KR20160049072A - Novel thermostable beta-glucosidase and the methods of preparation thereof - Google Patents

Abstract

The present invention relates to novel beta-glucosidase and a method for preparing the same. The present invention relates to beta-glucosidase isolated from T. pacificus, a gene encoding the beta-glucosidase, a recombinant vector including the gene, a host cell transformed by the recombinant vector, and a method for preparing beta-glucosidase by using the host cell. The beta-glucosidase according to the present invention shows hydrolysis activity on various pNP-glucopyranoside and oligosaccharide as well as laminarin. In particular, the beta-glucosidase shows exo-hydrolysis activity on laminarin, thereby being used to degrade laminarin in combination with endo-hydrolase.

Description

Novel thermostable beta-glucosidase and methods for producing the same are disclosed.

The present invention relates to a novel beta-glucosidase and a method for producing the same.

Research on renewable energy is actively being carried out in order to develop energy that can replace petroleum and coal in accordance with climate change convention and environmental regulations.

In the process of obtaining energy using bioenergy, grains such as sugarcane, corn and woody resources such as forestry and agricultural by-products were used. However, due to the limitation of cultivation area and difficulty in supplying and receiving it, production of biofuels using grain and woody resources There was difficulty.

In order to overcome these problems, marine algae with many fibers and polysaccharides are attracting attention as a new energy source. Such seaweeds contain various types of polysaccharides. In the brown algae such as mana, And 5.7% ~ 14.3%. Among the cells, it is composed of viscous polysaccharides such as alginic acid, fucoidan and laminarin, which is a storage polysaccharide.

Laminarin is the main skeleton of the β-1,3-glucoside bond, and is partially composed of β-1,6-glucosidic bonds, and alginic acid is D-mannuronic acid and L-glutaric acid are β- (MM), polyglutaric acid (GG), or a heteropolymer (MG) mixture of two components mixed together in homopolymer form.

Alginic acid and fucoidan, which are seaweed polysaccharides, are extracted from brown algae by processing methods such as alkaline, acid or enzyme treatment, and are used industrially as food additives and health food resources. However, due to the complicated structure of brown algae itself, It is difficult to use it as a substrate for fuel production, and the treatment of by-products and wastes generated in addition to currently used materials remains as a problem.

In KR2013-0097347 A, Bacillus megaterium , Pantoea agglomerans , Stenotrophomonas terrae , Microbacterium oxydans, Bacillus subtilis, Bacillus subtilis, The present invention relates to a method for decomposing by-products of brown algae processing and extraction using six complex microorganisms and complex microorganisms consisting of Bacillus bataviensis and Bacillus amyloliquefaciens .

In KR2013-0033635 A No. describes a micro tumefaciens oxy thiooxidans FS4 (Microbacteriumoxydans FS4) having a laminarin resolution.

The present inventors have found a novel beta-glucosidase capable of degrading laminarin and completed the present invention.

KR 2013-0097347 A KR 2013-0033635 A

It is a first object of the present invention to provide a novel beta-glucosidase.

A second object of the present invention is to provide a novel method for producing beta-glucosidase.

For the above purpose, a first aspect of the present invention is a novel beta-glucosidase of SEQ ID NO: 2 and functional equivalents thereof. "Functional equivalents" of the present invention include a portion of the glucosidase enzyme of SEQ ID NO: 2 Or the amino acid sequence variant in which all or part of the amino acid is deleted or added, all of which maintains the above enzyme function. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at a site that is not directly involved in the activity of the glucosidase enzyme.

A second aspect of the present invention provides a nucleic acid sequence encoding the glucosidase of SEQ ID NO: 2 and an equivalent nucleic acid sequence to the nucleic acid sequence. More specifically, the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 1.

An "equivalent nucleic acid sequence" includes the codon axis degenerate sequence of the glucosidase sequence. The term " codon degenerate sequence "means a nucleic acid sequence which is different from the sequence of naturally occurring but encodes a polypeptide having the same sequence as the naturally occurring glucosidase enzyme disclosed in the present invention.

