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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49

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]

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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

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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