Application of bacterial cellulose pellets in enzyme immobilization

Application of bacterial cellulose pellets in enzyme immobilization

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Journal of Molecular Catalysis B: Enzymatic 54 (2008) 103–108

Application of bacterial cellulose pellets in enzyme immobilization

Sheng-Chi Wu∗, Ying-Ke Lia

Department of Biotechnology, Fooyin University, 151 Chinhsueh Road, Ta-Liao Hsiang, Kaohsiung Hsien 831, Taiwan, ROC

Received 20 September 2007; received in revised form 25 December 2007; accepted 27 December 2007 Available online 4 January 2008

Abstract

Over recent years, there has been a growing interest in the use of cellulose materials in bioprocessing technologies. Bacterial cellulose which is the pure cellulose has unique physical properties which differ from those of plant cellulose and has therefore attracted attention as a new functional material. The applications of bacterial cellulose rarely use the pellet type but it has potential in enzyme immobilization since pellet form is usually used in this field. In this research, Glucoamylase which is widely used in the food industry was immobilized on bacterial cellulose beads after testing using various activation procedures. The results showed that the epoxy method with glutaraldehyde coupling was the best method. After comparison of the different types of bacterial cellulose beads for glucoamylase immobilization, the wet bacterial cellulose beads of the smallest size (0.5–1.5mm) were the best support. The immobilization of enzyme enhances its stability against changes in the pH value and temperature especially in the lower temperature region. The relative activity of the immobilized glucoamylase was still above 7% at pH 2.0 and it was the highest value in the literature. The relative activities were more than 68% in the lower temperature region even at 20◦C. Thus, bacterial cellulose beads are a practical potential support for the preparation of immobilized enzymes in industrial applications. © 2008 Elsevier B.V. All rights reserved.

Keywords: Glucoamylase; Immobilization; Bacterial cellulose beads; Activation; Stability

1. Introduction

Starch is the most abundant form of storage polysaccharide in plants. Hence, its application is very important in the food industries such as in the production of oligo by starch hydrolysis. Acid splitting is the traditional method for the production of glucose syrup. However, the procedures are not suitable for industrial mass production since the products are rather complicated and require high purification costs. And since the cost of using starch-hydrolyzed enzymes is lower and its procedures are much simpler, it has become the main method in starch hydrolysis [1,2]. A number of enzymes are used in starch hydrolysis for the production of glucose, fructose or maltose. Glucoamylase is one of the key enzymes used for starch processing which has extensive uses in the manufacture of crystalline glucose or glucose syrup, either as soluble or immobilized enzymes. The enzymehydrolyzes -1,4-and-1,6-glycosidiclinkagesofstarch to produce glucose [3]. In conventional enzymatic reactions, the soluble enzyme reacts with the substrate in the solution.

∗ Corresponding author. Tel.: +886 7 7811151x5703; fax: +886 7 7862707. E-mail address: dr009@mail.fy.edu.tw (S.-C. Wu).

After the completion of each batch of reactions, the enzymes are deactivated. Hence, the process would be more economical by using the immobilized systems since they allow the reuse of the enzymes. Immobilization of enzymes is generally carried out by adsorption or covalent coupling to solid matrices, as well as by entrapment or encapsulation in polymeric substances. Immobilization often results also in the improvement of enzyme stability under specific process conditions.

The application of the cellulose as a precursor for chemical modifications was exploited extensively even before its polymeric nature was determined and well understood [4].I nt he present work, glucoamylase is immobilized on bacterial cellulose which is an indigenous food of South-East Asia. Bacterial cellulose has unique physical properties which differ from those of plant cellulose and has therefore attracted attention as a new functional material. Therefore, it has been receiving constant attention during the past decade for the production of bacterial cellulose with its unique structure and set of properties such as excellent mechanical strength, ultra-fine fiber, biodegradability, and high crystallinity [5–8]. Its widespread field of application includes foods, acoustic diaphragms, production of unusually strong paper, and medical applications such as wound dressings and artificial skins [6]. Acetobacter was used in the present work

1381-17/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2007.12.021 sinceitcanproduceultrafinecellulosefibrils(50–80 nminwidth and 3–8nm in thickness). Cellulose fibrils are three dimensional network structures with a micrometer- to nanometer-scale [9].

