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Immobilization of #-galactosidases from Lactobacillus on chitin using chitin binding domain Mai Lan Mai Pham, Tatjana Leister, Hoang Anh Nguyen, Bien-Cuong Do, Tuan-Anh Pham, Dietmar Haltrich, Montarop Yamabhai, Thu-Ha Nguyen, and Tien-Thanh Nguyen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04982 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 23, 2017

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Journal of Agricultural and Food Chemistry

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Immobilization of β-galactosidases from Lactobacillus on chitin using a

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chitin-binding domain

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Mai-Lan Pham1,3+, Tatjana Leister3+, Hoang Anh Nguyen2, Cuong Do-Bien1, Anh Pham-

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Tuan1, Dietmar Haltrich3, Montarop Yamabhai4, Thu-Ha Nguyen3*, Tien-Thanh Nguyen1*

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School of Biotechnology and Food Technology, Hanoi University of Science and Technology.

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No.1, Daicoviet, Hanoi, Vietnam

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2

Faculty of Food Science and Technology, Vietnam National University of Agriculture.

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Trauquy, Gialam, Hanoi, Vietnam.

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3

Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of

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Natural Resources and Life Sciences, Vienna.

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Muthgasse 18, A-1190, Vienna, Austria

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Molecular Biotechnology Laboratory, Suranaree University of Technology.

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111 University Avenue, Nakhon Ratchasima Thailand

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+

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*

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Tien-Thanh Nguyen

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Phone/Fax : +84 3868 2470

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Email: [email protected]

equally contributed to this publication Corresponding author

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Thu-Ha Nguyen

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Phone: +43 1 47654-75251

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Email: [email protected]

25 1 ACS Paragon Plus Environment


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ABSTRACT

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Two β-galactosidases from Lactobacillus including a heterodimeric, LacLM type enzyme

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from L. reuteri L103 and a homodimeric LacZ type β-galactosidase from L. bulgaricus

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DSM 20081 were studied for immobilization on chitin using a carbohydrate-binding

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domain (chitin-binding domain, ChBD) from a chitinolytic enzyme. Three recombinant

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enzymes, namely LacLM-ChBD, ChBD-LacLM and LacZ-ChBD, were constructed and

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successfully expressed in L. plantarum WCFS1. Depending on the structure of the

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enzymes, either homodimeric or heterodimeric, as well as the positioning of the chitin-

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binding domain in relation to the catalytic domains, i.e. upstream or downstream of the

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main protein, the expression in the host strain as well as the immobilization on chitin

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beads were different.

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immobilization studies, thus a one-step immobilizing procedure could be performed to

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achieve up to 100% yield of immobilization without the requirement of prior purification

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of the enzyme. The immobilized-on-chitin enzymes were shown to be more stable than

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the corresponding native enzymes; especially the immobilized LacZ from L. bulgaricus

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DSM20081 could retain 50% of its activity when incubated at 37°C for 48 days.

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Furthermore, the immobilized enzymes could be recycled for conversion up to 8 times

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with the converting ability maintained at 80%. These results show the high potential for

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application of these immobilized enzymes in lactose conversion on an industrial scale.

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Keywords: β-galactosidase, Lactobacillus, immobilization, chitin- binding domain

Most constructs showed a high specificity for the chitin in

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INTRODUCTION

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Βeta-galactosidases (β-gal) (lactases, EC 3.2.1.23) are known as important enzymes for

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applications in the dairy industry,1 where they are used for lactose hydrolysis to produce

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low-lactose or lactose-free products as a response to lactose intolerance of consumers,

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which affects approximately 70% of the world population.2 Another useful property of β-

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gal is its transgalactosylation activity, by which health-promoting prebiotic galacto-

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oligosaccharides (GOS) can be formed from lactose.1, 3 Many studies have demonstrated

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the indirect health benefits of GOS, where GOS promote growth and activity of beneficial

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intestinal microbes in the host.4-6

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It is conceivable that β-gal from probiotic strains produce GOS that are also specific for

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these probiotics 7 and therefore β-gals from several probiotic Bifidobacterium spp.8, 9 and

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prominently Lactobacillus spp. such as L. reuteri, L. plantarum, L. sakei, L. pentosus, or

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L. bulgaricus have been studied pertaining to GOS production during the last decade.10-14

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Beta-galactosidases from Lactobacillus species are reported to be of two main types,

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consisting of either two different subunits (heterodimeric or LacLM type) or two identical

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subunits (homodimeric or LacZ type).15, 16 Two well-studied examples are the LacLM β-

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gal from Lactobacillus reuteri L10314 and the LacZ β-gal from Lactobacillus delbrueckii

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subsp. bulgaricus (L. bulgaricus) DSM 20081,16 both belonging to Glycoside Hydrolase

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family GH2.

