Lipase immobilized on walls of plastic beaker: Kinetic properties and application in washing of oil stained cloth

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1 Indi an Journal of Chemical Technology Vol. I 0. November 03, pp Lipase immobilized on walls of plastic beaker: Kinetic properties and application in washing of oil stained cloth S Arya, V Kalia, P Chandran & C S Pundir* Biochemistry Research Laboratory, Depanment of Bio-Sciences, M D University, Rohtak I, India Received 15 July 02; revised received 18 March 03; accepred 16 April 03 Commercial porcine pancreas lipase was immobilized covalently onto alkylamine glass-beads affixed on the inner wall of a plastic beaker by an adhesive. The immobilized enzyme retained 10.8% of the initial activity of free enzyme with a conjugation yield of 52 mg/g support. The optimum ph and incubation temperature were decreased, while time for linearity and Km for triolein of enzyme w.ere increased after immobilization. The utility of immobilized enzymes in removal of oil stain from cotton cloth by various detergents was tested by chemical method. All the detergents gave better washing in presence of immobilized lipase than that by detergent alone. Further, the washing by cheaper (non-enzymic) detergents in presence of immobilized lipase was almost similar to that by expensi, e (enzymic) detergents. The immobilized enzyme was used about times without any considerable loss of activity. Covalent immobilization of porcine pancreas lipase o nto free alkylamine glass-beads and its usefulness in removal of oil stain from cotton cloth has been reported earlier 1 Though, a better washing of cotton cloth es was found in presence of alkylamine glass bound lipase compared to the detergent alone, yet the handling of free alkylamine glass-beads and its reuse was a problem. There was always a chance of losing the glass-beads while removing the cotton cloth after washing and also during the change of detergent solution for next washing and removal of washing discard. Further, the washing i.e., removal of oil stain from cotton clothes was assessed only on visual basis. These problems were overcome in the present work by affixing th e alkylamine glass-beads on the wall of container before immobilization and employing a chemical method to evaluate the washing. Experimental Procedure Materials Zirconia coated alkylamine glass-beads (pore diameter 55 nm) was a gift from Dr H.H. Weetall, Environment Protection Group, Las Vegas, NV U.S.A.; lipase from porcine pancreas (40-70 U/mg protein) was from M/s SISCO Research Laboratory Pvt. Ltd., Mumbai. 'Lakme' long stay najj enamel of si lver white colour (754 dew drops) manufactured by Flora Cosmetics Ltd., Kundain, Goa (India) was *For correspondence ( pundircs@rediffmail.com; Fax: (9 1) ( 1262) ) purchased from local market. Gum-arabic, ol i ve-oi I and detergents were purchased from local market. A II other chemicals used were of AR grade. Assay of soluble lipase The porcine pancreas lipase was dissolved in 0.1 M tris buffer, ph 8.0 (I mg/ml). The activity of solu ble lipase was measured by titrati on of fatty acid. released from hydrolysis of fat by it as described by Sarita et al 1 One unit of lipase is defined as the amount or enzyme required to liberate Ill mole of free fatty acid from olive oil per min under the standard assay conditions (at 35 C and ph 8.0). Immobilization of lipase Oil affzxed glass-beads The inner side of a ml plasti c beaker was scratched with sand paper and then coated uni fo rml y upto the height of 1.5 em with a li ght coloured nai l paint (Lakme) with a brush. The powder or alkylamine glass-beads (300 mg) was sprinkled uniformly on thi s nail-paint layer and allowed it tn stand at room temperature (30±5 C) for 24 h. The lipase was immobilized on the affixed glass-beads through the process of glutaraldehyde coupling using the method of Guo et al.z with modificati ons. To JOO mg glass-beads affixed inside the pl astic beaker. : ml of 2.5% glutaraldehyde prepared in 0.1 M tri s HCI buffer, ph 8.0 was added and allowed to stand for 2 hat room temperature with gentle shaking after every 10 min. After 2h, the excess of glutaraldehyde was decanted and the glass-beads were washed

