JP2702677B2 - Thermostable β-glucosidase, β-glucosidase gene, vector containing the gene, and transformant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thermostable β-glucosidase, a β-glucosidase gene, a vector containing the gene, and a transformant. This β-glucosidase is an enzyme derived from a microorganism of the genus Cellvibrio and having improved heat resistance by recombining a part of its structural gene, and is capable of producing cellobiose efficiently by acting on cellooligosaccharides. it can.

[0002]

2. Description of the Related Art Generally, β-glucosidase is a kind of cellulase that degrades cellulose, and is known to be derived from bacteria, yeast, filamentous fungi and the like. The present enzyme is an enzyme that acts on cellooligosaccharides produced by decomposing cellulose to release glucose, and is an essential enzyme for completely decomposing cellulose to glucose.

[0003] Since different microorganisms producing the enzyme have different physicochemical properties of the obtained enzyme, it has heretofore been necessary to obtain β-glucosidase having desired properties.
We had to search for new producers. Further, a method for improving the heat resistance of the present enzyme has not been known yet.

[0004]

The β-glucosidase from Cellvibrio gilvus is:
It hardly acts on cellobiose, but acts on cellotriose, cellotetraose, cellopentaose and the like. That is, cellopentaose is decomposed into cellotetraose and glucose, cellotetraose is decomposed into cellotriose and glucose, and cellotriose is decomposed into cellobiose and glucose by the present enzyme, but cellobiose is hardly decomposed. Due to such characteristics of the present enzyme, cellobiose accumulates when the present enzyme is allowed to act on cellooligosaccharides.
However, since the heat resistance of the present enzyme is not so high as 30 ° C., it is required to improve the heat resistance to enhance the utility of the present enzyme.

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
As means for improving the heat resistance of β-glucosidase,
A part of a plurality of β-glucosidase genes having different origins was recombined (shuffled) and expressed to obtain β-glucosidase with improved heat resistance, thereby completing the present invention.

That is, the present invention firstly relates to a thermostable β-glucosidase having the following properties. (1) Action: acts on cellulose degradation products to produce glucose and cellobiose (2) Substrate specificity: degradability to cellobiose is lower than that to cellotriose, cellotetraose, and cellopentaose (3) Optimum pH : PH 6.0 (4) pH stability: stable at pH 4 to 9 (5) Optimal temperature: 40 to 45 ° C (6) Temperature stability: stable up to 35 ° C, 60% at 45 ° C
( 7) The enzyme gene has the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.
Having

Further, the present invention provides a chimeric β-glucosidase gene having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing,
The present invention relates to a plasmid containing the gene and Escherichia coli transformed with the plasmid.

Hereinafter, the present invention will be described in detail. The heat-resistant β-glucosidase of the present invention is a plant (almond) -derived β-glucosidase (trade name: β-glucosidase,
Cellotriose, compared to Wako Pure Chemical Industries, Ltd.)
The properties are different in that the degradability to cellobiose is lower than that to cellotetraose and cellopentaose.

As a means for improving the heat resistance of β-glucosidase, the present invention provides a method of creating a chimeric enzyme gene by recombining (shuffling) a part of a plurality of β-glucosidase genes having different origins and expressing the gene. It was adopted. The origin of the β-glucosidase gene used in the present invention may be any organism having an enzymatic activity of producing glucose and cellobiose by acting on a cellulose degradation product, and is preferably a microorganism, particularly a cell vibrio such as cell vibrio gilvas. Genus, those derived from the genus Agrobacterium such as Agrobacterium tumefacience, those derived from the genus Clostridium such as Clostridium thermocellum, Kluyveromyces fragilis, etc. Of the genus Culveromyces, those derived from the genus Saccharomycopsis such as Saccharomycopsis fibuligera (Saccharomycopsis fibuligera), also derived from the genus Butyrivibrio such as Butyrivibrio fibrisovens (Butyrivibrio fibrisovens) The Hansenula Anomala
nula anomala), those derived from the genus Luminococcus such as Ruminococcus albus, and those derived from the genus Schizophyllum commune (S
chizophyllum commune) and other species derived from the genus Schizophyllum, Aspergillus wenti
and those derived from Aspergillus genus such as i). Among them, those derived from the genus Servibrio are preferable because of the low degradability of cellobiose.