A third embodiment of the present invention is a recombinant vector comprising a gene encoding the novel beta-glucosidase of SEQ ID NO: 2. The term vector used in the present invention refers to a vector capable of transferring another nucleic acid to it Means a nucleic acid molecule. The term expression vector includes a plasmid, a cosmid or a phage capable of synthesizing a protein encoded by each recombinant gene carried by the vector. A preferred vector is a vector capable of self-replication and expression of the nucleic acid to which it is coupled.

A fourth embodiment of the present invention is a host cell transformed with a recombinant vector comprising a gene encoding the novel beta-glucosidase of SEQ ID NO: 2. The term "transformation" Or RNA is absorbed into the cell and the genotype of the cell is changed.

Suitable transforming cells include, but are not limited to, prokaryotes, fungi, plants, animal cells, and the like. Most preferably, Escherichia coli is used.

In a fifth aspect of the present invention, there is provided a method for producing a novel beta-glucosidase, which comprises culturing a recombinant vector transformed with a recombinant vector containing the gene encoding the novel beta-glucosidase of SEQ ID NO: 2 and then isolating beta-glucosidase from the host cell Beta-glucosidase. ≪ / RTI >

A fifth embodiment of the present invention is a method for hydrolyzing a β-1,3-linked polysaccharide using the β-glucosidase of SEQ ID NO: 2.

The present inventors newly identified a gene (Tpa-glu) encoding? -Glucosidase. The gene showed an ORF of 1,464 pairs encoding 487 amino acid residues and found that the deduced amino acid sequence is 77% identical to the β-glucosidase of Pyrococcus furiosus (Accession No. NP_577802). The gene was cloned and expressed in the Escherichia coli system. The recombinant protein was purified and characterized by metal affinity chromatography. Tpa-glu exhibited optimal activity at pH 7.5 and 75 ° C, and the heat resistance showed a half-life of 6 hours at 90 ° C. Tpa-glu exhibited hydrolytic activity against various pNP-glycopyranosides. PNP-? -Glucoglycopyranoside> pNP-? -Galactoglycopyranoside> pNP-? And Kcat / Km values in the order of lycoperianoside> pNP-? - xyloglucopyranoside. Furthermore, the enzyme exhibited exo-hydrolytic activity towards the β-1,3-linked polysaccharide (laminarin) and β-1,3- and β-1,4 linked oligosaccharides. This report is the first description of the enzyme of the ultra-warm archaebacteria exo-hydrolyzing activity towards the β-1,3-linked polysaccharide from the outside. This enzyme can be applied to the combination of? -1,3-endoglucanase necessary for saccharification of laminarin.

Figure 1 shows the gene cluster of the novel beta-glycosyl hydrolase in Thermococcus pacificus.
Figure 2 shows the phylogenetic relationship of several family 1 glycosyl hydrolases from hyperthermophilic archaea (A) and Tpa-glu and Pfu-glu. Tpa-glu is a beta-glucosidase from Thermococcus pacificus; Pfu-glu is a beta-glucosidase from Pyrococcus furiosus ; Pho-glu is a beta-glucosidase from P. horikoshii ; Tko-gly is a beta-glucosidase from T. kodakaraesis ; Pfu-man is a beta-mannosidase from P. furiosus .
Figure 3 shows the temperature (A), pH (B) and measured thermal stability (C) of Tpa-glu for thermal stability. The activity analysis was performed using the following buffers (50 mM each): sodium citrate, pH 4.0 -6.0 (?); Sodium phosphate, pH 6.0-8.0 (?); Tris-HCl, pH 8.0-8.5 (A). Tpa-glu was pre-incubated at 90 ° C and the remaining activity was measured at 75 ° C.
Figure 4 shows the results of measuring the β -1,4- (A) and β -1,3- links the (B) hydrolysis activity of the oligonucleotide Tpa-glu to the saccharide by thin layer chromatography. The reaction for 4 hours (PH 7.0) containing 1 μg enzyme and 1 mM substrate at 75 ° C. The product was analyzed by thin layer chromatography and the respective substrate is shown below : M, size marker; G2 glucose dimer; G3, Glucostreamer; G4, glucose tetramer; G5 glucose pentamer; G6 glucose hexamer.
Figure 5 shows the hydrolytic activity of Tpa-glu against laminarin by thin-layer chromatography. The reaction was carried out at 75 ° C for 0.5, 1, 2, 3 and 4 hours with 1 μg enzyme and 0.25% laminarin (PH 7.0), the product was determined by thin-layer chromatography, and the reaction time is shown in the line below: M, size marker; G2 glucose dimer; G3, Glucostreamer; G4, glucose tetramer; G5 glucose pentamer; G6 glucose hexamer.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples illustrate the present invention, and the contents of the present invention are not limited by the examples.

