Optimal immobilization of β-galactosidase from Pea (PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications

Bioresource Technology 100 (2009) 2667–2675

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Optimal immobilization of b-galactosidase from Pea (PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications Alka Dwevedi, Arvind M. Kayastha * School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India

a r t i c l e

i n f o

Article history: Received 14 October 2008 Received in revised form 23 December 2008 Accepted 23 December 2008 Available online 4 February 2009 Keywords: b-Galactosidase Sephadex Chitosan Immobilization Lactose hydrolysis

a b s t r a c t Response surface methodology (RSM) and centre composite design (CCD) were used to optimize immobilization of b-galactosidase (BGAL) from Pisum sativum onto two matrices: Sephadex G-75 and chitosan beads. The immobilization efficiency of 75.66% and 75.19% were achieved with Sephadex G-75 and chitosan, respectively. There was broad divergence in physico-chemical properties of Sephadex-PsBGAL and chitosan-PsBGAL. Chitosan-PsBGAL was better suited for industrial application based on its broad pH and temperature optima, higher temperature stability, reusability etc. Sephadex-PsBGAL and chitosanPsBGAL showed much variation in their catalytic properties with respect to soluble enzyme. About 50% loss in activity of Sephadex-PsBGAL and chitosan-PsBGAL were observed after 12 and 46 days at 4 °C, respectively. Chitosan-PsBGAL showed higher rate of lactose hydrolysis present in milk and whey at room temperature and 4 °C than Sephadex-PsBGAL. In both cases, lactose of milk whey was hydrolyzed at higher rate than that of milk. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Enzymatic hydrolysis of lactose by b-galactosidase (BGAL) has two main biotechnological applications; the utilization of whey, as glucose and galactose (the hydrolysates) having greater fermentation potential (Kosaric and Asher, 1985) and in the production of low lactose milk (and dairy products made from it) for consumption by lactose intolerant persons (Kretchmer, 1972). According to US patent 5334399, BGAL is being used for processing milk from yeasts Kluyveromyces fragilis, Kluyveromyces lactis and Candida pseudotropicalis. The enzymes are available either in liquid form, e.g., as solutions in water/glycerin, or in powder form. Recent approach is aiming to carry out lactose hydrolysis at lower temperatures (at 4 °C); higher temperatures lead to changes associated with browning due to maillard reaction, amadori rearrangement and direct caramelization (McBean and Speckmann, 1999). The maillard-type browning and sugar-amino type are most prevalent as they occur even at room temperatures due to low energy of activation than direct caramelization and are autocatalytic. Furthermore, low temperature hydrolysis is beneficial at industrial scale as fouling of reactor and microbial contamination are two major concerns to be taken into consideration to preserve the quality of milk. BGAL preparations, with operativity at lower temperatures could be much helpful in this respect. The activity of above specified BGAL preparations based on neutral lactase units (NLU) as determined by assay, falls in the range from approximately 1000–5000 NLU/g. * Corresponding author. Tel.: +91 542 2368331; fax: +91 542 2368693. E-mail address: [email protected] (A.M. Kayastha). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.12.048

It requires one gallon of 1000 NLU/g activity product to convert 1000 gallons of milk to a 70% lactose hydrolysis level in 24 h when the milk is stored at 4 °C. The food and drug administration (FDA), USA has determined that hydrolyzed lactose products must contain at least 70% less lactose than the non-hydrolyzed product for consumption by lactose intolerants. Lactose hydrolyzed, ultra-pasteurized or pasteurized milk containing at least 70% less lactose have been available in the USA for several years. Using immobilized BGAL with operativity at low temperatures would be industrially more valuable and cost effective. Industrially used BGAL preparations are either from bacterial or fungal sources, used for lactose hydrolysis however, there are no reports from plant sources. Widely distributed plant BGAL (Dey, 1984) could be a good substitute for lactose hydrolysis in industries due to easy availability, cost effectiveness and would be acceptable by GRAS [Generally recognized as safe, a FDA of USA]. Recently, biopolymers such as alginate, carrageenan, cellulose beads etc. (Martinsen et al., 1992; Jouenne et al., 1994; Roy and Gupta, 2003) are widely adapted for immobilization due to increasing demand from the consumer for so-called health foods, biodegradable, inexpensive, non-toxic and easy availability. The designing of efficient immobilized enzyme onto a matrix is a multivariate process involving many factors that could affect immobilization efficiency. The classical method of determining optimum conditions by varying one parameter while keeping the other at specified constant level is a single-dimensional, laborious and time-consuming method, often does not guarantee determination of optimal conditions (Wernimont, 1985). On the other hand, carrying out experiments with every possible factorial combina-

