Beta-glucosidase as well as coding gene and application thereof

Abstract

The invention provides beta-glucosidase as well as a coding gene and application thereof. The beta-glucosidase has cutting activity on lignocellulose, has remarkable advantages in degradation of cellulose and hemicellulose, has excellent enzyme activity, thermal stability, pH tolerance and multiple enzyme activities, and has potential application value in multiple fields of food, feed, textile, medical treatment, biological energy and the like.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering. More specifically, the present invention relates to β-glucosidase, its encoding gene, and applications. Background Art

[0002] In the biological world and industrial production, β-glucosidase, endoglucanase, filter paper enzyme, and hemicellulase play extremely crucial roles.

[0003] β-glucosidase belongs to the cellulase family and has other names including gentiobiase, cellobiase, and amygdalase. It can precisely act on β-D-glycosidic bonds, break them down into smaller molecules, and participate in various physiological functions of cells. It is widely distributed in the biological world and exists in various organisms such as archaea, bacteria, and eukaryotes, playing an important role in the basic metabolism and complex physiological processes of organisms. This enzyme family has a high degree of diversity and adaptability and can hydrolyze a variety of substrates, including alkyl, amino, or aryl β-D-glycosides, cyanogenic glycosides, disaccharides, and short oligosaccharides.

[0004] Endoglucanase acts on the β-1,4-glycosidic bonds inside cellulose, cutting long-chain cellulose molecules into shorter oligosaccharide fragments, which is an important initial step in the cellulose degradation process.

[0005] Filter paper enzyme can degrade the cellulose in filter paper, and its activity reflects the ability of the enzyme to act on natural cellulose substrates.

[0006] Hemicellulase can decompose hemicellulose into monosaccharides or oligosaccharides. Hemicellulose is an important component of plant cell walls, and its effective degradation is crucial for the comprehensive utilization of plant biomass.

[0007] It is particularly worth noting that β-glucosidase plays a key role as a rate-limiting enzyme in the cellulose degradation process. As the main component of plant cell walls, the degradation of cellulose is a key process for organisms to obtain energy and build their own structures. β-glucosidase converts cellulose into utilizable glucose by hydrolyzing cellobiose and soluble cellooligosaccharides, thus providing a stable energy source for the metabolism of organisms. In addition, this enzyme also participates in the hydrolysis processes of cellulose and hemicellulose, providing a potential raw material source for the production of bioethanol, which is of great significance for promoting the development of renewable energy.

[0008] However, the β-glucosidases currently used in industry mainly come from fungi such as Trichoderma and Penicillium. The enzymes from these sources have certain limitations in terms of enzyme activity, temperature sensitivity, and pH stability. Especially under high-temperature conditions, the activity of some enzymes will be affected, thus limiting the efficiency of cellulose degradation. In addition, some enzymes may also be inhibited by the end product glucose, further affecting their performance in practical applications.

[0009] To overcome these challenges, researchers are actively seeking innovative solutions. They use advanced technical means such as metagenomics to deeply explore the new β-glucosidase resources in uncultured microorganisms in the environment. These uncultured microorganisms may contain rich enzyme resources and have higher enzyme activity and stability. By combining modern biotechnology such as genetic engineering, researchers can optimize the structure and function of the enzyme, improve its enzyme activity and stability, and meet the requirements of industrial applications. This will help promote the application and development of β-glucosidase in the fields of biotechnology and industry and make important contributions to the sustainable development of our country. Summary of the Invention

[0010] To solve the above problems, the inventors screened 8 β-glucosidase activity genes from the rumen microorganisms of Tarim red deer, goitered gazelle, argali, sheep, cattle, and camels, as well as the monogastric microorganisms of Equus hemionus mongolicus through metagenomics technology.

[0011] Therefore, on the one hand, the present invention provides a β-glucosidase, which is selected from:

[0012] (1) a protein with an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8;

[0013] (2) a protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acids to the amino acid sequence of the protein in (1); and

[0014] (3) a protein with an amino acid sequence having at least 80% homology with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 and having the same function.

