UNIVERSITI PUTRA MALAYSIA

DETERGENCY STUDIES OF PROTEASE F1

GUNASEKARAN A/L THEKKAMALAI

FSAS 1996 19

DETERGENCY STUDIES OF PROTEASE Fl

GUNASEKARAN AIL THEKKAMALAI

MASTER OF SCIENCE UNIVERSITI PERTANIAN MALAYSIA

1996

DETERGENCY STUDIES OF PROTEASE Fl

By

GUNASEKARAN AIL THEKKAMALAI

Thesis Submitted in Fulfilment of the Requirements for the Degree of Master of Science in the Faculty of

Science ·and Environmental Studies, Universiti Pertanian Malaysia

February 1996

DEDICATION

This work is dedicated to my beloved father who passed away peacefutty

during the period of this study. Words just fail to express my heartfelt feelings of

,appreciation, gratitude and indebtedness for his excenent role in my life. If not for

him, I will not be the person I am today.

ACKNOWLEDGEMENTS

I wish to take this opportunity to express my heartfelt appreciation and

gratitude to the chainnan of my supervisory committee, Professor Dr. Abu Bakar

SaUeh, and committee members, Associate Professor Dr. Che Nyonya Abdul Razak,

Associate Professor Dr. Wan Md. Zin Wan Yunus and Dr. Salmiah Ahmad (PORIM)

for their invaluable guidance, comments, encouragement and constant support during

the period of this study and for their constructive criticism �f this manuscript during

its preparation.

My deep appreciation is also extended to Associate Professor Dr.

Kamaruzaman Ampon, Associate Professor Dr. Mahiran Basri, and Encik Ismail

Omar for their help, invaluable advice and constructive comments throughout 1he

course of this study.

I would also like say thanks to Associate Professor Dr. Tong Chow Chin, for

his advice and assistance on cellulase assay, Associate Professor Dr. Norhani I

Abdullah on her references about amylase assay and Associate Professor Dr. Noraini

Abd. Samad and Associate Professor Dr. Khatijah Yusoff on their permission to use

the centrifuge.

I am also grateful to all staff, graduate students and honours students of the

Department of Biochemistry and Microbiology, especially Raja Noor Za1iha,

Nonnayati, Rohaidab, Khanom, Mariam, Nizam, Rizal, Zainab, Faridah, Ramesh, Yu

iii

Yu, Ruby, Norhaizan, Sok Foon, Ridzuan, Ai Ai, Robert, Boon Leong, Ai Fei, Amy,

Jeevan and Fakah for their help in one way or another and Naidu Sannasi for helping

with the graphs in the thesis.

I would also like to express my appreciation to all staff of AOTC, PORIM,

especially Zahariah, Zuhairi, Siti, Rubaah, Parthiban, Zaitun, Ana and Rashid for their

help and favours in one way or another.

I would also like to express my heartfelt thanks to my mends Mr. and

Mrs.K.Palaniappan (Banting), Mr.G.Nadarajah (Taiping) and Miss S.ValIi (Malacca)

who gave moral support and encouragement and Mr. and Mrs. N.Kalaivanan

(Serdang) who also provided lodging for me and my family throughout the period of

study.

Also I would like to thank my late father, mother, brothers, sisters, parents-in­

law, brothers-in-law and sisters-in-law for their love, moral support and help and

especially Saras for also subsidising the expenses of this thesis.

I am also greatly indebted to UPM and PORIM for providing the facilities and

the Ministry of Science, Technology and Environment for financial backing through

IRP A (through Professor Dr. Abu Bakar SaIleh) to complete the study successfully.

Last but not least, I wish to express my greatest appreciation and gratitude to

my wife and son for their love, sacrifice, understanding and moral support.

