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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
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
5
6
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
7
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