Glutaraldehyde activation of polymer Nylon-6 for lipase immobilization: Enzyme characteristics and stability

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Bioresource Technology 99 (2008) 2566–2570

Glutaraldehyde activation of polymer Nylon-6 for lipase immobilization: Enzyme characteristics and stability Shweta Pahujani a, Shamsher S. Kanwar a, Ghanshyam Chauhan b, Reena Gupta a

a,*

Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, HP, India b Department of Chemistry, Himachal Pradesh University, Summer Hill, Shimla 171005, HP, India Received 10 December 2006; received in revised form 17 April 2007; accepted 20 April 2007 Available online 11 June 2007

Abstract An extracellular alkaline lipase of a thermo tolerant Bacillus coagulans BTS-3 was immobilized onto glutaraldehyde activated Nylon-6 by covalent binding. Under optimum conditions, the immobilization yielded a protein loading of 228 lg/g of Nylon-6. Immobilized enzyme showed maximum activity at a temperature of 55 °C and pH 7.5. The enzyme was stable between pH 7.5–9.5. It retained 88% of its original activity at 55 °C for 2 h and also retained 85% of its original activity after eight cycles of hydrolysis of p-NPP. Kinetic parameters Km and Vmax were found to be 4 mM and 10 lmol/min/ml, respectively. The influence of organic solvents on the catalytic activity of immobilized enzyme was also evaluated. The bound lipase showed enhanced activity when exposed to n-heptane. The substrate specificity of immobilized enzyme revealed more efficient hydrolysis of higher carbon length (C-16) ester than other ones. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Bacillus coagulans BTS-3; Lipase; Glutaraldehyde; Nylon-6 and Immobilization

1. Introduction Lipases (E.C. 3.1.1.3) occupy a place of prominence among biocatalysts owing to their multiple and novel multifold applications in oleo-chemistry, organic synthesis, detergent formulation and nutrition (Pandey et al., 1999). In the last few years, there has been an increasing interest in the use of enzymes for the biosynthesis of molecules in organic media (Gargouri et al., 2002; Castillo et al., 2003; Noel and Combes, 2003). Lipases have been successfully immobilized on a variety of matrices for performing esterification and trans-esterification reactions in organic solvents (Hiol et al., 2000). Immobilized lipases offer economic incentives of enhanced thermal and chemical stability, ease of handling, easy recovery and reuse relative to non-immobilized forms (Malcata et al., 1990; Kanwar et al., 2004). A particularly convenient, cheap and relatively inert matrix for immobilization of enzyme is Nylon-6. Unlike *

Corresponding author. Tel.: +91 177 2831948. E-mail address: [email protected] (R. Gupta).

0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.04.042

most other nylons, Nylon-6 is not a condensation polymer, but instead is formed by ring-opening polymerization. During polymerization, the peptide bond within each caprolactam molecule is broken; with the active groups on each side re-forming two new bonds as the monomer becomes part of the polymer backbone. Nylon-6 resembles natural polypeptides more closely; in fact, caprolactam would become an amino acid if it were hydrolyzed. Also it is non-toxic and readily available and can be obtained in a number of forms. The activation of nylon involved partial acid hydrolysis of the Nylon-6 surface to generate amino groups (and carboxyl groups), which could be coupled to proteins with glutaraldehyde (Sundaram and Hornby, 1970). Recently, Mucor javanicus lipase was effectively immobilized on silica nanoparticles activated by glycidyl methacrylate (Kim et al., 2006). Nylon-6 being largely non-porous is surface-bound with enzyme and it is difficult to compare enzyme kinetics and yields of immobilized enzyme due to the different forms of Nylon-6 used (mesh size or average bead diameter for example influence surface area per unit weight) (Zaidi

S. Pahujani et al. / Bioresource Technology 99 (2008) 2566–2570

et al., 1995). The aim of the work was to study the immobilization and biochemical characteristics of lipase of Bacillus coagulans BTS-3 immobilized on Nylon-6.

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enzyme activity and protein concentration (unbound) in the supernatant and matrix was assayed by standard methods; the relation between the matrix bound protein concentration and its corresponding activity was calculated.

