购物”的需求。如麦德龙商品种类中食品占40%，非食品占60%。食品类商品以时令果蔬、鲜肉、鲜鱼、奶制品、冷冻品、罐头、粮食制品、饮料、甜点为主，品种相对稳定。非食品领域的商品则按季节和顾客需要定期调整，涉及范围较广不仅包括日常生活用品、办公用品，还包括小型机械工具类产品。 三、经营理念 （一）拥有自有产权的店面 40年来，它无论在全球什么地方都是强调拥有自有产权的店面。与大型综合超市如家乐福与沃尔玛相比，麦德龙店对地点、面积的要求更严格。 （二）禁止1.2米以下的儿童进入卖场 作为一家大型仓储式商场，需要进行叉车作业，补充货品，而1.2米以下的儿童恰恰
溯源系统简明操作手册
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Make的短语
Make的短语,make的词组及常用俚语、习惯用语和日常用语总结 关键词：make的常用短语,make的词组 来源：澳典在线词典| 发布日期：2008-12-21 12:02 以make开头的短语和词组： make a Federal case out of sth. 过分强调某事, 过分夸大某事。 make a House (英下院)使出席议员达法定人数。 make a Virginia fence 摇摇晃晃地走。 make a balk of good ground 失去好机会。 make a balls of 把...搞糟。 make a bargain 成交。达成协议。 make a basket (篮球)投中一球。 make a beast of oneself 行同禽兽, 行为卑鄙。 make a bed 整理床铺。 make a beeline for [口]一直奔向...。抄近路向...走去。 make a bet 打赌。 make a bid for 出价买, 企图获得。 make a big deal out of 小题大作。 make a big fanfare 大吹大擂。 make a bitch of [口]弄坏, 弄糟。 make a blot for it 赶快逃走。 make a bolt for 急忙向...逃去。 make a bolt of it [俗]急逃, 奔去。 make a bow 鞠躬。 make a break for it [口](趁人不注意时)偷跑, 逃跑。 make a break of 连续得分。 make a brilliant figure 崭露头角。放异采, 引人注目, 超群出众。 make a buck 挣钱。 make a butt of 嘲弄[取笑]某人。 make a call 访问。到。 make a card 以一牌赢一墩。 make a career 向上爬, 谋求发迹。 make a caricature of 使滑稽化。 make a charge against 指控, 非难。 make a choice 做一选择。 make a chump out of 使...丢脸, 侮辱。 make a circuit 迂回, 绕道而行。 make a claim for 对（赔偿等）提出要求...。 make a claim to 认为...是属于自己的。 make a clean breast 坦白。 make a clean breast of 完全承认。全盘托出。 make a clean sweep of 彻底扫除。 make a collection for 为... 募捐。 make a companion of 与...作伴，与...为友。
Make构成的短语大集合
中学英语中由make构成的短语 1.make a decision 做出决定 I hope we can make a decision today. 2.make a plan for 为……订计划 Now it is much easier to make plans for your trips . 3.make a record 录制唱片 4.make fun of 取笑某人 5.make sentences 造句 6.make enemies 树敌 I know you have made a lot of enemies. 7.make ...from ... 用……制造…… But this time there will be no trees to make new soil from their leaves. 8.make a good effort 作很大努力 The people made a good effort. 9.make progress 取得进步 Are you making good progress? 10.make a suggestion 提出建议 Making suggestions and giving advice . He is making a suggestion. 11.make up 编出;编造；组成 Make up a dialogue ,using the following as a guide. 12.make a film 拍电影 They are going to make a film next month here. 13.make a journey 进行旅行 Why are you making this journey ? 14.make a study of 对……进行研究 When you do research ,you make a very careful study of a subject. 15. make a choice 做出选择 16.make use of 利用 People in Xinjiang are making use of wind for producing electricity 17.make ...into... 把……制成…… You'd better make the apples into juice for your baby 18.make a trip 进行旅行 What do you usually take along while you are making a trip ? 19.make no difference 对……没有关系；对……没有不同 It makes no difference because most of these places have been saying 'no' to us for quite a long time. (SB2A Lesson 6) 20.make noise 吵闹；发噪音 Mrs Cousins was making a lot of noise with the pans and did not hear anything unusual. 21.make money 赚钱 The Chinese government makes a lot of money from sales of tobacco every year. 22.make sure of 确保；确定
