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支付成功后，系统会自动生成账号（用户名为邮箱或者手机号，密码是验证码），方便下次登录下载和查询订单； 2、PDF文件下载后，可能会被浏览器默认打开，此种情况可以点击浏览器菜单，保存网页到桌面，既可以正常下载了。 105 J L Rombeau and D O Jacobs Nasoenteric Tube Feed ing in J L Rombeau and M D Caldwell eds Enteral and Tube Feeding W B Saunders Philadelphia Penn 1984 pp XX XX 106 A H McArdle et al A Rationale for Enteral Feeding as the Preferable Route for Hyperalimentation Surgery 90 613 623 1981 107 T Jones Enteral Feeding Techniques of Administration Gut 27 47 49 1986 108 C Page R Andrassay and J Sandier Techniques in Deliv ery of Liquid Diet Short Term and Long Term in M Deitel ed Nutrition in Clinical Surgery Williams conversely excessive time and effort may be expended in obtaining highly precise data to meet quick and dirty needs Understanding the basis of various kinds of assays will help in choosing the proper level of assay sophistica tion to avoid these two kinds of errors Characterization of Enzymes for Applications In industrial food applications the usual requirement of an enzyme is that it produce the desired functionality for the minimum cost This often implies that offerings by alter nate suppliers are assayed to find the best enzyme source for the process in hand The characterizing factors are 1 rate activity per gram of enzyme 2 pH optimum 3 temperature optimum 4 stability under conditions of use and 5 presence or absence of potentially deleterious side activities Rate By rate the food processor usually means the amount of modification obtained during the time allowed for enzyme action in the process This may be different from the initial rate of conversion of substrate to product as defined by an enzymologist An assay that measures the latter rate may be misleading if the modification occurs during extended incubation in the process pH Temperature The pH and temperature optimum curves published by suppliers usually confound the true influence of these factors on enzyme catalytic properties with the effect on enzyme stability Enzyme denaturation is influenced by numerous factors is usually irreversible and occurs with first order kinetics Optimum curves are constructed using assays that measure the amount of sub strate modification over a period of time and represent a summation of true rate effects plus denaturation during that time They should be used with caution Stability The presence of substrate stabilizes enzyme against denaturation thus the real optimum of interest is the stability of enzyme under the conditions of use time pH temperature substrate concentration inorganic ions organic solvents and so on An assay used to screen en zymes for a particular application should mimic as nearly as possible the actual use conditions to give a reliable es timate of cost benefit Side activities Preparing a pure isolated enzyme is cost prohibitive for food applications All commercial enzymes contain some activities other than those declared on the label for example fungal amylase usually contains some proteolytic activity a bacterial protease may also contain some xylanase and so on These side activities may pres ent a problem when the enzyme is used in certain food processing situations and assays should be applied that will detect them as well as the main enzyme of interest Characterization of Enzymes in Raw Materials Specificity Often enzymes with similar activities ie proteases are obtained from different sources and assays are used to characterize them in terms of units per gram If the different enzymes have varying specificity require ments the results may be misleading For example if two proteases have specificities corresponding to elastase and trypsin an assay based on azocollagen substrate will give a much higher value for the er enzyme while one based on casein will favor the latter If the protein to be modified is something quite different for example wheat gluten neither assay will give a reliable comparison Sub strate specificity may be of major importance as with pro teases or a negligible factor as with lipoxygenase It is best to assume that it is important until the contrary is established If an assay is used to monitor production of enzyme from biofermentation specificity may be overlooked Here the ideal is the quickest possible assay consistent with reason able accuracy making the assumption that enzyme speci ficity is constant This also depends on the conditions of biofermentation remaining constant particularly the pu rity of the microbiological innoculum being used A quick assay based on azocollagen may suffice even if the protease being made is similar to trypsin in its specificity Characterization of Enzyme Rate Parameters For a study made for the purpose of establishing basic en zymatic parameters catalytic rate constant affinity for substrate inhibitor binding etc the assay must provide appropriate velocity estimates In general this means the catalytic rate at time zero that is when the enzyme and substrate are first combined Assays involving