IV

TABLE OF CONTENTS

Page

ACKNOWLEDGE�ms............................................................................ iii

liST OF TABIES....................................................................................... viii

liST OF FIGlJRES.......... ............ ........ ......................... .... ...... ..................... ix

liST OF PLATES................... ................. . . ... . . . ... . . . . ... . . . . . . . . . . . . . .. . ........ . ... ...... xi

liST OF ABBREVIA nONS .............. ........................... ................... '" ........ xii

ABSTRACT................................................................................................ xiii

ABSTRAK .. ................................................................... !............................. xv

CHAPTER

1 INTRODUCTION................................................................. 1

2 UfERATURE REVIEW...................................................... 5

Detergent and their Ingredients............................................. 5 Surfactants ........... ........................................................... 6 Builders ......... ............. . ........................ ........................... 9 Bleaches ..................................................... .'..................... 13 Auxiliary Agents ...... . ....................................................... 14

The I Washing Process .................. ......................................... 14 Role of Water in Washing ................................................ 16 Water l-Iardness ....................... ........................................ 17 Soils .............................................................................. . . 18 'rextiles ............... ............................................................. 19 Physical Soil Removal...................................................... 21

Detergent Enzymes in General ............................................... 23

Protease ... ........ ................................................... .................. 26 Suitability of Serine Proteases for Detergents .................... 27 Commercial Detergent Proteases ................................. ..... 30 Parameters that Influence the PerfotmaDce of a Protease '" 32

v

3

4

Page

Fonnulating Enzymes into Detergents .................................... 35 Powdered Detergents ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 liquid Detergents . . . . . . .... . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Types of Fonnulations in which Enzymes are Found.......... 39 Composition of Enzyme Detergents ..... . ... .. ...... ... .............. 40

Cost Effectiveness of Enzymes........................... .................... 41

Biodegradability of Enzymes.............................. .................... 41

Effect of Enzyme Detergents to Health.................. . .. . . . . . . . . . . . . . . 44

The Enzymes used in the Project.................. .. ......... ........ ....... 46 Protease Fl . . . ... ........ ................ .................. .... ... ............... 46 Savinase .................. ... .................................................. . . . 47

MATERIALS AND MErnODS 48

Materials .. ........ ............. ................................. ...................... 48 Chen1ica1s I Materials .......... ................................. ..... ....... 48 Instru.tnents ................................... ................................... 50

Methods .... ......... ..... ... ...... ............. ....................................... 51 Enzyme Source........ ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Artificial lIard Water .............. ...... .................................... 51 Ammonia Buffer Solution.. .................... . . . . . . . . . . . . . . . . . . . . . . . . . . 54 TotallIardness Indicator ( THI) ........... .............................. 54 EDTA Solution (0.0 2 N) ........... ...................... .... ............ 54 Standardisation of EDT A ................................... ......... ..... 55 Standardisation of Artificial Hardwater .......... ......... ... ........ S5 Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S5 Enzyme Production.......................................................... 56 Assay of Enzyme Activity ....... . ......... .. .................... .......... 57 Washing Petfonnance Test ............................................... 59 Statistics and Calculations ... ..... ... . ... .. ..... .... .... ..... ... .... ... .... 61

RESULTS 63

Purity of Crude Protease Fl............. ...................... ......... ........ 63 Effect of the Various Parameters on Detergency...................... 63

Temperature .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Soil................................................................................. 69 Enzyme Concentration ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Water lIardness ... ............ ......... ... ........ ....................... ..... 75

vi

Page

pH .. ....... ............... ......... ... .... . ... ... .. ....... .... . . . . .. ...... . . . ........ 79 Enzymes, Surfactants and Builders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

5 DISCUSSION......... ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

6

Preparation of protease Fl.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Protease Fl versus Savinase .. ....... ...... ......... .... . . . . . . . . . . . . . . . . . . . . . . . 89 The Wash Test ................... .... ........ ......................... .... . . ... ..... 90 Soiled Cloths ............. ...... ................................. . . . . . . . ... . . . . . . . . . . 91 Soil Removal Calculation .. ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Effect of the Different Parameters on DetCrgency.................... 93

Enzyme . .................................... ............... .. ............ . . . ...... 93 Enzyme Concentration .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Soil (Soiled Cloth) ......................... ... ................. . ... .. . . .... ... 95 Water lIardness ... ....... ....... ............ .. .. .... ... .... . . . . . . . . . . . . . . . . . . . 95 Temperature .......................... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 pH .... ............................................ .... ........ ... ... ..... . .... . . .... 98 Enzymes and the Perfonnance of Surfactants and Builders.. 99

