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**Effect
of
hardness
and
detergent
on
enzymatic
catalysis
**

........... 2 Methods ......................... 9 ..........................................Table of Contents Technical University of Denmark Dept of Mathematics and Computer Summary .................................................................................................................................................................................................................................................................................................................... 1 Introduction ................................................................................................................................................................................................................................................................................................................................................................................................................................................................ 6 Conclusion .............. 1 Data ................................................ 3 Results ................................................................... 8 Appendix II .................................................................................................................... 7 Appendix I..................................................................................................... 4 Discussion ..................................................................................................

To optimize the effect of removing certain stains on textile surfaces the effect is analyzed by means of a laboratory experiment. The aim of the analysis is to describe and compare the performance of the enzymes and how different conditions affect enzyme performance. concentration of enzymes and enzyme type is of significant importance in stain removal processes. It is known that factors. The experiment measures how much protein is removed from a surface with “Surface Plasmon Resonance technology (SPR)”.Technical University of Denmark Dept of Mathematics and Computer 1 2 3 4 5 6 7 8 9 10 11 Summary Different enzymes improve textile washing. 1|Page . Adding more protein to samples also has an effect on protein removal. determining whether the day to day measurements are reliable and testing robustness of the experimental set-‐up. such as detergent and water hardness affect this catalyzation rate. and a range of enzymatic concentrations. and it is therefore of interest to quantify these effects. Enzymatic activity enhances this process by means of catalyzation. and translates it into a protein removal response. We wish to use statistical methods to examine differences and trends in the samples. We conclude that addition of detergent. Using the 15 nM enzyme concentration and addition of detergent shows that protein A removes proteins of surfaces significantly better than the other proteins included. A replication of each experiment yields a grand total of 160 different samples. hard and soft water. We construct a model using variance analysis that accounts for 90% of the variance in the data. This analysis includes factorial interactions. The conditions are addition of detergent. The performance of five enzymes was measured as the amount of protein removed from a surface when exposed to the factors hardness and detergent. and that addition of detergent yields the highest protein removal. These factors are some of the conditions that appear in normal laundry wash processes. but the effect is saturating. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Introduction Efficient protein removal is of vital importance in the textile cleaning industry. which is a biosensor that measures a resonance signal on a gold surface. We show that hardness of water has no significant influence on enzyme performance. and it is of interest to know performance under various conditions. The experiment is conducted using 5 different proteins under different conditions.

The output is response which is given as the amount of protein removed in RU (10-‐6 g m-‐2). Figure 1 shows the log10(Response) together with detergent and hardness. so each specific enzyme is labeled with a single date. 2|Page . C. A summary of all data included in our analysis can be seen in Table 1. De0. Data The dataset consists of 160 samples. for detergent and calcium respectively. Green = 7.5 nM and Blue = 15 nM. 2. Red = 2. Black = 0 nM. Ca+ and Ca0. The enzyme concentrations were tested under 4 different levels: 0 nM. Both these variables are binary and denoted De+. B.5 nM and 15 nM. No enzymes were run the same day.5 nM. with 80 different experimental combinations. D and E and analyzed one at a time. Increasing concentration increases the response. There is some indication in the data that the enzyme catalyzation gets saturated at the high concentrations (Figure 2). Increasing the concentration of enzymes in the samples also increases the response. but there is some saturation towards the high concentrations. 7. There does not seem to be a correlation between hardness and response. The enzymes were labeled A. B) The response on hardness of water. the result is sampled randomly into a variable called cycles.5 nM. with the experiment running for 2 consecutive days. extrapolation beyond existing data points is very uncertain.Technical University of Denmark Dept of Mathematics and Computer 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Figure 1: A) Boxplot of protein response with detergent+ and detergent0. and Figure 2: Response vs concentration of the 5 different enzymes. Detergent plus alone seems to have a positive effect on the response. After the experiment has run. This may potentially yield two problems: fitting the data as a linear model gives more imprecise results at high concentrations. The factors included in the data are hardness of water and addition of detergent.