Materials and methods for the following examples are as follows.

Strain and growth environment

Thermococcus pacificus was obtained from the German Collection of Microorganisms. The strain was routinely cultured at 85 ° C in YPS medium. E. coli DH5α and E. coli BL21-Rosetta ™ (DE3) pLys (Stratagene, La Jolla, CA, USA) were used for replication and overexpression of the Tpa-glu coding gene, respectively. E. coli strains were cultured at 37 ° C in Luria-Bertani (LB) medium supplemented with 50 ug / ml kanamycin.

Replication of the Tpa-glu gene

The approximate gene sequence of T. pacificus was analyzed by our group (not published). Genomic DNA from T. pacificus was prepared through a typical process. The Tpa-glu gene was amplified from genomic DNA by PCR using the following primers. : 5'-TAAGAAGGAGATATACATATGTATAAGTTTCCTAAAGATTT-3 '(sense primer comprising underlined NdeI site; SEQ ID NO: 3); 5'-CTCGAGTGCGGCCGCAAGCTTCCTCCTCACGAGGAAGTCTA-3 '(an antisense primer comprising an underlined HindIII site; SEQ ID NO: 4). The PCR product was digested with NdelI / HindIII and ligated into the pET-24a (+) vector digested with NdeI / HindIII. Further, the conjugate product was transformed into E. coli DH5α cells. Positive transformants were screened using restriction enzyme digests, and copies were identified by DNA sequencing.

Enzyme analysis

The appropriate pH and temperature for Tpa-glu was determined as substrate using p-Nitrophenoyl-glucopyranoside (pNP-glu). The pH for the enzyme was measured at pH 4.0 to 8 using a buffer solution of 50 mM sodium citrate (pH 4.0-6.0), sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH 8.0-8.5) 80 ° C. The temperature range for the enzyme was measured at 60-100 ° C under standard conditions. All reaction mixtures (200 μl) containing 0.5 μg enzyme and 1 mM pNP-glu were incubated for 10 minutes. Absorbance of the liberated p-nitrophenol was measured at 405 nm using an Asys UVM 340 microplate reader (Biochrom, UK). All reactions were repeated three times. The thermal stability of Tpa-glu was incubated with pNP-glu at 75 ° C and 90 ° C for several periods, and the remaining activity was measured using pNP-glu as a substrate.

To determine the kinetic parameters of Tpa-glu against several pNP-glycopyranosides (竜 = 8.4 mM -1 cm cm-1 of pNP at 405 nm), the standard reaction mixture was added to a final volume of 1 ml with 50 mM sodium Phosphate (pH 7.0), 0.25-5 mM pNP-glycopyranoside and H2O. The reaction mixture was preheated at 75 ° C for 10 min before the addition of the enzyme (1-3 μg). The reaction was carried out at 405 nm for 2 minutes at 70 ° C using a UV-2600 spectrophotometer (Shimadzu, Kyoto). glucosidase unit (U) was defined as the amount required to promote the production of 1.0 μmol of pNP in 1 minute. Initial velocities were obtained directly from the initial slope of the time course plots. Km and Kcat values were calculated using the Michaelis-Menten equation. The standard reaction conditions included 50 mM sodium phosphate (pH 7.0), 0.25-7 mM substrate, and water in a final volume of 200 μl to determine the kinetic properties of Tpa-glu against cellobiose and laminaria bios. The reaction mixture was preheated at 75 ° C for 10 min before addition of the enzyme (2 μg), the reaction was performed at 75 ° C for 1-10 min and the reaction was initiated by adding 800 μl of 0.1% dinitrosalicylic acid (DNS ) Was added and stopped. The reaction mixture was incubated for 5 minutes at 100 ° C. Absorbance was measured at 540 nm using an UV-2600 spectrophotometer and converted to the amount of glucose liberated using a standard curve. One unit (U) of β-glucosidase activity was defined as the amount of enzyme required to catalyze the production of 1.0 μmol of glucose per minute.