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tion of the test variables is impractical because of the large number of experiments required (Haaland, 1989). In order to overcome these problems, optimization studies have been done using response surface methodology (RSM), a statistically designed experimental protocol in which several factors were simultaneously varied. This multivariate approach has advantages in terms of reductions in the number of experiments, improved statistical interpretation possibilities and reduced time requirements from overall analysis. RSM has been found to be much successful and economical during optimization of various industrial processes (Ren et al., 2008; Deepak et al., 2008). The present work reports the immobilization of PsBGAL from Pisum sativum onto two matrices: Sephadex G-75 and chitosan beads using glutaraldehyde. Both Sephadex and chitosan have very good biocompatibility, low toxicity, chemically inert and high hydrophilicity. Various factors implicated in immobilization of PsBGAL onto Sephadex and chitosan beads were optimized using statistical experimental design. Furthermore, a comparison study involving physico-chemical properties and efficiency in lactose hydrolysis present in milk and milk whey were carried out. 2. Methods 2.1. Materials and enzyme All chemicals, buffers and other reagents were of analytical grade. Unless stated all the chemicals were purchased from Sigma Chem. Co. (St. Louis, MO, USA). Milli Q quality water with resistance of higher than 18 MX was used throughout the experiments. Pea (P. sativum var. arvense AP-3) seeds were generously provided by the Indian Institute of Vegetable Research (IIVR), Varanasi, India. PsBGAL was purified from soaked pea seeds (in 25 mM sodium phosphate buffer, pH 6.8 overnight at 4 °C) using following steps, discussed here briefly1: Buffers used in various steps of purification contained 1 mM dithiothreitol, 1 mM phenylmethanesulphonylfluoride and 0.02 mM ethylenediaminetetraacetic acid unless stated otherwise. Seeds were crushed using kitchen blender in 25 mM sodium phosphate buffer, pH 6.8 (buffer A). Extract prepared was filtered using two layers of muslin cloth, centrifuged at 8420g for 20 min at 4 °C. Supernatant was collected and precipitated using 40–55% ammonium sulfate and centrifuged. Pellet was dissolved in a minimum volume of buffer B [25 mM sodium phosphate buffer, pH 6.1 containing 2 M (NH4)2SO4] and dialyzed against the same. Enzyme was applied onto Octyl Sepharose-4B equilibrated with buffer B, and later enzyme elution was carried by 25 mM sodium phosphate buffer, pH 6.1 containing 1 M (NH4)2SO4 at 25 °C. High specific activity fractions were pooled, concentrated and dialyzed against 50 mM Tris-HCl, pH 8 (buffer C). Enzyme was applied to DEAESephacel equilibrated with buffer C and eluted with NaCl gradient from 0 to 0.5 M. High specific activity fractions were pooled, concentrated and dialyzed against 50 mM sodium acetate buffer, pH 5. Final preparation was 910 fold purified with a specific activity of 77.33 lmoL min1 mg1 and was found to be homogenous on SDS–PAGE.1 2.2. Immobilization of PsBGAL on Sephadex Immobilization was carried out by the method of Khare et al. (1991) with minor modifications. Sephadex grades ranging from G 25 to G 75 were tried for immobilization of PsBGAL. Gel beads 1 The protocol for PsBGAL isolation from pea is part of a manuscript entitled ‘‘A bGalactosidase from Pea (Pisum sativum L.) Seeds (PsBGAL): Purification, Stabilization, Catalytic energetics, Conformational heterogeneity and its Significance” (Dwevedi, A; Kayastha, A. M., communicated to ‘‘Phytochemistry”).

of Sephadex were swollen overnight and packed in a vial (500 lL). Packed vials were equilibrated at pH ranging from 6 to 7 using 50 mM sodium monobasic–sodium dibasic phosphate buffer. Equilibrated vials were treated with optimum% (v/v) of glutaraldehyde (prepared in equilibration buffer) for 30 min at room temperature. Gel beads were washed for five times with equilibration buffer to remove excess glutaraldehyde. Varying amount (lg) of PsBGAL was added to glutaraldehyde treated gel beads and incubated overnight at 4 °C. Gel beads were centrifuged for 10 min at 8,420g, at room temperature (27 °C). Supernatant containing unbound protein was discarded. The process was repeated five times to completely wash off any unbound proteins. Immobilized PsBGAL preparation was stored at 4 °C in 25 mM sodium phosphate buffer (pH 7). 2.3. Immobilization of PsBGAL on chitosan beads It was carried out by the method described by Krajewska et al. (1990) with minor modifications. Chitosan (1%) was prepared in 1% acetic acid solution by heating at 45 °C, while stirring the solution. This solution was poured into a syringe, with its nozzle of diameter 5 mm and allowed to drop into 1 M KOH solution, which was continuously stirred for 4 h at room temperature for hardening of chitosan beads. Beads of uniform shape and size (diameter 5 mm) were obtained and then filtered using Whatman filter paper, washed and stored in phosphate buffer (used for optimization of immobilization) at 4 °C, until used. Beads were activated using glutaraldehyde ranging from 1% to 5% (v/v) (prepared in 50 mM sodium phosphate buffer at pH ranging from 6 to 7) overnight, at room temperature (27 °C). Activated chitosan beads were washed for five times with buffer used for optimization to remove any unattached glutaraldehyde. Varying amounts (lg) of PsBGAL were added to activate chitosan beads and incubated overnight at 4 °C. Beads were washed 4–5 times with buffer used for optimization to remove unbound protein. Chitosan-PsBGAL beads were stored at 4 °C in 25 mM sodium phosphate buffer pH 7 until used. 2.4. Protein assay Protein was assayed by the method of Bradford (1976) using the commercial Bradford reagent, calibrated with crystalline bovine serum albumin. 2.5. Enzyme assay 2.5.1. Soluble enzyme Activity towards o-nitrophenyl-b-D-galactopyranoside (ONPG) was estimated in 500 lL of reaction mixture containing 50 mM glycine HCl (pH 3.2), 20 mM ONPG and enzyme with a final concentration of 5 lg mL1. Reaction was terminated by addition of 20 mM sodium tetraborate (1.5 mL) (note: sodium tetraborate is used for both reaction termination and color development) after incubation of 5 min at 37 °C and absorbance was recorded at 405 nm. One unit of BGAL activity was defined as the amount of enzyme required to release 1 lmoL of o-nitrophenol produced per min per mL at 37 °C (Extinction coefficient of o-nitrophenol: 4.05  103 M1 cm1). Activity towards lactose was estimated in 50 lL of reaction mixture containing 50 mM lactose prepared in 50 mM acetate buffer (pH 4) and enzyme with a final concentration of 25 lg mL1. Reaction was stopped after 10 min by heating the reaction mixture in a boiling water bath for 5 min. Glucose released was estimated using a commercially available kit (Span Diagnostics Ltd., Mumbai) based on Glucose oxidase–Peroxidase (GOD–POD) method (Keilin and Hartree, 1952). Reaction mixture (20 lL) was added to (500 lL) glucose reagent, color was