With Detergent (Det+) or without (Det0). Det0 Ca+. 2. with the exception of a single outlier: Enzyme B. In order to deal with this we log transformed the response variable before making the model. C.3 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 Methods Our first approach was to assume the level of response is given as a function of the various factors and enzymes.Technical University of Denmark Dept of Mathematics and Computer 53 54 55 Table 1: Table of variables and response variable in the model. Initially we assumed that there was no bias in the response measurements for neither cycle nor days. if this is needed in the future.4 Levels 10 34 5 6. D. These figures revealed some inconsistencies in variance. have constant variance and are normal distributed. 3|Page . B. E Variance 33.0 154254. This gave acceptable distributions. An ANCOVA analysis of data was conducted using two-way interactions. Hardness: with ½(Ca+) or without (Ca0) Variable Name Variables Run Date Cycle Enzyme Type Enzyme Concentration Detergent Hardness Output Response Type Categorical Categorical Categorical Continuous Categorical Categorical Continuous Mean Categories 3/12/2008 5/12/2008 … 1. and Cooks distance for the observations (Appendix 1).3 Det+. To verify this we inspected the fit with residuals. a QQ-normality plot. αi is the intercept of the model with factor i. as this allowed us to interpolate between levels. Ca0 2 2 434. When subsetting data and only test for one factorial variable we use a regular ANOVA 𝑌!" = 𝛼! + 𝜀!" (2) The assumptions for these models are that residuals are independent. 3 … 34 A. β is the slope in the model and Xj are the observations in the data. ε is noise. Concentration was used as a continuous variable. The equation for ANCOVA is 𝑌!" = 𝛼! + 𝛽! 𝑋! + 𝜀!" (1) Where Y is the Response variable.

1. we used a more graphical approach. As we already concluded that detergent significantly influenced data. Enzyme B is apparently a little less efficient relative to the others when detergent is added. Enzyme Concentration and Detergent.Technical University of Denmark Dept of Mathematics and Computer 75 76 77 78 concentration = 0. The model showed that Hardness had no significant effect (p > 0.5.327 0. enzyme type and concentration significantly influence protein removal.001.30). Det0 and Ca0 looks biased in the response.14. All statistical analysis was performed using R 2.33E-‐14*** 2. Detergent.01. Significance levels: *** p < 0.932 15. 79 80 81 82 83 84 85 86 87 88 89 90 Results We tested a multi-way ANCOVA model with log-response as a function of the continuous variable concentration. * p < 0. and was thus removed from the data set.54E-‐03** 3. Hardness and all their two way interactions. P-values and a summary of the model are shown in table II.31E-‐44*** 3. Figure 3 shows that some of the enzymes react slightly different to addition of detergent. This concludes that detergent. Enzyme Type Enzyme Concentration Detergent Enzyme:Detergent Enzyme Concentration:Detergent Degrees of Sum of P-‐value freedom squares 4 1 1 4 1 3.33E-‐27*** 1. and the 3 categorical variables Enzyme. Table 2: Results from the ANCOVA model including Enzyme type.768 1. scripts and analysis are documented in Appendix II. ** p < 0. The final model explains 89% of the variation in the data. The sum of squares value shows that the most important factor to remove protein is detergent.61E-‐13*** 91 92 93 94 95 96 97 Secondly we wanted to determine whether or not the interactions where the same for all enzymes included in the model.711 2. The interactions are denoted with a “:”.29) on response. As the difference between the curves are not the same with and without detergent implies that not all the proteins interact the same way with the factor.611 31. After removing Hardness as a factor the model showed that the two-way interaction between Enzyme type and Enzyme concentration had no effect either (p > 0. 4|Page .

