Introduction


Enzymes are biological catalysts. A catalyst is anysubstance which makes a chemical reaction go faster, without itself beingchanged. A catalyst can be used over and over again in a chemical reaction: itdoes not get used up. Enzymes are very much the same except that they can beeasily denatured by some means. All enzymes are made of protein; that is whythey are sensitive to heat, pH and heavy metal ions. Unlike ordinary catalysts,they are specific to one chemical reaction. An ordinary catalyst may be usedfor several different chemical reactions, but an enzyme only works for onespecific reaction. Enzymes must have the correct shape to do their job. Enzymeschange their shape if the temperature or pH changes, so they have to have theright conditions.


Conventional chemical processes are generally severe andfibre damage may occur. However, enzymes are characterized by their ability tooperate under mild conditions. As a result processes may take place withoutadditional harm to the fibre. Enzymes are also readily biodegradable andtherefore potentially harmless and environmentally friendly. This chapterdiscusses various properties of enzymes and applications of cellulases intextile processing.


Properties of the enzymes


a) Enzymes accelerate reaction:


Enzymes accelerate a particular chemical reaction bylowering the activation energy for the reaction. They achieve this by formingan intermediate enzyme substrate complex, which alters the energy of thesubstrate such that it can be more readily converted in to the product. Theenzyme itself is released unaltered at the end of the reaction, thus acting asa catalyst. It can be schematically represented by the following equation:


Substrate + Enzyme → Substrate-enzyme complex →Substrate + Enzyme


Enzymes have an excellent catalytic power. They acceleratereactions, which are often undetectable in the absence of enzyme, by enormousamounts, sometimes several million fold. An outstanding example of thiscatalytic power is demonstrated by the enzyme triose phosphate isomerase. Thisenzyme accelerates the isomerisation of glyceraldehyde 3 phosphate thousandtimes compared with the rate in the presence of acetate ions.


Another example of the catalytic power of enzymes is theactivity of peroxidase, an enzyme employed in the textile industry. Onemolecule of this enzyme can convert five million molecules of hydrogen peroxideto water and oxygen in one minute.


Enzymes do not alter the equilibrium position of thereactions they catalyze. The energy profile for a typical reaction shows thatin order to proceed from reactants to products an energy barrier (∆G*) must be overcome. Fromthe transition state theory of rate, the relationship between the rate constant(k) and the free energy of activation (∆G*)can be expressed as


K= kT/h.e-∆G*/RT = kT/h. e-∆H*/RT. e∆S*/R


Where,


k= Boltzman constant

h= Plancks constant

T= absolute temperature

R= Gas constant

∆H* & ∆S* are enthalpy and entropy ofactivation respectively.


 

In order to speed up the reaction at constant temperature, the value of ∆G* must be lowered. The function of a catalyst is therefore, to lower this energy barrier. This is possible to be achieved via the rapid formation and decomposition of an intermediate, which is represented by a local minimum. In all cases it may be noted that the catalyst does not alter the magnitude of free energy change (∆G) and therefore does not cause a shift in the equilibrium between reactants and products; it merely increases the rate at which the equilibrium is attained. The same consideration also applies to enzymatic catalysis.


If in a conversion of a substrate (A) to a product (B) the concentration of B is two folds greater than that of A when equilibrium is reached in the absence of enzyme, then the same ratio of B is produced when the enzyme is present. One of the most important pieces of information about an enzyme is its specific activity, which describes the enzymatic strength towards a particular substance. Enzyme activity is a measure of substrate molecules converted into product in a unit of time, per molecules of enzyme when the enzyme is fully saturated with the substrate. This is a key value for determining an enzymes selling price, its dosage and thus, its cost in actual use. In most instances specific activity is expressed as μ mol of substrate per minute per mg of enzyme protein.


b) Enzymes are specific in their action:

Most enzymes have a high degree of specificity and will catalyze a reaction with only one or a few substrates. There are exceptions and some proteases have a fairly low specificity to protein substrates. However, one particular enzyme will only catalyze a specific type of reaction. This property is the basis of a method of classification of enzymes. There are six classes of enzymes:


  • Oxidoreductases
  • Transferases
  • Hydrolases
  • Lyases
  • Isomerases
  • Ligases


Oxidoreductuses catalyze oxidation & reduction of the substrate. e.g. oxidases, dehydrogenases, hydroperoxidases. Transferases catalyze the transfer of particular group from one substrate to another.e.g. Methyl transferases, Amino transferases. Hydrolases cause hydrolysis reactions and are mostly used in textile industry. They catalyze the hydrolysis of ester, ether, peptide etc. Lyases cleave a covalent bond like C-C,C-O,C-N etc in the substrate and form a double bond in it. Isomerases change the substrates to their isomers by intermolecular rearrangement. e.g. cis trans isomerases and ligases catalyse the union of two small molecules to from a bigger molecule.