TLC analysis

(2-6 units), laminarlyl oligosaccharides (2-6 monomers), and laminarin were used in order to confirm the hydrolytic activity against oligosaccharides and polysaccharides. The product was purified on silica gel 60 F254 (Merck, Darmstadt, Germany) and butanol: acetic acid: water (2: 1: 1) to give a final concentration of 50 mM sodium phosphate (pH 7.0), 1 mM substrate, The plates were dried in a hood and stained with 0.03% N- (1-naphthyl) -ethylenediamine and 5% H2SO4 in methanol. The plates were incubated in the hood for 10 min Lt; RTI ID = 0.0 > 110 C. < / RTI >

<Example 1> Gene structure and sequence analysis of Tpa-glu

Thermococcus pacificus (optimal temperature 80-88 ° C), an extremely thermophilic archaebacteria, can grow on a variety of substrates such as peptides and starches (Miroshnichenko et al . 1998). On the basis of the gene analysis of the strain, the inventors newly identified a gene encoding GH1 [beta] -glucosidase, which is located in a new family of glycoside hydrolases consisting of cellulose, laminarin and agarase enzymes (Fig. One). The gene revealed an ORF of 1,464 pairs encoding 487 amino acid residues.

The amino acid sequence of Tpa-glu deduced therefrom showed 77% identity with β-glucosidase from Pyrococcus furiosus (Bauer et al . 1996) and 54% identity with β-glycosidase from Sulfolobus solfataricus Cebellis et al ., 1990), 40% identity with β-glycosidase from T. kodakaraensis (Ezaki et al ., 1999) and 33% identity with β-glucosidase from P. horikoshii (matsui et al . 2000). We have analyzed the phylogenetic tree of Group 1 glycosyl hydrolase from super-thermophilic archaea. Degree. As can be seen in 2A, there are three branches. One includes P. horikoshii β-glucosidase, a membrane protein whose polypeptide length is known to be about 13% shorter than others. The second branch contains β-glycosidase of T. kodakaraensis and β-mannosidase of P. furiosus . Previous reported results indicate that β-glycosidase of T. kodakaraensis exhibits two functions, β-glycosidase and β-mannosidase, which are related to conserved regions. Tpa-glu is located in the third branch, and contains β-glucosidase of P. furiosus and β-glycosidase of Sulfolobus solfataricus .

We performed a pair of structure-based alignments between Tpa-glu and P. furiosus β-glucosidases. Tpa-glu showed high affinity at all sites except for one large difference from the β-glucosidase of P. furiosus . Tpa-glu has an insertion sequence of 12 amino residues (299 to 310) at the ring site between a-helical 12 of? -Glucosidase of? Fusiosus and? -Strand d9 (Fig. 2B). However, these extended rings are also observed in other hyperthermophilic archaeal β-glucosidases and show varying sizes and sequences (data not shown).

Example 2 Expression and Characterization of Tpa-glu

To produce Tpa-glu for activity analysis, constructs containing the coding type of Tpa-glu in the pET-24a (+) vector were expressed in E. coli . The recombinant protein was soluble and purified using metal affinity chromatography TALON (TM). The purified Tpa-glu revealed a massive protein with a molecular mass of 58 kDa (data not shown). The effect of pH on the hydrolytic activity of Tpa-glu was investigated in the range of pH 4.0 to 8.5. The assay with pNP-glu as substrate showed optimal activity at pH 7.5 (Fig. 3A). The effect of temperature on the hydrolytic activity was revealed in the range of 60-100 ° C, and the optimal polymerase activity of Tpa-glu was found to occur at 75 ° C (Fig. The heat resistance of Tpa-glu was tested by measuring the decrease in hydrolytic activity after pre-incubation at 75 ° C and 90 ° C. The half-life of the enzyme at 75 ° C and 90 ° C was 16 hours and 6 hours, respectively (Fig.