** p < 0. we also tested the response compared to the random variable.00E-‐05 *** 8. Significance levels: *** p < 0. with the optimal conditions for protein removal. 98 99 100 101 102 103 104 105 106 107 108 The next step was to investigate if one enzyme was significantly removing proteins better than the others. The summary of all the enzymes can be seen in Table 3.Technical University of Denmark Dept of Mathematics and Computer Figure 3: log10 response vs enzyme concentration for the 5 different enzymes. with a possible minimum of 1391.5. Table 3 The 5 enzymes compared to enzyme A with 15nM added. In comparison the worst enzyme was D with a mean protein removal of 663.87E-‐05 *** 109 110 111 112 113 114 5|Page Analysis of experimental bias To investigate if there was any systematic error in the data sampling.15E-‐06 *** 1.5% 1392 893 932 605 1003 97. Given is also the mean and the 95% confidence interval.01.7 and a maximum of 1676. To do so we split up the data set into a smaller group.39E-‐10 *** 8. Furthermore we tested day to day variation by testing the samples which has 0 nM concentration of enzymes against one another.5% 1676 1076 1122 728 1208 P-‐value 3. Structural differences between the two plots suggest stronger interactions between some enzymes with detergent than others.5 RU. which also states the p-values compared to enzyme A.2 on a 95% confidence interval. B) without detergent added. Enzyme A Enzyme B Enzyme C Enzyme D Enzyme E Estimate 1527 980 1023 663 1101 2. with detergent added and 15nM enzyme added. ANOVA shows that all the other enzymes are significantly different from A. . A) with detergent added to the sample.001. With an ANOVA-test we were able to determine that enzyme A was the best enzyme with a mean protein removal of 1527.3 RU. cycle. * p < 0.

and comparing it to a model with concentration as a continuous variable . which yielded the possibility of using the variable as a factor or as a continuous variable. This was done in order to be able to interpolate between experiments with different concentration. As all cycles are sampled randomly. in spite of the fact that using it as a factor gave a significantly better model. In order for our analysis to be valid in terms of hardness and detergent in the water. and therefore the credibility of the results.0013). The addition of the reference samples would also be helpful in this particular problem. The analysis shows that enzyme types are significantly different (p < 0. If the main purpose of the experiment is to determine the catalytic capabilities of enzymes. Using concentration as factor instead of continuous variable Enzyme concentration was given at four different levels. there should be no significant difference between the cycles. and comparing them with an ANOVA shows that using concentration as a factor gives a significantly better fit (p < 1. and with concentration 0.47).31E-21). using hardness. Concentration was used as a continuous variable instead of a factor. In order to make a better model it should be considered to make a model more appropriate than a linear fit for enzyme kinetics. this strongly indicates experimental bias in the setup. which apparently looks more like a Michaelis-Menten saturation model.Technical University of Denmark Dept of Mathematics and Computer 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 To test if cycle sampling influenced the deterioration of proteins we made an ANOVA with cycle as explanatory variable. Making a model with concentration as a factor. or sampling error at the day enzyme B was analyzed. The day-to-day runs were explored by an ANCOVA analysis. Notably the sample or experimental bias within day B yields high uncertainty. More data would have given us more samples to do the statistically analysis and check for errors within the different groups. The implication and possible solutions of this are evaluated in the discussion. In the previous section we have used it as a factor. 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 Discussion The few data points for each group give large statistical uncertainty in the validation of the data. 6|Page . the addition of less detergent would be appropriate. in order to be able to extrapolate the model. As the samples should be equal (no enzyme added). This turns out to be true as the difference between cycles is insignificant (p > 0. With the data set available it would be reasonable to adjust data to an expected mean with 0 enzyme added. Enzyme type and detergent as explanatory variables. as it turns out to be the main factor explaining protein degradation from surfaces. we assume that the concentrations of these two factors are the same in all observations.

enzyme concentration and enzyme type has a highly significant positive effect. All five types of enzyme have a significant reaction with detergent. Their interactions are also significant. We also conclude that enzyme A has the highest catalytic capabilities under optimal conditions. with the interaction between concentration and enzyme type omitted. Oppositely detergent. 7|Page .Technical University of Denmark Dept of Mathematics and Computer 148 149 150 Future work with the data would include a more thorough analysis of interactions with the individual enzymes. and that enzyme D is the worst enzyme for removing proteins. but to what degree is important knowledge. as well as with concentration. 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 Conclusion We conclude that hardness does not have an effect on protein degradation in our experiment.