The present inventor has selected a β-glucosidase gene derived from Agrobacterium tumefaciens as a β-glucosidase gene derived from Cervibrio gilvas and a gene for recombining a part of the gene. Comparison of the amino acid sequences of the enzymes translated from these genes revealed that the amino acid homology was high (20% or more). Further, β-glucosidase derived from Agrobacterium tumefaciens is suitable for the purpose of the present invention because it is heat-resistant.

Hereinafter, β derived from Servibrio gilbas
The present invention will be described by way of a specific example in which gene recombination is performed using a glucosidase gene and a β-glucosidase gene derived from Agrobacterium tumefaciens, and a chimeric enzyme is created therefrom.

(1) Preparation method of plasmid [0012] The method of Kashiwagi et al. [Journal of Fermentation and Bio
Engineering, vol. 75, pp. 159-165 (1993)] to prepare a plasmid pCG5 containing a β-glucosidase gene derived from Cervibrio gilvas. Also, Castle L.
A. et al.'S method [Journal of Bacteriology, vol. 174, 1478-
1486 (1992)], a plasmid pcbg1 containing a β-glucosidase gene derived from Agrobacterium tumefaciens was prepared.

(2) Assay of Recombinant Region of Gene In the present invention, the assay of the region that recombines the β-glucosidase gene can be performed by the following method.
The homology of the amino acid sequence of β-glucosidase is compared, and the homology is usually 15 to 100%, preferably 30%.
An arbitrary region of １００100% is selected. This region exists at a plurality of locations. The length of the selected region is 10-5 amino acids
The number of DNA bases in the recombination region may be 30 to 1500 bases. This region is preferably identical to both pCG5 as a DNA fragment acceptor and pcbg1 as a DNA fragment donor.
It may be shifted by 1 to 30 residues in the NA base.

(3) Restriction enzyme treatment Next, a restriction enzyme that cuts the DNA encoding the selected amino acid region is selected, and the recombinant region of the plasmid containing the β-glucosidase gene is cut. When selecting a restriction enzyme, care must be taken so that the translation frame from DNA to amino acid does not shift at the junction of the completed chimeric gene. PCG is the receptor
It is necessary to select one or two types of restriction enzyme that does not cut the site other than the selected region as the restriction enzyme that cuts No. 5. However, it is also possible to cut only the selected region by partially decomposing the receptor plasmid, even if an enzyme that cuts about 1 to 3 sites other than the selected region is used. The end of the recombination region of the receptor plasmid cut in this manner is blunted by a conventional method and then dephosphorylated. On the other hand, the restriction enzyme that cleaves the donor plasmid only needs to be able to cleave the selected region, and may be able to cleave portions other than the selected region.

(4) Agarose gel electrophoresis The DNA fragment of the donor plasmid after digestion with restriction enzymes was obtained from Sambrook, J., Fritsch, EF and Maniatis,
T. "Molecular Cloning, A Laboratory Manual 2
Edition ", Chapter 6.6, Vol. 1 (1989), separated by agarose gel electrophoresis, and
Separate the NA fragment. Then, the obtained DNA
The ends of the fragment are blunted by a conventional method.

(5) Method for Preparing Chimeric Enzyme Gene The DNA fragments prepared in the above (3) and (4) are mixed, and a ligation reaction is performed to prepare a plasmid containing the chimeric enzyme gene. The structure of the thus prepared plasmid containing the chimeric enzyme gene CH-15 is shown in FIG. 1 together with the restriction sites of the plasmid pCG5 and the plasmid pcbg1.

(6) Transformation of Escherichia coli Using a plasmid containing a chimeric enzyme gene,
k, J., Fritsch, EFand Maniatis, T. "Molecular Cl
oning, A Laboratory Manual 2nd edition ", Chapter 1.74, Vol.1
(1989) according to the method described in (1989). The transformed Escherichia coli has been deposited with the National Institute of Advanced Industrial Science and Technology, and the accession number is FERM.
P-14614. Then, 1 mM fluorescent substrate (4
-Methylumbelliferyl-β-glucoside)
The transformed E. coli was spread on a B agar medium, and 20 to 3
After culturing at 7 ° C for 1 to 3 days, usually at 30 ° C for 1 day,
The presence or absence of the expression of the chimeric enzyme gene is confirmed using the enzyme activity as an index. The nucleotide sequence of the obtained chimeric enzyme gene is confirmed by decoding using Tack Dye Primer Cycle Sequencing Kit (manufactured by PerkinElmer).
Also, the amino acid sequence is determined from this base sequence.