2 Arya eta/.: Applicati on of immobilized lipase in washing of oil stained cloth thoroughly with 0.1 M tris HCI buffer ph 8.0 until the ph of the washing discard was 8.0. Lipase solution ( ml) was added to the glutaraldehyde activated glass-beads and kept at 4 C for 48 h with occasional shaking. The unbound enzyme was decanted and tested for activity and protein. The glass-beads were washed 3-4 times with the same buffer, until no activity of enzyme was detected in the washing. The enzy me protein bound to glass-beads was estimated by determining the loss of protein from the solution during immobilization using the method of Lowry et ai. Assay of immobilized enzyme The reacti on mixture containing 5 ml of olive-oil emul sion and 5 ml of tris HCI buffer (0.1 M, ph 8.0) in a ml glass beaker was preincubated at 35 C for I 0 min in a water-bath shaker. Then this reaction mixture was transferred to the plastic beaker contmlllng immobilized lipase onto affixed alkylamine glass-beads termed as 'reaction beaker'. In case of control, the plastic beaker contained only gl utaraldehyde activated affixed alkylamine glass beads. After incubation at 35 C for min. 10 ml acetone and methanol mixture (I : 1) was added to both test and control flask to stop the reaction and the reaction mi xture was then titrated against N NaOH using phenolphthalein as indicator and volume of NaOH consumed in titration was noted. Application of affued immobilized lipase The affixed immobilized lipase was used for removal of oil stain from cotton cloth in presence of solution of commercial detergents. For thi s purpose, the cotton cloth pieces (size: 4.5x4.5 em) were stained with 0.2 ml mustard oil. The stock solutions of eight commercially available detergents namely Surf Excel, Aerial Compact, Rin Shakti, Surf Ultra, Nirma, OK, Rin Supreme and Wheel in di stilled water (2.0 g/l) were prepared. Three stained cloth pieces were taken for each detergent. One piece was washed with detergent alone, second pi ece was washed with detergent in affixed immobilized lipase while th e third piece was washed with detergent and free lipase solution at 30 C for min with continuous stirring. Similarly, one stained piece was washed in di still ed water while other was washed in distilled water and immobilized lipase. Each washed cloth piece was rinsed two times manually with distilled water. The washing performance in each case was determined as follows: the content of oil retained in test cloth after washing, was extracted by dipping the washed cloth piece in 10 ml of petroleum ether for min with gentle stirring. The fat extract was transferred to a ml round bottom distillation flask containing 25 ml alcoholic KOH (0.5 M). The flask was attached to a reflux condenser and the mixture was reflu xed in a boiling water bath for 30 min. The flask was removed. cooled to room temperature (30 C) and th e mi xture was titrated against 0.5 M HCl us1ng I CJr phenolphthalein as an indicator. The blank was set up similarly but no oil was taken in it. The volume o f HCI consumed in each titration was noted down. Reusability and storage To reuse the affixed immobilized lipase, th e ' reaction beaker' was rinsed with di stilled water 3--+ times and used in the next washing. The 'reaction beaker' was filled with distilled water to cover immobilized lipase and stored at 4 C for 3 mont hs when not in use. Results and Discussion Commercially available porcine pancreas lipase has been immobilized covalently onto alkylamine glassbeads affixed on the inner side of a pl asti c beaker with a fixative. The immobilized enzyme retained about 10.8% of the initial activity of free enzyme with a conjugation yield of 52 mg/g of support (Table I). Kinetic properties of immobilized lipase A comparison of some kinetic properties of lipase coupled to affixed alkylamine glass with those of free enzyme and enzyme bound to free alkylamine glassbeads is given in Table 2. The maximum acti vity of Table l- lmmobilizati on of porcine pancreas lipase onto alkylamine g lass-beads affixed inside a pl astic beaker with nai l paint Lipase added to 300mg Lipase coupled to % Lipase Total ac ti vity Total activit y Retention o f Conju gation affixed glass-beads 300mg affixed coupled added (Jlmo le (Jlmole FFA/min) specific yie ld (mg) glass-beads (mg) FFA/min) activity (mg./g) [Sp. ac ti vity of free lipase was 3.86 J.!M FFNmin/mg. Sp. activity of immobilized lipase was J.!M FFA/min/mg. The enzy me ac ti vit y is defined as the amount of enzyme required to produce I J.Jmo le FFA/min under th e standard assay conditions.! FFA =Free fatty acid Data are the mean o f three replicates 599