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The recombinant β-gals from L. reuteri L103 and L. bulgaricus DSM20081 were

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successfully developed and overexpressed in food-grade Lactobacillus hosts, 16-18 yielding

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remarkably high levels of activity, for example ca. 23 kU to 53 kU per L of fermentation

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broth, respectively.16,

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recombinant β-gals from L. reuteri L103 and L. bulgaricus DSM 20081 were studied in

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The lactose conversion towards GOS of both native and


Journal of Agricultural and Food Chemistry 16, 19-21

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detail both in batch and in continuous bioreactors.14,

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achieved were approximately 38% of total sugars (at a lactose conversion rate of 80% and

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an initial lactose concentration of ~200 g/L) using LacLM from L. reuteri L103,20 or 50%

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GOS for recombinant LacZ from L. bulgaricus DSM 20081 (at 90–95% of lactose

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conversion and an initial lactose concentration of ~200 g/L).16 These GOS mixtures

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contained mainly (non-lactose) disaccharides, trisaccharides and tetrasaccharides, in

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which the transferred galactosyl moiety is attached via β-1,3 and β-1,6-linkages. These

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structures are important for potential prebiotics.20

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In order to study the application of these enzymes in more detail we aimed at

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immobilization of these two β-gals. To date, there have been no efforts on immobilization

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of β-gal from L. reuteri L103, even though β-gals are well-studied enzymes in term of

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immobilization.22 Immobilization will enable the reuse of these biocatalysts, and it might

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also contribute to stabilization of the two β-gals, since their thermostability could be a

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limiting factor for their application.23

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The chitin-binding domain (ChBD) is part of some chitin or chitosan-degrading enzymes,

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chitinases or chitosanases, where it is responsible for the tight binding of the enzyme onto

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the substrate, thus increasing the hydrolytic activity. 24, 25 The ChBD of Bacillus circulans

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WL-12 chitinase A1 was one of the most well studied carbohydrate-binding domains. It

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belongs to the carbohydrate-binding module family 12 of the CAZY (carbohydrate-active

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enzyme) database. The three dimensional structure of this ChBD have been determined by

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NMR.26 It consists of 45 amino acids, Ala655-Gln699, including several hydrophobic and

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aromatic residues with low solvent accessibility, thus forms the rigid and compact

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structure.26 Two β-sheets is composed of five strands including Thr660-Tyr662, Gln666-

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Tyr670, Lys673-Cys677, His681-Ser683 and Trp696-Leu698 residues.26 This ChBD has

Maximum GOS yields


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been reported to bind only insoluble or crystalline chitin but not to the chito-

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oligosaccharide, soluble derivatives of chitin or other polysaccharides.27 Based on the

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high affinity to chitin, ChBD from WL-12 have been successfully applied for

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immobilization of several enzymes such as D-hydantoinase 28 or levansucrase 29 on chitin

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material. The procedure for the simultaneously purification and immobilization was a

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simple mixing the enzyme solution to the insoluble chitin without the requirement of

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purified enzyme.

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immobilization methods. For example, the covalent binding method immobilization

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required the hash condition to create the bond between the enzyme molecule to the

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support material.23 The absorption method bases on the weak forces like Van der Waals,

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hydrophobic interaction or hydrogen interaction, thus the immobilized enzymes are

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loosely bound to support materials.30

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This study focuses on the immobilization of heterodimeric L. reuteri L103 LacLM and

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homodimeric L. bulgaricus DSM20081 LacZ β-gal on chitin using ChBD of Bacillus

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circulans WL-12 chitinase A1. The recombinant enzymes were fused to the ChBD via

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DNA-based molecular methods. The biochemical characteristics of these fusion enzymes

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in comparison to the native enzymes are shown, thus the effects of the ChBD and the

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immobilization on the properties of these enzymes are elucidated.

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MATERIALS AND METHODS

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Chemicals and reagents

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All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated and

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were of the highest quality available. The endonucleases were purchased from New

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England BioLabs (Ipswich, MA) and used as recommended by the supplier. T4 DNA

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ligase was from Fermentas (Vilnius, Lithuania).