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developed for 10 min at 37 °C and absorbance was recorded at 505 nm. One unit of enzyme activity is defined as 1 lmoL of glucose released per min per mL at 37 °C. 2.5.2. Immobilized enzyme About 500 lL of Sephadex-PsBGAL and single bead of chitosanPsBGAL were used during routine assay. Activity towards ONPG was estimated by incubating immobilized PsBGAL in 20 mM ONPG solution prepared in 50 mM glycine–HCl buffer (pH 3.2) for 5 min at 37 °C. About 1.5 mL of 20 mM sodium tetraborate was added for color development and absorbance was recorded at 405 nm. One unit of activity was defined as 1 lmoL of o-nitrophenol produced per min per mL at 37 °C. Activity towards lactose was estimated by incubating immobilized PsBGAL in 50 mM lactose solution prepared in 50 mM acetate buffer (pH 4) for 10 min at 37 °C. 20 lL of reaction mixture was taken for glucose estimation using glucose reagent as described in case soluble enzyme assay. One unit of enzyme was defined as 1 lmoL of glucose released per min per mL at 37 °C.

The optimum pH of PsBGAL (soluble and immobilized) were studied with respect to ONPG and lactose as substrates by varying the pH of assay buffer in the range of 1–5 and 2–7, respectively. In all cases, 50 mM buffers were used: KCl–HCl (pH 1–2), glycine–HCl (pH 2–4), acetic acid–sodium acetate (pH 4–6) and sodium phosphate (pH 6–7). The optimum temperature of PsBGAL (soluble and immobilized) were studied by assaying using ONPG in the temperature range 30–100 ± 1 °C with the incubation period of 10 min for immobilized (Sephadex and chitosan) and 2 min for soluble enzyme (due to poor thermostability of soluble PsBGAL at higher temperatures). The activity as a function of substrate concentration was measured for soluble and immobilized PsBGAL using substrates: ONPG and lactose by varying concentration (ONPG: 0.5–20 mM and lactose: 1–50 mM) under similar assay conditions as described in section on enzyme assays. The Km and Vmax were determined by using Lineweaver–Burk plot with the help of SigmaPlot 8.0 software. 2.9. Storage stability

2.6. Immobilization efficiency The efficiency of immobilization was evaluated for chitosanPsBGAL and Sephadex-PsBGAL using following relation:

Immobilization efficiencyð%Þ ¼

Specific activity of immobilizedPsBGALðU=mgÞ  100 Specific activity of solublePsBGALðU=mgÞ

Enzyme units were calculated using ONPG as substrate. Specific activity of soluble PsBGAL was 77.33 lmoL min1 mg1 during calculation. Specific activity of immobilized enzyme was calculated by subtracting the specific activity of washed fractions from the specific activity of enzyme added. 2.7. Experimental design and statistical analyses Experimental design and statistical analyses reported in this paper were generated using STATGRAPHICS Centurion XV (StatPoint Inc., USA). Effect of various parameters (including pH,% glutaraldehyde, amount of enzyme, number of chitosan beads in case of chitosan-PsBGAL, different grades of Sephadex in case of SephadexPsBGAL) on immobilization of PsBGAL onto chitosan beads and Sephadex were optimized using 2^4 factorial experimental design (Montogomery, 2001). This design assesses the influence of the main factors, as well as their interaction. The four factors were studied in case of chitosan-PsBGAL and their levels were: glutaraldehyde (1% and 5%; v/v), PsBGAL (10 and 50 lg), pH (6 and 7) and number of chitosan beads (diameter 5 mm) (1 and 5), whereas in case of Sephadex-PsBGAL factors were: glutaraldehyde (1% and 5%; v/v), PsBGAL (10 and 50 lg), pH (6 and 7) and Sephadex grade (G-25 and G-75). The response variable was immobilization efficiency (%) in each case. Based on the results of factorial design, RSM was applied in two phases. The steepest ascent method was applied in order to investigate out of the initial experimental region, along the path of steepest ascent until no further increase in response was observed (Montogomery, 2001). The steepest ascent method allowed coming closer to the optimum point and locating a new experimental region. Finally and as third stage, in this new region a routable central composite design was performed, in order to determine optimum conditions for immobilization.

Storage stability of chitosan-PsBGAL and Sephadex-PsBGAL were studied for a period of 50 days at 4 °C and the residual activity was checked from time to time, with ONPG as substrate by the method described in section on enzyme assays. 2.10. Reusability The immobilized PsBGAL (Sephadex-PsBGAL and chitosanPsBGAL) were stored in 25 mM sodium phosphate buffer (pH 7) at 4 °C, were reused 15 times over a period of 15 days and the residual activity was measured with ONPG as substrate. After assay, immobilized preparation was washed with sodium phosphate buffer and then stored at 4 °C. Furthermore the immobilized enzyme, which showed better stability, was reused for prolonged periods. 2.11. Lactose hydrolysis Ten gram of defatted powdered milk (Nestlé) was dissolved in 100 ml of distilled water (1: 10; w/v). This ratio was chosen due to turbidity at higher concentration which made it difficult to assay spectrophotometrically. Same fraction was then precipitated by 2 N HCl until pH was dropped to 4.5, and centrifuged at 8,420g for 5 min at 4 °C and used as milk whey. 50 lL of above specified 1:10 milk and milk whey were incubated with immobilized PsBGAL (500 lL of Sephadex-PsBGAL and single bead of chitosanPsBGAL) for different time interval at room temperature and 4 °C. The glucose content in treated milk and whey were estimated using GOD–POD method as described in section on enzyme assays, by taking out 20 lL aliquots of the milk and milk whey. % Lactose hydrolysis was calculated by the formula:

%Lactose hydrolysis Glucose present without treatment with immob: BGAL  100 Glucose present after treatment with immob: BGAL %Lactose unhydrolyzed ¼ 100  %lactose hydrolysis ¼

A plot was generated with log% lactose unhydrolyzed versus time, and rate constant of lactose hydrolysis was determined using the slope of the plot using relation:

2.8. Steady state kinetics

Slope ¼ For all steady state kinetics studies, concentration of soluble enzyme and amount of immobilized enzyme (Sephadex and chitosan) were same as described under section on enzyme assays.

k ; 2:303

where rate constant k ¼

2:303 t

log

100 100  x

where ‘x’ is lactose unhydrolyzed. Therefore, time required for 50% . lactose hydrolysis is given by: t 1=2 ¼ 0:693 k

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3. Results and discussion 3.1. Optimization of immobilization of PsBGAL onto Sephadex 3.1.1. Screening through factorial design 2^4 Table 1 shows the design performed using factors pH,% glutaraldehyde, amount of enzyme, Sephadex grade and response was immobilization efficiency (%). Table 2 shows the analysis of Student ‘t’ test of the model obtained for Sephadex-PsBGAL. The first-order model adjusts well to the experimental data, only 8.5% of the total variation was not explained by the model (R2 = 0.9150). As shown in the Table 2, factors including glutaraldehyde, amount of enzyme, type of Sephadex grade have significant effect (P < 0.05) than pH on immobilization efficiency of Sephadex-PsBGAL. Interactions between factors: amount of enzyme versus Sephadex grade and glutaraldehyde versus Sephadex grade were most significant with respect to other interactions (Table 2). According to Table 1, immobilization efficiency was higher at pH 7 than at pH 6 (Table 1: exp. 16:14, 13:2, 12:5, 1:6, 3: 8, 9:15, 10:7, 11:4), moreover Sephadex G-75 was much better than G-25 (Table 1, at pH 7: exp. 1:9, 3:12, 10:16, 11:13). Thus, with the model of Table 2, it can be predicted that the immobilization efficiency of PsBGAL depends on type of Sephdex grade, amount of enzyme and glutaraldehyde (%).

Table 1 Factorial design (2^4) matrix and experimental results of dependent variable immobilization efficiency of Sephadex-PsBGAL. S. no.

Glutaraldehyde (v/v)

Amount of enzyme (lg)

pH

Sephadex grade

Immobilization efficiency (%)a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1.0 1.0 5.0 1.0 5.0 1.0 5.0 5.0 1.0 5.0 1.0 5.0 1.0 5.0 1.0 5.0

10.0 50.0 10.0 50.0 10.0 10.0 50.0 10.0 10.0 50.0 50.0 10.0 50.0 50.0 10.0 50.0

7.0 6.0 7.0 6.0 6.0 6.0 6.0 6.0 7.0 7.0 7.0 7.0 7.0 6.0 6.0 7.0

75 25 75 75 25 75 75 75 25 75 75 25 25 25 25 25

49.98 39.87 48.98 59.23 55.67 40.12 69.73 45.63 35.67 72.41 59.67 58.45 43.25 60.12 35.56 61.21

3.1.2. Steepest ascent method This method is used to search out best conditions for maximum immobilization efficiency of PsBGAL onto Sephadex from initial screening experimental region. According to experimental screening design, Sephadex G-75 was found to be better suited for immobilization than G-25. In case of Sephadex-PsBGAL, enzyme immobilization was based on the clumping of enzyme molecules to large sized aggregate in the presence of glutaraldehyde (Mateo et al., 2004). These large sized aggregate are obstructed to move outside Sephadex matrix due to limitation of pore size of specific grade of Sephadex. Glycosylation nature of PsBGAL1 is an important limiting factor in the formation of large sized aggregate. Thus, Sephadex G-25 having larger pore size has higher probability of enzyme leaching. Sephadex grades higher than G-75 have lower immobilization efficiency as found during experimentation (data not shown). It could possibly be due to blockage of catalytic sites by large sized aggregates due to smaller pore size of higher Sephadex grades. Thus, glutaraldehyde and amount of enzyme were used towards path of the steepest ascent. The most suitable operational conditions found to be used for further optimization using centre composite design (CCD) were: 3.8–5.2% glutaraldehyde would be best suited for maximum immobilization efficiency of PsBGAL in the range of 38– 52 lg (found using experimentation, data not shown) at pH 7 using Sephadex G-75. 3.1.3. Centre composite design (CCD) To efficiently explore the best condition obtained by steepest ascent method, a rotable central composite experimental design (2^2 and a star point; used to estimate curvature) with two centre point was constructed (data not shown). Based on the experimental results obtained by CCD, data were fitted to second degree polynomial equation as given by:

Immobilization efficiency ¼ 293:549 þ 84:8088  A þ 8:3778  B  12:1674  A2 þ 0:496  A  B  0:1204  B2

a Obtained immobilization efficiency has standard error of 6 ±2% to corresponding values.

