Applied

Statistics and Statistical Software, 02441

Effect of hardness and detergent on enzymatic catalysis

Table of Contents

Technical University of Denmark Dept of Mathematics and Computer

Summary .............................................................................................................................................. 1 Introduction .......................................................................................................................................... 1 Data ...................................................................................................................................................... 2 Methods ................................................................................................................................................ 3 Results .................................................................................................................................................. 4 Discussion ............................................................................................................................................ 6 Conclusion ........................................................................................................................................... 7 Appendix I............................................................................................................................................ 8 Appendix II .......................................................................................................................................... 9

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Summary
Different enzymes improve textile washing, and it is of interest to know performance under various conditions. The performance of five enzymes was measured as the amount of protein removed from a surface when exposed to the factors hardness and detergent. We construct a model using variance analysis that accounts for 90% of the variance in the data. We show that hardness of water has no significant influence on enzyme performance, and that addition of detergent yields the highest protein removal. Adding more protein to samples also has an effect on protein removal, but the effect is saturating. 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. We conclude that addition of detergent, concentration of enzymes and enzyme type is of significant importance in stain removal processes.

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Introduction
Efficient protein removal is of vital importance in the textile cleaning industry. Enzymatic activity enhances this process by means of catalyzation. It is known that factors, such as detergent and water hardness affect this catalyzation rate, and it is therefore of interest to quantify these effects. These factors are some of the conditions that appear in normal laundry wash processes. To optimize the effect of removing certain stains on textile surfaces the effect is analyzed by means of a laboratory experiment. The experiment measures how much protein is removed from a surface with Surface Plasmon Resonance technology (SPR), which is a biosensor that measures a resonance signal on a gold surface, and translates it into a protein removal response. The experiment is conducted using 5 different proteins under different conditions. The conditions are addition of detergent, hard and soft water, and a range of enzymatic concentrations. A replication of each experiment yields a grand total of 160 different samples. We wish to use statistical methods to examine differences and trends in the samples. The aim of the analysis is to describe and compare the performance of the enzymes and how different conditions affect enzyme performance. This analysis includes factorial interactions, determining whether the day to day measurements are reliable and testing robustness of the experimental set-up.

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Figure 1: A) Boxplot of protein response with detergent+ and detergent0. Detergent plus alone seems to have a positive effect on the response. B) The response on hardness of water. There does not seem to be a correlation between hardness and response.

Data
The dataset consists of 160 samples, with 80 different experimental combinations. The enzymes were labeled A, B, C, D and E and analyzed one at a time, with the experiment running for 2 consecutive days. No enzymes were run the same day, so each specific enzyme is labeled with a single date. After the experiment has run, the result is sampled randomly into a variable called cycles. The output is response which is given as the amount of protein removed in RU (10-6 g m-2). The factors included in the data are hardness of water and addition of detergent. Both these variables are binary and denoted De+, De0, Ca+ and Ca0, for detergent and calcium respectively. Figure 1 shows the log10(Response) together with detergent and hardness. The enzyme concentrations were tested under 4 different levels: 0 nM, 2.5 nM, 7.5 nM and 15 nM. Increasing the concentration of enzymes in the samples also increases the response. There is some indication in the data that the enzyme catalyzation gets saturated at the high concentrations (Figure 2). This may potentially yield two problems: fitting the data as a linear model gives more imprecise results at high concentrations, and
Figure 2: Response vs concentration of the 5 different enzymes. Black = 0 nM, Red = 2.5 nM, Green = 7.5 nM and Blue = 15 nM. Increasing concentration increases the response, but there is some saturation towards the high concentrations.

extrapolation beyond existing data points is very uncertain. A summary of all data included in our analysis can be seen in Table 1. 2|Page

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Table 1: Table of variables and response variable in the model. With Detergent (Det+) or without (Det0). Hardness: with (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, 2, 3 34 A, B, C, D, E

Variance 33.0 154254.4

Levels 10 34 5

6.3

Det+, Det0 Ca+, Ca0

2 2

434.3

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Methods
Our first approach was to assume the level of response is given as a function of the various factors and enzymes. Initially we assumed that there was no bias in the response measurements for neither cycle nor days. Concentration was used as a continuous variable, as this allowed us to interpolate between levels, if this is needed in the future. An ANCOVA analysis of data was conducted using two-way interactions. The equation for ANCOVA is

!" = ! + ! ! + !"