3 Indian J. Chem. Techno!., November 03 Table 2- Kinetic parameters of free porcine pancreas lipase and lipase bound to free and affixed alkylamine glass-beads Parameters Free lipase Lipase conjugated to free alkylamine glass-beads Lipase conjugated to affixed alkylamine glass-beads Optimum ph Temperature for max imum activity " (Kcal/mole) Time of incubation Saturating concentration of triolein substrate (mm) Km for triolein Data are the mean of three replicates C min 50mM 4.242x 10-3 M mol/min C min C min loomm x l0-1 M 1.33 mol/min 1 --Free --o-tmmobilized --~r- Affixed immobilized 1 --Free-D-Immobilized --l>-affixed immobilized ~ 80 ;; ~ <(... ~ 60 Q) 0:: "# ph Temp ("C) 50 Fig.!-Effect of ph on free lipase and lipase immobilized onto free and affixed alkylamine glass-beads on wall of a plastic beaker. affix ed alkylamine glass bound lipase was attained at ph 6.8, which is slightly lower than that of enzyme bound to free glass-beads (ph 7.1) and much lower than that of free enzyme (ph 7.5) (Fig. 1). It might be displaced upon immobilization particularly when the support material is charged. The affixed immobilized enzyme showed maximum activity at 30 C which is equal to that of enzyme bound free glass-beads and lower to that of free enzyme (35 C) (Fig. 2). The energy of activation (.) of the immobilized enzyme was increased to Kcallmole compared to free enzyme (2.05 Kcallmole) and almost equal to lipase conjugated to free glass-beads (5.404 Kcal/mole). The time of incubation was increased to 50 min in comparison to that of free lipase (15 min) and lipase conjugated to free alkylamine glass-beads ( min) (Fig. 3), which might be due to diffusion of the Fig. 2-Effect of temperature on free lipase and li pase immobilized onto free and affixed alkylamine glass-beads on wa ll of a plastic beaker. substrate from bulk to the active center of the immobilized enzyme. The substrate (tri olein ) concentration required for the maximum acti vity or saturation of lipase was increased from mm (for free enzyme) to 0 mm after immobilizati on. K 111 value as calculated from Lineweaver-Burk Pl ot was increased for affixed immobilized enzyme ( x10-3 M) as compared to free enzyme (4.242 x lo --~ M ) indicating the decreased affinity of the enzy me towards the substrate (triolein) after immobilizati on. The Vmax was changed from ~-tmollmin (free enzyme) to 1.33 ~-tmol/min after immobilizati on to affixed glass beads. The changes in kinetic propert ies of enzyme after immobilization are controlled by four factors: Change in enzyme conformation and its microenvironment, steric effects and bulk and diffusional effects

4 Arya el a/.: Application of immobilized lipase in washing of oi l stained cloth 1 --Free -o-lmmobilized Affixed immobilized c 80 :;: u <( ~ 60 ~ Qi a::: 40 cf Time of incubation (min) Fig. 3-Effect of time of incubation on free lipase and lipase immobilized onto free and affixed alkylamine glass-beads on wall of a plastic beaker. Storage and reusability The immobilized lipase on glass-beads affixed inside the plastic beaker showed no noticeable loss of its activity during its regular uses for about times over a period of 3 months, when stored in distilled water at 4 C. Application of afftxed immobilized lipase The oi I stained cotton cloth pieces were washed with detergents alone and in presence of free and immobilized lipase. The residual oi l in cloth pieces was extracted in petroleum ether, saponified with 0.5 M alcoholic KOH and the unused KOH was titrated against 0.5 M HCl using phenolphthalein as an indicator. The amount of HCl used