Most enzymes have a maximum activity at an optimum temperature. The reaction rate increases with increasing temperature until the optimum temperature is reached. Above this value enzyme activity decreases rapidly until a point when the enzymes become permanently deactivated by denaturization. Temperature below the optimum range decreases the enzyme activity without damaging the protein structure. Lower ranges of temperature can be used with longer times of exposure to the substrate.


Enzymes also have an optimum pH and its activity decreases sharply on both sides of the optimum. So, that small pH fluctuation will not affect greatly the reaction rate. Since they are protein molecules, enzymes can be denatured by extremes of pH and temperature or by excessive stirring. The methods used in the textile industry to stop enzymatic reactions after the desired treatment is carried out are based on this characteristic. An increase in pH or temperature renders the enzymes inactive and harmless to textile substrate.


Enzyme treatments are usually carried out in a buffered media. Precautions have to be taken with the temperature of the treatment liquor to obtain maximum enzyme efficiency. The presence of enzyme inhibitors, such as heavy metals, ionic detergents, cross linking agents and formaldehyde based products has also to be prevented. The efficiency of an enzyme catalyzed reaction depends upon enzyme concentration, substrate concentration, duration of reaction, temperature of reaction , pH of the system, presence of activators and presence of inhibitors.


 

Enzyme activators and inhibitors


Some of the bivalent metallic cations activate certain enzymes as for example Ca++, Sr++, Mg++, Zn++, Co++ etc sensitize the substrate towards enzymatic attack. Some of the chemicals like alkalies, antiseptics, acid liberating agents tend to inhibit the enzymes activity. Enzymes activity is inhibited by blocking certain useful groups. The inhibitors possess certain affinity to the enzymes and thus there occurs a competition between the substrate and the inhibitor to combine with the enzyme which retards the reaction. Heavy metal cations like lead, mercury, copper, iron are lethal to enzymes and their effects are detrimental. Reducing and oxidizing agents also acts as a inhibitors of enzymes.


c) Enzymes can replace hazardous chemicals:


Quite a number of chemicals used in textile chemical processing are known to pose various environmental problems and hazardous to the persons working with, if not rigidly controlled. The use of an enzyme can often replace a number of such chemicals which are toxic and are better avoided where possible. The use of an enzyme catalyzed reaction may permit much shorter times for a certain process to occur and milder conditions to be employed, thus being both safer and more cost effective.


d) Enzymes are biodegradable:


After completion of an enzymatic reaction the enzymes when released in drain water get decomposed to amino acids by various proteolytic enzymes secreted by micro organisms present in sewerage plants which are then available to reenter the food chain.


Mechanism of enzyme action: Lock & Key theory


Enzymes have active centers, which are the points where substrate molecule can join. Just as a particular key fits into a lock, a particular substrate molecule fits into the active site of the enzyme. The substrate forms a complex with the enzyme. Later the substrate molecule is converted into the product and the enzyme itself is regenerated (Fig.1).


Fig. 1 Lock & Key model of enzyme specificity


The process continues until the enzyme is poisoned by a chemical bogie (Fig.2) or inactivated by extremes of temperature, pH or by other negative conditions in the processing environment.


Fig. 2 Active site of enzyme blocked by poison molecule


 

Various enzymes used in textile processing:


Amylases: Which convert amylose or amylopectin polymers , commonly referred to as starch in to water soluble shorter chain sugars (Starch desizing)


Pectinases: Which hydrolyse pectins consisting of linear polymers of galacturonic acid (bio-scouring replacing caustic)


Lipases: Which hydrolyse fats and oils into alcohol and organic acids.


Proteases: Which catalyse splitting protein molecules, and in the extreme may break the protein into the component amino acids.

Catalases or peroxidases: Which catalyse the decomposition of peroxide, also known as peroxide killer.

Cellulases: Which catalyse the hydrolysis of cellulosic materials (bio-singeing or bio-polishing).