(7) Purification of Chimeric Enzyme Escherichia coli (FERM P-14614) transformed with a plasmid containing the chimeric enzyme gene was isolated from ampicillin (5.
0 μg / ml) in an LB medium containing 20 to 37 ° C.
The cells are cultured at 30 ° C. for 24 hours for 24 days, and the obtained cells are sonicated. The crushed liquid is centrifuged, and the obtained supernatant is subjected to ion exchange column chromatography (SP Sepharose column, manufactured by Pharmacia) to purify the chimeric enzyme (thermostable β-glucosidase). Through the above process, the thermostable β-glucosidase of the present invention can be obtained.

[0019]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited by these examples. Example 1 (1) Assay of Recombination Site Amino acid sequence of enzyme translated from β-glucosidase gene derived from Cervibrio gilvas and amino acid sequence of enzyme translated from β-glucosidase gene derived from Agrobacterium tumefaciens Were compared, and a region having a high amino acid homology was examined. The result is shown in FIG. The region where the amino acid homology is 35%, that is, the amino acid sequence derived from Servibrio gilbas,
The 52nd region and the 759th to 818th region of the amino acid sequence derived from Agrobacterium tumefaciens were selected, and restriction enzymes that cut that region were selected.
Specifically, StyI as a restriction enzyme that cleaves the β-glucosidase gene derived from Cervibrio gilvas,
AvaI was selected as a restriction enzyme that cleaves the β-glucosidase gene derived from Agrobacterium tumefaciens. At this time, care must be taken not to shift the translation frame from DNA to amino acids.

(2) Degradation by Restriction Enzyme After cutting the plasmid pCG5 containing the β-glucosidase gene derived from Cervibrio gilvas with the restriction enzyme StyI, the ends were blunted using a DNA blunting kit (Takara Shuzo). And dephosphorylated using alkaline phosphatase (A). In addition, the plasmid pcbg1 containing the β-glucosidase gene derived from Agrobacterium tumefaciens was digested with the restriction enzyme AvaI, and then digested with Sambrook, J., Fritsch, EF and Maniat.
is, T. "Molecular Cloning, A Laboratory Manual 2
Edition ", Chapter 6.6, Vol. 1 (1989), followed by agarose gel electrophoresis followed by 647 bp DNA
Fragments were obtained, and the ends were blunted using a DNA branding kit (Takara Shuzo Co., Ltd.) (B).

(3) Preparation of chimeric enzyme gene, its expression, and confirmation of chimeric enzyme gene The previously prepared (A) and (B) were mixed, and the ligation reaction was performed using a DNA ligation kit (Takara Shuzo Co., Ltd.). Then, a plasmid containing the chimeric enzyme gene was prepared (FIG. 1). Using the plasmid thus obtained, Sa
mbrook, J., Fritsch, EF and Maniatis, T. "Molecu
lar Cloning, A Laboratory Manual 2nd edition ", 1.74
E. coli was transformed according to the method described in Chapter, Vol. 1 (1989). Next, the transformed Escherichia coli was spread on an LB agar medium containing 1 mM of a fluorescent substrate (4-methylumbelliferyl-β-glucoside), and cultured at 30 ° C. for 1 day.
The presence or absence of the expression of the chimeric enzyme gene was confirmed using the enzyme activity as an index. The nucleotide sequence of the obtained chimeric enzyme gene was read using Tack Dye Primer Cycle Sequencing Kit (manufactured by PerkinElmer), confirmed, and shown in SEQ ID NO: 1 in the sequence listing. In addition, the amino acid sequence was also determined from this nucleotide sequence, and is shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence homology between β-glucosidase from Cervibrio gilvas and the resulting chimeric enzyme is 9
The homology between Agrobacterium tumefaciens-derived β-glucosidase and the amino acid sequence of the obtained chimeric enzyme was 34%.