Table 2 Analysis of Student ‘t’ test for response of dependent variable immobilization efficiency of Sephadex-PsBGAL by factors including glutaraldehyde (%), amount of enzyme (lg), pH and Sephadex grade as well as their interactions. Source

Sum of squares

Df

Mean Square

F-ratio

P-value

A: glutaraldehyde B: amt of enzyme C: pH D: Sephadex grade AB AC AD BC BD CD Total error Total (corr.)

740.52 569.18 35.076 195.65 12.3377 0.945756 0.945756 4.52626 204.705 5.02881 56.7605 2002.62

1 1 1 1 1 1 1 1 1 1 5 15

740.52 569.18 35.076 195.65 12.3377 0.945756 177.889 4.52626 204.705 5.02881 11.3521 –

65.23 50.14 3.09 17.23 1.09 0.08 15.67 0.40 18.03 0.44 – –

0.0005 0.0009 0.1391 0.0089 0.3449 0.7844 0.0108 0.5555 0.0081 0.5352 – –

R2 = 0.9717; R2adj = 0.9150 (Df); Df = degree of freedom; Standard error of Est. = 3.36929.

where A: Glutaraldehyde (v/v), B: Amount of enzyme. The statistical significance of the model equation was evaluated by Student ‘t’ test (Table 3) which adjusts well to the experimental data obtained by CCD (R2 = 0.9646; R2adj = 0.9204). The response surface and contours (at the base) also indicated the same result (Fig. 1A). Finally a verification experiment was performed under the previous operational conditions. The highest immobilization efficiency was achieved in the verification experiment corresponding to 75.66% very close to the value predicted by model based on CCD (75.38%). Optimal conditions for glutaraldehyde and amount of enzyme were found to be 4.4% (v/v) and 43.8 lg of PsBGAL, respectively. Therefore, it can be summarised

Table 3 Results of the analysis by Student ‘t’ test of the central composite rotatory design for response of dependent variable (% immobilization efficiency of Sephadex-PsBGAL) by factors: glutaraldehyde (%) and amount of enzyme (lg) and their interactions. Source

Sum of squares

Df

Mean square

F-ratio

P-value

A: glutaraldehyde B: amount of enzyme AA AB BB Total error Total (corr.)

11.3118 10.0314 42.2996 6.1504 41.4005 3.15826 89.2443

1 1 1 1 1 4 9

11.3118 10.0314 42.2996 6.1504 41.4005 0.789565 -

14.33 12.70 53.57 7.79 52.43 -

0.0194 0.0235 0.0019 0.0493 0.0019 -

R2 = 0.9646; R2adj = 0.9204 (Df); Df = degree of freedom; Standard error of Est. = 0.888575.

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Table 4 Factorial design (2^4) matrix and experimental results of dependent variable immobilization efficiency of chitosan-PsBGAL.

A

Efficiency (%) Immobilization

78 76 74 72 70 68 66

/v )

5.2 5.0 4.8 4.6 4.4

(v

64

60

4.2 46

44

42

of En

zyme

4.0 40

µg) (µ

38

3.8

ta

48

Amou nt

Gl u

50

ra ld

eh y

de

62

S. no.

Glutaraldehyde (v/v)

Amount of enzyme (lg)

pH

Number of beads

Immobilization efficiency (%)a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

5.0 5.0 5.0 1.0 1.0 5.0 1.0 5.0 5.0 5.0 1.0 1.0 5.0 1.0 1.0 1.0

50.0 50.0 10.0 50.0 50.0 10.0 10.0 10.0 50.0 10.0 10.0 10.0 50.0 50.0 10.0 50.0

7.0 7.0 7.0 7.0 6.0 7.0 7.0 6.0 6.0 6.0 6.0 7.0 6.0 7.0 6.0 6.0

1 5 5 5 1 1 5 1 1 5 1 1 5 1 5 5

62.78 54.65 37.28 60.18 67.12 38.98 39.94 41.10 60.35 31.85 51.20 48.79 49.96 64.57 40.01 56.19

a Obtained immobilization efficiency has standard error of 6±2% to corresponding values.

ncy (%) Immobilization Efficie

B Table 5 Analysis of Student ‘t’ test for response of dependent variable immobilization efficiency of chitosan-PsBGAL by factors including glutaraldehyde (%), amount of enzyme (lg), pH and Sephadex grade as well as their interactions.

80 75 70 65 60 55 50 40

Am 42 ou 44 nt 46 of En 48 zy 50 m e ( 52 µg )

0.5 1.0

v) e (v/ ehyd d l a ar 1.5

2.0

Glut

Fig. 1. Response surface plot and contour plot (base of plot) for immobilization efficiency (%) of PsBGAL onto Sephadex G-75 (A) and chitosan (B) as a function of amount of enzyme (lg) and glutaraldedyde (%).

that immobilization efficiency of 75.66% was achieved using Sephadex G-75 at 4.4% (v/v) glutaraldehyde, pH 7 when 43.8 lg of PsBGAL was used. RSM was found to be much useful leading to higher percent of immobilization efficiency as reported earlier (Khare et al., 1991). 3.2. Optimization of immobilization of PsBGAL onto chitosan beads 3.2.1. Screening through factorial design 2^4 Table 4, showed experimental design based on 2^4 factorial, using four factors viz. glutaraldehyde, amount of enzyme, number of beads and pH. Table 5, shows the analysis of Student ‘t’ test of the model obtained by experimental factorial matrix of chitosanPsBGAL. The first-order model adjusts well to the experimental data, only 2.49% of the total variation was not explained by the model (R2 = 0.9751). Factors including glutaraldehyde, amount of enzyme and number of beads were most significant while interaction between number of beads versus pH was most significant with respect to interactions between other factors at P < 0.05 (Table 5).