in titration of each case is given in Table 3, which is directly proportional to the amount of unused KOH, which in turn reflects the residual oi l in cloth piece after washing. Hence, more the HCl used and more the unused KOH, lesser the residual oi l and better the washing performance. Two types of detergents were tested, expensive (enzymic) detergents such as Surf Excel and Ariel Compact and non-enzymic detergents such as Surf Ultra, Rin Shakti, Nirma, OK, Rin Supreme and Wheel. The washing performance of detergents in presence of immobilized lipase or free lipase was found in the following order: Surf Excel> Ariel Compact> Rin Supreme> Rin Shakti> Surf Ultra> Nirma> OK> Wheel> Distilled water. Similar order Table 3-Evaluation of washing performance by titrimetri c determination of residual oil content in the cotton c loth after washing with various detergents alone and in the presence of free/immobilized porcine pancreas lipase on inner wall o f plasti c beaker Detergents used (2g/L) None (Distilled water) 14.5 Surf Excel 16.3 Ariel Compact 16.1 Rin Shakti 15.7 Surf Ultra 15.4 Nirma 15.1 OK 14.9 Rin Supreme 15.7 Wheel 14.7 Data are the mean of three replicates Amount of 0.5 M HCI used in titration (ml) Without With With lipase immobili zed free lipase lipase R of washing performance was obtained by detergents alone. The combinations of free/ affixed immobilized lipase with any one of the d~tergents gave better washing than that by detergent alone. Although washing of cotton clothes by detergents gave better results in presence of free lipase compared to that by immobilized lipase, free lipase cannot be preferred over immobilized one, due to its exhaustible property. The amount of HCI consumed in washing by nonenzymic detergents such as Rin Shakti and Rin Supreme+immobilized lipase was almost similar to that by enzymic detergents alone such as Surf Excel or Aerial revealing the same washing performance or cheaper detergents in presence of immobilized lipase as that of expensive detergents. The immobilized lipase was reused about times in such washings without any considerable loss of activity. Further th e lipase bound to affixed glass-beads had no handling problem and no risk of loss of support, as experienced in case of enzyme coupled to free glass beads 1 Lipase is well-known to hydrolyse the fats present in the oil and thus removes the oil stain from the cloth freely and rapidly. The lipase has been employed along with the alkaline proteases in the detergents. Generally. lipase in the free form is not safe as this might be attacked by proteases and inhibited by surfactants 5. Lipase immobilized to affixed alkylamine glass-beads in the detergent solution is generally susceptible ro inhibitory effects of surfactant and protease. Thus, th e use of plastic beaker bound lipase in washing of oil stained clothes by detergents has not only increased their washing efficiencies without consuming th em in 601

5 Indi an J. Chem. Techno!., November 0."1 the process but also made cheaper detergents equivalent to expensive detergents alone for washing purpose. Conclusion Commercial porcine pancreas lipase has been immobilized onto alkylamine glass-beads affixed on inner base of a plastic beaker. There were some changes in the kinetic properties of enzyme after immobilization. The immobilized enzyme was employed in washi ng of oi l stained cotton clothes by vari ous detergents. The washing by cheaper (non-enzymic) detergents in presence of immobilized enzyme was almost similar to that by expensive (enzymic) detergents. The immobilized enzyme was reused about times without any loss of its activity. Thi s work could be exploited for improving the washing performance of washing machines by immobi lizing the lipase on the inner walls of a washing chamber. References Sarita Malik V & Pundir C S. Indian J Che111 Tcdutnl. 7 (00) Guo J A, Mo P S & Li G X. Appl Bioche111 Biotccluwl. 23 (1990) I 5. 3 Lowry 0 H, Rosenbrough N J, FarrA L & Randal R J..I!Jinl Che111, 193 ( I 951) Vieth W R & Yenkatasubramani an K, Che111 Techno/. 4 ( i<j 7-l) Andree N, Muller W R & Schmid R D. Appl!Jiocltnlt Biotechnol, 2 ( I 980) 2 I