28, 29

Moreover, this method can overcome the disadvantages of other



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Bacterial strains and media

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The bacterial strains, plasmids and primers used in this study are listed in Tables 1 and 2.

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Lactobacillus plantarum WCFS131 was grown in MRS medium (Oxoid, Basingstoke,

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UK) at 37°C without agitation. Escherichia coli NEB5α (New England BioLabs,

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Ipswich, MA) as cloning host was cultivated in Luria-Bertani (LB) medium at 37°C with

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shaking at 200 rpm. Agar media were prepared by adding 1.5% agar to the respective

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media. Unless otherwise stated, the antibiotic concentrations were 5 µg/mL or 200 µg/mL

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of erythromycin (Erm) for Lactobacillus or E. coli, respectively, and 100 µg/mL of

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ampicillin for E. coli.

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Oligonucleotide primers for PCR amplification were supplied by VBC-Biotech Service

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GmbH (Vienna, Austria). The appropriate endonuclease restriction sites were introduced

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in the forward and reverse primers (Table 2).

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Plasmid construction and transformation

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The DNA amplification was performed with Phusion High-Fidelity DNA polymerase

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(Finnzymes, Espoo, Finland). Plasmid DNA from E. coli was purified using the Gene

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Elute plasmid Miniprep kit (Sigma, St. Louis, MO). DNA was purified with the

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WizardRSV Gel and PCR Clean-up system kit (Promega, Madison, WI). The pJET1.2

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plasmid (CloneJET PCR cloning kit, Fermentas) was used for sub-cloning when

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necessary.

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Three recombinant fusion proteins were constructed. Two are based on LacLM from L.

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reuteri L103, and the chitin-binding domain ChBD was attached upstream of LacL

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(termed ChBD-LacLM) or downstream of LacM (termed LacLM-ChBD). The third

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fusion protein was based on LacZ of L. bulgaricus DSM20081 with ChBD linked

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downstream of LacZ (termed LacZ-ChBD).


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For construction of the expression plasmids of the fusion proteins LacLM-ChBD or LacZ-

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ChBD, the fragment of chbd of Bacillus circulans WL-12 chitinase A1 was amplified

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from the plasmid pTxB1 (New England BioLabs) with the primer pair F1 and R1 (Table

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2). After double digestion with XhoI and Acc65I, this fragment was ligated to the

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fragment of pSIP403,32 which contained gusA as the original reporter gene and was

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treated by the same endonucleases, resulting in plasmid pSCBD-gusA (Table 1, Figure 1).

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The gusA gene was then excised by double digestion with NcoI and XhoI to obtain the

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empty vector pSCBD. This empty vector was ligated to the NcoI- and XhoI-fragment of

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lacLM (encoding LacLM from L. reuteri L103) from pHA103.115 resulting in pSCBDlac1

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(Table 1, Figure 1). Similarly, lacZ was amplified from pJETlacZ16 by PCR with the two

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primers F4, R4 (Table 2), and then digested by BsmBI and XhoI prior to ligation into

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empty pSCBD resulting in pSCBDlac3 (Table 1, Figure 1).

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For the construction of the expression plasmid for ChBD-LacLM, chbd from pTxB1 was

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amplified with the two primers F2 and R2, resulting in a fragment of F2-chbd-R2. The

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lacLM gene was PCR-amplified from the lacLM template in pTH103.1 using the F3 and

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R3 primers resulting in the fragment F3-lacLM-R3. Because of the 18 nt-complimentary

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sequence between F3 and R2 (Table 2), the fragment F2-chbd-R2 could be used as a mega

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forward primer in combination with R3 as reverse primer to amplify the F3-lacLM-R3

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template, resulting in the fragment F2-chbd-lacLM-R3. The PCR product from this

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amplification was digested by BsaI and XhoI and ligated to NcoI-XhoI digested pSIP403,

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resulting in pSCBDlac2 (Table 1, Figure 1).

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This strategy resulted in expression vectors, in which the target genes, lacLM or lacZ

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linked to the chbd sequence, are controlled by the inducible promoter PsppA, similar to gusA

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in the original pSIP403 vector.32 These plasmids were then electroporated into competent



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cells of L. plantarum WCFS1 as previously described.33 The transformants carrying the

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plasmids were determined and screened by using a colony PCR amplification of the target

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fusion genes.