&Lt; Example 3 > Kinetic characteristics of Tpa-Glu

pNP-? -glucopyranoside (pNP-glu), pNP-? -galactopyranoside (pNP-gal), pNP-? -mannopyranoside (pNP- The hydrolytic activity of Tpa-glu against various pNP-glycopyranosides, such as pNP-xyl and β-dacarbacide, such as cellobiose and laminalia bios, was tested. Tpa-glu exhibited hydrolytic activity against various pNP-glycopyranosides, exhibiting a Km value of 0.58 mM in pNP-glu and the highest affinity. The highest catalytic efficiency (Kcat / Km value) of Tpa-glu was obtained when pNP-glu was used as a substrate, followed by pNP-gal, pNP-man and pNP-xyl (Table 1). Furthermore, the hydrolytic activity of Tpa-glu against? -Deracarbide such as Celobiose and laminalia bios was also tested. Tpa-glu showed a Km value of 2.61 mM in the lamina bios and showed the highest affinity, which is lower than the 22.48 mM value found in the case of cellobiose. The Kcat / Km value of Tpa-glu against laminaria bios was 3.97-fold higher than that of cellobiose and 7.02-fold lower than that of pNP-glu (Table 1). The properties of this broad substrate specificity of Tpa-glu were similar to those of the hyperthermophilic archaea [beta] -glucosidase and [beta] -glycosidase that have already been characterized and reported.

temperament K _m (mM) K _cat (s ^-1 ) K _cat / K _m (s ^-1 mM ^-1 ) pNP- [ beta] -glucopyranoside  0.58 64 110.34 pNP- ? -galactopyranoside 10.36 58   5.62 pNP- beta -mannopyranoside  5.06  6   1.18 pNP- ? - xylopyranoside  6.47    1.18   0.18 Cellobiose 22.48 89   3.96 Laminaribiose  2.61 41  15.71

Example 4 Substrate Specificity of Tpa-glu

The hydrolytic activity of Tpa-glu was tested for various polysaccharides such as avicel, cellulose, mannan, laminarin and xylan. Interestingly, Tpa-glu exhibited strong exo-hydrolytic activity on laminarin in polysaccharides and did not hydrolyze avicel, cellulose, mannan and xylan (data not shown). Β-glucosidase of P. furiosus showed very little activity in laminarin. As already reported, β-glucosidase (Sacellaris et al. 1997) of the mesophilic Cellovibrio mixtus hydrolyzes laminarin as a substrate. This substrate specificity of Tpa-glu was different from β-glucosidase and β-glycosidase in hyperthermophilic archaea.

To answer these questions, the inventors examined the hydrolytic activity of Tpa-glu on β-1,3, - and β-1,4-linked oligosaccharides and maninarin through TLC analysis Respectively. Tpa-glu exhibited hydrolytic activity against all oligosaccharides and monosaccharide as final product from oligosaccharide (Figs. 4A and B). This result indicates that Tpa-glu hydrolyzes the exo-type. Moreover, Tpa-glu exhibited substrate specificity higher than that of β-1, 4-linked oligosaccharides for β-1,3-linked oligosaccharides, and these results are consistent with kinetic data. Moreover, As shown in Fig. 5, Tpa-glu exhibited highly efficient hydrolytic activity against laminarin and produced the major end product monosaccharide according to the reaction time. The characteristic of Tpa-glu that hydrolyzes the polysaccharide, laminarin, is distinguished from the previously reported characteristics of? -Glucosidase and? -Glycosidase of hyperthermophilic archaea. It is not clear why Tpa-glu has a unique substrate specificity, although Tpa-glu is very similar to β-glucosidase in P. furiosus with respect to the primary structure. The ring site of Tpa-glu was assumed to be important for substrate specificity of the enzyme. As mentioned above, the constituent of the ring site is located in the AC 2 complex of the quadruplet constituent of β-glucosidase of P. furiosus . In addition, it has been reported that it is involved in ion-pair networks with linkage sequences from the second and fifth βα units in β-glycosidases of S. solfataricus , the network occurring at the quadruple interface between carboxy terminal ends, And involves the interaction of individual ion pairs. The inventors predicted that the substrate specificity of Tpa-glu would be related to the ionic interaction between the ring site and the fourth barrel helix at the quaternary interface. To understand the substrate specificity of Tpa-glu, additional investigations are needed, such as structural or mutational analysis of the protein.

In the above, replication and characteristics of genetic encoding of the thermostable β-glucosidase of T. pacificus have been described. The purified Tpa-glu showed hydrolytic activity against laminarin as well as various pNP-glycopyranosides and oligosaccharides. The exo-hydrolyzing activity of laminarin of Tpa-glu can be used as a cell wall component in combination with β-1,3-endoglucanase in the glycosidation of brown algae containing laminarin.