SU1\4MARY, CONCLUSION AND RECOMMENDATIONS

101

Swrunary .................... ......... ........... .. ............. ...... ...... .... ...... . 101 Conclusion .................. ........ ............ .. ......... ...... ..... . .. .. ........... 104 Recommendations...... ....... .... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

BffiUOGRAPHY.................................................................. 108

APPENDICES....................................................................... 114

A Flow charts showing protease, lipase, ceHulase and ex-amylase assays ........ ... ...... ... ....... .. . ....... . .. . .. . .. . ........ .... ... 115

B Preparation of pH buffers........................... ... .... ........... .. 116 C Additional table ... ................ . ..... .... ............ . .... . ....... ....... 117 D Samples of swatches used in the project ............... ...... . ... 118 E Abstract of poster presented at conference . . .................... 119

VITA.................................................................................... 120

vii

LIST OF TABLES

Table Page

1. Historical review of the preparation and use of detergent enzytnes ....................................................................... 3

2. Surfactants of various ionic nature ............ . . . .................. 7

3. Auxiliary agents and their role in detergents . . . . . . . . ......... ... 15

4. IInportant types of soil . ... .... ...... .... .... ...... ................ ....... 19

5. Different types of fibres used in textiles............................ 20

6. Interfacial properties affected by adsorption of detergent ingredients ................ ..................... ............................... 22

7. The different types of proteases . ......... . . . . . . . . . . . . . . . . . . .. . . . . . . . 27

8. Requisite properties for detergent enzytnes ..................... 29

9. The suitability of proteases for use in detergent preparations ....................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

10. Comparison of detergent properties of Esperase, Alcalase and Savinase .............. ..... .............. ................... :. . . . . . . . . . . . 30

11. Composition of an enzytne detergent ... ................. . .......... 42

12. Frame formulations for powdered heavy-duty detergents... 43

13. Frame fonnulations for Jiquid heavy-duty detergents......... 44

14. Activities of other enzytnes in crude Protease Fl ............. 63

15 Units currently used for expressing the hardness of water.. 117

viii

LIST OF FIGURES

F�e Page

1. Stepwise removal of a particle P from a substrate in a smfactant solution by electrical forces ........... ........................................... 21

2. Decomposition of triglycerides by lipase .................. ..... ............. 25

3. Parameters which influence the perfomtance of a protease under wash ....................... ......... . .......................... �............................ 32

4. Decomposition of blood stain with unspecific and specific protease respectively ............................................ .... ...... ......... 34

S. The effect of the enzymes on soiled cloth AS12-o30 at various temperatures........... ......... ................. ........................................ 64

6. The effect of the enzymes on soiled cloth AS12-049 at various temperatures ........................................................................... ..

1. The effect of the enzymes on soiled cloth CSI-013 at various 65

temperatures...... ... .............. ....... ................. ...... .................. ...... 61

8. The effect of the enzymes on soiled cloth CS9..()()4 at various temperatures............................................................................. 68

9. The effect of the enzymes on different soiled cloths at RT... . .... . . . 10

10. The effect of the enzymes on different soiled cloths at SO°C........ 11

11 The effect of the enzymes on different soiled cloths at 10°C.. . . . . . . 12

12. The effect of different concentrations of Protease Fl on soiled cloth AS12-o49 at selected temperatures..................................... 13

13. The effect of different concentrations of Savinase on soiled cloth AS 12-049 at selected temperatures............................................. 74

ix

14. The effect of the enzymes on soiled cloth AS 12-049 at RT in different water hardnesses. . . .. . . . . . . ... ... . . . . . . . . . . . . ... . . . . .. . . . . . .. . . . . . . . . . . . . . 76

1 5. The effect of the enzymes on soiled cloth AS12-049 at 50°C in different water hardnesses. . . . . . . .. . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . 77