(4) Purification of Chimeric Enzyme Escherichia coli containing a plasmid containing the chimeric enzyme gene was cultured in an LB medium containing ampicillin (50 μg / ml) at 30 ° C. for 24 hours, and the obtained cells were disrupted by ultrasonication. did. The crushed liquid was centrifuged, and the obtained supernatant was subjected to ion exchange column chromatography (SP Sepharose column, manufactured by Pharmacia) to purify the chimeric enzyme.

(5) Characteristics of Chimeric Enzyme β-Glucosidase (C) derived from Servibrio gilbas
G) β from Agrobacterium tumefaciens
-The characteristics of the glucosidase (AT) and the chimeric enzyme (CH) are shown in Figs. In the figure, ■ is CH
, ● indicates CG, and ▲ indicates AT. As is evident from FIG. 3, the optimum pH is CG at pH 6.2 to pH 6.0.
4. AT showed pH 7.2-7.4 and CH showed pH 6.0. Further, as shown in FIG.
Are pH 4-9, AT is pH 5-10, CH is pH 4-9
It is. The optimum temperature is shown in FIG. As is clear from this figure, CG is 35 ° C, AT is 60 ° C, and CH is 40 to 4 ° C.
5 ° C., and CH was 5-10 ° C. better than CG. As for the temperature stability, as shown in FIG. 6, CG was stable up to 30 ° C., AT was 55 ° C., CH was up to 35 ° C., and CH maintained 60% activity even at 45 ° C. .

(6) Utilization of Chimeric Enzyme When the chimeric enzyme was allowed to act on cellobiose, cellotriose, cellotetraose, and cellopentaose, it did not act on cellobiose as effectively as β-glucosidase derived from Cellvibrio gilbas. , And the heat resistance increased by 5 to 10 ° C. As a model of a cellulose degradation product, a 20 mM phosphate buffer (pH: 1%) containing each component of cellobiose, cellotriose, cellotetraose, and cellopentaose at a concentration of 1% (W / V).
6.2) When the enzyme was allowed to act at 40 ° C. for 1 day,
2% (W / V) cellobiose and 1% as reaction products
(W / V) glucose and 1% (W / V) of other oligosaccharides were obtained. When β-glucosidase derived from Agrobacterium tumefaciens was allowed to act under the same conditions, about 0.5% (W / V) cellobiose was present in the reaction product. From the above, it was found that the present enzyme has improved heat resistance and is suitable as an enzyme for producing cellobiose by acting on cellooligosaccharides.

[0025]

According to the present invention, a plurality of β of different origins
-A part of the glucosidase gene is recombined to obtain a chimeric enzyme gene, which can be expressed to obtain a thermostable β-glucosidase.