Applications of Cellulases


Bio-polishing


Bio-polishing, a technique first adopted by the Danish Firm Novo Nordisk for the finishing treatment of cellulosic fabrics with cellulase enzymes. The main objectives of the bio-polishing is to upgrade the quality of the fabric by removing the protruded fibres from the surface and modification of the surface structure of the fibre, thereby making it soft and smooth. In conventional process protruded fibres are removed by singing process and smoothness imparted by chemical treatment. The conventional methods are temporary, fibres return on the surface of the fabric and chemicals are removed after few washing and fuzz is formed. The fuzz on the surface spoils the fabric appearance and generates customers dissatisfaction whereas bio- polishing is permanent and it not only keeps the fabric in good condition after repeated washing but also enhances feel, colour, drapeability etc consequently products become more attractive to the customer and fetch better prices. The bio-polishing treatment offers the following advantages:


  • Improved pilling resistance.
  • A clearer, lint and fuzz-free surface structure.
  • Improved drapeability and softness.
  • The effects are durable
  • Slight improvement in absorbency
  • Fashionable effects on fabric like distressed look of denim


Examples of some cellulases are Aspergillus niger, Trichoderma longibrachiatum, Fusarium solani and Trichoderma viride. The enzymes are biomolecules of about 20 amino acids with molecular weight ranging from 12,000 to 1,50,000 and therefore they are too large to penetrate the interior of a cellilosic fibre. Hence, only 1,4 β-glucosidic bonds at the surface of cellulose fibre are affected. This results in removal of surface hairs which are responsible for improvement in the hand and feel of the fabric due to surface etching.


Cellulases are multi-component enzyme systems and a complex mixture of :


Endoglucanases(EG) or Endocellulases

Cellobiohydrolases(CBH) or Exocellulases

Cellobiases or β-glucosidases


All these components work synergistically together. EG hydrolyse cellulose randomly along the chains, preferentially the amorphous region. CBH attack the chain ends and produce primarily cellobiose coupled with the enzyme. The cellubiose and any small chain oligomers produced by CBH are then hydrolysed by the third enzyme β-glucosidase into glucose.


The finishing effects delivered by cellulases are always obtained in process where strong mechanical agitation of fabric is provided during the treatments (for example, rotating drum washers and jets). Research studies revealed that increasing the degree of mechanical agitation increases the extent of the hydrolysis, and it seems to increase EG activity relative to CBH activity in a total crude mixture. Further studies with EG- and CBH-rich preparations also indicate that with higher levels of mechanical agitation an EG-rich preparation causes relatively higher weight loss.


 

In another study EG-enriched cellulase was found less aggressive than the whole cellulase and resulted in less fabric damage. The whole cellulase was found to be best for treatment of cotton and lyocell fabrics and the CBH enriched cellulase was more suitable for linen, viscose and delicate cotton knits. A combination of pectinase and cellulase significantly improves water wetting and retention properties, to a level similar to those of commercially scoured cotton fabrics.


Minimizing fabric strength loss is particularly important for linen and viscose rayon fabrics. Linen is a very strong fibre it is easily weakened by cellulase treatment. Viscose rayon is a weak fibre particularly when wet, therefore it is highly susceptible to damage if enzymatic hydrolysis is not carefully controlled. On both viscose and lyocell, cellulase alters the handle and drapeability and removes surface fuzz. Cellulose also reduces the tendency of viscose to pill and reduces fibrillation of lyocell, but on cellulose acetate the effects tend to be less marked.

Cellulases are usually classified by the pH range in which they are more effective and, accordingly, acid cellulase, neutral cellulase and alkaline cellulase, commercial products are available.


Acid Cellulases


This type of cellulases normally work in the pH range of 4.5-5.5.However, they can be further classified based on pH sensitivity. The three different types of acid cellulases and their corresponding operating pH are: standard acid cellulases: pH 4-6.5, modified acid cellulases : pH 5-6.5 and endo-enriched acid cellulases : 4.5-6.5


Acid cellulases in general, are more active on cotton cellulose compared to the neutral cellulases, hence an effective surface etching is obtained in shorter time. However, severe decomposition may occur if not properly controlled and it may result in lower tear and busting strength of the treated fabrics. Moreover, acid cellulases have a relatively short shelf life, not exceeding 2-3 months. The stability is low when storage temperature exceeds 700 Fahrenheit. This may lead to inconsistent storage results during summer months. Besides these, acid cellulases extract substantial amount of dyestuff from the fabric, particularly indigo dyes (this property is exploited in denim washing) which may in turn deposit on the white portion of the fabric. This phenomenon is known as back staining.


Neutral Cellulase


These cellulases work best at a pH 6. They are less reactive and therefore require longer treatment time compared to the former class. Strength loss and back staining problems are less with this class. Neutral cellulose powders have a longer shelf life and good thermal stability.


Alkali Cellulase


Alkali stable cellulase can be incorporated in household detergent formulations for effective stain removal.


