7
`
`Biochemical Engineering Journal 6 (2000) 7–11
`
`a-Amylase immobilized on bulk acoustic-wave sensor by UV-curing coating
`(cid:3)
`Deliang He, Yan Cai, Wanzhi Wei, Lihua Nie, Shouzhuo Yao
`College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China
`
`Received 1 June 1999; accepted 24 March 2000
`
`Abstract
`A new method for immobilization of a-amylase by UV-curing coating is proposed in this paper. The immobilization procedure of
`UV-curing coating on piezoelectric quartz crystal is simple and convenient, and causes less loss of enzymatic activity. The activity of the
`immobilized a-amylase is monitored by a technique based on bulk acoustic-wave (BAW) sensor. The frequency shift of BAW sensor can
`reflect the degree of hydrolysis of starch by the immobilized a-amylase. It is appropriate for the immobilized a-amylase to hydrolyze
`the soluble starch under pH 7.0 condition, which is similar to that of the free a-amylase. Kinetic parameters (the Michaelis constant, Km,
`and the maximum initial rate Vmax) of the enzymatic hydrolysis of starch by the immobilized a-amylase are estimated by using a linear
`
`method of Lineweaver–Burk plot. KmD12.7 mg ml−1 and VmaxD15.9 Hz min
`−1. And the experimental results show that the immobilized
`a-amylase entrapped by the UV-curing coating retains adequate enzymatic activity and can be reused more than 50 times under certain
`experimental conditions. © 2000 Elsevier Science S.A. All rights reserved.
`Keywords: a-Amylase; Immobilization; UV-curing coating; Bulk acoustic-wave sensor
`
`1. Introduction
`
`Enzymes can be immobilized in a variety of water-soluble
`and water-insoluble matrices with little or no immediate
`loss of their catalytic activity [1]. Immobilization of sol-
`uble enzymes on or in organic or inorganic matrices per-
`mits researchers to add, remove, and reuse them at will. It
`is economical for industrial processors to substitute soluble
`enzymes with immobilized enzyme systems in some case.
`These are of interest, and importance, for theoretical reasons
`and industrial applications.
`Many methods of enzyme immobilization have been
`reported in the literature, such as: adsorption [2], adsorp-
`tion and cross-linking [3], cross-linking [4], ion-exchange
`resins [5], entrapment [6], microencapsulation [7], copoly-
`merization [8], and covalent attachment [9]. Entrapment
`is an important immobilization method for enzymes. Bay-
`han immobilized a-chymotrypsin via physical entrapment
`within large, uniformly spherical, and thermally reversible
`poly(N-isopropylacrylamide) [poly(NIPAM)] beads [10].
`Baran et al., [11] immobilized beta-galactosidase by entrap-
`ment in the bulk of the poly(2-hydroxyethylmethacrylate)
`(pHEMA) membranes, the storage stability of the enzyme
`was found to increase upon immobilization. Nakao et al.
`
`(cid:3)
`
`Corresponding author. Tel.: C86-731-8822286; fax: C86-731-8824525.
`E-mail address: szyao@mail.hunu.edu.cn (S. Yao)
`
`1369-703X/00/$ – see front matter © 2000 Elsevier Science S.A. All rights reserved.
`PII: S 1 3 6 9 - 7 0 3 X ( 0 0 ) 0 0 0 6 8 - 1
`
`[12] used calcium alginate gel beads to entrap glucose oxi-
`dase as well as palladium particles to decompose hydrogen
`peroxide formed together with gluconic acid. Abdel-Naby
`et al. [13] immobilized alkaline protease from Bacillus my-
`coides on various carriers by different methods including:
`physical adsorption on hitoson, ionic binding on Amber-
`lite IR-120, covalent binding on chitin, and entrapment in
`2% cross-linked polyacrylamide. Ortega [14] immobilized
`b-glucosidase by entrapment in both calcium alginate and
`polyacrylamide gels. Centonze et al. [15] reported a glucose
`biosensor based on entrapment of glucose oxidase (GOD)
`in a poly(o-phenylenediamine) (PPD) film synthesized onto
`a conducting organic salt (COS) electrode. Besides, many
`materials were used for enzyme entrapment, such as: poly-
`mers, silicone rubber, silica gel, starch, etc.