[0026]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2256 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Cellvibrio gilvus and Agrobacterium tumefa
ciens Direct origin Plasmid name: CH-15 Sequence characteristics Characteristic symbol: mat peptide Location: 1..2256 Characteristic determination method: P sequence ATG CAT CGC AAG AAC CAC GCC GCC GCC GCG GCG CGC ACC GCC CTG GTC 48 Met His Arg Lys Asn His Ala Ala Ala Ala Ala Arg Thr Ala Leu Val 1 5 10 15 GCG GCC ATT GCC GCA GCC TTC CCG CTG TTC ACC CCC GGC GCC TTC GCC 96 Ala Ala Ile Ala Ala Ala Phe Pro Leu Phe Thr Pro Gly Ala Phe Ala 20 25 30 GCG CCC GAG CCG GCC GAG CCG GCC CAG AAG CCC TGG CTG GAT GCT TCC 144 Ala Pro Glu Pro Ala Glu Pro Ala Gln Lys Pro Trp Leu Asp Ala Ser 35 40 45 CTG GAC GCC GAC CAG CGT GCC CGC CTG GCC GTG CAG GCC ATG ACC CAG 192 Leu Asp Ala Asp Gln Arg Ala Arg Leu Ala Val Gln Ala Met Thr Gln 50 55 60 CAG GAG AAG CTG CGC TGG GTG TTC GGC TAT TTC GGT CAC GAT TTC GGC 240 Gln Glu Lys Leu Arg Trp Val Phe Gly Tyr Phe Gly His Asp Phe Gly 65 70 75 80 AAG AGC AAG AAG CAT CCG GAT GCA CTG CCT CAG TCG GCC GGC TAC ATC 288 Lys Ser Lys Lys His Pro Asp Ala Leu Pro Gln Ser Ala Gly Tyr Ile 85 90 95 CCG GGC ACG CCG CGC CTG GGT CTG CCG GCA TTG TTC GAG ACC GAT GCG 336 Pro Gly Thr Pro Arg Leu Gly Leu Pro Ala Leu Phe Glu Thr Asp Ala 100 105 110 GGC CAG GGC GTG GCC TCG CAG TCC GGC GCC AAC GTG CGC GAG CGC ACC 384 Gly Gln Gly Val Ala Ser Gln Ser Gly Ala Asn Val Arg Glu Arg Thr 115 120 125 GCC TTG CCC TCG GGC CTG AGC ACC GCC TCG ACC TGG GAC CCG AAG GTG 432 Ala Leu Pro Ser Gly Leu Ser Thr Ala Ser Thr Trp Asp Pro Lys Val 130 135 140 GCC TAC GCC GGC GGC GCC ATG ATC GGC TCG GAG GCG CGC GCT TCC GGC 480 Ala Tyr Ala Gly Gly Ala Met Ile Gly Ser Glu Ala Arg Ala Ser Gly 145 150 155 160 TTC AAC GTC ATG CTG GCA GGT GGC GTG AAC CTG CAG CGC GAG CCG CGC 528 Phe Asn Val Met Leu Ala Gly Gly Val Asn Leu Gln Arg Glu Pro Arg 165 170 175 AAC GGC CGC AAC TTC GAA TAC GCG GGC GAG GAT CCG CTG CTG GCC GGC 576 Asn Gly Arg Asn Phe Glu Tyr Ala Gly Glu Asp Pro Leu Leu Ala Gly 180 185 190 ACC ATG ATC GGC CAG GCA ATC AAG GGC GTG GAA TCG AAC AGG ATC ATC 624 Thr Met Ile Gly Gln Ala Ile Lys Gly Val Glu Ser Asn Ar g Ile Ile 195 200 205 TCG ACG CTC AAG CAC TTC GTG CTG AAC GAC CAG GAA ACC GGC CGC AAC 672 Ser Thr Leu Lys His Phe Val Leu Asn Asp Gln Glu Thr Gly Arg Asn 210 215 220 GAG CTC GAT GCG CGC ATC GAC AAG GCG GCG CTG CGC ATG TCC GAC CTG 720 Glu Leu Asp Ala Arg Ile Asp Lys Ala Ala Leu Arg Met Ser Asp Leu 225 230 235 240 CTG GCG ATG GAG CTG GCG CTG GAG CAG TCC GAC GCC GGC TCG GTG ATG 768 Leu Ala Get Glu Leu Ala Leu Glu Gln Ser Asp Ala Gly Ser Val Met 245 250 255 TGC GCC TAC AAC CGC CTC AAC GGA CCG TAT ACC TGC GAA CAT CCG TGG 816 Cys Ala Tyr Asn Arg Leu Asn Gly Pro Tyr Thr Cys Glu His Pro Trp 260 265 270 CTG CTG AGC GAG GTG CTC AAG CGC GAC TGG GGT TTC CGC GGT TAT GTC 864 Leu Leu Ser Glu Val Leu Lys Arg Asp Trp Gly Phe Arg Gly Tyr Val 275 280 285 ATG TCC GAC TGG GGC GCC ACC CAC AGC ACC GTG GCC GCC GCC AAC AGC 912 Met Ser Asp Trp Gly Ala Thr His Ser Thr Val Ala Ala Ala Asn Ser 290 295 300 GGC CTG GAC CAG CAG TCC GGC CAG GAA TTC GAC AAG TCC CCG TAC TTC 960 Gly Leu Asp Gln Gln Ser Gly Gln Glu Phe As p Lys Ser Pro Tyr Phe 305 310 315 320 GGC GGC GCG CTG GAA GAA GCC GTC AAG ACC GGG GCC GTG CCG CAG AAG 1008 Gly Gly Ala Leu Glu Glu Ala Val Lys Thr Gly Ala Val Pro Gln Lys 325 330 335 CGC CTG GAC GAC ATG GTC ACC CGC ATC GTG CGC ACC ATG TTC GGC AAG 1056 Arg Leu Asp Asp Met Val Thr Arg Ile Val Arg Thr Met Phe Gly Lys 340 345 350 GGC GTG GTC GAC AAC CCG CTC AAG CCG GGC GTC GCG ATC GAC TTC GCC 1104 Gly Val Val