[0015] In a specific embodiment, the β-glucosidase is selected from proteins having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, preferably the protein having an amino acid sequence as shown in SEQ ID NO:1.

[0016] In a specific embodiment, the β-glucosidase further has one or more of the following activities: endoglucanase activity, filter paper enzyme activity and hemicellulase activity, wherein the protein encoded by SEQ ID NO:1 has β-glucosidase activity: 27.089 U / mg - 21435.592 U / mg; the proteins encoded by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 have β-glucosidase activity: 672.317 - 829.896 U / ml; the proteins encoded by SEQ ID NO:2 and SEQ ID NO:8 have endoglucanase activity: 0.701 - 1.4 U / ml; the proteins encoded by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8 have filter paper enzyme activity: 0.07 - 3.365 U / ml; and / or the proteins encoded by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 genes have hemicellulase activity: 2.548 - 130.66 U / ml.

[0017] Another aspect of the present invention also provides a gene encoding the β-glucosidase according to the present invention.

[0018] In a specific embodiment, the gene is selected from:

[0019] (1) DNA having a nucleotide sequence as shown in any one of SEQ ID NOs: 9 - 32, preferably DNA as shown in SEQ ID NO:9;

[0020] (2) A DNA molecule that hybridizes with the DNA described in (1) under stringent conditions; and

[0021] (3) A DNA molecule having at least 80% homology with the DNA described in (1).

[0022] Another aspect of the present invention also provides a recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium containing the gene according to the present invention.

[0023] In a specific embodiment, the cell line is a eukaryotic or prokaryotic cell line, and / or the recombinant bacterium is a bacterium or a fungus.

[0024] In a specific embodiment, the recombinant bacterium is Escherichia coli, Bacillus subtilis or yeast.

[0025] Another aspect of the present invention also provides the use of the gene according to the present invention or the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium according to the present invention in the production of β-glucosidase. In a specific embodiment, the use is for non-disease treatment and non-disease diagnosis applications.

[0026] In a specific embodiment, the β-glucosidase is used in the fields of food, feed, textile, medical treatment or bioenergy (such as the preparation of cellulosic ethanol).

[0027] Another aspect of the present invention also provides the use of the β-glucosidase according to the present invention, the gene according to the present invention or the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium according to the present invention in the degradation of cellulose or hemicellulose. In a specific embodiment, the use is for non-disease treatment and non-disease diagnosis applications.

[0028] The β-glucosidase of the present invention has cleavage activity against lignocellulose, has significant advantages in the degradation of cellulose and hemicellulose, has excellent enzyme activity, thermal stability, pH tolerance and / or multiple enzyme activities, and has potential application values in multiple fields such as food, feed, textile, medical treatment and bioenergy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] From the following detailed description in conjunction with the drawings, the above features and advantages of the present invention will become more obvious, wherein:

[0030] Figure 1 It is a schematic diagram of the structure of the recombinant plasmid of the present invention.

[0031] Figure 2 It is an SDS-PAGE electrophoresis diagram of the recombinant protein expression in the present invention.

[0032] Figure 3 It is the determination of the thermal stability of the β-glucosidase encoded by the ML117-1 gene.

[0033] Figure 4 It is the determination of the optimal reaction temperature of the β-glucosidase encoded by the ML117-1 gene.

[0034] Figure 5 Determination of the activity of β-glucosidase encoded by the ML117-1 gene at different pH values.

[0035] Figure 6 Determination of the effect of different metal ions on the activity of β-glucosidase encoded by the ML117-1 gene.

[0036] Figure 7 Protein gel diagram of the induced expression of proteins encoded by the ML-4, EHL-18, PY-3, MY2-2-K, N3F-4-K, T4F-1-K, and MGYL-2 genes.

[0037] Figure 8 Histogram of the enzyme activity of proteins encoded by the ML-4, EHL-18, PY-3, MY2-2-K, N3F-4-K, T4F-1-K, and MGYL-2 genes.