16. The effect of the enzymes on soiled cloth AS12-049 at 70°C in different water hardnesses............... . . . . . ... . ... . . . . . ... . . . . . . . ... . . . . . . . . . . . . . 78

17. The effect of the enzymes on soiled cloth AS12-049 at RT at various pH's............................................... ........................... . ... 81

18. The effect of the enzymes on soiled cloth AS12-049 at SO°C at various pH's. . . . . . . . . . ... . . ... . . . . . . . . . . . . . . . . . . . . . . .. .. ... . . . . . . . . . . . . .. . . . . . . . . . . ....... 82

19. The effect of the enzymes on soiled cloth AS12-049 at 70°C at various pH's.. . . . . . ... ...... ...... ... .. .... . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . 83

20. The effect of LAS and enzymes on soiled cloth AS12-049 at selected telnperatures...... ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

21. The effect ofFAS and enzymes on soiled cloth AS 12-049 at selected telnperatures... . . . . . . . . . . ...... . . . . . . . . . . ... . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 8S

22. The effect of STPP and enzymes on soiled cloth AS12-049 at selected temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,.. . . . . . . . . . . . . 86

23. The effect of zeolite and enzymes on soiled cloth AS12-049 at selected telnperatures...... . ... . . . ... . . . ... . . . . . . . . . . . . . . . . . . ... . . . . . . ...... . . . . . . . . . . 87

24. The effect of FAS, zeolite and enzymes on soiled cloth AS12-049 at selected temperatures.. .......... . . . . . . . ... . . ... . ... .... . . . . . ... . . . . . . . ..... 88

x

Plate

1. Terg-o-tometer

LIST OF PLATES

2. Macbeth Colour Eye 3000 Spectrophotometer

xi

Page

51

53

AES

AI

AOS

APMSF

CAS no.

CM C

CMS

E.C.no.

EDTA

FAS

FWA

LAS

NTA

PMSF

PORIM

RT

STP (STPP)

TIll

Tris

SDS-PAGE

%SR

LIST OF ABBREVIATIONS

Alcohol ether sulphates

Active ingredient

Alpha olefin sulfonate

AmidinophenyJmeth.ane sulfonyl fluoride

Chemical Abstract Service munber

Carboxymethyl cellulose

Carboxymethyl starch

Enzyme Classification munber

Ethylene diamine tetraacetic acid

Fatty alcohol sulfate

Fluorescent whitening agents

Linear a1kylbenzene sulfonate

Nitrilotriacetic acid

Phenyhoethanesulphonyl fluoride

Palm on Research Institute of Malaysia

Room temperature

Sodium triphosphate (Sodium tripolyphosphate)

Total hardness indicator

Tris(hydroxymethyl)aminomethane

Sodium dodecyl sulphate - polyacrytamide gel electrophoresis

Percentage soil removal

xii

Abstract of thesis submitted to the Senate of Universiti Pertanian Malaysia in fulfillment of the requirements for the degree of Master of Science.

Chairman

Faculty

DETERGENCY STUDIES OF PROTEASE Fl

By

GUNASEKARAN AIL THEKKAMALAl

February, 1 996

Professor Abu Bakar Salleh, Ph.D.

Science and Environmental Studies

Protease FI, a thermostable alkaline protease extracted from Bacillus

stearothermophilus strain Fl was investigated for its soil removing properties. In this

study the general purity of crude Protease Fl was first determined. Protease Fl was

found to be free of lipase, cellulase and a.-amylase activities.

The washing performance of crude Protease Fl was measured in terms of

percentage soil removal and compared to a commercial enzyme, Savinase. The effects

of various factors such as types of soil, washing temperature, enzyme activity, water

hardness, pH, types of surfactants and builders on the washing performance of

Protease Fl were measured.

The washing performance was determined by stirring a type of soiled cloths in

1 liter solution of enzyme (with a fixed amount of activity) for 10 minutes, followed

by 2x3 minutes rinsing. The difference in the whiteness of the soiled cloth befote and

after washing indicate the degree of soil removal (or detergency). Among the various

xiii

types of soiled cloth studied, the one soiled with oil, pigment and milk (AS12) pvc a

better contrast when washed by the enzymes than the cloihs soiled by btood or the

egg.

