AGARASE-3,6-ANHYDRO-L-GALACTOSIDASE-ARABINOSE ISOMERASE ENZYME COMPLEX AND METHOD FOR PRODUCTION OF TAGATOSE FROM AGAR USING THE SAME

Abstract

The present disclosure relates to an enzyme complex of arabinose isomerase, agarase and 3,6-anhydro galactosidase and a method for producing tagatose by degrading agar using the same. By using the enzyme complex according to the present disclosure, agar obtained from marine biomass can be degraded effectively and useful physiologically active substances such as tagatose can be obtained effectively therefrom.

Claims

1. An agarase complex wherein: a fusion protein 1 in which a monosaccharide convertase and a dockerin module are bound; a fusion protein 2 in which agarase and a dockerin module are bound; and a fusion protein 3 in which 3,6-anhydro-L-galactosidase and a dockerin module are bound; are linked via dockerin-cohesin binding by a mini scaffold protein comprising a cohesin module.

2. The enzyme complex according to claim 1, wherein the monosaccharide convertase is arabinose isomerase derived from Lactobacillus.

3. The enzyme complex according to claim 2, wherein the arabinose isomerase comprises amino acid sequence of SEQ ID NO 1.

4. The enzyme complex according to claim 1, wherein the dockerin is derived from cellulase.

5. The enzyme complex according to claim 4, wherein the dockerin is encoded nucleotide sequence of SEQ ID NO 35.

6. The enzyme complex according to claim 4, wherein the cellulase is selected from a group consisting of endo--1,4-glucanase B, endo--1,4-xylanase B and exo-glucanase S.

7. The enzyme complex according to claim 1, wherein the agarase is derived from one selected from a group consisting of Pseudomonas, Saccharophagus and Aleromonas.

8. The enzyme complex according to claim 7, wherein the agarase is -agarase.

9. The enzyme complex according to claim 8, wherein the -agarase is encoded nucleotide sequence of SEQ ID NO 26.

10. The enzyme complex according to claim 1, wherein the 3,6-anhydro-L-galactosidase is derived from Zobellia.

11. The enzyme complex according to claim 10, wherein the 3,6-anhydro-L-galactosidase is encoded nucleotide sequence of SEQ ID NO 36.

12. The enzyme complex according to claim 1, wherein the mini scaffold protein is one selected from a group consisting of mini cellulose-binding protein A (mCbpA), Clostridium thermocellulm-derived mini scaffold protein (mCipA) and Clostridium cellulolyticum-derived mini scaffold protein (mCipC).

13. The enzyme complex according to claim 12, wherein the mini scaffold protein is mini cellulose-binding protein A (mCbpA).

14. The enzyme complex according to claim 13, wherein the mini cellulose-binding protein A is encoded nucleotide sequence of SEQ ID NO 17.

15. A method for producing tagatose by degrading biomass using the enzyme complex according to claim 1.

16. The method according to claim 15, wherein the biomass is agar derived from red algae.

17. A method for preparing an enzyme complex, comprising: preparing a fusion protein 1 by linking a dockerin module to arabinose isomerase; preparing a fusion protein 2 by linking a dockerin module to -agarase; preparing a fusion protein 3 by linking a dockerin module to 3,6-anhydro-L-galactosidase; preparing a mini scaffold protein having a cohesin module; and preparing an agarase complex by binding the cohesin module to the dockerin modules by quantifying the fusion proteins 1-3 and the mini scaffold protein of the step (d) to the same concentration and proportion and mixing them in a binding solution comprising 25 mM calcium chloride (CaCl.sub.2)).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0045] FIG. 1A and FIG. 1B show schematic views of a recombinant vector prepared according to the present disclosure (1A) and the expression of the vector in E. coli (1B).

[0046] FIG. 2 shows the activity of converting D-galactose to D-tagatose using an enzyme complex prepared according to the present disclosure.

[0047] FIG. 3 shows the activity of degrading agar and converting to tagatose of an enzyme complex prepared according to the present disclosure.

[0048] FIG. 4A and FIG. 4B show the activity of an enzyme complex prepared according to the present disclosure for substrates (4A: purified gar, 4B: agar).

BEST MODE

[0049] Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples.

EXAMPLES

Example 1. Expression Vector for Novel Enzyme Complex

[0050] In order to prepare a novel enzyme complex, a vector and a transformant including genes encoding the components of the enzyme complex were prepared.

[0051] 1.1 Lactobacillus-Derived Arabinose Isomerase Expression Vector

[0052] For cloning of proteins for producing tagatose from an agar degradation product as a substrate, a vector expressing the arabinose isomerase gene was prepared first.