Technical University of Denmark Dept of Mathematics and Computer 181 182 183 184 185 186 187 188 8|Page .179 180 Appendix I Figure 1 shows the residuals of the log transformed analysis.

table('SPR. data = mydata) m <.read.'0')) # Make Enzyme a factor for extra model determination ##################################### Make a linear model of all variables ####################### fm <. # Script for case 1 detergent Technical University of Denmark Dept of Mathematics and Computer ################################# Load and view data ########################### setwd("~/My Dropbox/02441 Applied Statistics and Statistical Software/Case_1") # Set working directory graphics.factor(mydata$EnzymeConc .factor(mydata$Cycle.'2.mydata[-14.txt'.3]) # Log response for equal variance mydata <.log10(mydata[.lm(lResponse ~ (Enzyme+EnzymeConc+DetStock)^2 .'factor')) #################################### Manipulate data for cleaner analysis ######################### mydata$Cycle <.lm(lResponse ~ (Enzyme+EnzymeConc+DetStock+CaStock)^2 .189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 Appendix II R-code used to calculate statistics in the report.lm(lResponse ~ (Enzyme+EnzymeConcF+DetStock)^2-Enzyme:EnzymeConcF.'numeric'. 'numeric'.] # Remove outlier 14 mydata$EnzymeConcF <.5'.summary(fm) # See and assign summary anova(fm) # Make ANCOVA to see #### Leave calcium out of the final model.'factor'.'factor'.fmfac.'7. data = mydata) summary(fm1) an <. 'factor'.anova(fmfac) aF anf <. header = TRUE.5'. colClasses = c('factor'. data = mydata) summary(fmfac) aF <. also the interaction betweeen EnzCon and Enz ##### fm1 <.off() mydata <.levels = c('15'.Enzyme:EnzymeConc.anova(fm1) an ##### Use concentration as a factor to compare the two models ######## fmfac <.anova(fm1. levels = 1:34) # Put cycles conscutively mydata$lResponse <. test = 'F') anf #### Using Concentration as a factor describes data significantly better ####################### #### Show graphically that the data have equal variance and are normally distributed #### 9|Page .

which = 1:4) # plot the model.2. col = cols[4]) points(lResponse ~ EnzymeConc. data = mydata.6] == 'Det+'.5] == '15') # Make a linear model.2)) # 4 subplots. data = mydata. data = mydata .subset = mydata[.off() cols <.subset = mydata[.summary(fmHigh) # Put summary into a variable ints <.off() # Remove existing graphics windows(width = 7) # Make a new window 7 inches wide par(mfrow=c(2.4] == 'A'. data = mydata.subset = mydata[.lm(lResponse ~ Enzyme -1.5] == '0') summary(fm0) anova(fm0) ############################ There is significantly differences between the days with 0 enzyme ### ########################## See if the enzymes differ under optimal conditions #################### mydatadet <. data = mydata. data = mydatadet.confint(fmHigh) # Calculate the 95% confidence anova(fmHigh) # There is difference among enzymes mens <. data = mydata. col = cols[3]) points(lResponse ~ EnzymeConc. and get all intercepts (remove -1 for more usable model).4] == 'A'.mydata[mydata[. subset = mydatadet[.1)) # Make margins as small as possible plot(fm1.subset = mydata[. col = cols[5]) ############################ Looks as a saturating function ################################# ########################## Test if the days differ with 0 protein added ##################### fm0 <. type = 'n') points(lResponse ~ EnzymeConc. data = enzA) summary(fmA) anova(fmA) 10 | P a g e .1:5 plot(lResponse ~ EnzymeConc.4] == 'D'.229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 Technical University of Denmark Dept of Mathematics and Computer graphics. data = mydata. with simplified cooks distance ################################ See the response as a function of enzyme concentration ##### graphics.subset = mydata[.4] == 'E'.lm(lResponse ~ (Enzyme+CaStock+DetStock)^2.mydata[mydata[.4.lm(lResponse ~ (DetStock + EnzymeConc)^2. in a 2by2 matrix par(mar = c(5.subset = mydata[.fmHigh$coefficient # Retrieve means ####################### A is significantly better than all the other enzymes ###################### ####################### Do the enzymes interact the same way between effects ######### enzA <.4] == 'B'. col = cols[2]) points(lResponse ~ EnzymeConc.] # Find all the data with Det+ fmHigh <. col = cols[1]) #abline(afm[1].] fmA <. Subset 15 nM p <. afm[2]) points(lResponse ~ EnzymeConc.4] == 'C'.