2. Methods 2.5. Immobilization kinetics 2.1. Microorganism and lipase The thermophilic and alkaliphilic lipase-producing bacterium was originally isolated from the kitchen waste of a sweet shop through direct enrichment method using normal saline (0.89%, v/v) under shaking conditions. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE–Sepharose column chromatography. Its molecular weight was 31 kDa on SDS– PAGE (Kumar et al., 2005). 2.2. Immobilization of lipase on Nylon-6 The purified lipase from B. coagulans was immobilized on Nylon-6. Increasing amount of protein (2.28–22.8 g) was added to a fixed amount of matrix (50 mg). Nylon-6 particles were partially hydrolyzed with 6 N hydrochloric acid for 30 min, rinsed with distilled water and contacted with 2.5% glutaraldehyde solution prepared in potassium phosphate buffer at pH 7.0 for 1 h. Finally, the glutaraldehyde-treated Nylon-6 beads were rinsed with excess of buffer and contacted with a purified enzyme at 4 °C for 12 h. 2.3. Enzyme assay Lipase assay was performed by a colorimetric method (Winkler and Stuckmann, 1979). The stock solution of pNPP (20 mM) was prepared in iso-propanol. The reaction mixture comprised of 75 ll of p-NPP stock solution and 5 ll of crude/ purified enzyme or 50 mg of immobilized enzyme. The final volume of this reaction mixture was made to 3 ml with 0.05 M tris buffer, pH 8.5 for free and 7.5 for bound lipase with Triton X-100 (0.4%, v/v) and gum acacia (0.1%, w/v). The test tubes were incubated for 10 min at 55 °C under continuous shaking (120 rpm) in water-bath-incubator. Appropriate control with a heatinactivated enzyme (5 min in boiling water bath) was included with each assay. The absorbance of p-nitrophenol released was read at A410 nm (Shimadzu UV/Visible spectrophotometer, Japan). Each of the assays was performed in duplicate and mean values were presented. The enzyme activity was defined as lmol(s) of p-nitrophenol released per min by 1 ml of free enzyme or per gram of immobilized enzyme (weight of matrix included) under standard assay conditions. The protein was assayed by a standard method (Lowry et al., 1951). 2.4. Effect of protein concentration on immobilization Increasing amounts of protein (2.28–22.8 lg) were incubated with fixed amount of the Nylon-6 matrix (50 mg). The

A study was conducted to find out the rate of immobilization of the lipase on the Nylon-6. The activity of (unbound) lipase and free protein was checked in the supernatant (which had already been withdrawn periodically) by standard methods. 2.6. Biochemical characterization of Nylon-6 immobilized lipase 2.6.1. Effect of temperature and pH on the activity The immobilized enzyme (50 mg matrix) was taken in potassium phosphate buffer, pH 8.5 and assayed for residual lipase activity at different temperatures 35, 45, 55, 65 and 75 °C and for pH same amount of enzyme was assayed in tris buffer (0.1 M) at different pH values (6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5). 2.6.2. Stability profile of immobilized enzyme The thermostability of immobilized enzyme was studied by incubating the biocatalyst at 55, 60, 65 and 70 °C for 2 h in a water bath-shaker (120 rpm). Likewise to determine stability at varying pH, the immobilized enzyme was separately preincubated in 0.1 M tris buffer at pH 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5 for 2 h at 55 °C and the residual lipase activity was determined under standard assay conditions. 2.6.3. Reusability of immobilized enzyme for hydrolytic activity towards 4-NPP Immobilized enzyme was washed thrice with 0.1 M tris buffer (pH 8.5) and enzyme activity was assayed at 55 °C for 10 min repeatedly till the hydrolytic activity towards p-NPP was 25% of the original activity. 2.6.4. Determination of Km and Vmax The assay of bound enzyme was carried out using different concentrations of 4-NPP (5, 10, 15, 20 and 25 mM). A Lineweaver–Burke curve was used to calculate Km and Vmax of the enzyme. 2.6.5. Effect of organic solvents on immobilized lipase activity The effect of various alcohols (methanol, ethanol, isopropanol, iso-butanol, acetone and chloroform) as well as alkanes (n-pentane, n-hexane and n-heptane) was studied on activity of immobilized lipase. The bound lipase (50 mg) was separately incubated with each of the selected solvents at 55 °C for 30 min. Thereafter, the solvent was removed by decantation and immobilized biocatalyst was assayed for residual lipase activity.