Protease Fl washed better than Savinase at an the temperatures studied. The

difference in performance was fOlUld to be greatest at 700C. washing efficacy was

found to increase with increase in concentration of the enzymes lUl1il a certain level

beyond which a drop in the percentage soil removal was observed. The enzymes

perfonned better at lower water hardncsscs. Generally Protease Fl was more sensitive

to water hardness than Savinase. At room temperature (RT) and SOOC, Savinase

perfonned better at all pH's. However, at 70°C and pH 7 and 8, Protease Fl

perfonned better.

When fonnulated with swfactants or builders, Protease Fl was f01md to

enhance the perfonnance of swfactants while Savinase, the builders. The best

washing perfonnance was when enzymes, smfactant and builders were fonnulated

together and the washings canied out at RT and 500C.

xiv

Abstrak tesis yang dikemukakan kepada Senat Universiti Pertanian Malaysia sebagai ntemenuhi syarat untuk mendapatkan Ijazah Master Sains.

KAJIAN PENCUCIAN OLEH PROTEASE F1

Oleh

GUNASEKARAN AIL THEKKAMALAI

Pengerusi

Fakulti

Februari, 1996

Professor Abu Bakar Salleh, Ph.D.

Sains dan Pengajian Alam Sekitar

Kajian mengenai ciri-ciri keupayaan mencuci oleh Protease F l, enzim

protease alkali termostabil yang dihasilkan oleh Bacillus stearothermophilus teiah

dilakukan. Dalam kajian ini ketulenan Protease F l kasar secara am ditentukan dan

didapati tiada aktiviti enzim-enzim lipase, sellulase dan a-amilase dalam Protease F l

kasar.

Keupayaan mencuci oleh Protease F l telah diukur dad segi peratus kotoran

yang ditanggalkan dan dibandingkan dengan satu enzim komersil, Savinase. Prestasi

mencuci oleh beberapa faktor seperti jenis kotoran, suhu, aktiviti enzim, keliatan air,

pH dan jenis surfaktan dan 'builder', telah diukur.

Prestasi mencuci telah ditentukan dengan mengacau sejenis bin kotor dalam 1

liter larutan enzim ( dengan sejumlah aktiviti tertentu) selama 10 minit, diikuti oleh

bilasan 2x3 minit. Perbezaan dalam kebersihan pada kain kotor sebelum dan selepas

mencuci menunjukkan tahap penanggalan kotoran( atau pencucian). Di antara jenis

xv

kain kotor yang dikaji, bin yang dicemari minyak, pigmen dan 8USU (AS12) memberi

perbezaan yang lebih jelas hila dibasuh oleh kedua enzim daripada bin yang dikotori

darah atau telur. Protease Fl mencuci lebih baik daripada savinase pada sernua BUbu

yang dikaji. Perbezaan dalam prestasi didapau paling besat pada 70°C. Keupayaan

mencuci didapati mening1cat dengan meningkatnya kepekatan enzi.m sehingga ke tahap

tertentu. Kepekatan enzim yang lebih dari tahap itu menjejaskan pencucian dan

mengw-angkan penangga1an kotoran. Enzim-enzim itu mencuci lebih baik pada

keliatan air yang rendah. Sccara am Protease Fl lebih sensitif daripada Savinase

kepada keliatan air. Pada subu biIik dan SO°C, Savinase mencuci lebih baik pada

semua pH. Walaubagaimanapun, pada 700C dan pH 7 dan 8, Protease Fl mencuci

lebih baik.

Apabila difonnulasi dengan surfaktan dan 'builder', enzim Protease Fl

didapati meningkatkan keupayaan mencuci oleh surfaktan dan Savinase meningkatkan

keupayaan mencuci oleh 'builder'. Prestasi pencucian terbaik berlaku apabila enzim­

enzim, surfaktan dan 'builder' difonnulasi bersama dan pencucian di1akukan pada

subu bi1ik dan sooc.

xvi

CHAPTERt

INTRODUCfION

Detergents belong to the group of consumer products which are indispensable

for the maintenance of cleanliness, health and hygiene. Their economic importance

worldwide is considerable, although consumption varies markedly from country to

country.