(1)

Where Y is the Response variable, i is the intercept of the model with factor i. is the slope in the model and Xj are the observations in the data. is noise. When subsetting data and only test for one factorial variable we use a regular ANOVA !" = ! + !" (2)

The assumptions for these models are that residuals are independent, have constant variance and are normal distributed. To verify this we inspected the fit with residuals, a QQ-normality plot, and Cooks distance for the observations (Appendix 1). These figures revealed some inconsistencies in variance. In order to deal with this we log transformed the response variable before making the model. This gave acceptable distributions, with the exception of a single outlier: Enzyme B, 3|Page

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concentration = 0, Det0 and Ca0 looks biased in the response, and was thus removed from the data set. All statistical analysis was performed using R 2.14.1, scripts and analysis are documented in Appendix II.

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Results
We tested a multi-way ANCOVA model with log-response as a function of the continuous variable concentration, and the 3 categorical variables Enzyme, Detergent, Hardness and all their two way interactions. The model showed that Hardness had no significant effect (p > 0.29) on response. 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 > 0.30). This concludes that detergent, enzyme type and concentration significantly influence protein removal. The sum of squares value shows that the most important factor to remove protein is detergent. The final model explains 89% of the variation in the data. P-values and a summary of the model are shown in table II.
Table 2: Results from the ANCOVA model including Enzyme type, Enzyme Concentration and Detergent. The interactions are denoted with a :. Significance levels: *** p < 0.001, ** p < 0.01, * p < 0.5.

Enzyme Type Enzyme Concentration Detergent Enzyme:Detergent Enzyme Concentration:Detergent

Degrees of Sum of P-value freedom squares 4 1 1 4 1 3,932 15,611 31,327 0,711 2,768 1,33E-14*** 2,33E-27*** 1,31E-44*** 3,54E-03** 3,61E-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. As we already concluded that detergent significantly influenced data, we used a more graphical approach. Figure 3 shows that some of the enzymes react slightly different to addition of detergent. 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. Enzyme B is apparently a little less efficient relative to the others when detergent is added.

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Technical University of Denmark Dept of Mathematics and Computer

Figure 3: log10 response vs enzyme concentration for the 5 different enzymes. A) with detergent added to the sample, B) without detergent added. Structural differences between the two plots suggest stronger interactions between some enzymes with detergent than others.

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The next step was to investigate if one enzyme was significantly removing proteins better than the others. To do so we split up the data set into a smaller group, with the optimal conditions for protein removal; with detergent added and 15nM enzyme added. 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, with a possible minimum of 1391.7 and a maximum of 1676.2 on a 95% confidence interval. In comparison the worst enzyme was D with a mean protein removal of 663.5 RU. The summary of all the enzymes can be seen in Table 3, which also states the p-values compared to enzyme A.
Table 3 The 5 enzymes compared to enzyme A with 15nM added. ANOVA shows that all the other enzymes are significantly different from A. Given is also the mean and the 95% confidence interval. Significance levels: *** p < 0.001, ** p < 0.01, * p < 0.5.

Enzyme A Enzyme B Enzyme C Enzyme D Enzyme E

Estimate 1527 980 1023 663 1101

2.5% 1392 893 932 605 1003

97.5% 1676 1076 1122 728 1208

P-value 3,15E-06 *** 1,00E-05 *** 8,39E-10 *** 8,87E-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, we also tested the response compared to the random variable, cycle. Furthermore we tested day to day variation by testing the samples which has 0 nM concentration of enzymes against one another.

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To test if cycle sampling influenced the deterioration of proteins we made an ANOVA with cycle as explanatory variable. As all cycles are sampled randomly, there should be no significant difference between the cycles. This turns out to be true as the difference between cycles is insignificant (p > 0.47). The day-to-day runs were explored by an ANCOVA analysis, using hardness, Enzyme type and detergent as explanatory variables, and with concentration 0. The analysis shows that enzyme types are significantly different (p < 0.0013). As the samples should be equal (no enzyme added), this strongly indicates experimental bias in the setup, or sampling error at the day enzyme B was analyzed. Using concentration as factor instead of continuous variable Enzyme concentration was given at four different levels, which yielded the possibility of using the variable as a factor or as a continuous variable. In the previous section we have used it as a factor, in order to be able to extrapolate the model. Making a model with concentration as a factor, and comparing it to a model with concentration as a continuous variable , and comparing them with an ANOVA shows that using concentration as a factor gives a significantly better fit (p < 1.31E-21). The implication and possible solutions of this are evaluated in the discussion.