incubation for a fixed length of time present some difficulties for this purpose Progress curves in which the concentration of product is measured at intervals during the incubation are valid and are not used as often as they might be to determine enzymatic parameters THEORETICAL ASPECTS OF ENZYME ASSAYS Properly judging the nature of the desired assay requires a certain amount of theoretical understanding This does not have to be in great depth the requisite fundamentals are easily grasped and applied The effort will be repaid by improved enzyme assays for routine work and process de sign 1 Assay Characteristics A sound assay regardless of the level of sophistication re quired will have 1 linear dependence on enzyme concen tration 2 adequate consideration of pH and temperature effects 3 appropriate accuracy 4 adequate sensitivity and 5 speed and ease of perance Unfortunately many assays emphasize the last factor at the expense of the other four Enzyme Linearity Enzyme linearity is paramount A curved plot of assay response versus amount of enzyme used indicates that some chemical physical and or en zymological factors have been overlooked In fixed time as says the ation of product is often not strictly linear with time because of substrate depletion or product inhi bition and this nonlinearity is more pronounced at higher enzyme concentrations Occasionally the chemical reaction used to quantitate the amount of product ed is not stoichiometric leading to a nonlinear plot of for example spectrophotometric absorbance versus enzyme amount Numerous nonlinear assays found in the literature indi cate an incomplete understanding of the system being used for the assay Many assays are linear over a limited range of activity These may be used particularly if they meet the require ment for ease of use if care is taken to ensure that mea surements are made only within the linear range Temperature pH Elevated temperatures and extremes of pH contribute to enzyme denaturation during the assay Temperature optimum curves are always due to this phe nomenon and activity decreases at high or low pH also may be due to enzyme instability The pH also may affect enzyme catalytic activity influencing the ionization state of the active site and possibly the substrate 2 4 A sound assay will take these factors into account Accuracy Sensitivity The required levels of accuracy more properly precision and sensitivity should be care fully considered Measurements of a amylase activity may have a coefficient of variation of 2 or of 10 depending on whether the assay is a replicated colorimetric one using a modified substrate 5 or a viscometric one using gelati nized ground grain 6 For standardizing a purified amy lase for use in bread production the er would be ap propriate while for identifying bins of wheat that has been subjected to sprouting the latter is quite ade quate Likewise assays at almost any level of sensitivity may be constructed Using a fluorescent substrate pico molar concentrations of trypsin may be assayed 7 while in monitoring the production of microbial protease a sim ple protein based assay 8 will do the job although it is some five orders of magnitude less sensitive Convenience Speed and ease of perance should be the last factors considered These are important in many industrial contexts but a fast easy inadequate assay will only result in the rapid generation of much useless data After linearity precision enzyme stability and sensitivity are established then steps may be taken to increase out put Initial Rates Most assays measure the rate of ation of product from substrate that is d P dt This measure ment may be of the initial rate d P dt at the initiation of the reaction or of the amount of product ed during a fixed time of incubation of substrate with enzyme Fixed time assays are convenient in that a large number of sam ples may be run simultaneously Unfortunately they are also more prone to complications leading to the nonline arity mentioned above Accurate initial rate measure ments are not contaminated by effects such as substrate depletion product inhibition or enzyme denaturation However they are not always easy to obtain A relatively simple of finding the initial rate is the following Set up the assay system take samples at various times t and measure product concentration P at each time Usu ally there is a slight downward curve of the plot of P ver sus t Fig 1 that makes the determination of the tangent d P ldt at zero time difficult Using a simple program Ba sic or even most spreadsheet programs today are capable of this fit the data points with a least squares quadratic curve P a b X t c X t2 The first derivative gives the desired initial rate with rea sonable accuracy d P ldt b 2c X and d P dt t 0 b Determining Rate Parameters Michaelis Menten Parameters In designing assays it is useful to know the maximum rate obtainable with a given amount of enzyme Vmax and the concentration of sub strate that gives half that rate M