`(1,4-a-D-glucanglucanohydronase,
`a-Amylase
`EC
`3.2.1.1) exists extensively in plants, animals and micro-
`organisms. In the human body, it is present in fluids includ-
`ing serum, saliva, urine, etc. It is well known that a-amylase
`is applied widely in food industry, beer production, and
`other drink manufactures. But once used, a-amylase can-
`not retrieve easily from the reaction systems. So, it is very
`valuable to employ immobilized a-amylase to supercede
`free a-amylase. UV-curing coating was selected to immo-
`bilize a-amylase on bulk acoustic-wave (BAW) sensor by
`entrapment in this experiment. UV-curing coatings, which
`are generally formulated from the following components:
`Reactive Surfaces Ltd. LLP
`Ex. 1026 (Rozzell Attachment G)
`Reactive Surfaces Ltd. LLP v. Toyota Motor Corp.
`IPR2016-01914
`
`

`

`8
`
`D. He et al. / Biochemical Engineering Journal 6 (2000) 7–11
`
`prepolymer, monomer, photoinitiators and additives, cover
`many fields of applications [16–20]. These coatings, based
`on free-radical or cationic polymerization of unsaturated
`monomers, can solidify quickly after UV-light radiation
`in a few seconds. They will not pollute environment as a
`result of no solvents and cause less damage to the catalytic
`activity of enzyme.
`Some published methods have been applied to deter-
`mine the catalytic activity of a-amylase, such as spec-
`trophotometry [21,22], fluorimetry [23], amperometry
`[24], electrophoresis [25,26],
`isoelectric focusing [27],
`chromatography [28] and immunological method [29].
`a-amylase hydrolyzes internal a-1,4 linkage of starch (glu-
`cose residues in a-1,4 linkage) to yield maltose, maltotriose,
`and a-dextrin [30]. The viscosity of the starch solution
`has obvious change during the hydrolysis of starch by
`a-amylase. Therefore, the enzymatic hydrolysis of starch by
`immobilized a-amylase could be monitored by BAW sensor
`based on the viscosity change of the starch solution [31].
`The BAW sensor has been extensively used as a kind of
`highly sensitive chemical and biological sensor in various
`fields, since it oscillated successfully in liquid phase in the
`1980s [32–36]. The resonant frequency of the piezoelectric
`quartz crystal (PQC) changes with the viscosity and density
`change of the liquid, the following equation is effective under
`certain experimental conditions:
`
`(cid:18)
`
`1F D −F 3=2
`
`s
`
`(cid:17)L(cid:26)L
`(cid:25) (cid:22)Q(cid:26)Q
`
`(cid:19)1=2
`
`(1)
`
`Fig. 1. Schematic representation of the experimental assembly.
`
`plied with 5 V by a d.c. voltage regulator (Model JWY-30B,
`Shijazhuan Electronic Factory No. 4). The detection cell
`was placed in a self-made air-bath chamber which was ther-
`mostated at 20(cid:6)0.2
`(cid:14)
`C. Frequency changes were measured
`with a digital counter (Model SS3341A, Shijazhuan Elec-
`tronic Factory No. 4). The tested solution was stirred with
`a magnetic stirrer (Shanghai Electro-communication Instru-
`mentation Factory). The UV-curing machine used for the
`immobilization of a-amylase was provided by the Machin-
`ery and Electric Factory of Hunan University. The working
`conditions were arranged in Table 1.
`
`2.2. Reagents and chemicals
`
`−1) was ob-
`a-Amylase (from Bacillus subtilis, 300 U mg
`tained from Shanghai Boao Biological Technical Company.
`One activity unit is the amount of amylase that liberates
`(cid:14)
`C.