麦德龙仓储超市管理案例
麦德龙仓储超市管理案例 一、背景情况 二十世纪九十年代，仓储式超市经营模式的创始者，总部设在德国的麦德龙（METRO）将仓储式超市——这种影响世界商业发展的新型业态带入中国，其所特有的新战略、新思路和新理念，对国内零售业的发展有很好的借鉴作用。 麦德龙集团成立于1964年，位于当今世界500强的前50位，在国际商业企业中排名第三，也是世界上最大的现付自运制的商业连锁公司。1995年麦德龙集团旗下的荷兰公司与上海锦江集团合资成立上海锦江麦德龙集团，并于1996年在上海开设了第一家大型仓储式会员制连锁店，截至2001年上海锦江麦德龙集团已开设了15家连锁超市，其中仅上海地区就开设了4家，各卖场拥有的会员数均超过11万，营业额排名位于全国连锁业百强第九位，销售额达49.4亿元。 二、麦德龙仓储式超市的客户管理 仓储式超市实际上就是以零售的方式来从事批发业务。超市既是服务中心又是配送批发中心。这种差异化的市场定位使麦德龙成为了专业客户的超级仓库。 （一）有限的目标顾客户 绝大多数普通超市的目标消费群是无差异性的，即服务对象是全体普
通消费者，消费者短期内的重复购买率高，但每次的购买量不大，多为临时性随机性消费行为。而仓储式超市的目标消费群比较明确，麦德龙针对“有限”客户，即只对工商领域的经营者、群体消费层实行会员制，会员必须是具有法人资格的企事业单位，且不收取会员管理费用。因此，麦德龙仓储式超市直接为企事业单位、中小零售商、宾馆、餐厅、部队、工厂、学校等法人团体服务。 经过三十多年的经营发展，麦德龙自创了一套适合C&C体制的商品信息管理系统和会员信息管理系统，使内部管理成功实现了信息化。最初，麦德龙的服务人员将会员填写的《客户登记卡》的资料输入微机，创建顾客的初始资料。会员信息管理系统自动记录顾客每一次的购买情况，根据各类客户的购买频率和消费结构，准确分析出客户需求的动态发展趋势，使麦德龙能对顾客需求变化迅速做出反应，及时调整商品结构和经营策略，最大程度地满足顾客需求。 麦德龙根据客户规模和购买量将客户分“ABC”三类，A类是他们的重点客户，经常能进行长达一二小时的经营分析和策略研究。由于他们不仅关心自己的经营状况，还能关心客户的经营状况，在服务上是全方位的，所以就能使大批的客户成为其稳定的市场。麦德龙还专门为其成立“客户顾问组”，对客户的消费结构进行分析，向客户（特别是中小企业）提供特色咨询服务。同时，与主要的客户进行沟通，向他们提出采购建议，帮助客户降低采购成本。通过提供全方位服务，麦德龙不仅拥有了大批的稳定客户，还及时掌握了市场需求动态，从
关于Make的一些词组
Make 一、make do with sth 将就使用例：If we can’t get limes,we’ll have to make do with lemons. 如果我们没有酸橙，只好将就一下用柠檬。 二、make for 1.朝…移动或走例：He made for the pig.他向那头猪走去。2.非常适合（协调）； 天造地设例：Jim and Alice are made for each other. Jim和Alice是天生一对。 三、make sb/sth into sb/sth 使变成；使转变为例：She made her spare room into an office. 她 把她家多余的房间改成办公室。 四、make sth of sb/sth 懂得；理解例：What do you make of Colin’s letter? Conlin的信你看得 明白么？ 五、make off (with sth) 匆忙离开，逃走（如偷了东西后）例：Jason’s made off with my wallet! with my wallet! Jason偷走了我的钱包！ 六、make sb/sth out 1,理解；明白I can’t make out what he is saying.我不懂他在说什么。2. 看出；听出；辨认出I could hardly make out anything in the thick fog.在浓雾中我几乎什么也看不见。 3. 开出，写出The teacher has made out a list of reference books.老师已开出了一份参考书单。4.（informal）暗示；建议例：You make me out to be a liar.你暗示我不要说谎。 5. （informal）试图建立或者证明例：Jason made out that he was innoc ent. Jason试图证明自己是无辜的。 七、make up 1.化妆例：It didn’t take her long to make up.她很快就化妆好了。2. 构成；组 成例：How many countries make up the European Union? 欧盟由多少个国家组成？ 3. 编造；捏造；虚构例：Jason made up an excuse in order to escape the punishment. 为了逃避惩罚Jason编造了一个借口。4.补充；补足；凑足例：We need one more person to make up our football team.我们还需要一个人才可以凑足一个足球队。5.make up for sth 补救； 弥补例：Her enthusiasm makes up for her lack of experience. 她的热诚弥补了她经验的不足。6.和解，和好例：James and Kobe quarreled, but made up after a while.詹姆斯和科比吵架，但一会儿就和好了。 八、be made of 由…制成（原材料显而易见的情况）be made from由…制成（原材料不易 看出的情况）be made in 在…制造 九、make over 1.转让，移交例：He has made over his property to a hospital.他已经将财产转 交给了一家医院。2.修改，改造例：He asked the tailorto make over his trousers.他叫裁缝修改他的裤子。