[0053] Referring to the base sequence of the arabinose isomerase (LsAraA) gene from the gDNA of bacteria in the genus Lactobacillus, primers were designed and synthesized such that the Sac I recognition sequence was inserted to the 5 end of a forward primer (SEQ ID NO 2) and the Hind III recognition sequence was inserted to the 5 end of a reverse primer (SEQ ID NO 3). Then, PCR was conducted using the synthesized primers. As a result, the PCR band of the 1479-bp arabinose isomerase (LsAraA) gene (SEQ ID NO 4) was observed (result not shown).

[0054] Then, the arabinose isomerase (LsAraA) gene was purified, ligated to the E. coli expression vector pColdII, and transformed into E. coli BL21 using the restriction enzymes Sac I and Hind III. The transformant was named BL21/LsAraA. Then, the ligated recombinant plasmid DNA was isolated from the transformant. The recombinant plasmid vector was named pColdII LsAraA.

[0055] 1.2 Preparation of Lactobacillus-Derived Arabinose Isomerase Expression Vector and Transformant Fused with Dockerin

[0056] An expression vector and a transformant wherein the arabinose isomerase enzyme of Example 1.1 was fused with a cellulase-derived dockerin domain were prepared.

[0057] A. Arabinose Isomerase Gene Fragment

[0058] Referring to the base sequence of the arabinose isomerase (LsAraA) gene from the genomic DNA of bacteria in the genus Lactobacillus, primers were designed and synthesized such that the Sac I recognition sequence was inserted to the 5 end of a forward primer (SEQ ID NO 5) and the 10-bp sequence at the N-terminal of the dockerin moiety of the endo--1,4-glucanase B gene derived from Clostridium cellulovorans was inserted to the 5 end of a reverse primer (SEQ ID NO 6). Then, PCR was conducted using the synthesized primers. As a result, the PCR bands of the Sac I recognition sequence capable of recognizing the dockerin moiety and the 1521-bp arabinose isomerase (LsAraA) gene were observed (result not shown).

[0059] B. Dockerin Fragment

[0060] In addition, referring to the base sequence of the dockerin moiety of the endo--1,4-glucanase B gene from the gDNA of Clostridium cellulovorans, primers were designed and synthesized such that the 10-bp sequence at the C-terminal of the arabinose isomerase (LsAraA) gene was inserted to the 5 end of a forward primer (SEQ ID NO 7) and the Kpn I recognition sequence was inserted to the 5 end of a reverse primer (SEQ ID NO 8).

[0061] The 10-bp sequence at the N-terminal of the dockerin moiety of the endo--1,4-glucanase B gene is represented by SEQ ID NO 9, and the 10-bp sequence at the C-terminal of the arabinose isomerase (LsAraA) gene is represented by SEQ ID NO 10.

[0062] Then, PCR was conducted using the synthesized primers. As a result, the PCR band of the dockerin moiety of the 195-bp endo--1,4-glucanase B gene (SEQ ID NO 11) was observed.

[0063] C. Preparation of Fusion Protein Expression Vector and Transformant

[0064] The gene amplification product of the arabinose isomerase (LsAraA) gene and the dockerin domain of cellulase obtained above was subjected to electrophoresis on 0.8% agarose gel, and the DNA fragments on the agarose gel were recovered using a gel extraction kit (GeneAll).

[0065] Using the two recovered DNA fragments, primers were designed and synthesized such that the Sac I recognition sequence was inserted to the 5 end of a forward primer (SEQ ID NO 12) and the Kpn I recognition sequence was inserted to the 5 end of a reverse primer (SEQ ID NO 13).

[0066] Then, overlap PCR was conducted using the recovered DNA fragments in order to link the arabinose isomerase (LsAraA) gene with the dockerin domain of cellulase. The overlap PCR reaction was conducted at 94 C. for 2 minutes followed by 10 cycles of 94 C. for 30 seconds, 52 C. for 25 minutes and 72 C. for 5 minutes, finally at 72 C. for 5 minutes. As a result of the PCR, a PCR band of the bacterium-derived expansin gene linked with the 1671-bp dockerin domain of cellulase (SEQ ID NO 14) was observed (result not shown).

[0067] After purifying the dockerin-fused arabinose isomerase LsAraA Doc gene, a recombinant expression vector was prepared by ligating the SacI and KpnI restriction enzyme sequences to the E. coli expression vector pColdII. Then, a transformant was prepared by transforming E. coli BL21 with the expression vector. Then, the plasmid DNA of the ligated recombinant expression vector was isolated from the transformant. The recombinant vector was named pColdII LsAraA-Doc (FIG. 1A), and the E. coli transformant was named BL21/LsAraA-Doc.

