DSpace at VNU: Biochemical studies on the immobilized lactase in the combined alginate-carboxymethyl cellulose gel

1.Effect of weight ratio of carboxymethyl cellulose to sodium alginate to the yield of lactase immobilization A and the total and specific activity of the biocata-lyst B and C... 2.Scanni

Thi Hai Anh Mai1, Van Nguyen Tran, Van Viet Man Le∗

Department of Food Technology, Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, Viet Nam

a r t i c l e i n f o

Article history:

Received 2 June 2012

Received in revised form 24 February 2013

Accepted 2 March 2013

Available online 14 March 2013

Keywords:

Alginate

Carboxymethyl cellulose

Enzyme activity

Immobilization

Immobilized enzyme

Lactose

a b s t r a c t

Additionofcarboxymethylcellulosetoalginategelsignificantlyimprovedtheyieldoflactase immo-bilizationandthetotalandspecificactivityofthefixedenzyme.Itwasduetoadecreaseinprotein lossduringthegelbeadformationintheenzymeimmobilizationprocedureandanincreasein spe-cificsurfaceareaofthegelbeads.Whentheweightratioofcarboxymethylcellulosetosodiumalginate

inthegelsupportwas1.0:1.5,theyieldoflactaseimmobilizationachieved58.2%andthisvaluewas 14.2%higherthantheyieldoflactaseimmobilizationinalginategel.Theimmobilizedlactaseinthe combinedalginate–carboxymethylcellulosegelexhibitedhigherthermalandpHstabilitythanthefixed enzymeintheconventionalalginategel.However,theMichaelis–Mentenconstant(Km)increasedfrom 99.57mM(fixedlactaseinalginategel)to107.24mM(fixedlactaseinalginate–carboxymethyl cellu-losegel)whiletheapparentturnovernumber(Kcat)andthespecificityconstant(Kcat/Km)oftheboth immobilizedbiocatalystswerestatisticallysimilar

© 2013 Elsevier B.V All rights reserved

1 Introduction

stabil-ity[3,4].␤-Galactosidasecanbeusedintwoforms:inthesoluble

opera-tion[5,6]

∗ Corresponding author Tel.: +84 8 38 64 62 51; fax: +84 8 38 63 75 04.

E-mail addresses: lvvman@hcmut.edu.vn , lvvman2003@yahoo.com (V.V.M Le).

1 Present address: Faculty of Agriculture and Forestry, Tay Nguyen University, 567

Le Duan Street, Buon Ma Thuot City, Daklak Province, Viet Nam.

1369-703X/$ – see front matter © 2013 Elsevier B.V All rights reserved.

[15–17]

2 Materials and methods

A



2.3.6 Mechanicalpropertiesofgelbeads

3 Results and discussion

[27]

poly-mer[15–17].Ourresultsprovedthatthehigherthecarboxymethyl

40 44 48 52 56 60 64

Weigh rao of c arbo xylmethyl cellulo se to alginate (w/w)

A

0.4 0.6 0.8 1.0 1.2 1.4

Weigh rao of c arbo xylmethyl cellulo se to alginate (w/w)

B

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

Weigh rao of c arbo xylmethyl cellulo se to alginate (w/w)

C

Fig 1.Effect of weight ratio of carboxymethyl cellulose to sodium alginate to the yield of lactase immobilization (A) and the total and specific activity of the biocata-lyst (B and C).

Fig 2.Scanning electron micrograph of the surface (A and C) and the cross-section (E) of alginate gel bead, of the surface (B and D) and the cross-section (F) of the combined alginate–carboxymethyl cellulose gel bead (the weight ratio of carboxymethyl cellulose to sodium alginate in the combined gel was 1.0:1.5).

Table 1

Mechanical properties of the gel beads with the immobilized lactase (n = 5).

Different small letters in the same column mean significant difference at P < 0.05.

* The weight ratio of carboxymethyl cellulose to sodium alginate in the gel beads was 1.0:1.5.

0

10

20

30

40

50

60

70

80

90

100

Time (min)

Fig 3.Thermal denaturation of the soluble and entrapped lactase The enzyme

activity measured at the initial moment was taken as 100%: (· · ·) free lactase; (—)

lactase entrapped in alginate–carboxymethyl cellulose gel; (- - -) lactase entrapped

in alginate gel: () 55 ◦ C, (䊉) 60 ◦ C, and () 65 ◦ C.

0 10 20 30 40 50 60 70 80 90 100

Time (min)

Fig 4. pH denaturation of soluble and entrapped lactases The enzyme activity measured at the initial moment was taken as 100%: (· · ·) free lactase; (—) lactase entrapped in alginate–carboxymethyl cellulose gel; (- - -) lactase entrapped in algi-nate gel: pH () 4.5, (䊉) 5.5, and () 6.5.

Table 2

Half-lives (t 1/2 ) and inactivation rate constant (k) of the free and immobilized lactase in alginate–carboxymethyl cellulose gel and alginate gel at three different temperatures.

carboxymethyl cellulose gel

Lactase entrapped in alginate gel

Table 3

Half-lives (t 1/2 ) and inactivation rate constant (k) of the free and immobilized lactase in alginate–carboxymethyl cellulose gel and alginate gel at three different pH values.

carboxymethyl cellulose gel

Lactase entrapped in alginate gel

Apparent K m and K cat values of the free and immobilized lactase at pH 4.5 and temperature 55◦C.

Free lactase Immobilized lactase in alginate–carboxymethyl cellulose gel Immobilized lactase in alginate gel

Different small letters in the same row mean significant difference at P < 0.05.

Table4presentssomekineticparametersofthefreeandfixed

4 Conclusions

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