Source

Sum of squares

Df

Mean square

F-ratio

P-value

A: glutaraldehyde B: amount of enzyme C: pH D: beads AB AC AD BC BD CD Total error Total (corr.)

162.881 1344.14 5.51076 262.683 6.77301 8.22256 2.16826 3.73456 0.507656 21.8323 15.1981 1833.65

1 1 1 1 1 1 1 1 1 1 5 15

162.881 1344.14 5.51076 262.683 6.77301 8.22256 2.16826 3.73456 0.507656 21.8323 3.03962 –

53.59 442.21 1.81 86.42 2.23 2.71 0.71 1.23 0.17 7.18 – –

0.0007 0.0000 0.2360 0.0002 0.1957 0.1609 0.4369 0.3181 0.6997 0.0438 – –

R2 = 0.9917; R2adj = 0.9751 (Df); Df = degree of freedom; Standard error of Est. = 1.74345.

Effect at pH 6 was better than pH 7 (Table 4) due to decrease in the density of surface amino groups at higher pH which binds to glutaraldehyde, important during enzyme immobilization (Krajewska, 1991). Therefore, pH 6 was chosen as optimum for rest of experiments. On studying the effect of number of beads on immobilization efficiency, single bead was most effective in producing higher immobilization efficiency (Table 4). Therefore, for further optimization of immobilization of PsBGAL onto chitosan bead, two factors including amount of enzyme and glutaradehyde were standardized. 3.2.2. Steepest ascent method The factors including amount of enzyme and glutaraldehyde were taken for steepest ascent method. The operational conditions determined by the method to be further used for CCD were: Glutaraldehyde (0.2–2.0%), Amount of enzyme (38–52 lg) at pH 6 using single chitosan bead (data not shown). Here lower% glutaraldehyde was required for enzyme immobilization. In chitosanPsBGAL, glutaraldehyde is the sandwich adhesion molecule that binds enzyme to the matrix. Here, only limited amount of glutaraldehyde is required for covering the whole surface of chitosan bead whereas in case Sephadex-PsBGAL, enzyme immobilization depends on its aggregation which requires larger% glutaraldehyde.

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Table 6 Results of the analysis by Student ‘t’ test of the central composite rotatory design for response of dependent variable (% immobilization efficiency of chitosan-PsBGAL) by factors: glutaraldehyde (%) and amount of enzyme (lg) and their interaction. Sum of squares

Df

Mean square

F-ratio

P-value

A: glutaraldehyde B: amount of enzyme AA AB BB Total error Total (corr.)

35.1812 8.59894 262.441 9.15063 3.43528 0.796842 347.899

1 1 1 1 1 4 9

35.1812 8.59894 262.441 9.15063 3.43528 0.199211 –

176.60 43.17 1317.40 45.93 17.24 – –

0.0002 0.0028 0.0000 0.0025 0.0142 – –

R2 = 0.9977; R2adj = 0.9948 (Df); Df = degree of freedom; Standard error of Est. = 0.4463.

3.2.3. Central composite design After obtaining optimal region for immobilization of PsBGAL onto chitosan, experimental CCD (2^2 and a star point) with 2 centre point was constructed (data not shown). Data obtained by CCD was fitted to second degree polynomial equation as given by:

Immobilization efficiency ¼ 51:9465 þ 54:6211  A þ 3:8322  B  13:47  A2  0:4033  A  B  0:0347  B2 where A: Glutaraldehyde (v/v), B: Amount of enzyme. Analysis by Student ‘t’ test (Table 6), supports CCD with R2 = 0.9917; R2adj = 0.9751. Response surface plot with contour at the base (Fig. 1B) showed the interaction of amount of enzyme with glutaraldehyde and their effect on immobilization efficiency which indicates same results as obtained by CCD. Finally a verification experiment was performed in the described operational conditions, optimum conditions were found to be 1.3% (v/v) of glutaraldehyde was used to immobilize 47.6 lg of PsBGAL with immobilization efficiency of 75.19% (data not shown). Obtained immobilization efficiency was almost near to the value predicted by the model which was 74.92%. Summarizing the optimum conditions for immobilization of PsBGAL onto chitosan beads: Single bead equilibrated with 1.3% of glutaraldehyde at pH 6 was able to immobilize 47.6 lg of enzyme. 3.3. Steady-state kinetics 3.3.1. Optimum pH Sephadex-PsBGAL showed a shift towards acidic side (sharp pH optima at 2.8) with ONPG while no change with lactose as compare to soluble PsBGAL (Fig. 2). Chitosan-PsBGAL has broad pH optima with ONPG (2.8–3.2) and lactose (4–4.4) with respect to soluble enzyme (Fig. 2). Optimum pH depends on factors such as pKa of amino acid groups present at the active site of the enzyme, microenvironment around the active site and the solvent polarity of the solution (Price and Stevens, 2000). Most of the effects of perturbation of pH observed in immobilized enzyme preparations may be explained by considering the distribution of protons throughout the system and analyzing the factors that lead to relative accumulation or depletion of protons in the microenvironment around the enzyme. The pH optima obtained in case of ONPG with soluble and immobilized PsBGAL were lower than lactose due to greater solvent polarity of the former solution. Sephadex-PsBGAL has lower pH optima (in case of ONPG) with respect to soluble and chitosan-PsBGAL due to free movement constraints of solutes by van der Waals forces, most importantly hydrogen bonds imposed by the matrix leading to formation of unstirred layer (‘Nernst’ layer) around the immobilized enzyme. On hydrolysis of ONPG to onitrophenol and galactose by Sephadex-PsBGAL, released galactose