`1.0 mg of maltose from starch in 3 min at pH 6.9 at 20
`Soluble starch powder was the product of Peng Count Junle
`Chemical Factory. A series of starch solutions were prepared
`−1 phosphate buffer solution,
`by dissolving starch in 0.1 mol l
`then were stored in a refrigerator before use. The UV-curing
`coating was obtained from Weisheng High-Technical Com-
`pany of Hunan University. The prepolymer (WS-6810-1)
`0
`-bisphenyl phenol
`used in the UV-curing coating was p,p
`A epoxide acrylic resin, its structural formula is shown in
`Fig. 2. All other chemicals were of analytical grade. Freshly
`doubly distilled water was used throughout.
`
`2.3. Immobilization of (cid:11)-amylase by the UV-curing coating
`
`The Au plated piezoelectric quartz crystal (PQC) was
`cleaned with chloroform and acetone and dried in the air.
`The a-amylase powder and the UV-curable coating were
`mixed thoroughly according to the weight proportion of 3:7.
`Thereafter, 0.3 mg of the admixture was daubed on one side
`
`where 1F is the frequency shift of the crystal, Fs the reso-
`nant frequency, (cid:17)L the viscosity of the liquid, (cid:26)L the density
`of the liquid, (cid:22)Q the shear modulus of the quartz crystal, and
`(cid:26)Q the density of the quartz crystal. Based on relationship 1,
`the BAW sensor can be used as a viscosity and density detec-
`tor just like the technique presented in this paper. According
`to the response of BAW sensor, the enzymatic hydrolysis
`process can be monitored. The effect of pH on the enzy-
`matic activity of a-amylase immobilized by UV-curing coat-
`ing is studied by BAW sensor. And kinetic parameters (the
`Michaelis constant, Km, and the maximum initial rate Vmax)
`are estimated by using the Lineweaver–Burk plot [37,38].
`Also, the reusing times of the immobilized a-amylase by
`UV-curing coating is studied by BAW sensing technique.
`
`2. Experimental
`
`2.1. Apparatus
`
`The experimental assembly employed is shown in Fig. 1.
`A 9-MHz AT-cut crystal (JA-5 model, diameter 12 mm) with
`Au electrode (diameter 5.5 mm) was presented by the Peking
`Factory No. 707. One side of the crystal was positioned at
`the bottom of a well-type cell. The IC-TTL oscillating circuit
`made in Xiangtan Printed Circuit Factory of Hunan was sup-
`
`Table 1
`Working conditions of the UV-curing machine
`
`Power of UV
`lamp (kW)
`
`3
`
`Radiation
`intensity
`−1)
`(W cm
`80
`
`Distance of
`two lamp
`(cm)
`10
`
`Solidification
`rate
`−1)
`(m min
`8
`
`

`

`D. He et al. / Biochemical Engineering Journal 6 (2000) 7–11
`
`9
`
`Fig. 2. Structural formula of p, p
`
`0
`
`-bisphenyl phenol A epoxide acrylic resin.
`
`of the PQC, then the PQC with the admixture was rotated
`at a rate of 20 rpm for 30 min to spread the mixture uni-
`formly on the PQC surface. After these processes, the PQC
`covered with a thin glass cup was put into the UV solidifi-
`cation operation machine. So a thin film of UV-curing coat-
`ing including a-amylase was coated on the PQC surface. A
`series of BAW sensor with immobilized a-amylase within
`UV-curing coating were prepared.
`
`2.4. Frequency shift measurements of the enzymatic
`hydrolysis of starch
`
`The BAW sensor coated with a thin UV-curing coating
`−1 phosphate buffer solution
`film was steeped into 0.1 mol l
`(pH 6.9) for 5 min. Then, 1.0 ml starch solution was taken out
`from the stock and put into the detection cell and the starch
`solution was hydrolyzed by the immobilized a-amylase on
`one side of PQC. At the same time, the resonant frequency
`of BAW sensor was recorded. The frequency shifts with
`time were measured under different pH values and concen-
`trations of starch solutions. The frequency shifts were also
`recorded while the BAW sensor was reused to hydrolyze the
`starch solutions with identical concentration. The frequency
`shifts were measured three times, and the mean values were
`calculated.