[0068] 1.3 Confirmation of Expression of Novel Enzyme Complex in Transformant

[0069] In order to investigate protein expression in the transformant obtained in Example 1, purification and SDS-PAGE were performed using His-Tag.

[0070] Expression was induced in the E. coli transformant with IPTG at 16 C. for 12 hours using 1 mM IPTG. After creating a condition where the arabinose isomerase enzyme and the dockerin-fused arabinose isomerase enzyme can be expressed, the cells were cultured at 16 C. for 12 hours under shaking and then centrifuged. The cells were lysed by sonication and then centrifuged. Proteins obtained by concentrating the supernatant (Millipore, Amicon 10 kDa cutoff) was loaded onto SDS-PAGE. Then, the proteins were analyzed by western blot using the His-tag attached at the N-terminal. As a result, the arabinose isomerase enzyme and the dockerin-fused arabinose isomerase enzyme were observed at the expected locations (FIG. 1B).

Example 2. Construction of Arabinose Isomerase Enzyme Complex with Mini Cellulose-Binding Protein Linked and Analysis of Activity for Galactose Substrate

[0071] 2.1 Construction of Expression Vector for Enzyme Complex

[0072] For cloning of the mini cellulose-binding protein A gene having a cellulose-binding module (CBM) and two cohesin modules of cellulose-binding protein A which is the primary scaffolding subunit of Clostridium cellulovorans, primers were synthesized such that the BamHl recognition sequence (ggatcc) was inserted to the 5 end of a forward primer (SEQ ID NO 15) and the XhoI recognition sequence (ctcgag) was inserted to the 5 end of a reverse primer (SEQ ID NO 16) referring to the base sequence. As a result, a 1659-bp PCR band containing the mCbpA gene (SEQ ID NO 17) which is a part of the cellulose-binding protein A gene derived from Clostridium cellulovorans was observed (result not shown).

[0073] 2.2 Confirmation of Enzyme Complex Formation

[0074] In order to confirm the formation of a complex through binding between the arabinose isomerase linked with the dockerin module of the endo--1,4-glucanase B gene and the mini cellulose-binding protein mCbpA, the two proteins were mixed and incubated at low temperature and then incubated to induce complex formation. For the complex formation, the mini scaffold protein and the arabinose isomerase were quantitated both to 10 nmol and then mixed in a binding solution containing 25 mM CaCl.sub.2). For binding between the cohesin module and the dockerin module, reaction was conducted at 4 C. for 24 hours.

[0075] The formation of a complex through binding between the arabinose isomerase enzyme linked with the dockerin module of the endo--1,4-glucanase B gene and the mini cellulose-binding protein mCbpA was confirmed by measuring increased tagatose conversion activity. As a result of measuring tagatose conversion activity for mCbpA, arabinose isomerase and arabinose isomerase-mCbpA using galactose as a substrate, the degradation activity was increased in the order of mCbpA (M), arabinose isomerase (LsAraA, L) and arabinose isomerase-mCbpA (LM) (FIG. 2). Because mCbpA is an inactive protein with no tagatose conversion activity, the increased activity is due to the enzyme complex formation.

[0076] In addition, since the fusion protein with the mini cellulose-binding protein mCbpA showed higher tagatose conversion activity than arabinose isomerase (LsAraA) alone, it was confirmed that mCbpA improves the tagatose conversion activity of arabinose isomerase (LsAraA).

Example 3. Construction of Enzyme Complex Expression Vector for Agar Degradation Product

[0077] For production of tagatose from the less expensive substrate agar, a fusion protein including agarase and dockerin and a fusion protein including 3,6-anhydro-L-galactosidase and dockerin were designed.

[0078] 3.1 Preparation of Enzyme Complex (Fusion of -Agarase and Dockerin)

[0079] A. Isolation of -Agarase Gene

[0080] In order to bind -agarase to the dockerin gene, primers for processing both ends of the -agarase gene were prepared. Primers were designed and synthesized such that the Sac I recognition sequence was inserted to the 5 end of a forward primer (SEQ ID NO 18) and the referring to the base sequence of the -agarase AgaB gene from the genomic DNA of bacteria in the genus Zobellia and a 10-bp sequence at the N-terminal was inserted to the 5 end of a reverse primer (SEQ ID NO 19) referring to the base sequence of the dockerin moiety of the endo--1,4-glucanase B gene derived from Clostridium cellulovorans. Then, PCR was conducted using the synthesized primers. The PCR reaction was conducted at 95 C. for 5 minutes, followed by 30 cycles of 95 C. for 1 minute, 52 C. for 1 minute and 72 C. for 2 minutes, finally at 72 C. for 5 minutes. As a result, a 1005-bp PCR band containing the -agarase gene (SEQ ID NO 20) was observed (result not shown).