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<110> Korea Institute of Ocean Science & Technology <120> Novel thermostable beta-glucosidase and the methods of          preparation thereof <130> PN140029 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1464 <212> DNA <213> Thermococcus pacificus <400> 1 atgtataagt ttcctaaaga ttttctcttt ggatattcct ggtccgggtt ccagtttgag 60 atgggactcc ccgggagcga ggttccaaac agcgactggt gggtatgggt tcatgacatt 120 gaaaacatag caacgggtct cgttagtggg gacctgccgg agaacgggcc cgcttactgg 180 gatctctata agaaagacca cgatatagcg gaaagcctcg ggatggacgc gattagggga 240 ggaatagagt gggcgagaat cttccccaag ccaacgtttg acgtgaaagc ccgcgttgag 300 aaggatgaag agggcaacat agtctcggtg gaagtccctg aaagttcaat taaggaactc 360 gaaaaaatag ccgacatgaa ggccctcgag cactaccgtg agatctacgc ggactggaaa 420 gagagaggaa agacattcat actcaacctc taccactggc cattgcccct gtggctccat 480 gatcctctta aggtaagaaa actcggaccc gatagagctc cagcaggatg gcttgacgat 540 aagagtgttg ttgagttcgc caagtttgcg gcattcgttg cataccacct cgatgacctc 600 gttgacagct ggagcacgat gaacgagcca aacgtcgtgt atcagaacgg ctacacaagg 660 ccgaccagtg gttttccacc gggatatctg agctttgagg ccgaaagaaa ggcaaagatg 720 aacctcatcc aggcccatgc ccgggcctac gatgttatca aagagtactc tgacaaagat 780 gtggggataa tatacgcata cacatggccc gacccgctca gggaagaggt cgaagatgaa 840 gtgcgggcga taagggagag ggaactctac agcttcgttg atgccgtcca ctttgggaaa 900 gccgccgaca tcgaagaaag ggacgacctc cagggaaagg tggactggct cggcgtgaac 960 tactactcaa ggctggcctt tgacagggtg aacggtcacg tcctccctgt gtcgggttat 1020 ggattctccg gagagagggg tggctacgcc aggtccggaa gggcctgcag cgacttcggg 1080 tgggagattt accccgaagg ccttgaaaag ctccttaagg accttgcaaa aagatacggc 1140 ctccccatga tgataacaga gaatggcata gccgatgccg ctgacaggta ccgctcccac 1200 taccttgtaa gccacctcag ggccgtttac gaggccatga aagagggcgc cgatgtgagg 1260 ggctacctcc actggtcgct aaccgataac tatgaatggg cccagggctt caggatgagg 1320 tttgggctcg tatacgtgga tatggggacc aagaaacgct atttacgtcc aagtgctctt 1380 gttttcagag aaatagcgac cagaaaagag attccggaag aacttgagca cctttccagc 1440 ttagacttcc tcgtgaggag gtag 1464 <210> 2 <211> 487 <212> PRT <213> Thermococcus pacificus <400> 2 Met Tyr Lys Phe Pro Lys Asp Phe Leu Phe Gly Tyr Ser Trp Ser Gly   1 5 10 15 Phe Gln Phe Glu Met Gly Leu Pro Gly Ser Glu Val Pro Asn Ser Asp              20 25 30 Trp Trp Val Trp Val His Asp Ile Glu Asn Ile Ala Thr Gly Leu Val          35 40 45 Ser Gly Asp Leu Pro Glu Asn Gly Pro Ala Tyr Trp Asp Leu Tyr Lys      50 55 60 Lys Asp His Asp Ile Ala Glu Ser Leu Gly Met Asp Ala Ile Arg Gly  65 70 75 80 Gly Ile Glu Trp Ala Arg Ile Phe Pro Lys Pro Thr Phe Asp Val Lys                  85 90 95 Ala Arg Val Glu Lys Asp Glu Glu Gly Asn Ile Val Ser Val Glu Val             100 105 110 Pro Glu Ser Ser Ile Lys Glu Leu Glu Lys Ile Ala Asp Met Lys Ala         115 120 125 Leu Glu His Tyr Arg Glu Ile Tyr Ala Asp Trp Lys Glu Arg Gly Lys     130 135 140 Thr Phe Ile Leu Asn Leu Tyr His Trp Pro Leu Pro Leu