100

Lactose

100 80 60 40 20

% Relative Activity

Source

% Relative Activity

2672

80

0

2

3

4

5

6

7

pH

60

40

20

1

2

3

4

5

pH Fig. 2. Optimum pH of soluble PsBGAL (h), Sephadex-PsBGAL (s) and chitosanPsBGAL (N) using ONPG as substrate. Inset showed optimum pH using lactose as substrate. % Relative activity was determined using relation: enzymatic activity corresponding to different pH/maximum enzymatic activity corresponding to particular pH  100. Vertical bars represent standard error during independent experiments.

gets accumulated around immobilized enzyme than o-nitrophenol, due to higher number of hydrogen bonds present in the former. Higher pKa of galactose increases the pH around the immobilized enzyme than that present in bulk phase leading to lowering of pH optima towards acidic side. Chitosan-PsBGAL has broad pH optima (with ONPG and lactose) with respect to soluble enzyme (Inset Fig. 2) due to large quantity of enzyme units immobilized per unit surface area of chitosan bead (Zulu effect; Portaccio et al., 2003) with respect to later ones. During routine assay with ONPG, 5, 87.6 and 727.64 lg mL1 were used in case of soluble enzyme, Sephadex-PsBGAL and chitosanPsBGAL, respectively. Broadening of pH optima is explained as: At the steady state, the rate of inward substrate diffusion at any point is equal to the rate of removal in the form of product by the enzyme. Due to high intrinsic specific activity of chitosanPsBGAL, the substrate concentration gradient through the immobilized enzyme will be steep and consequently the substrate may not penetrate to the centre of the immobilized enzyme particle. There is reduction in enzyme activity in the presence of constraints (like change in pH) and thus the substrate concentration gradient becomes less steep. This allows the substrate to penetrate further into the immobilized enzyme particle. More enzymes have now become available to the substrate; in effect to the enzyme concentration which has been raised. Two factors therefore work antagonistically on the reaction rate: change in pH reducing the rate while the rise in effective enzyme concentration will tend to increase the rate, so moderating the effect of the pH change. Broadening of pH optima during enzymes immobilization on chitosan have been reported earlier (Okuma et al., 2000; Wentworth et al., 2004). 3.3.2. Optimum temperature During an assay for 10 min, Sephadex-PsBGAL has sharp temperature optima at 55 °C, while chitosan-PsBGAL has broad temperature optima lying in the range 60–65 °C (Fig. 3). Soluble PsBGAL has temperature optima at 60 °C during an assay for 2 min due to its susceptibility at higher temperatures (Fig. 3). Sephadex-PsBGAL has lower temperature optima than chitosanPsBGAL due to solubilization of matrix at higher temperatures.

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100

Log % Residual Activity

2.0

% Relative Activity

80

60

40

20

1.5

1.0

0.5

0 30

40

50

60

70

80

90

100

o

Temperature ( C)

0.0 0

30

60

90

120

150

Time (min) Fig. 3. Optimum temperature of soluble PsBGAL (h), Sephadex-PsBGAL (s) and chitosan-PsBGAL (N). Vertical bars represent standard error during independent experiments. % Relative activity was determined using similar relation given in Fig. 2.

Chitosan-PsBGAL has broad temperature optima, due to higher local enzyme concentration (Zulu effect) as discussed in the section on optimum pH. The shift of optimum temperature of BGAL immobilized on various matrices have been reported in the range 50– 60 °C (Khare et al., 1991; Tanriseven and Dogan, 2002; Zhang et al., 2008).

Fig. 4. Thermal stability of chitosan-PsBGAL (d) and Sephadex-PsBGAL (s) at 50 °C. Values obtained corresponding to log% residual activity at different incubation time (min) were fitted through linear fitted regression program using SigmaPlot 8.0, with R2 equals to 0.9903 and 0.9986 in case of chitosan-PsBGAL and Sephadex-PsBGAL, respectively and P < 0.001 in both cases.

been widely reported (Chang and Juang, 2001; Reddy and Kayastha, 2006; Zhang et al., 2008) with extent of stabilization depending on type of interaction between enzyme and matrix. 3.5. Storage stability and reusability

3.3.3. Kinetic parameters There was no significant change in Km of chitosan-PsBGAL but it was almost doubled in case of Sephadex-PsBGAL with respect to soluble PsBGAL with ONPG and lactose as substrates. There was a drastic decrease in Vmax by approx. 79% and 86% (with respect to soluble PsBGAL) in case of chitosan-PsBGAL with substrates ONPG and lactose, respectively. Sephadex-PsBGAL showed a decrease of Vmax by approx. 45% and 57% (with respect to soluble PsBGAL) with substrates ONPG and lactose, respectively (data not shown). Obtained Km and Vmax for Sephadex-PsBGAL and chitosan-PsBGAL suggest that there were diffusional constrains in the former case while enzyme has undergone a conformational change during immobilization onto chitosan bead (Seong et al., 1981). 3.4. Thermal stability Sephadex-PsBGAL has t1/2 of 22 min, while chitosan-PsBGAL has t1/2 of 116 min at 50 °C, respectively (Fig. 4). It can be concluded that chitosan-PsBGAL is more thermostable than SephadexPsBGAL. This was due to solubilization of Sephadex at higher temperatures leading to lower thermostability of Sephadex-BGAL than chitosan-PsBGAL. Thermostability of an enzyme depends on factors like protein concentration, size of protein, and exposure of hydrophobic domains (Spassov et al., 1995). Thermostabilization of enzyme using glutaraldehyde has been known since last decades (Mohapatra and Hsu, 1994). Enzyme immobilization using glutaraldehyde has been an effective tool for its thermostabilization due to introduction of additional linkages led to restriction of free movement at higher temperatures. Therefore, amide linkages present in enzyme are protected from disruption leading to stability of enzyme at higher temperatures. Furthermore, due to Zulu effect enzyme denaturation under drastic conditions (pH, temperature etc.) is barely detectable during an assay at normal substrate concentration. Enzyme thermostabilization during immobilization has