[0038] Sequence Listing Description

[0039] The specific sequences used in the embodiments of the present invention are as follows:

[0040] SEQ ID NO:1 - β-glucosidase encoded by ML117-1

[0041] MGMFKVHYLHKNGKVLSYEQWETNYQKEVLTLVKKKLKGILLSVVLCAQLILPSTTVEAAQTFYNNTTGNEDGYDYELWKDNGSTSMTIKGGGNFECWWENIGNALFRKGKKFDCTQTYQQIGNISIDFGVDYQPNGNSYLCVYGWTRDPLVEYYIVESWGTWRPPGATSKGTITVDGGTYDVYETTRVNQPSIDGDTTFQQYWSVRTSKRTSGTISVTEHFKAWESMGMKMGKLYEAALNVEGYQSSGWADVYKNDISVGGSISSGSGSSSSDNSSSAAPGTAQGTRVECETMSINGQYAGTISSPFSGVALYANDESVSYTQNFTSGTHDFTLSGASDGSNMAKVDLVIGGENKGTFYFGDANHAEYTIKNVTHGTGNQKIELVVNDDDGNWDAYVDALIIGGSGVGSDSSGSTSGGTTSGGTTSGGTTSGTTNTSDSMECENMTITGQYAGTISSPFSGVALYANDESCKYTQYFSNGTHNFTLRGCSDSSNMAKVDLKIGGETKGTFYFGDSYPAEYTIENVSHGTGNQTIELVVTADDGNWDAYLDCLTISGTSTSTGTTTGGTSGATKMVALTFDDGPSTTTTPQVLDILEKYDVVATFFLIGQQVNSDTMSIMQRQIAMGCELGNHSYTHVDMSNLSATDIKNQIEWTSSAIKNTVNYDVKFFRPPYLGTSNTMYQNIDLAFIQGIGCNDWESSVSASQRANTVLSSVKDGSIVLLHDFQGNTNTVQALPTIIEGLKNQGYTFVTLSELFEAKGVNPNQEYKIWSNVND

[0042] β-glucosidase encoded by SEQ ID NO:2-ML-4

[0043] MKRLKTRFDERLRVMALALLVSCSSTPLWAQGLKDAYKGYFMIGVAVNQRNVTNASQSALVKQEFNSMTAENDMKPEPTEPREGEFNWESADRIANFARQNGIKLRGHCLMWHSQIGKWMYEDNPTKEVFFKRMRNHIHTIVNRYKDVIYCWDVVNEAITDDRNASDPYRQSPLYKIAGDEFIAKAFEYAREADPKALLFYNDYNECDPVKSKRIFEMVKKMKAAGVPIDGIGMQGHYNIYGPTEKEVDDAIKLYKQVVNHIHVTELDIRINEEMGGQLRFSREGQTVSDSIKQHLADQYARVFRVFRNHKDVVKCVTFWNLSDRDSWLGARNYPLPFDTEYKPKLAYEYIKDMKAPKWDMPKKPARPKRNNRGGAQAERPPFNPDLAFPENPNIKEDFKPSVKNQPGQQYPQVNSQGYVRFRVELPEAKSVVVSLGLGGRGGTKLHKTYDGSWVGTTEGPMDEGFHYYHLTVDGGVVNDPGAMNYFGSTRWESGIEIPAHDQDFYAVKNVPHGNVQQVLFWSESTKTTRRAFVYTPPQYGKNKKQKYPVLYLQHGWGEDETAWSNQGHANLIMDNLIAEGKIEPFIIVMTYGMTNEVRFGGLRQFTAEAFEKVLVDELVPYIDANFQTRADKEHRAMAGLSMGGFETKLITLRRPEVFGYYGLMSGGQYAPTDIKDKKQVKLIFQSCGSKENPDAIKKSTEALKAAGHNAVGYVSEGTAHEFLTWRRSLKELALLLFK