Traditional production of bacterial cellulose was in stationary culture conditions and a thick, gelatinous membrane is accumulated on the surface of a culture medium, whereas under agitated culture conditions, produced cellulose is almost in the form of a fibrous suspension. The bacterial cellulose produced by the airlift reactor formed unique pellets, which were also much larger and different from the fibrous bacterial cellulose form produced in the stirred-tank reactor [10]. The applications of bacterial cellulose rarely use the pellet type but it has potential in enzyme immobilization since pellet form is usually used in this field. Therefore,thepellettypeofbacterialcellulosewasproducedand testedforenzymeimmobilizationinthepresentwork.Thelargescale production of the pellet form of bacterial cellulose in the fermentor was established recently using the conventional airlift reactor in 50 L [1] and modified airlift reactor in 20 L [12]. Thus, the application of bacterial cellulose pellet is expected to expand even more.

The immobilization of glucoamylase on different types of bacterial cellulose beads was investigated in the present work. Bacterial cellulose beads were produced by the strain of Acetobacter xylinum in a shaking flask with baffle. A range of immobilization chemistries were also examined for active bacterial cellulose with the aim of maximizing both the amount of immobilized biocatalyst and the retention of enzymatic activity. Thermal, pH, and storage stability of immobilized enzymes were also studied and compared with free enzymes. The results are expected to have practical importance for further applications.

2. Materials and methods 2.1. Materials

Glucoamylase (amyloglucosidase, exo-1,4- -glucosidase,

EC 3.2.1.3, from Aspergillus niger), soluble starch (from potato, as substrate in determining enzyme activity according to Zulkowsky) and 3,5-dinitro salicylic acid were purchased from Sigma Chemical Co. Glucose and peptone were obtained from Merck Co. Ltd. Germany while yeast extract was obtained from Sigma Chemical Co. For the activated reagents, 1,4-butanediol diglycidyl ether and EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; C8H17N3·HCl) were obtained from Sigma Chemical Co., glutaraldehyde was purchased from

Fluka. The other chemicals were of analytical grade.

2.2. Microorganism and cultivation conditions

The microorganism, A. xylinum subsp. Sucrofermentans

BPR2001, used in this study was purchased from Japan Collection of Microorganisms (RIKEN, Saitama, Japan). The culture medium for its maintenance contained (%, w/v): mannitol, 2.5; yeast extract, 0.5; peptone, 0.3; agar, 1.5 at pH 5.5. The culture medium used for the production of bacterial cellulose beads was the Hestrin & Schramn (HS) medium consisting of 2.0% (w/v) glucose, 0.5% (w/v) yeast extract, 0.5% (w/v) peptone, 0.27%

(w/v) Na2HPO4·12H2O and 0.115% (w/v) citric acid monohydrate [12]. Prior to sterilization at 121 ◦C, the pH value of the medium was adjusted to 5.0. The organisms were grown in a 500mL flask containing 300mL of the HS medium and then cultivated at 30◦C and 120rpm. The cultivation time was at least 48h and based on the size of bacterial cellulose beads for the desired experiments.

2.3. Preparation of bacterial cellulose beads

To obtain the bacterial cellulose, samples from the culture broth were centrifuged at 9100rpm for 30min. The cellulose beads were obtained by dissolving the cells with 0.1N NaOH at 90◦C for 30min and then washed twice with deionized water.

2.4. Activation

2.4.1. Epoxy method (M1) [13]

The bacterial cellulose beads (1g) were suspended in 1.5mL of1MNaOHandthen0.2mLof1,4-butanedioldiglycidylether was added. The mixture was stirred at 60◦C for 2h and the final product was washed successively with distilled water.