There was no loss in activity of chitosan-PsBGAL till 40 days, while Sephadex-PsBGAL was stable for only 10 days at 4 °C (data not shown). Higher stability of chitosan-PsBGAL was due to higher local enzyme concentrations than Sephadex-PsBGAL as discussed in previous sections. Improved storage stability by immobilization has been reported by various workers (Chang and Juang, 2001; Reddy and Kayastha, 2006). There was almost negligible loss after 10 washes in case of chitosan-PsBGAL while a loss of 70% was observed in case of Sephadex-PsBGAL after 10 washes (data not shown). With repeated use of immobilized enzyme, the strength of binding between the matrix and enzyme get weakened, leading to loss in activity. Frequent encountering of substrate into the active site causes its distortion, thus reduces its catalytic efficiency. These results indicate that enzyme is bound to chitosan with higher strength, due to which it was not leached out with repeated washing. 3.6. Hydrolysis of lactose in milk and whey From the plot shown in Fig. 5A, B, it was found that t1/2 for lactose hydrolysis by chitosan-PsBGAL was much lower than that of Sephadex-PsBGAL. t1/2 (h) of chitosan-PsBGAL at 4 °C: Milk?57.43 ± 1.13, Whey?9.29 ± 0.12; at 27 °C: Milk?5.17 ± 0.07 Whey?4.66 ± 0.03. t1/2 (h) of Sephadex-PsBGAL at 4 °C: Milk? 1732.5 ± 5.21, Whey?69.18 ± 1.32; at 27 °C: Milk?99.97 ± 2.02, Whey?33.36 ± 0.75. Lactose of whey was better hydrolyzed in both cases (Sephadex-PsBGAL and chitosan-PsBGAL) than milk lactose. It was due to the fact that optimum pH of Sephadex-PsBGAL and chitosan-PsBGAL lie in acidic range (see section on optimum pH) leading to better lactose hydrolysis in whey (pH 4.5) than in milk (pH 6.4). Li et al. (2007) have reported that BGAL from K. lactis immobilized onto cotton fabric hydrolyzed 90% lactose present in milk

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A

Log % Lactose Unhydrolyzed

Log % Lactose Unhydrolyzed

B

A

2.0

1.9

1.8

1.7

1.6

2.0

1.9

1.8

1.5 1.7 0

2

4

6

8

Incubation Time (h)

0

5

10

15

20

25

30

Incubation Time (h)

Fig. 5. (A) Lactose hydrolysis by chitosan-PsBGAL. (B) Lactose hydrolysis by Sephadex-PsBGAL. Values obtained corresponding to log% lactose remained at different incubation time (h) were fitted through linear regression program available at SigmaPlot 8.0 with R2 > 0.99 and P < 0.001. Filled and open circle represent lactose hydrolysis present in whey and milk at room temperature, respectively. Filled and open triangles represent lactose hydrolysis present in whey and milk at 4 °C, respectively. Values obtained corresponding to log% lactose remained at different incubation time (h) were fitted through linear regression program available at SigmaPlot 8.0 with R2 > 0.99 and P < 0.001.

within 120 min and 30.23% lactose hydrolysis present in whey in 43.7 min, at 37oC. Roy and Gupta (2003) have reported hydrolysis of lactose present in milk and whey by over 90% and about 60% within 5 h at 30 °C, respectively by LactozymTM immobilized on cellulose beads. It can be concluded that chitosan-PsBGAL has desirable properties like good stability, reusability, broad temperature and pH optima. Furthermore, with very high hydrolyzing ability for milk and milk whey at 4 °C and room temperature, it can be commercially exploited by milk based industries. 4. Conclusions Statistically designed experimentation for optimum immobilization of PsBGAL onto Sephadex and chitosan proved to be much economical due to lower enzyme requirement, time sparing and efficient. The process is helpful in determination of key factor during enzyme immobilization, due to which maximization of immobilization efficiency was achieved easily. Furthermore, subsequent exploitation of immobilized PsBGAL in hydrolyzing lactose present in milk and whey make it commercially worthful. Acknowledgement One of us (A.D.) would like to thank to Council of Scientific and Industrial Research (CSIR), New Delhi for financial assistance in the form of Junior and Senior Research Fellowships. References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chang, M.-Y., Juang, R.-S., 2001. Stability and catalytic kinetics of acid phosphatase immobilized on composite beads of chitosan and activated clay. Process Biochem. 39, 1087–1091. Deepak, V., Kalishwaralal, K., Ramkumarpandian, S., Venkatesh Babu, S., Senthilkumar, S.R., Sangiliyandi, G., 2008. Optimization of media composition for nattokinase production by Bacillus subtilis using response surface methodology. Bioresour. Technol. 99, 8170–8174. Dey, P.M., 1984. Biochemistry of the multiple forms of glycosidases in plants. In: Meister, A. (Ed.), Advances in Enzymology and Related Areas of Molecular Biology, vol. 56. John Wiley and Sons, Inc., New York, pp. 180– 186.

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