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Evaluation of the expression of recombinant β-galactosidases

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This experiment was performed following a previous article.18

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Fermentation

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In order to obtain sufficient amount of the enzymes for characterization, immobilization

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and application, L. plantarum WCFS1 harboring different plasmids was cultivated in 1

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liter of medium to obtain larger amounts of the recombinant enzymes. The bacterial cells

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were induced at OD600nm ~0.3 and harvested at OD600nm ~6. The cell pellets were

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disrupted by homogenization with a French Press (Aminco, Silver Spring, MD). The cell-

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free extract, clarified by ultracentrifugation (Beckman, USA) at 30000 × g for 20 min,

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4°C, was used either directly for immobilization on chitin beads (New England BioLabs)

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or for a single-step affinity purification using the p-aminobenzyl 1-thio-β-D-

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galactopyranoside (ABTG) resin (Sigma) as described in a previous study.14

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Immobilization on chitin beads

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Before use, the chitin beads were washed three times with sodium phosphate buffer (50

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mM, pH 6.5) and re-suspended in the same buffer. Five hundred microliters of chitin bead

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suspension were mixed with the same volume of diluted cell-free crude extracts of 1000

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U/mL of oNPG activity. The immobilization experiments were carried out at 4°C with

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gentle agitation for 1 to 18 hours. Chitin beads were separated from the supernatant by

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filtration and rinsed with sodium phosphate buffer. The supernatant and wash solutions

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were collected and pooled for SDS-PAGE analysis and protein measurement. Chitin

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beads were re-suspended in buffer for further studies.


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The immobilization yield (IY) and the activity retention (AR) were calculated according

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to Klein et al.34 The IY (%) is defined as the ratio of immobilized activity in relation to

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total applied activity, in which the immobilized activity was determined by subtraction of

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the residual activity in the supernatant after immobilization from the total applied activity.

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The AR (%) is the percentage of activity measured on chitin beads in relation to the

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theoretical immobilized activity.

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The amount of chitin-bound protein was indirectly estimated by subtraction of the protein

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concentration in the supernatant after immobilization from that in the crude extract (prior

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to the immobilization).

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Influence of pH and temperature on activity and stability of the immobilized enzymes

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The pH optimum of β-gal activity was evaluated in Briton-Robinson buffer 17 with the pH

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ranging from 4 and 9. To determine the pH stability, the immobilized enzymes were

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incubated at 30°C or 37°C in Briton-Robinson buffer of different pH values. At various

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time intervals, the residual activities were measured with the oNPG assay. The

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inactivation constants kin were obtained by linear regression of ln(residual activity) versus

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time. The half-life values τ1/2 were calculated using τ1/2 = ln(2)/ kin 35.

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The optimum temperature for hydrolysis activity of β-gals with both substrates lactose

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and oNPG was determined in the range of 20°C and 80°C. For estimation of the kinetic

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thermostability, enzymes were incubated in sodium phosphate buffer 50 mM pH 6.5 at

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different temperatures ranging from 20°C to 80°C. At various time intervals, residual

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activities were measured with oNPG as substrate and plotted versus the incubation time.

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The half-life values of thermal inactivation (τ1/2) were similarly calculated as above.

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Steady-state kinetic measurements



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To estimate the kinetic parameters of recombinant and fusion β-gals for lactose and

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oNPG, substrate concentrations were varied from 1 to 25 mM for oNPG, and 10 to 600

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mM for lactose.14 The enzyme assays were performed for 10 min at 30°C in 50 mM

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sodium phosphate buffer (pH 6.5). The kinetic parameters were calculated by using the

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Henri-Michaelis-Menten model and non-linear least-square regression.

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Reusability of the immobilized enzymes

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To assess the possibility of recycling the immobilized enzyme preparations for

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conversion, immobilized β-gal LacLM L. reuteri L103 was added to sodium phosphate

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buffer (50 mM, pH 6.5) containing 600 mM lactose and maintained at 30°C and 600 rpm

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agitation for the conversion of lactose. Every 24 hours, the chitin beads carrying the

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enzyme were filtered off and rinsed with buffer, and reused in another batch conversion

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experiment under identical conditions. The filtrates from every conversion cycle were

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collected to determine the glucose concentration by using a commercial D-glucose assay

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kit (GOPOD Format, Megazyme, Wicklow, Ireland). The glucose concentration in the

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filtrate from the first conversion was taken as 100% of converting ability of the

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immobilized enzyme preparation.

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Immobilized LacZ from L. bulgaricus DSM 20081 was also tested for its reusability but

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using the substrate oNPG. After 5 minutes of incubation with oNPG at 30°C, chitin beads

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carrying LacZ were separated from the liquid phase and reused in the next conversion

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experiments. The concentration of oNP in the liquid phases after the conversion was

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measured with a spectrophotometer at 420 nm and used to calculate the residual

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converting ability of enzyme in each repeating conversion.