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2.6.6. Substrate specificity of immobilized lipase Each of the selected substrate, 4-nitrophenyl palmitate, 4-nitrophenyl laurate, 4-nitrophenyl caprylate and 4-nitrophenyl formate (20 mM) prepared in iso-propanol was used to assay the hydrolytic activity of the bound lipase at 55 °C after 10 min incubation.

ecules of water (Rahman et al., 2005). Previously when lipase from a Bacillus GK8 was immobilized on silica, it took only 30 min to bind maximally (Dosanjh and Kaur, 2002). In another study, lipase from B. coagulans BTS-1 showed maximum immobilization onto silica after 20 min of incubation (Kanwar et al., 2004).

3. Results and discussion

3.2. Characteristics of immobilized enzyme

3.1. Immobilization and immobilization kinetics of lipase on Nylon-6

An increase of four times in the activity of immobilized lipase was observed when temperature was raised from 45 to 55 °C. Optimum temperature of bound enzyme (55 °C) was also the optimum temperature of free enzyme (Kumar et al., 2005). A further increase in the temperature resulted in a marked decrease in the activity of bound lipase. In another study, the optimum temperature for immobilized lipase from a mutant strain of Corynebacterium (Roy et al., 2004) and silica-immobilized lipase from the B. coagulans MTCC-6375 (Kanwar et al., 2005) was found to be 50 °C. Immobilization of lipase from C. rugosa on chitosan showed optimum reaction temperature of 30 °C (Hung et al., 2003) while immobilization of lipase from same organism on kaolin showed highest activity at 40 °C (Rahman et al., 2005). The bound enzyme showed 88% residual activity when incubated at 55 °C for 2 h while residual activity decreased at higher temperatures, which might be due to the disturbance of globular structure of the protein by heat. Earlier, free enzyme preparation showed 50% residual activity at 55 °C (Kumar et al., 2005). In our recent study, the enzyme when immobilized on activated polyethylene, showed 60% residual activity at 70 °C (Kumar et al., 2006). Thus, the immobilized preparations were much more stable than the soluble enzymes at higher temperatures. Lipase from Bacillus thermocatenulatus when immobilized on hydrophobic supports, maintained 100% of the activity at 65 °C (Palamo et al., 2004). Bacillus GK8 lipase when immobilized on HP-20 beads retained complete activity at 60 °C (Dosanjh and Kaur, 2002). The immobilized enzyme was fairly stable within a pH range of 7.5–9.5 with optimum pH 7.5. The free enzyme was stable within a pH range of 8.0–10.5 with optimum pH 8.5 (Kumar et al., 2005). Bacillus sp. lipase was stable in the pH range of 7.0–10.0 with optimum pH 8.0 (Dosanjh and Kaur, 2002). Lipase from a mutant strain of Corynebacterium sp. was immobilized and pH for the assay of immobilized lipase was found to be the same (8.0) as that of a soluble enzyme (Roy et al., 2004). In contrast, the immobilized lipase of C. rugosa was stable in the pH range of 5.0–8.0 with optimum pH 9.0 (Hung et al., 2003).

The lipase of B. coagulans BTS-3 (total protein 11.4 lg) when incubated with support (50 mg) showed maximum binding efficiency (70%) for the protein (lipase) with 1.2 U/g of enzyme activity (Fig. 1). Previously, a protein loading of approximately 15 mg/g was reported for lipase immobilized on Nylon-6 (Zaidi et al., 2002). Recently, it was found that 77% of protein in supernatant from Candida rugosa was immobilized onto kaolin (Rahman et al., 2005). The immobilization kinetics revealed that the lipase was optimally immobilized on Nylon-6 within 1 h of incubation (Fig. 2) and only little free (unbound) lipase could be detected in the supernatant. The lipase molecules might be immobilized on the exposed surface and within the upper layer of the support by the chemisorption (adsorption) as well as chemical activation (glutaraldehyde) of Nylon-6 as the protein molecules of lipase replace the molProtein

70

Bound protein (%)

1.4

Lipase

1.2

60

1

50

0.8

40 0.6

30

0.4

20

Bound lipase (U/g)

80

0.2

10 0 0

2.28

5.7

11.4

17.1

0 22.8

Total protein (microgram)

80

1.4

70

1.2

60

1

50

0.8

40

0.6 30 20 10

Protein (%)

0.4

Lipase (U/g)

0.2

Bound lipase (U/g)

Bound protein (%)

Fig. 1. Effect of protein concentration on immobilization of lipase from Bacillus coagulans BTS-3 on Nylon-6.

0

0 0

10

30

60

120

180

240

300

Time (min)

Fig. 2. Immobilization kinetics of lipase from Bacillus coagulans BTS-3.