make用法及短语总结
make用法及短语 一、make的复合宾语。英语中make一词用法甚多，是使用频率最高的动词之一，而make 作使役动词的用法也很常见。意思是“使成为”、“使作为”、“使变成”，其后的复合宾语（即宾语+宾语补足语）有以下表达方式： 1. make +宾语+名词（作宾语补足语） Most pop singers make music their career.大多数流行歌手把音乐当作他们的职业。 Bill Gates’Microsoft makes him a phenomenon in the business world. 比尔·盖茨的微软使他成为商界的一个奇才。 What makes the ocean such a great place to live? 究竟是什么东西使大海成为如此优越的生活场所呢? European football is played in 80 countries，making it the most popular sport in the world. 80个国家踢欧式足球，使它成为世界上最受欢迎的运动。 After all，what makes a new invention such a wonderful thing is that it allows us to do something we could not do before． 毕竟，一项发明之所以成为如此奇妙的事情就在于它可以让我们做以前不能做的事。 2. make +宾语+不带to的不定式（作宾语语补足语） Nobody made us go to bed at a certain time.没有人让我们在某一固定时间就寝。 Pop music makes people feel easy and forget about the real world；rock music makes people think about the world and how to make their life better． 流行音乐令人松弛安心，忘记这真实的世界，而摇滚乐使人思考这个世界和如何改善自己的生活。 Nothing can make me turn against my country．什么也不能使我背叛我的祖国。 Love may create the wonder，and may make people become satisfied and optimistic． 爱可以创造奇迹，可以让人变得满足和乐观。 注意：但改成被动语态时，则要加to。 Children should be made to understand the importance of saving water． 应该让孩子们理解节水的重要性。 3. make +宾语+形容词（作宾语补足语） But I was happy to help because I knew it was to realize our dream of making black and
make用法及短语总结
m a k e用法及短语总结 Document serial number【NL89WT-NY98YT-NC8CB-NNUUT-NUT108】
m a k e用法及短语一、make的复合宾语。英语中make一词用法甚多，是使用频率最高的动词之一，而make作使役动词的用法也很常见。意思是“使成为”、“使作为”、“使变成”，其后的复合宾语（即宾语+宾语补足语）有以下表达方式： 1. make +宾语+名词（作宾语补足语） Most pop singers make music their career. 大多数流行歌手把音乐当作他们的职业。 Bill Gates’ Microsoft makes him a phenomenon in the business world. 比尔·盖茨的微软使他成为商界的一个奇才。 What makes the ocean such a great place to live 究竟是什么东西使大海成为如此优越的生活场所呢 European football is played in 80 countries， making it the most popular sport in the world. 80个国家踢欧式足球，使它成为世界上最受欢迎的运动。 After all， what makes a new invention such a wonderful thing is that it allows us to do something we could not do before．毕竟，一项发明之所以成为如此奇妙的事情就在于它可以让我们做以前不能做的事。 2. make +宾语+不带to的不定式（作宾语语补足语） Nobody made us go to bed at a certain time. 没有人让我们在某一固定时间就寝。 Pop music makes people feel easy and forget about the real world； rock music makes people think about the world and how to make their life better． 流行音乐令人松弛安心，忘记这真实的世界，而摇滚乐使人思考这个世界和如何改善自己的生活。 Nothing can make me turn against my country．什么也不能使我背叛我的祖国。
溯源系统简明操作手册