Use of enzymes in detergents was first described by Otto Rohm. He had

found that fabrics could be cleaned more easily and at lower temperatures when

penetrated with fat and protein digesting enzymes (Dambmann et al.,1971). However

such enzyme-containing detergents failed to play a major role in the following decades

since the only available proteolytic enzyme preparauon, a pancreatic extract obtained

from slaughtered animals, was too sensitive to the alkaline and oxidative components

of detergents. In 1932, enzymes were utilized in a soap composition and were found

to enhance the cleansing action of soap gready (McCarty, 1971).

Preparation of proteolytic enzymes by fennentation using specific strains of

bacteria (Bacillus subtilis, later on Bacillus licheniformis) became possible in the

early 60's. These enzymes were highly resistant to aJkaJi (active over a broader pH

spectnun, from 7.5 to 10), stable to oxidizing agents of the perborate type and

showed adequate stability at temperatures as high as ca.65°C for the time period

required by nonnal wash processes (Jakobi and Lohr, 1987).

1

2

Commercial production of detergent enzymes experienced rapid expansion in

the years that followed. By 1968, detergent enzymes had already taken root in

detergent fonnulations in Europe while in the united States they just started appearing

in their detergent powders after an initial resistance to their incorporation into

detergents (Davidsohn and Milwidsky, 1986) due to inhalation of enzyme dust which

resulted in allergenic reactions in workers.

The practical use of enzymes in detergent fonnulations was facilitated by two

major developments. One was the reduction of builders such as sodiwn

tripolyphosphate in detergents to solve environmental problem. The other was the

move towards lower washing temperatures as a result of the growing use of synthetic

fibres and to save energy (Starace, 1981; Maase and Tilburg,1983; KrOsmann and

Bercovici, 1991). Both these factors contributed significantly towards higher detergent

enzyme consumption since the addition of enzymes partially compensated for the loss

in detergency suffered as a result of the two developments.

Gradual development over the years has made enzymes an indispensable

ingredient in detergents (Dambmann et a1., 1971). Having been marketed for more

than 25 years, they have now become well established as nonnal ingredients in both

powder and liquid detergents all over the world (Christensen et al., 1986). They

accoWlt for approximately 25% of the total wortdwide enzyme production and

represent one of the largest and most successful commercial large scale applications of

modern biotechnology (Godfrey and Reichett, 1986).

Table 1 provides a brief historical review of the production and application of

detergent enzymes.

Table 1

Historical review of the preparation and use of detergent enzymes

Year

1913

Enzyme

Otto Rohm claims the use oftIyptic enzymes for detergents

Enzyme-containing detergents

detergents con1Bining pancreatic enzymes

, 1927 optimized detergents containing pancreatic enzymes

1960 Alcalase

Post - 1960 Microbial proteases made available on a commercial scale by Novo Industri, Copenhagen

1968

1969

1970

1972

1975

Other producers followed, e.g., Maxatase from Gist en Spiritusfilbrieken N.V., Delft; Nagase from Nagase Co.; MonJase 1 10 from Monsanto; Esperase from Novo

Additional microbial enzymes suggested for use in deteIgents (amylases, lipases, pectinases, nucleases, oxidoreductases, etc.)

first commercial product containing microbial proteases (presoak and wash pretreatment agent)

first heavy duty detergent with microbial proteases (presoak and wash pretreatment agent)

microbial pfoteases contained in 80% of aD detergents in the Fedem1 Republic ofGennany

severe setback of addition of microbial proteases due to public criticism (the C<aDetgy debate"

enzymes in detergents declared to be safe by the German Federal Health Agency marlcet share of enzyme­containing detezgents stabilizes in Gennany at 800,1,

Source: Adopted and modified from Berg and Boeck (1976) cited by Jakobi and Lbhr (1987).