2.4.2. Epoxy with EDC coupling method (M2)

The bacterial cellulose beads (1g) produced by the epoxy method using 1,4-butanediol diglycidyl ether were mixed with 2.5mL of distilled water and 1g glycine at 60◦C and pH 12 for 2h. After that, the beads were activated with 3mL of 0.1% EDC at 25 ◦C for 1 h. Finally, the activated bacterial cellulose beads were washed successively with distilled water.

2.4.3. Epoxy with glutaraldehyde coupling method (M3)

The bacterial cellulose beads (1g) produced by the epoxy method using 1,4-butanediol diglycidyl ether were mixed with 0.5mLofdistilledwaterand0.5mLof30%(w/v)ammonia.The mixture was stirred at 60◦C for 2h. After washing with distilled water, the beads were mixed with 1mL of 25% (w/v) aqueous glutaraldehyde for 2 h. The glutaraldehyde-activated bacterial cellulose beads were washed successively with distilled water.

2.4.4. Epoxy with glutaraldehyde and EDC coupling method (M4)

The bacterial cellulose beads (1g) produced by the glutaraldehyde method was mixed with 3mL of 0.1% EDC and stirred for 1h at room temperature. The product was washed successively with distilled water.

2.5. Immobilization of glucoamylase

Glucoamylase immobilization was carried out by adding the bacterialcellulosebeads(1g)toasuspensionof3mLglucoamylase (0.1%) in 5mM sodium phosphate at pH 4.5 and 25◦C for 60min. After immobilization, the derivatives were washed with distilled water.

S.-C. Wu, Y.-K. Lia / Journal of Molecular Catalysis B: Enzymatic 54 (2008) 103–108 105 2.6. Estimation of enzyme activities

The activity of the free and the immobilized glucoamylase were determined by estimating the amount of glucose produced by enzyme. The hydrolysis reactions were carried out in the phosphate buffer with 1% of the substrate. In present study, starch was chosen as the substrate. After adjusting the pH to 4.5, the mixture was incubated at 60◦C for 3min. The liberated glucose was measured using DNS (3,5-dinitrosalicylic acid) method. One unit of enzyme is defined as the amount of enzyme which releases reducing carbohydrates equivalent to 1 mol glucose from soluble starch per min.

The kinetic parameters (Michaelis constant Km and max- imum rate Vmax) were calculated by measuring the rates of reaction at various substrate concentrations. The values were

substituted into the Hanes–Woolf equation to obtain Km and Vmax.

2.7. pH and thermal stability of immobilized glucoamylase

The pH stability of the immobilized glucoamylase was studied by incubating the immobilized enzyme at 25◦C in buffers of varying pH (3.5–6.5) for 1h and then determining the hydrolytic activity at the optimum pH and temperature. Relative activities were calculated as the ratio of the activity of immobilized enzyme after incubation to the activity at the optimum reaction pH.Thethermalstabilityofglucoamylasewastestedbyincubatingtheimmobilizedenzymeatvaryingtemperatures(20–90◦C) and determining the activity at its optimum reaction temperature. Relative activities were calculated as mentioned above and plotted against temperature.

3. Results and discussions 3.1. Comparison of different activate methods

Fouractivatemethodswereselectedfromliteraturetoinvestigatewhethertheenzymescouldbemosteffectivelyimmobilized on solid phases for practical applications or not. Among the methods, the glutaraldehyde and EDC coupling methods were derived from the epoxy method and the results are shown in Table 1. The epoxy method with glutaraldehyde coupling was the best method and its relative activity was at least 15% more than the others. Therefore, M3 procedure was chosen in the following experiments since its highest relative activity after immobilization.

Table 1 Relative activities of immobilized enzymes for the different activated methods

Method Relative activity (%)

M1 (Epoxy method) 85.3 M2 (Epoxy with EDC coupling method) 68.9 M3 (Epoxy with Glutaraldehyde coupling method) 100

M4 (Epoxy with Glutaraldehyde and

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