溯源系统简明操作手册201311 版 目录 一、系统网址https://www.wendangku.net/doc/677830255.html,（注意：网址中无www） ------------------------------------------ 2 二、选择浏览器必须使用火狐或IE7、IE8、IE9浏览器------------------------------------------- 2 ( 一 )火狐下载地址https://www.wendangku.net/doc/677830255.html,/zHfrob5（注意大小写）--------------------------------------------------- 2 ( 二 ) IE 兼容性设置IE7 、 IE8、 IE9 浏览器（必须打开兼容性视图）----------------------------------------- 2 三、系统注册------------------------------------------------------------------------------------------------------------------------------ 3 四、进货登记（初次登记）---------------------------------------------------------------------------------------------------------- 4 五、完善基础数据----------------------------------------------------------------------------------------------------------------------- 5 ( 一 )供应商资料录入---------------------------------------------------------------------------------------------------------------- 5 ( 二 )采购品资料录入---------------------------------------------------------------------------------------------------------------- 6 ( 三 )采购品与供应商关联--------------------------------------------------------------------------------------------------------- 6 六、进货登记（日常登记）---------------------------------------------------------------------------------------------------------- 7 七、进货登记（批量导入）---------------------------------------------------------------------------------------------------------- 8 八、进货登记（数据共享方式）---------------------------------------------------------------------------------------------------- 9 ( 一 )选择或确认协议供应商------------------------------------------------------------------------------------------------------ 9 ( 二 )选择或确认协议门店------------------------------------------------------------------------------------------------------- 10 ( 三 )供应商配送登记-------------------------------------------------------------------------------------------------------------- 10 ( 四 )门店自动收货----------------------------------------------------------------------------------------------------------------- 11 九、其他信息的维护------------------------------------------------------------------------------------------------------------------ 11 十、供应商公示（新功能）-------------------------------------------------------------------------------------------------------- 11 ( 一 )准备工作：-------------------------------------------------------------------------------------------------------------------- 12 ( 二 )公示设置------------------------------------------------------------------------------------------------------------------------ 13 ( 三 )公示效果------------------------------------------------------------------------------------------------------------------------ 14 十一、技术支持--------------------------------------------------------------------------------------------------------------------- 14 ( 一 )溯源密码重置----------------------------------------------------------------------------------------------------------------- 14 ( 一 )电话报修------------------------------------------------------------------------------------------------------------------------ 15 ( 二 )网上报修------------------------------------------------------------------------------------------------------------------------ 15