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Discussion
The few data points for each group give large statistical uncertainty in the validation of the data, and therefore the credibility of the results. More data would have given us more samples to do the statistically analysis and check for errors within the different groups. Notably the sample or experimental bias within day B yields high uncertainty. With the data set available it would be reasonable to adjust data to an expected mean with 0 enzyme added. The addition of the reference samples would also be helpful in this particular problem. In order for our analysis to be valid in terms of hardness and detergent in the water, we assume that the concentrations of these two factors are the same in all observations. Concentration was used as a continuous variable instead of a factor, in spite of the fact that using it as a factor gave a significantly better model. This was done in order to be able to interpolate between experiments with different concentration. In order to make a better model it should be considered to make a model more appropriate than a linear fit for enzyme kinetics, which apparently looks more like a Michaelis-Menten saturation model. If the main purpose of the experiment is to determine the catalytic capabilities of enzymes, the addition of less detergent would be appropriate, as it turns out to be the main factor explaining protein degradation from surfaces. 6|Page

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Future work with the data would include a more thorough analysis of interactions with the individual enzymes. All five types of enzyme have a significant reaction with detergent, as well as with concentration, but to what degree is important knowledge.

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Conclusion
We conclude that hardness does not have an effect on protein degradation in our experiment. Oppositely detergent, enzyme concentration and enzyme type has a highly significant positive effect. Their interactions are also significant, with the interaction between concentration and enzyme type omitted. We also conclude that enzyme A has the highest catalytic capabilities under optimal conditions, and that enzyme D is the worst enzyme for removing proteins.

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Appendix I
Figure 1 shows the residuals of the log transformed analysis.

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Appendix II
R-code used to calculate statistics in the report.
# 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.off()

mydata <- read.table('SPR.txt', header = TRUE, colClasses = c('factor', 'factor','numeric','factor', 'numeric','factor','factor'))

#################################### Manipulate data for cleaner analysis ######################### mydata$Cycle <- factor(mydata$Cycle, levels = 1:34) # Put cycles conscutively mydata$lResponse <- log10(mydata[,3]) # Log response for equal variance mydata <- mydata[-14,] # Remove outlier 14 mydata$EnzymeConcF <- factor(mydata$EnzymeConc ,levels = c('15','7.5','2.5','0')) # Make Enzyme a factor for extra model determination ##################################### Make a linear model of all variables ####################### fm <- lm(lResponse ~ (Enzyme+EnzymeConc+DetStock+CaStock)^2 , data = mydata) m <- summary(fm) # See and assign summary anova(fm) # Make ANCOVA to see #### Leave calcium out of the final model, also the interaction betweeen EnzCon and Enz #####

fm1 <- lm(lResponse ~ (Enzyme+EnzymeConc+DetStock)^2 - Enzyme:EnzymeConc, data = mydata) summary(fm1) an <- anova(fm1) an ##### Use concentration as a factor to compare the two models ######## fmfac <- lm(lResponse ~ (Enzyme+EnzymeConcF+DetStock)^2-Enzyme:EnzymeConcF, data = mydata) summary(fmfac) aF <- anova(fmfac) aF anf <- anova(fm1,fmfac, test = 'F') anf #### Using Concentration as a factor describes data significantly better #######################

#### Show graphically that the data have equal variance and are normally distributed ####

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Technical University of Denmark Dept of Mathematics and Computer

graphics.off() # Remove existing graphics windows(width = 7) # Make a new window 7 inches wide par(mfrow=c(2,2)) # 4 subplots, in a 2by2 matrix par(mar = c(5,4,2,1)) # Make margins as small as possible plot(fm1, which = 1:4) # plot the model, with simplified cooks distance

################################ See the response as a function of enzyme concentration ##### graphics.off() cols <- 1:5 plot(lResponse ~ EnzymeConc, data = mydata, type = 'n') points(lResponse ~ EnzymeConc, data = mydata,subset = mydata[,4] == 'A', col = cols[1]) #abline(afm[1], afm[2]) points(lResponse ~ EnzymeConc, data = mydata,subset = mydata[,4] == 'B', col = cols[2]) points(lResponse ~ EnzymeConc, data = mydata,subset = mydata[,4] == 'C', col = cols[3]) points(lResponse ~ EnzymeConc, data = mydata,subset = mydata[,4] == 'D', col = cols[4]) points(lResponse ~ EnzymeConc, data = mydata,subset = mydata[,4] == 'E', col = cols[5]) ############################ Looks as a saturating function #################################

########################## Test if the days differ with 0 protein added ##################### fm0 <- lm(lResponse ~ (Enzyme+CaStock+DetStock)^2, data = mydata ,subset = mydata[,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 <- mydata[mydata[,6] == 'Det+',] # Find all the data with Det+ fmHigh <- lm(lResponse ~ Enzyme -1, data = mydatadet, subset = mydatadet[,5] == '15') # Make a linear model, and get all intercepts (remove -1 for more usable model). Subset 15 nM p <- summary(fmHigh) # Put summary into a variable ints <- confint(fmHigh) # Calculate the 95% confidence anova(fmHigh) # There is difference among enzymes mens <- fmHigh$coefficient # Retrieve means