These are the funda mental parameters in the Michaelis Menten M M rate Figure 1 Nonlinearity of product ation with time due to various factors Initial rate for the theoretical reaction is 0 5 Fit ting the nonlinear rates with a second degree polynomial the cal culated initial rates are Substrate depletion 0 51 Product inhi bition 0 51 Enzyme denaturation 0 44 If the curves are fitted to a third degree polynomial the calculated rates are 0 50 0 50 and 0 49 respectively Source Ref 1 p 93 equation v Vmax S KM S where S is substrate concentration and i is the actual rate d P dt The usual procedure is to measure v at several concentrations S and then calculate Vmax and KM either by applying a computer program 9 such as HYPER Fig 2a or by fitting a straight line to one of the linear transs of the M M equation The usual trans is the double reciprocal or Lineweaver Burk plot l v Wmax Ku Vmax l S where l v is plotted versus 1 S Fig 2b From statistical considerations this is the least desirable trans to use 11 A better plot is the Hanes plot of S v versus S Fig 2c S v KMJVmax l Vmax S The points are spaced along the x axis at the same intervals as in the M M plot rather than being crowded together near the y axis The larger experimental errors inherent in the smaller values of v at low S have less influence on the linear least squares regression line The Hanes plot should be used in treating v S data graphically the use of the unsatisfac tory Lineweaver Burk plot should be discontinued The M M equation is a differential equation that is i equals d P dt at the instantaneous value of S usually taken as the initial substrate concentration If v is only available as P It from a fixed time assay then the value taken for S for the above calculations should be the av erage of the substrate concentration at the beginning and end of the incubation period S 0 S t 2 This approx imation gives estimates of Vmax and KM that are much closer to the true values than if the initial value of S is used 12 Inhibitors Two types of enzyme inhibitors are of interest to food sci entists 7 low affinity inhibitors and 2 high affinity in hibitors The er are effective in the millimolar to mi cromolar concentration range and readily dissociate from the enzyme an example is inorganic phosphate inhibiting phytase The latter are effective in the nanomolar to pi comolar concentration range and are bound tightly to the enzyme an example is soybean trypsin inhibitor For low affinity inhibitors the dissociation constant K1 is a useful parameter to know for high affinity inhibitors the amount present is usually of more concern Inhibition Model A general equilibrium model of inhi bition is shown in Figure 3 If the parameter a is very large so that the species EIS does not exist the inhibition is termed competitive inhibitor competes with S for the en zyme The effect shown in Figure 4a is that Vmax is un changed and KM increases If a I and O EIS is ed but does not proceed to product P noncompetitive inhibition results As shown in Figure 4a Vmax decreases and KM is unchanged If a is greater than 1 but not ex tremely large mixed inhibition occurs These types are di agnosed by comparing the Hanes plots in the absence and presence of inhibitor Figure 4b Inhibitor Constant If the inhibition is competitive KM is determined from the ratio of Kapp the apparent value of K in the presence of inhibitor of concentration to KM no inhibitor present Kapp KM I I JK1 If inhibition is noncompetitive the ratio of true maximum velocity to apparent maximum velocity in the presence of inhibitor gives Vmax Vapp 1 1 I JK1 Determining K1 a and in the cases of partial and mixed inhibition is too complex to be discussed here High Affinity Inhibitors Measuring the concentration of high affinity inhibitors eg soy trypsin inhibitor is rela tively straightforward 13 Trypsin is mixed with aliquots of soy meal extract and after a brief incubation for for mation of the inhibitor enzyme complex the amount of un inhibited enzyme remaining is measured by a simple as say The rate of reaction is plotted versus the size of the extract aliquot the straight line through the data obtained at lower levels of inhibition intersects the x axis at a point where the amount of enzyme equals the amount of inhib itor present Fig 5 14 In the example shown 1 2 mL of soy meal extract contained a molar amount of trypsin in hibitor equal to the number of moles of trypsin used in each assay tube If the absolute amount of trypsin is established by a titration assay the amount of soy trypsin inhibitor can be expressed in absolute molar units rather than ar bitrary trypsin inhibitor units 15 Conversely if the ab solute concentration of high affinity inhibitor is known this serves to measure the molar amount of en zyme present Endogenous Inhibitors Endogenous inhibitors may be present in crude extracts of materials containing the en zyme being assayed resulting in a marked nonlinearity in the assay Fig 6a The uninhibited rate may be found