[0081] B. Processing of -Agarase Gene

[0082] Referring to the base sequence of the dockerin moiety of the endo--1,4-glucanase B gene from the gDNA of Clostridium cellulovorans, primers were designed and synthesized such that a 10-bp sequence at the C-terminal of the -agarase AgaB gene from the genomic DNA of bacteria in the genus Zobellia was inserted to the 5 end of a forward primer (SEQ ID NO 21) and the NotI recognition sequence was inserted to the 5 end of a reverse primer (SEQ ID NO 22). Then, PCR was conducted using the synthesized primers. The PCR reaction was conducted at 95 C. for 5 minutes, followed by 30 cycles of 95 C. for 1 minute, 52 C. for 1 minute and 72 C. for 2 minutes, finally at 72 C. for 5 minutes. As a result, a 211-bp PCR band containing the dockerin moiety of the endo--1,4-glucanase B gene (SEQ ID NO 23) was observed (result not shown).

[0083] C. Construction of Enzyme Complex ( Agarase-Dockerin) Expression Vector

[0084] The gene amplification product of the agarase AgaB gene and the dockerin domain of the cellulase obtained above was subjected to electrophoresis on 0.8% agarose gel. The DNA fragments on the agarose gel were recovered using a gel extraction kit (GeneAll).

[0085] Then, overlap PCR was conducted using the recovered DNA fragments in order to link the agarase AgaB gene with the dockerin domain of cellulase. The overlap PCR reaction was conducted at 94 C. for 2 minutes, followed by 10 cycles of 94 C. for 30 seconds, 52 C. for 25 minutes and 72 C. for 5 minutes, finally at 72 C. for 5 minutes. From the recovered two DNA fragments, primers were designed and synthesized such that the SacI recognition sequence was inserted to the 5 end of a forward primer (SEQ ID NO 24) and the NotI recognition sequence was inserted to the 5 end of a reverse primer (SEQ ID NO 25). After conducting PCR at 95 C. for 5 minutes, followed by 30 cycles of 95 C. for 1 minute, 52 C. for 1 minute and 72 C. for 2 minutes, finally at 72 C. for 5 minutes, a 1225-bp PCR band containing the chimeric -agarase AgaB gene derived from Zobellia galactanivorans with the dockerin domain of cellulase linked (SEQ ID NO 26) was observed as the PCT product (result not shown).

[0086] Then, after cleaving the dockerin-fused chimeric -agarase AgaB gene and the AgaB Doc gene, E. coli BL21 was transformed by ligating to the E. coli expression vector pET22b(+) with SacI and NotI. Then, the ligated recombinant plasmid DNA was isolated from the transformant. The recombinant vector was named pET22(+) AgaB-Doc, and the E. coli transformant was named BL21/AgaB-Doc.

[0087] 3.2 Preparation of Enzyme Complex (Fusion of 3,6-Anhydro-L-Galactosidase and Dockerin)

[0088] A. Isolation of 3,6-Anhydro-L-Galactosidase Gene

[0089] For cloning of the dockerin domain of cellulase for 3,6-anhydro-L-galactosidase with dockerin bound, primers were designed and synthesized such that the EcoRI recognition sequence was inserted to the 5 end of a forward primer (SEQ ID NO 27) referring to the base sequence of the 3,6-anhydro-L-galactosidase AhgA gene from the genomic DNA of bacteria in the genus Zobellia and a 10-bp sequence at the N-terminal was inserted to the 5 end of a reverse primer (SEQ ID NO 28) referring to the base sequence of the dockerin moiety of the endo--1,4-glucanase B gene derived from Clostridium cellulovorans. Then, PCR was conducted using the synthesized primers. The PCR reaction was conducted at 95 C. for 5 minutes, followed by 30 cycles of 95 C. for 1 minute, 52 C. for 1 minute and 72 C. for 2 minutes, finally at 72 C. for 5 minutes. As a result, a 1224-bp PCR band containing 3,6-anhydro-L-galactosidase (SEQ ID NO 29) was observed (result not shown).