[0044] β-glucosidase encoded by SEQ ID NO:3-EHL-18

[0045] MSKRKFISMLAALMTGLSTVTMLPFVPTSTLAADVVYNNFESSYGGWYGNADNVVLTTETEIGYQNSKAMKVTGRYSSADGAASSKGLYLTGGTKYDYNVKVYSENEEKFTVTLMYIDEKTEEKTTVKLISEDVKANSWANLSAAFEAPVGTYEYLLTITTDSANDFIFDDVKITTQKSANVAHAANEKGLKDVFASYFRIGNTLNNETVKNSSLMAMYLKEYNSVTCGNQMKPDYLMVQSESKNDNVAISLKSCAAIMDFCVQNNLGMRGHTLVWHSQTPSWFFKKDFKNDGAWVDKNTMDKRLDNYIKNVFSAIETQYPTLDLYAYDVCNECIANSQELFTNNNGIREPGDHKVKNGTSAWVQIYKDNSYVEKAFAIARKYAPEGCELYYNDYNEYWDGKRDRIYELCKPLYEKGLLDGIGMQSHISANATGFGGTDSYIKAMKKFLSIGCDVQITELDVSIENGKYTLQNQADKYEAIFKAAMNWNEAPQSKGRITAVCMWGLYDTLSWIGAEDKPLLYDDNVKPKPAHEALMKLVPQSEWGEVITPPADIEPDSNGWYFNSVFEESTDGWEARGSANILQSGRKAYEGEESLLVENRTSSWHGAAFTLNPRAFKVGKSYSFSVNTTYLEGDDTDKFYLKLQYTNSDGDTKYATIAEATAIKGQWVQLTNTNYKLPDDAADMKIYVETAESTNNFYIDDAVGATAGTIIKGAVTVEKYILGDIDNDGIINVFDMILARKGLVEGFTSTREKNSADIDKNGKYEINDAVLLQSYILGKIKKFK

[0046] β - glucosidase encoded by SEQ ID NO:4 - PY - 3

[0047] MKYYRILFLAAAATALVSCAMDELKDYEVEKPQSIVQYEYLNDYDVLKNYVDRTASPDFKLGAALAASDFMAGGQVYALAASNFDEMTAGNDMKYASVVADNGDMSFDNVRNFVNAAADAGMTVYGHTLAWHSQQNNKYLNSLIADKEIEIEEGATDTVVDAEFDYTKMSAWYYWGQGPDGSTRGIVDGVFQSYLPEAIPNFWEFQYHVADGIPWVAGRSYKITMMIRASAAAKFTLAAGTWDGQAGGEIEVGTEWQEVTATRNISVDGAGFVMFQSGNFGGTIEMQWLKVTHEEAKAVSWWTDVISNGDAEGDAVANFVSTHVGATNGPADIVDGAGVDGTRAFVVSSAGGGTNSWDTQFFVYADRPFEEGDKVKLAFDYRADVANGAESQAHSTPGGYIHWDAGAAINFTTDWQHFEKTIVVSSTVSPGGEFQTFAWNLDVGAPAAPVNKYYFDNIVFAIEESGNTIPQTPEEKRDTLIGAMDRWIKGMMEVTAEKVSAWDAVNEAISGGDGDGDGWYDLQSAANGDPANNFYWQDYLGNEEYVRIVIAKARQYYAEFGGTAPLRLFINDYNLESDWDDNKKLKSLIHWIEVWESDGVTKIDGIGTQMHVSCYENPQTQASKEEHVVKMLELLAATGKLVKISELDMGYVDANGQTVPTSSMTEEQHKAMAAYYKFIVSKYFEIIPAAQQYGITQWCLTDASGELGTGWRGGEPVGLWDLNYSRKHTYAGFAEGLKGNK