The Bio-polishing Technology


Biopolishing or bio finishing can be performed continuously and in batch form but the treatment conditions are more easily controlled in batch processes for which winches, jiggers, jet or over flow machines are suitable. In principle, biofinishing can be carried out along with any other stage of textile finishing, with dyeing for example provided that both processes are subject to identical conditions.

However, it is best carried out after bleaching and before dyeing. The conditions for bio polishing are as follows:

The bio-polishing process requires:


Enzyme Dosage: 1-5% on fabric weight (depending on the activity of the enzyme)

Liquor Ratio: 5-15 liters / Kg. of fabric.

Time: 60-120 minutes (depending upon the amount of hydrolysis required).

Temperature: 50-600 C.

pH : 4.5-5.5 (For acid stable cellulase)


After treatment, the enzyme must be deactivated either by alkali treatment at pH 9-10 or by increasing the temperature to 70-800C and giving a treatment for 10 minutes.


 

Bio-Denim Washing:


Another use of cellulase enzyme is in the fading of denims. Denims are manufactured from indigo dyed warp yarns. The dyes are mainly absorbed on the surface of the fibre, a phenomenon technically termed as ring dyeing. The fibre surface etching with cellulase enzymes results in exposure of the undyed core of the fibres which gives a faded look to the denim. The dye removal is further facilitated by the mechanical abrasion.


Earlier the effect was obtained by washing denim with pumice stones. Pumice stones are soft, light and porous in nature. About 1-2 Kg pumice stones per pair of jeans were used to get the desired worn out look. Though stone washing gives the desired result but it has got several disadvantages. The major problem with stone washing is that lot of sludge gets deposited in the effluent tank due to worning of pumice. The sludge has to be separated from effluent water and disposed off. The use of stones was, therefore, replaced by cellulase enzymes.


When indigo dye is released to the wash liquor during washing, the solution turns dark blue. Indigo dye has two amino groups which are capable of getting protonated in an acidic media. Due to protonation, the dyestuff gains an overall positive charge on the contrary; cellulose maintains its negative charges in an acidic media. Positive and negative charges attract one another in solution. Therefore, in acidic pH the affinity of indigo for cotton increases. Some of this indigo redeposit on the whiter parts of the denim fabric which spoils the colour contrast of the stone wash effect. This phenomenon is known as back staining.


Back staining problem is more evident with acid cellulases. The use of neutral cellulases is recommended to control the back staining problem because of their better control in decolouration effect and resistance to backstaining. Some auxiliary chemicals help in controlling the back staining effect e.g. Sandoclear IDS Liq is claimed to be very efficient in removing back-staining. Treatment with proteases during rinsing or at the end of the cellulase washing step results in significant reduction of back staining and improved contrast.


The use of enzyme for denim washing has the following advantages over pumice stone washing.


  • Superior garment quality
  • Increased load handling (30 35%)
  • Environment friendly processing
  • Less damage to seams, edges and badges
  • Extra softener not required
  • Less equipment wear
  • Easy handling of floors and sewers
  • No handling of pumice/ ceramic stones


Further Reading


  1. D. P. Chattopadhyay , J . K Sharma & R B Chavan , Indian Journal of Fibre & Textile Research, June 2000, pp.121-129
  2. D. P. Chattopadhyay, K. N. Chatterjee, I. Bhadra and Rucheera Gumber, Man-Made Textiles in India, November 1997, pp 452-458.
  3. A. K. Patra & D. P. Chattopadhyay, Textile Asia, August 1998, pp 46-48
  4. S V. Chikkodi, Textile Research Journal, December 1995, pp 564-569
  5. Andersen, International Dyer, September 1995, pp 10-15.
  6. S. B. Pawar, K. D. Shah & G. R. Andhorikar, Man-Made Textiles in India, April 2002, pp 133-138.
  7. Rekha R, Man-Made Textiles in India, October 2002, pp 398-401
  8. S. R. Shukla, U.Sharma & K. S. Kulkarni, Colourage, February 2000, pp 19-24.
  9. A. Kumar, Mee Young yoon and Charles Purtell, Textile Chemist & Colourist,. April 1997, pp 37-42
  10. Pia sarkka, ITB Dyeing/Printing/Finishing, February 1995, pp 62-66.
  11. B Schmitt & A. K. Prasad, October 1998, pp 20-24.
  12. G. Nalankilli, Colourage, October 1998, pp 17-19.
  13. K. Wong, X. M. Tao, C. W. M. Yuen & K. W. Yung, Textile Asia, March 1997, pp No. 47-52.

About the Author:


The author is associated with the Department of Textile Chemistry in the Faculty of Technology & Engineering in M. S. University, Vadodara , India.



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