3.3. Reusability of immobilized enzyme for hydrolytic activity towards 4-NPP Immobilized enzyme retained 85% of its original hydrolytic activity after 8th cycle (Table 1). In a previous study, a Bacillus lipase immobilized on CNBr-activated-Sepharose 4B retained full activity even after 13 cycles (Dosanjh

S. Pahujani et al. / Bioresource Technology 99 (2008) 2566–2570 Table 1 Reusability of immobilized lipase for hydrolytic activity towards 4-NPP Cycle no.

Activity (U/g)

Residual activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13

1.22 1.19 1.16 1.14 1.11 1.09 1.07 1.05 0.98 0.87 0.72 0.35 0.25

100 97.5 95.0 93.4 90.9 89.3 87.7 86.0 80.3 71.3 59.0 28.5 20.4

and Kaur, 2002). Recently, C. rugosa lipase was immobilized in the polymer of polyvinyl alcohol (PVA), alginate and boric acid and showed nearly complete retention of activity in reuse up to 10 cycles (Dave and Madamwar, 2006). 3.4. Km and Vmax of the immobilized lipase The Km for immobilized lipase was 4 mM and Vmax 10 lmol/min/ml. The Vmax of immobilized lipase was found to be higher than that of free lipase (0.72 lmol/ min/ml). The comparable Km values for Nylon-6 immobilized lipase and free lipase (3.8 mM) indicated almost same affinity towards the substrate. For a Pseudomonas cepacia lipase, Km and Vmax values of 12 mM and 30 lmol/min, respectively, were reported with p-NPP (Pancreac’h and Baratti, 1996). The Km and Vmax values of 18.0 mM and 96.1 U/mg protein, respectively for C. rugosa lipase immobilized on chitosan were reported (Hung et al., 2003). 3.5. Effect of different organic solvents on the catalytic activity

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iso-propanol increased the activity of bound enzyme while ethanol, methanol and iso-butanol inhibited the activity. The supports might trap and prevent the disruption of the enzyme-bound water essential to maintain the threedimensional structure of the enzyme for catalysis as the polar solvents tend to strip water from enzyme molecule. Therefore, enzyme might be more stable in organic solvents than in water, and this was the reason why they were used as reaction media in enzymatic activities. In our earlier study, the same enzyme when immobilized on glutaraldehyde-activated-polyethylene showed enhanced activity in presence of n-hexane (Kumar et al., 2006) 3.6. Substrate specificity The higher C-length (C:16) ester (4-NPP) was more efficiently (60–70%) hydrolyzed by bound enzyme than other esters. 4-NPP was also more efficiently hydrolyzed by free enzyme (Kumar et al., 2005). This indicated a preferential specificity of B. coagulans lipase towards longer carbon chain length substrates, as reported previously for a thermoalkaliphilic lipase from Bacillus sp. (Sunna et al., 2002). In another study, a lipase from psychrotrophic Pseudomonas sp. displayed highest activity towards C:10 acyl groups of 4-nitrophenyl esters (Rashid et al., 2001). Also, immobilized lipase from B. coagulans BTS-1 was more hydrolytic to a medium C-length ester than shorter or longer C-length esters (Kanwar et al., 2004). 4. Conclusion In the present study, a lipase from B. coagulans BTS-3 immobilized on glutaraldehyde activated Nylon-6 retained good lipase activity at pH 7.5 and temperature 55 °C; and also showed efficient hydrolysis of substrate p-NPP during repetitive catalysis in aqueous phase. It showed enhanced activity when exposed to n-heptane and iso-propanol. Acknowledgements

The activity of immobilized lipase was enhanced in presence of n-heptane and decreased in acetone as compared to the control (Table 2). Amongst alcohols, an exposure to

The financial support from Department of Biotechnology, Ministry of Science and Technology, Government of India to Department of Biotechnology, Himachal Pradesh University, Shimla (India) is thankfully acknowledged.

Table 2 Effect of organic solvents on the activity of immobilized enzyme

References

Solvent

Activity (U/g)

Relative activity (%)

Buffer Methanol Ethanol iso-Propanol iso-Butanol Acetone Chloroform n-Pentane n-Hexane n-Heptane

1.20 0.56 0.53 1.28 0.79 0.19 1.38 0.75 0.13 1.88

100 46 44 106 65.8 15.8 115 62.5 10.8 156

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