`
`3. Results and discussion
`
`3.1. Typical frequency shift curves in different starch
`solutions
`
`The viscosity of the starch solution has obvious change
`when the immobilized a-amylase by UV-curing coating is
`used to hydrolyze the starch. This will result in the resonant
`frequency shift of the BAW sensor. The typical frequency
`shift curves of the enzymatic hydrolysis of starch by the im-
`mobilized a-amylase are shown in Fig. 3. The resonant fre-
`quency of PQC decreased with time and 1F is diverse in dif-
`ferent concentration starch solutions. The frequency change
`of BAW sensor means the viscosity of the starch solution
`has varied. That is to say that starch has been hydrolyzed
`partly by the immobilized a-amylase. So the response of
`BAW sensor can reflect the enzymatic hydrolysis of starch
`and the activity of the immobilized a-amylase.
`
`Fig. 3. Typical response curves of BAW sensor during the enzymatic
`hydrolysis of a series of starch solutions.
`
`3.2. Effect of pH on the immobilized (cid:11)-amylase
`−1 starch so-
`One mil1iliter each of 5.0 and 7.5 mg ml
`lutions was hydrolyzed by the immobilized a-amylase on
`the BAW sensor, respectively. The pH of the starch solu-
`tions varied from 6.0 to 8.0. The frequency shift of 10 min
`(1F1DF0−F1, F0 is the initial frequency of BAW sensor,
`F1 the frequency of BAW sensor after 10 min) was recorded
`to describe the pH effect on the enzymatic activity of the
`immobilized a-amylase. When an enzyme is immobilized
`either within a matrix or on the surface of a carrier, the ef-
`fect of pH on the enzymatic activity of the enzyme may
`change, depending on the nature of the carrier [39]. It is
`necessary to know the influence of pH on the activity of the
`immobilized a-amylase. The effect of pH on the activity of
`the immobilized a-amylase is shown in Fig. 4. It can be
`found that the maximum response (1F1) appears when pH
`is (cid:25)7.0. So, it is appropriate for the immobilized a-amylase
`
`Fig. 4. The effect of pH on the activity of the immobilized a-amylase in
`−1 starch solutions.
`5.0 and 7.5 mg ml
`
`

`

`10
`
`D. He et al. / Biochemical Engineering Journal 6 (2000) 7–11
`
`Fig. 5. The dependence of the reciprocal of enzymatic hydrolysis rate on
`the reciprocal of concentration of starch.
`
`Fig. 6. The curves of the frequency shift (1F1) of BAW sensor vs. the
`−1 starch solutions.
`used times in 5.0 and 7.5 mg ml
`
`The frequency shift of 10 min (1F1) was recorded every
`time to check the activity loss of the immobilized a-amylase
`while it was reused. The a-amylase can be reused after it
`was immobilized, but the ability to hydrolyze the starch
`will decrease along with the increasing of the used times.
`The curves of the frequency shift (1F1) and used times are
`shown in Fig. 6. The response decreased rapidly within six
`used times, while it had less variation from 8 to 35 used
`times. Afterwards, 1F1 declined increasingly. That is to
`say, the apparent activity of the immobilized a-amylase
`within the UV curing coating decrease while they were
`reused, this is because a-amylase may dissolve into the
`starch solution and the enzymatic activity may declined
`while the BAW sensor covered with UV-curing coating in-
`cluding a-amylase was reused. However, it is obvious that
`the a-amylase immobilized by UV-curing coating can be
`reused many times to hydrolyze the starch. The lifetime of
`the immobilized a-amylase can be estimated according to
`the response of BAW sensor when it is reused to hydrolyze
`the starch.
`
`4. Conclusion
`
`A new method for immobilization of a-amylase on PQC
`by UV-curing coating is proposed. It can be seen that the
`immobilization procedure of UV-curing coating is simple
`and convenient, and causes less enzymatic activity loss.