Asp Asn Pro Leu Lys Pro Gly Val Ala Ile Asp Phe Ala 355 360 365 GCC AAC GGC GGT CAG CCG CCA GAC GGC GAA GAG GGC ATG GTC CTG CTC 1152 Ala Asn Gly Gly Gln Pro Pro Asp Gly Glu Glu Gly Met Val Leu Leu 370 375 380 AAG AAC GAA GGC CGC CTC CTG CCG CTC GCG AAA ACC GTG CGC ACG ATC 1200 Lys Asn Glu Gly Arg Leu Leu Pro Leu Ala Lys Thr Val Arg Thr Ile 385 390 395 400 400 GCC GTC ATT GGC GGA CAT GCG GAT GCC GGC GTG CTC TCG GGC GGC GGC 1248 Ala Val Ile Gly Gly His Ala Asp Ala Gly Val Leu Ser Gly Gly Gly 405 410 415 TCG TCC CAG GTG TAC CCA GTG GGC GGC ATC GCG GTC AAG GGC TTG TTG 1296 Ser Ser Gln Val Tyr Pro Val Gly Gly Ile Ala Val Lys Gly Leu Leu 420 425 430 CCT GCC ACC TGG CCG GGT CCG GTG GTG TAC TAC CCG TCC TCG CCG CTG 1344 Pro Ala Thr Trp Pro Gly Pro Val Val Tyr Tyr Pro Ser Ser Pro Leu 435 440 445 CGC GCG ATC CAG GCC CAG GCC CCG AAT GCG AAA GTC GTG TTC GAC GAC 1392 Arg Ala Ile Gln Ala Gln Ala Pro Asn Ala Lys Val Val Phe Asp Asp 450 455 460 GGC CGC GAC CCA GCC CGC GCC GCG CGC GTG GCC GCT GGG GCC GAC GTC 1440 Gly Arg Asp Pro Ala Arg Ala Ala Arg Val Ala Ala Gly Ala Asp Val 465 470 475 480 GCC CTG GTC TTC GCC AAC CAG TGG ATC GGC GAG GCC AAC GAC GCC CAG 1488 Ala Leu Val Phe Ala Asn Gln Trp Ile Gly Glu Ala Asn Asp Ala Gln 485 490 495 ACG CTC GCG CTG CCG GAC GGC CAG GAA GAG CTG ATC ACG TCG GTG GCG 1536 Thr Leu Ala Leu Pro Asp Gly Gln Glu Glu Leu Ile Thr Ser Val Ala 500 505 510 510 GGC GCC AAC GGG CGC ACC GTG GTC GTG CTG CAG ACC GGC GGA CCG GTC 1584 Gly Ala Asn Gly Arg Thr Val Val Val Leu Gln Thr Gly Gly Pro Val 515 520 525 ACC ATG CCC TGG CTG GCG CGC GTT CCG GCC GTG CTG GAA GCC TGG TAT 1632 Thr Met Pro Trp Leu Ala Arg Val Pro Ala Val Leu Glu Ala Trp Tyr 530 535 540 CCG GGC ACC AGC GGC GGC GAG GCG ATC GCG AAT GTG CTG TTT GGC GCG 1680 Pro Gly Thr Ser Gly Gly Glu Ala Ile Ala Asn Val Leu Phe Gly Ala 545 550 555 560 GTC AAT CCG TCC GGC CAC CTG CCG GCC ACC TTC CCG CAA TCC GAG CAG 1728 Val Asn Pro Ser Gly His Leu Pro Ala Thr Phe Pro Gln Ser Glu Gln 565 570 575 CAA CTG CCG CGC CCG AAA CTC GAT GGC GAT CCG AAG AAC CCC GAG CTG 1776 Gln Leu Pro Arg Pro Lys Leu Asp Gly Asp Pro Lys Asn Pro Glu Leu 580 585 590 CAG TTC GCC GTC GAC TAC CAC GAA GGT GCG GCG GTC GGC TAC AAG TGG 1824 Gln Phe Ala Val Asp Tyr His Glu Gly Ala Ala Val Gly Tyr Lys Trp 595 600 605 TTC GAC CTG AAG GGC CAC AAG CCG CTG TTC CCG TTC GGG CAC GGC CTG 1872 Phe Asp Leu Lys Gly His Lys Pro Leu Phe Pro Phe Gly His Gly Leu 610 615 620 TCC TAC ACA ACC TTC GCG TAC TCC GGT CTG TCC GGC CAG CTC AAG GAT 1920 Ser Tyr Thr Thr Phe Ala Tyr Ser Gly Leu Ser Gly Gln Leu Lys Asp 625 630 635 640 GGC CGC CTG CAC GTG CGC TTC AAG GTG ACC AAC AC C GGC AAC GTG GCC 1968 Gly Arg Leu His Val Arg Phe Lys Val Thr Asn Thr Gly Asn Val Ala 645 650 655 GGC AAG GAC GTG CCG CAG GTG TAC GCC GCG CCG ATG TCC ACC AAA TGG 2016 Gly Lys Asp Val Pro Gln Val Tyr Ala Ala Pro Met Ser Thr Lys Trp 660 665 670 GAG GCG CCG AAG CGC CTG GCC GCC TGG AGC AAG GTC GCC CTG CTG CCG 2064 Glu Ala Pro Lys Arg Leu Ala Ala Trp Ser Lys Val Ala Leu Leu Pro 675 680 685 GGC GAG ACC AAG CCG GGC GCG ACC GGT ACG GCG GTG CTG AAG ATC GCT 2112 Gly Glu Thr Lys Pro Gly Ala Thr Gly Thr Ala Val Leu Lys Ile Ala 690 695 700 CCT CGC GAC TTG GCT TAC TTC GAT GTC GAG GCC GGT CGT TTC CGG GCT 2160 Pro Arg Asp Leu Ala Tyr Phe Asp Val Glu Ala Gly Arg Phe Arg Ala 705 710 710 720 GAT GCG GGC AAG TAC GAG CTG ATC GTG GCG GCC AGC GCC ATC GAT ATC 2208 Asp Ala Gly Lys Tyr Glu Leu Ile Val Ala Ala Ser Ala Ile Asp Ile 725 730 735 CGG GCA AGC GTA AGT ATT CAC TTG CCG GTC GAT CAT GTG ATG GAG CCG 2256 Arg Ala Ser Val Ser Ile His Leu Pro Val Asp His Val Met Glu Pro 740 745 750