Trp Leu His 145 150 155 160 Asp Pro Leu Lys Val Arg Lys Leu Gly Pro Asp Arg Ala Pro Ala Gly                 165 170 175 Trp Leu Asp Asp Lys Ser Val Val Glu Phe Ala Lys Phe Ala Ala Phe             180 185 190 Val Ala Tyr His Leu Asp Asp Leu Val Asp Ser Trp Ser Thr Met Asn         195 200 205 Glu Pro Asn Val Val Tyr Gln Asn Gly Tyr Thr Arg Pro Thr Ser Gly     210 215 220 Phe Pro Pro Gly Tyr Leu Ser Phe Glu Ala Glu Arg Lys Ala Lys Met 225 230 235 240 Asn Leu Ile Gln Ala His Ala Arg Ala Tyr Asp Val Ile Lys Glu Tyr                 245 250 255 Ser Asp Lys Asp Val Gly Ile Ile Tyr Ala Tyr Thr Trp Pro Asp Pro             260 265 270 Leu Arg Glu Glu Val Glu Asp Glu Val Arg Ala Ile Arg Glu Arg Glu         275 280 285 Leu Tyr Ser Phe Val Asp Ala Val His Phe Gly Lys Ala Ala Asp Ile     290 295 300 Glu Glu Arg Asp Asp Leu Gln Gly Lys Val Asp Trp Leu Gly Val Asn 305 310 315 320 Tyr Tyr Ser Arg Leu Ala Phe Asp Arg Val Asn Gly His Val Leu Pro                 325 330 335 Val Ser Gly Tyr Gly Phe Ser Gly Glu Arg Gly Gly Tyr Ala Arg Ser             340 345 350 Gly Arg Ala Cys Ser Asp Phe Gly Trp Glu Ile Tyr Pro Glu Gly Leu         355 360 365 Glu Lys Leu Leu Lys Asp Leu Ala Lys Arg Tyr Gly Leu Pro Met Met     370 375 380 Ile Thr Glu Asn Gly Ile Ala Asp Ala Ala Asp Arg Tyr Arg Ser His 385 390 395 400 Tyr Leu Val Ser His Leu Arg Ala Val Tyr Glu Ala Met Lys Glu Gly                 405 410 415 Ala Asp Val Arg Gly Tyr Leu His Trp Ser Leu Thr Asp Asn Tyr Glu             420 425 430 Trp Ala Gln Gly Phe Arg Met Arg Phe Gly Leu Val Tyr Val Asp Met         435 440 445 Gly Thr Lys Lys Arg Tyr Leu Arg Pro Ser Ala Leu Val Phe Arg Glu     450 455 460 Ile Ala Thr Arg Lys Glu Ile Pro Glu Glu Leu Glu His Leu Ser Ser 465 470 475 480 Leu Asp Phe Leu Val Arg Arg                 485 <210> 3 <211> 41 <212> DNA <213> Tpa-Glu forward primer <400> 3 taagaaggag atatacatat gtataagttt cctaaagatt t 41 <210> 4 <211> 41 <212> DNA <213> Tpa-Glu reverse primer <400> 4 ctcgagtgcg gccgcaagct tcctcctcac gaggaagtct a 41

Claims (9)

The isolated protein of SEQ ID NO: 2. The isolated glucosidase of SEQ ID NO: 2 and functional equivalents thereof. A separate nucleic acid sequence encoding the protein of SEQ ID NO: 2. 4. The nucleic acid sequence according to claim 3, wherein the nucleic acid sequence is SEQ ID NO: A recombinant vector comprising the nucleic acid sequence according to claim 3. A cell transformed with the recombinant vector according to claim 5. A method for producing glucosidase enzyme, which comprises culturing the transformed cells according to claim 6, and isolating the glucosidase enzyme from the cultured cells. A method for hydrolyzing a? -1,3-linked polysaccharide using the β-glucosidase of SEQ ID NO: 2 or a functional equivalent thereof 9. The hydrolysis method according to claim 8, wherein the polysaccharide is laminarin.

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