as follows 1 Let e be the amount of enzyme in the largest aliquot of extract used in making the plot of Figure 6 X is the fraction of that aliquot used for each of the other points Substrate depletion Product inhibition Denaturation Theoretical line Figure 3 General equilibrium model for reversible enzyme in hibition Source Ref 1 p 38 The measured rate at each point is vt A plot is made of XIvi versus X Fig 6b From the y axis intercept calculate the uninhibited rate that is I intercept equals the true rate due to enzyme concentration e This is useful for comparing enzyme amounts from different sources and during enzyme purification until the inhibitor has been re moved SPECIFIC ENZYME ASSAYS Several assays for enzymes most commonly measured by food scientists will be described These are only examples of the wide range of ingenious s that have been re ported for following the rate of product ation by food related enzymes In most cases factors such as pH tem perature activating ions time of reaction and detection s may be adjusted to fit the specific needs of the project in hand Thus these should be considered as start ing points for designing assays to meet particular require ments not the only way to measure the enzyme activity under investigation Proteases and Peptidases Substrates Protein substrates for proteases are most often hemoglobin or casein and must be completely soluble in buffer Casein for protease assays is designated nach Hammarsten while hemoglobin for protease assay is usually of high quality Casein precipitates below pH 6 so it is used at neutral to alkaline pH Hemoglobin must be denatured before use either by treatment with acid if the assay is at acidic pH 16 or urea neutral to alkaline pH assay 17 Gelatin sometimes used in viscometric assays is quite heterogeneous so lot to lot reproducibility is a con cern Proteins may be derivatized to fit assay needs Diazo tized protein allows measurement of solubilized peptide with a visible range colorimeter 18 If the amino groups freed during hydrolysis are quantitated using for example Figure 2 Determining Vmax and KM from experimental data using three different s a HYPER computer program Vmax 58 55 KM 17 44 Std Dev 1 65 b Lineweaver Burk plot ymax 49 60 KM 12 85 Std Dev 2 54 c Hanes plot Vmax 58 96 KM 17 56 Std Dev 1 66 Source Data from Ref 10 Substrate a Rate Inhibitor ml Figure 5 Plot of enzyme rates in presence of high affinity soy trypsin inhibitor Source Data from Ref 14 TNBS trinitrobenzenesulfonic acid the e amino groups of lysine give a high blank value that may be removed by making the succinyl 19 or V Af dimethyl 20 derivative of the protein The latter is preferable for trypsinlike pro teases Small synthetic molecules are also useful for assaying proteases These give a change in spectrophotometric ab sorbance as they are hydrolyzed so a continuous assay with its advantages is possible Table 1 lists a number of small molecule substrates Most of these are applicable to serine and or sulfhydryl proteases with two exceptions FAGLA is a substrate for metalloprotease neutral prote ase and Z Gly Phe is a carboxypeptidase substrate Acidic protease may be assayed using a chromogenic peptide Aminopeptidase is usually assayed using an amino acid derivative such as L leucine naphthylamide Protease Assays Most assays with protein substrates involve incubation for a set length of time stopping the reaction with TCA trichloroacetic acid and measuring the amount of soluble peptide Buffered TCA 0 11 MTCA 18 g L 0 22 M sodium acetate 18 g L and 0 33 M acetic acid 19 8 g L gives superior enzyme linearity compared Figure 6 for finding uninhib ited rate in the presence of endogenous inhibitor Source Ref 1 p 109 Figure 4 Hyperbolic and Hanes plots showing different modes of in hibition Substrate concentration a Enzyme xL a No Inhibitor Endogenous Inhibitor No inhibitor Noncompetitive Competitive Mixed No inhibitor Competitive Noncompetitive Rate Rate Rate vt with a simple aqueous TCA solution 8 Quantitation may be direct absorbance of filtrate at 275 nm or colorimetric Folin Lowry 28 bicinchoninic acid 29 or TNBS 3O An example of a casein based assay is the following 8 To 5 mL casein 12 mg mL in 0 03 M phosphate buffer pH 7 5 add 1 mL enzyme solution and after 10 min reaction add 5 mL of buffered TCA After 30 min the mixture is filtered and absorbance at 275 nm is measured For Folin Lowry quantitation to 1 mL of nitrate add 1 mL alkaline buffer 1M Na2CO3 0 25 M NaOH 0 4 mL copper reagent 0 1 CuSO4 5H2O 0 2 NaK tartrate mix and allow to stand 10 min Then add 0 75 mL diluted phenol reagent Folin Ciocalteau reagent diluted with 3 volumes H2O mix wait 10 min and measure absorbance at 700 nm ver sus an appropriate reagent blank A plot of log absorbance versus log protein is linear over the range 3 to 400 jug of protein eg bovine serum albumin standard 31 An assay using TNBS to measure freed amino groups 30 uses 7V Af dimethyl 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