lm(lResponse ~ (DetStock + EnzymeConc)^2.5.5.4] == 'B'.tapply(mydata$lResponse.lm(lResponse ~ (DetStock + EnzymeConc)^2. data = enzC) summary(fmC) anova(fmC) enzD <. xlab = 'Enzyme' .4. ylim = c(0.] fmD <.mydata[mydata[.] fmB <.height= 2) par(mar=c(2.levels(mydata$EnzymeConcF) enZ <.mydata[mydata[.mydata$Enzyme).] fmE <.4] == 'E'.0.off() cols <. ylab = 'log10 Response'. beside = TRUE.lm(lResponse ~ (DetStock + EnzymeConc)^2.4] == 'C'. data = enzB) summary(fmB) anova(fmB) enzC <.5) for (i in 1:4) 11 | P a g e .mydata[mydata[.1:4 windows(width = 3.matrix(0.8.] lvl <.levels(mydata$Enzyme) Detline <. data = enzE) summary(fmE) anova(fmE) ############################ They are all significantly correlated with both effects ######## ########################### Plot different figures ########################################## graphics.] fmC <.mydata[mydata[.4.270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 Technical University of Denmark Dept of Mathematics and Computer enzB <.6] == 'Det+'.mean) barplot(dat.lm(lResponse ~ (DetStock + EnzymeConc)^2.3)) abline(0.4] == 'D'.0) # Bar plot of concentration vs response for all 5 enzymes #################### Detergent figures ####################################### mydatadet <.5.list(mydata$EnzymeConc.0.mydata[mydata[. data = enzD) summary(fmD) anova(fmD) enzE <. col = cols.5)) dat <.

ylab = '') for (i in 1:5) { points(lvl.mydatadet[mydatadet[.1)) plot(lResponse ~ EnzymeConc.j] <.3.j] <. type = 'o'.3.5] ==lvl[i]. data = mydatadet . ylim = c(0.5] ==lvl[i].lvl colnames(Detline) <. labels = 'B') ## Detergent plots! ########### Make a box plot of response vs addition of detergent and hardness ################ 12 | P a g e .mydatadet[mydatadet[. type = 'n'.Detline[.i].311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 Technical University of Denmark Dept of Mathematics and Computer for (j in 1:5){{ sub1 <.enZ mydatadet <.sub1[. ylab = 'log10 Response (RU)') for (i in 1:5) { points(lvl.] Detline2[i.sub1[.matrix(0.4).mean(subset(sub1$lResponse. data = mydatadet .5) for (i in 1:4) for (j in 1:5){{ sub1 <.2.2)) par(mar= c(4.5.3.4). col = i. xlab = 'Enzyme Concentration (nM)'.mean(subset(sub1$lResponse.i]. pch = 16) } legend('bottomright'.mydata[mydata[.2. horiz=TRUE. main = 'Without detergent'. type = 'n'. xlab = 'Enzyme Concentration (nM)'.4] == enZ[j])) }} rownames(Detline) <. ylim = c(0.5.6] == 'Det0'.enZ. cex = 0. fill = 1:5.3.off() windows(width=7. type = 'o'.2.3.8. main = 'With detergent'.3.] Detline[i. title = 'Enzyme') text(0.enZ graphics.lvl colnames(Detline2) <.4.] Detline2 <.5) par(mfrow=c(1.4] == enZ[j])) }} rownames(Detline2) <. labels = 'A') plot(lResponse ~ EnzymeConc. col = i.4. pch = 16) } text(0.3.Detline2[.

data = mydata.1. offset=2) 13 | P a g e . xlab = 'Hardness'. ylab = 'log10 Response (RU)') text(mydata$DetStock[1].labels = 'A') plot(lResponse ~ CaStock.1.8. pos = 4.5. data = mydata. labels ='B'.0. ylab = '') text(mydata$CaStock[1].5) par(mfrow=c(1.off() windows(width=7.4.1)) plot(lResponse ~ DetStock.352 353 354 355 356 357 358 359 360 361 362 363 Technical University of Denmark Dept of Mathematics and Computer graphics. height = 2.2)) par(mar=c(2.5.

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