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Lactose hydrolysis and transgalactosylation experiments


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The transformation of lactose was carried out in batch mode. Both chitin-immobilized I-

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LacLM-ChBD and purified soluble LacLM-ChBD were added at equal concentrations of

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1.5 Ulactose/mL to sodium phosphate buffer (50 mM, pH 6.5) containing 10 mM MgCl2 and

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205 g/L lactose. Lactose conversion experiments were performed at 30°C for 24 hours at

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600 rpm of agitation.

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The chitin-immobilized β-gal of L. bulgaricus was applied in different amounts ranging

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from 1.7 to 9.7 Ulactose/mL to sodium phosphate buffer (50 mM, pH 6.5) containing 10

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mM MgCl2 and 50 g/L or 205 g/L lactose. This enzyme preparation was also used for

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conversion of lactose in ultra-high-temperature treated whole cow milk. The incubation

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temperature was varied from 37 to 60°C.

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In all conversion experiments, samples were periodically withdrawn, heated at 99°C for 5

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minutes and further analyzed for lactose, galactose, glucose and galacto-oligosaccharides

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(GOS) present in the samples. Qualitative analysis of sugar was performed by TLC as

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described previously by Nguyen and co-workers.14 Furthermore, samples from conversion

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experiments were quantitatively analyzed by High Performance Liquid Chromatography

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(HPLC) (Dionex, Germany) equipped with a refractive index detector and an Aminex

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HPX-87K (300 mm x 7.8 mm) carbohydrate analysis column (Bio-Rad, CA, USA). The

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chromatographic separation was performed at 80°C with ultra-pure water used as eluting

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solvent at a flow rate of 0.5 mL per min. The concentration of saccharides was calculated

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by interpolation from external standards. Total GOS concentration was calculated by

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subtraction of the quantified saccharides (lactose, glucose, galactose) from the initial

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lactose concentration. The GOS yield (%) was defined as the percentage of GOS

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produced in the samples compared to initial lactose.

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β-galactosidase assay



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β-Galactosidase activity was determined using o-nitrophenyl-β-D-galactopyranoside

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(oNPG) or lactose as substrates following Nguyen et al. (2006).14 In brief, the activity

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assays were performed in 50 mM sodium phosphate buffer of pH 6.5 at 30°C, and the

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final substrate concentrations in the 10-min assays were 22 mM for oNPG and 600 mM

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for lactose. One unit of oNPG activity was defined as the amount of enzyme releasing 1

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µmol of oNP per minute, whereas one unit of lactase activity was defined as the amount

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of enzyme releasing 1 µmol of D-glucose per minute under the given conditions.

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Protein measurement

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Protein concentration was determined by using the method of Bradford36 with bovine

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serum albumin, BSA (Sigma) as standard.

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SDS-PAGE analysis

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For visual observation of the expression level of the recombinant β-gals in L. plantarum

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WCFS1 and the effectiveness of the immobilization, cell-free extracts, liquid phases and

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chitin beads (after immobilization) were analysed by SDS-PAGE using MOPS as running

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buffer.16

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Statistical Analysis

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All experiments and measurements were performed at least in duplicate, and the data are

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given as the mean ± standard deviation when appropriate. Student’s t test was used for the

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comparison of data with significance value ∝ = 0.05.

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RESULTS AND DISCUSSIONS

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Plasmid construction and expressions of recombinant β-galactosidases in L.

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plantarum WCFS1


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In this work, the three expression plasmids pSCBDlac1, pSCBDlac2 and pSCBDlac3,

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containing the sequences of the three recombinant fusion proteins LacLM-ChBD, ChBD-

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LacLM and LacZ-ChBD, respectively, were constructed and successfully electroporated

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into L. plantarum WCFS1. The transcription of the encoding sequences lacLM-chbd,

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chbd-lacLM and lacZ-chbd is regulated by the inducible promoter PsppA in these plasmids

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(Figure 1). The expression levels of the different constructs and the recombinant β-

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galactosidases in L. plantarum WCFS1 were investigated.

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Induced cells formed intracellular LacLM-ChBD at around 32 U per mL of fermentation

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broth with a specific activity of ca. 179 U per mg protein (Table 3). ChBD-LacLM was

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expressed in 3-fold lower yields (p

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