`Comparison between the two immobilization methods of
`a-amylase is shown in Table 2. Furthermore, compared with
`the spectrophotometric and fluorimetric method, the pro-
`posed method based on BAW sensor is convenient as no
`extra reagents such as chromogen or fluorescein is needed,
`and so it is simpler. Meanwhile it is fit for the continuous
`monitoring and study of the dynamics of the enzymatic hy-
`drolysis process of starch. It is appropriate for the a-amylase
`immobilized with UV-curing coating to hydrolyze the starch
`under pH 7.0 condition, which is similar to that of free
`a-amylase used in this experiment. And kinetic parameters
`(Km and Vmax) are obtained by using the Lineweaver–Burk
`
`plot, KmD12.7 mg ml−1 and VmaxD15.9 Hz min
`−1. The im-
`mobilized a-amylase can be reused to hydrolyze the 5.0
`
`entrapped by UV-curing coating to hydrolyze the starch un-
`der the pH 7.0 condition, which is showing little difference
`to that of the free a-amylase. This implies that the nature
`of UV-curing coating has little influence on the enzymatic
`activity of a-amylase. So the succeeding experiments were
`carried out under pH 7.0.
`
`3.3. Estimation of kinetic parameters of the immobilized
`(cid:11)-amylase
`
`The frequency shift of BAW sensor can mirror the viscos-
`ity change during the enzymatic hydrolysis of starch. The
`initial hydrolysis rate could be gained by linear fitting the
`response of BAW sensor within 3 min. Kinetic parameters of
`the enzymatic reaction can be estimated by the direct linear
`method of the Lineweaver–Burk plot [37,38] of the initial
`hydrolysis rates from BAW sensor:
`D Km
`C 1
`1
`[S]
`Vmax
`Vmax
`where V and Vmax are the initial hydrolysis rate and maxi-
`−1, Km the Michaelis constant
`mum hydrolysis rate in Hz min
`
`−1, and [S] the starch concentration in mg ml−1.
`in mg ml
`Thus, a plot of the reciprocal of initial rate and the reciprocal
`of starch concentration for the immobilized a-amylase on
`PQC should give a straight line. Furthermore, the intercept
`gives 1/Vmax and the slope is Km/Vmax, from which Vmax
`and Km can be calculated. In this work, the concentration
`−1. The exper-
`range of starch was from 1.2 to 10.0 mg ml
`imental temperature was controlled at 20(cid:6)0.2
`(cid:14)
`C. The de-
`pendence of 1/V and 1/[S] is shown in Fig. 5, it can be seen
`that 1/V is linear to 1/[S]. By linear fitting according to Eq.
`(2) (nD8, rD0.982), the kinetic parameters are obtained, Km
`
`−1 and Vmax 15.9 Hz min−1. The value of Km
`is 12.7 mg ml
`
`
`is higher than that of free a-amylase (e.g. 6(cid:2)10−4 g ml−1)
`after a-amylase is entrapped into UV-curing coating.
`
`1 V
`
`(2)
`
`3.4. Reusing times of the immobilized (cid:11)-amylase
`
`Two BAW sensor with immobilized a-amylase were
`−1 starch
`reused to hydrolyze 1 ml of 5.0 and 7.5 mg ml
`solutions (pH 7.0), respectively. Each was reused 50 times.
`
`

`

`D. He et al. / Biochemical Engineering Journal 6 (2000) 7–11
`
`11
`
`Table 2
`Comparison of two immobilization methods of a-amylase
`
`Method
`
`Main materials
`
`Immobilization conditions
`
`Procedure
`
`Industrialization
`
`Cross-linking
`Entrapment
`
`APTESa, TEAb, IPDc
`UV-curing coating
`
`Low temperature and long reaction time
`Room temperature and short
`operating time
`
`Three step, intricate
`One step, simple and
`convenient
`
`Difficult to achieve
`Easy to bring into
`effect
`
`Reference
`
`[31]
`This paper
`
`a Aminopropyltriethoxysilane.
`b Triethylamine.
`c Isophthaloyl dichloride.
`
`−1 starch solutions for 50 times at a operationand 7.5 mg ml
`
`of 10 min. It could be prognosticated that the immobilized
`a-amylase by UV-curing coating could apply for industrial
`purpose, UV-curing coating could be used to immobilize
`some other enzyme, and some other enzymes could be stud-
`ied after immobilized on PQC.
`
`Acknowledgements
`
`This work was financially supported by the National Sci-
`ence Foundation of China and the Project of Hunan Science
`and Technology Commission.
`
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