####################### A is significantly better than all the other enzymes ######################

####################### Do the enzymes interact the same way between effects ######### enzA <- mydata[mydata[,4] == 'A',] fmA <- lm(lResponse ~ (DetStock + EnzymeConc)^2, data = enzA) summary(fmA) anova(fmA)

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Technical University of Denmark Dept of Mathematics and Computer

enzB <- mydata[mydata[,4] == 'B',] fmB <- lm(lResponse ~ (DetStock + EnzymeConc)^2, data = enzB) summary(fmB) anova(fmB)

enzC <- mydata[mydata[,4] == 'C',] fmC <- lm(lResponse ~ (DetStock + EnzymeConc)^2, data = enzC) summary(fmC) anova(fmC)

enzD <- mydata[mydata[,4] == 'D',] fmD <- lm(lResponse ~ (DetStock + EnzymeConc)^2, data = enzD) summary(fmD) anova(fmD)

enzE <- mydata[mydata[,4] == 'E',] fmE <- lm(lResponse ~ (DetStock + EnzymeConc)^2, data = enzE) summary(fmE) anova(fmE) ############################ They are all significantly correlated with both effects ########

########################### Plot different figures ##########################################

graphics.off() cols <- 1:4 windows(width = 3.5,height= 2) par(mar=c(2.5,4.5,0.8,0.5)) dat <- tapply(mydata$lResponse,list(mydata$EnzymeConc,mydata$Enzyme),mean) barplot(dat, beside = TRUE, xlab = 'Enzyme' , ylab = 'log10 Response', col = cols, ylim = c(0,3)) abline(0,0) # Bar plot of concentration vs response for all 5 enzymes

#################### Detergent figures ####################################### mydatadet <- mydata[mydata[,6] == 'Det+',] lvl <- levels(mydata$EnzymeConcF) enZ <- levels(mydata$Enzyme) Detline <- matrix(0,4,5) for (i in 1:4)

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Technical University of Denmark Dept of Mathematics and Computer

for (j in 1:5){{ sub1 <- mydatadet[mydatadet[,5] ==lvl[i],] Detline[i,j] <- mean(subset(sub1$lResponse,sub1[,4] == enZ[j])) }} rownames(Detline) <- lvl colnames(Detline) <- enZ

mydatadet <- mydata[mydata[,6] == 'Det0',] Detline2 <- matrix(0,4,5) for (i in 1:4) for (j in 1:5){{ sub1 <- mydatadet[mydatadet[,5] ==lvl[i],] Detline2[i,j] <- mean(subset(sub1$lResponse,sub1[,4] == enZ[j])) }} rownames(Detline2) <- lvl colnames(Detline2) <- enZ

graphics.off() windows(width=7,3.5) par(mfrow=c(1,2)) par(mar= c(4,4,2,1)) plot(lResponse ~ EnzymeConc, data = mydatadet , type = 'n', main = 'With detergent', ylim = c(0.5,3.4), xlab = 'Enzyme Concentration (nM)', ylab = 'log10 Response (RU)') for (i in 1:5) { points(lvl,Detline[,i], type = 'o', col = i, pch = 16) } legend('bottomright',enZ, fill = 1:5, horiz=TRUE, cex = 0.8, title = 'Enzyme') text(0.2,3.3, labels = 'A')

plot(lResponse ~ EnzymeConc, data = mydatadet , type = 'n', main = 'Without detergent', ylim = c(0.5,3.4), xlab = 'Enzyme Concentration (nM)', ylab = '') for (i in 1:5) { points(lvl,Detline2[,i], type = 'o', col = i, pch = 16) } text(0.2,3.3, labels = 'B') ## Detergent plots!

########### Make a box plot of response vs addition of detergent and hardness ################

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Technical University of Denmark Dept of Mathematics and Computer

graphics.off() windows(width=7, height = 2.5) par(mfrow=c(1,2)) par(mar=c(2.5,4.5,0.8,1)) plot(lResponse ~ DetStock, data = mydata, ylab = 'log10 Response (RU)') text(mydata$DetStock[1],1,labels = 'A') plot(lResponse ~ CaStock, data = mydata, xlab = 'Hardness', ylab = '') text(mydata$CaStock[1],1, labels ='B', pos = 4, offset=2)

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