3

Protease Fl , a thennostable enzyme produced by Bacillus st£arothermophilus

has high temperature stability and is active at alkaline pH's (Abdul Ralnnan,1993). It

4

was fOWld to have soil removing properties comparable to the established detergent

enzyme Savinase (Cheah, 1994). The objcc1ive of this study is to confinn this finding

and to detennine if Protease Fl has suitable properties to be used as a detergent

enzyme.

CHAPTER 2

LITERATURE REVIEW

Detergents and their Ingredients

A detergent is strictly anything that cleans, including soap and even water, but

the word is normally used only for synthetic detergents which are referred to as

surfactant or syndet in the USA whilst in Europe the corresponding tenn is tenside

(for tension active materials).

A detergent is defined as a formulation comprising essential constituents

(builders, boosters, fillers and auxiliaries), which is specially devised to promote the

development of detergency (Davidsohn and Milwidsky, 1986). The tenn detergency

refers to the theory and practice of dirt removal from solid surfaces by surface

chemical means (Shaw, 1985; Adamson, 1990).

The two categories of detergents on the market in various parts of the world

are household detergents (for domestic laundries) and institutional detergents (for

industrial and commercial use). Among the household detergents are heavy-dUty or

all-pwpose detergents, specialty detergents, laundry aids and aftertreatment aids.

Generally, detergents for household and institutional use are very complex

formulations containing several different types of substances which can be categorized

into the following major groups: surfactants, builders, bleaching agents and auxilliuy

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agents. The different components have specific ftmctions in the washing process,

,although to some extent they have synergistic effccts. Apart from these substances,

certain additives are made necessary by the production process, whereas other

materials are introduced to improve product appearance.

Surfactants

Sutfactants constitute the most important group of detergent ingredients and

are present in all types of detergents. Generally these are water-soluble surface active

agents consisting of a hydrophobic portion (usually a long alkyl chain) attached to

hydrophilic or solubility-enhancing fimctional groups. They can be classified as

anionic, cationic, nonionic or amphoteric, depending on the charge present in the

molecule after dissociation in aqueous solution. Table 2 provides an overview of the

various classes of surfactants.

Anionic surfactants are the most common agents in detergents designed for

laundry, dishwashing, and general cleansing while nonionic surfactants being efficient

wetting agents and effective emulsifiers are also finding broad USe at home and in

industIy (McKenzie, 1978). Cationic sutfactant use is largely restricted to

aftemeatment aids because of the fimdamental incompatibility of these materials with

anionic surfactants. Amphoteric surfactants, known for their extreme skin kindness,

have found an increasing market in the manufacture of toiletries and cosmetics

(Palicka, 1991).

When added to water the sutfactant molecules align themselves along the

interphase (air-water boundary) such that the hydrophilic ends face the water and the

hydrophobic ends away from the water. Such an adsotption of sutfactants along the

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interphase helps reduce the surface tension and improves its weUing properties

(W ooUau, 1985). A solution which has sufficiently s1roDg wetting properties in re1a1ion

to the substrate can penetrate under the soil and gradually case it away as a droplet.

Tablel

Surfactants of various Ionic nature

Surfactant FonnuJa Ionic nature

AlkylsuJfonates R-So;""Na+ Anionic

Dialkyldimethyl ��-t-C1\]a- Cationic ammonium chlorides

Alkyl poly( ethylene RO-(CH1-cH1-O) JI Nonionic glycol) ethers

Betaines R-tC1\1-"O

Amphoteric

Source: Adapted from Juobi and Whr (1987).

Surfactant properties are influenced by the structure of the hydrophobic

residue. Adsorption and wash effectiveness gcneraJly increase with increasing chain

length. For example ionic surfactants bearing n-allcyl groups show a tinear relationship

between the number of carbon atoms in the surfactant molecules and the logarithm of

the amount of surfactant adsorbed on activated carbon or bo8n (Jakobi and Whr,

1987).

Surfactants with little branching in their alkyl chains generally show good

wash effectiveness but relatively poor wetting characteristics, whereas more highly

branched surfactants are good wetting agents but have unsatisfactory detergency. For