[Brief description of the drawings]

FIG. 1 shows a β-glucosidase gene on a plasmid and restriction enzyme cleavage sites.

FIG. 2 shows homology and recombination region of β-glucosidase gene on a plasmid.

FIG. 3 is a graph showing the optimum pH of various enzymes.

FIG. 4 is a graph showing the pH stability of various enzymes.

FIG. 5 is a graph showing optimum temperatures of various enzymes.

FIG. 6 is a graph showing the temperature stability of various enzymes.

[Explanation of symbols]

In FIG. 2, white portions indicate regions where homology is not recognized, and black portions and hatched portions indicate regions with high homology.

 ──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Takeshi Tokuyasu 1-1-603-303, Azuma, Tsukuba-shi, Ibaraki (56) Reference APPL. MICROBIOL. BI OTECHNOL. (1988), 28: 380-386 APPL. ENVIRON. MICR OBIOL. , VOL. 54, NO. 12 (1988), p. 3147-3155

Claims (4)

(57) [Claims] 1. A thermostable β-glucosidase having the following properties. (1) Action: acts on cellulose degradation products to produce glucose and cellobiose (2) Substrate specificity: degradability to cellobiose is lower than that to cellotriose, cellotetraose, and cellopentaose (3) Optimum pH : PH 6.0 (4) pH stability: stable at pH 4 to 9 (5) Optimal temperature: 40 to 45 ° C (6) Temperature stability: stable up to 35 ° C, 60% at 45 ° C
( 7) The enzyme gene has the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.
Having 2. A chimeric β-glucosidase gene having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. 3. A plasmid containing the chimeric β-glucosidase gene according to claim 2. 4. An Escherichia coli transformed with the plasmid according to claim 3.