[0090] B. Isolation of Dockerin Gene

[0091] Referring to the base sequence of the dockerin moiety of the endo--1,4-glucanase B gene from the gDNA of Clostridium cellulovorans, primers were designed and synthesized such that a 10-bp sequence at the C-terminal of the 3,6-anhydro-L-galactosidase AhgA gene from the genomic DNA of bacteria in the genus Zobellia was inserted to the 5 end of a forward primer (SEQ ID NO 30) and the Hind III recognition sequence was inserted to the 5 end of a reverse primer (SEQ ID NO 31). Then, PCR was conducted using the synthesized primers. The PCR reaction was conducted at 95 C. for 5 minutes, followed by 30 cycles of 95 C. for 1 minute, 52 C. for 1 minute and 72 C. for 2 minutes, finally at 72 C. for 5 minutes. As a result, a 211-bp PCR band containing the dockerin moiety of the endo--1,4-glucanase B gene (SEQ ID NO 32) was observed.

[0092] C. Construction of Enzyme Complex (Galactosidase-Dockerin) Expression Vector

[0093] The gene amplification product of the 3,6-anhydro-L-galactosidase AhgA gene and the dockerin domain of cellulase obtained above was subjected to electrophoresis on 0.8% agarose gel, and the DNA fragments on the agarose gel were recovered using a gel extraction kit (GeneAll).

[0094] Then, overlap PCR was conducted using the recovered DNA fragments in order to link the 3,6-anhydro-L-galactosidase AhgA gene and the dockerin domain of cellulase. From the recovered two DNA fragments, primers were designed and synthesized such that the EcoR I recognition sequence was inserted to the 5 end of a forward primer (SEQ ID NO 33) and the HindIII recognition sequence was inserted to the 5 end of a reverse primer (SEQ ID NO 34). After conducting PCR at 95 C. for 5 minutes, followed by 30 cycles of 95 C. for 1 minute, 52 C. for 1 minute and 72 C. for 2 minutes, finally at 72 C. for 5 minutes, a 1383-bp PCR band containing the chimeric 3,6-anhydro-L-galactosidase AhgA gene (SEQ ID NO 36) derived from Zobeffia galactanivorans with the dockerin domain of cellulase linked (SEQ ID NO 35) was observed (result not shown).

Example 4. Analysis of Activity of Enzyme Complex (Dockerin Complex) for Agar Degradation Product

[0095] Expression was induced in the BL21/AgaB-Doc and BL21/AhgA-Doc recombinant strains using 1 mM IPTG at 16 C. for 12 hours. After centrifugation, the cells were lysed by sonication and then centrifuged. Proteins were obtained by concentrating the supernatant (Millipore, Amicon 10 kDa cutoff).

[0096] Tagatose conversion activity was investigated using agar as a substrate. More specifically, purified agar was degraded using dockerin-agarase (cAgaB), dockerin-3,6-anhydro-L-galactosidase (cAhgA), dockerin-agarase (cAgaB) or dockerin-3,6-anhydro-L-galactosidase (cAhgA), and then tagatose conversion activity was analyzed after adding the dockerin-arabinose isomerase fusion protein (LsAraA).

[0097] As seen from FIG. 3, the combination of dockerin-agarase (cAgaB) and dockerin-3,6-anhydro-L-galactosidase (cAhgA), i.e., cAgaB/cAhgA+LsAraA, showed higher tagatose conversion activity for the purified agar.

Example 5. Preparation of Enzyme Complex

[0098] Finally, the enzyme complex according to the present disclosure (-agarase-3,6-anhydro-L-galactosidase-arabinose isomerase) was prepared as follows. The mini scaffold protein, -agarase, 3,6-anhydro-L-galactosidase and arabinose isomerase were quantitated to the same concentration of 10 nmol and same proportion and then mixed in a binding solution containing 25 mM CaCl.sub.2). Then, reaction was conducted at 4 C. for 24 hours for binding between the cohesin module and the dockerin module.

Example 6. Analysis of Activity of Enzyme Complex for Various Agar Substrates

[0099] The activity of the complexes consisting of -agarase-3,6-anhydro-L-galactosidase-arabinose isomerase and mCbpA with various compositions was analyzed using various agar (purified agar and red algae agar) substrates.

[0100] As seen from FIG. 4A and FIG. 4B, the -agarase-3,6-an hydro-L-galactosidase-arabinose isomerase enzyme complex showed higher tagatose conversion efficiency than agarase or arabinose isomerase alone for both the purified agar (4A) and the red algae agar (4B). In the figure, C stands for control, B for -agarase, A for 3,6-anhydro-L-galactosidase, M for mini scaffold protein, and L for arabinose isomerase.