[0048] β - glucosidase encoded by SEQ ID NO:5 - MY2 - 2 - K

[0049] MRQTKRLALAAVIAMSTTATWAQGLKDAYKDYFMIGVAVNQRNVSNTQQQDLIRREFNSMTAENDMKPEPTEPREGQFNWENADRIANFARQNNIKLRGHCLMWHSQIGRWMLGDNPTKEVFYQRMRKHIQAIVTRYKDIVYCWDVVNEAIEDNPNATDPYRQSPMYKLCGDEFIEKAFQYAREADPNALLFYNDYSTVDPHKRDRIYNMVKKMKAKGVPIDGIGMQAHYNIYFPTEDLLDQAITKFKSIVKHIHITEFDIRVNEEMGGGLQFSREGINVTDSVKQHLADQYARCFRVFRKHRDVIDCVTFWNLGDRDSWLGAANYPLPFDTEYKPKLAYEYIRDMKAPVWNLPPRPARKPRPQGQQRRRGGEQAQRPAFNPDMAFPEDPNVKEDFRPSTKNQPGQQFPMVNSQGIVRFRVEIPDAQAVSVSLGLGGQGGTPLHRAYDNSWVGQTAGPMDEGFHYYHLTVDGGTLNDPGAQNYYGSCRWESGIEIPAHDQDFYAEKNVPHGQVSEVRFWSQSTQSLKRAFVYLPPTYGKNKKEKFPVLYLQHGWGENEYAWSNQGHANLIMDNLLAEGKTKPFIIVMTYGMTNDVRFGGIGQFTAKEFETVLVDELVPYIDSHFQTIAKKDGRAMAGLSMGGIETKLITLRRPEVFGYYGLLSGGQYEPKDIKSKDQVKLIFQSCGSKERPDGIRQSTEALKAAGINAVSYVSEGTAHEFLTWRRSLKEMAPLLFR

[0050] β - glucosidase encoded by SEQ ID NO:6 - N3F - 4 - K

[0051] MKKGLVKLIAGALAGMFLLSGCDALPKGSDGPREVPQTITADFTKKENKGNWVTYGDCKVTLKDGTAVVTERKSSNSGVAVPCPDYRGNTIKTTAQATTENDSLTLSMRYEIFGNVTYVNIAGGQPDASGAVSITGTVEIPANAENALIYLEANDVRDYTVSSFTVSIEGEFNNLTGVPVESLQDPTQTASLAAAYADYFKFGVASPATVMTNTNDGFRKLITTQFNSVTPENELKPDSVLDAATTLADPAKYNECPAIHFDAAKPILDFCKENGIQMRGHTLVWYSQTPSWLFYENYDVNGNLASRELMLKRMENYIDQVMNWCEENYPGVIYAWDVVNEAAADDGGMRDCYWKQTIGDDYVAKAFEFARKHGPAGVQLFYNDYNEYQTSKQDDILAFLKPIAEAGNLDGMGMQSHISSGLNVDNYVEAAKRYADELGVVIHITELDVTAPKSVNPMYDQGVYMKKFFTAIIEADKAGVPIECVTVWGLTDDMSWKSATKPLLFYGNLSPKPAYEGVMCAINGGEVAKPADYVEPVSDTTPFTEDYEDQSFIGGPRYSSVQKVVEGGHDSGFCLQNTEGYAEYDGYSIDVSQYAGHVIHVSFWIKSKADVCKCTADIDGTWPNIAEVQTGSDEWVFGEGDFEIEEGATLTIYFESSDMSPFFLDDLEIKAMN

[0052] β-glucosidase encoded by SEQ ID NO:7-T4F-1-K

[0053] MFSKDFIWGCASSAYQIEGGAFEGGRGESIWDVFSHTPGKIHEGHTGDVACDFYHRYKEDIALMRRMGIRAYRFSLSWSRILPEGTGRVNGEGIAFYNAVIDELLKNGIEPYITLYHWDFPQALQERGGWVSQESVGWFAEYAAVVSRAFSDRVTNFITLNEPQCFTGIAHLHCEHAPGYKLEPKAMFQMVHNVLKAHGAAVVALRENAVRPIKVGYAPTCGMVYPRTETPEDIEACRRYLFSCPDDLSNWTWNVPWFSDPVFLGKYPEDGLKKYAPYLPEITPEDMALISQPLDFMGENIYNGIMLEADEQGNPRYVDRVPGFPHTGNNWPVTPECFRWGLKFLYERYKTPIYVTENGMCCRDVLSSDGRVHDPQRIDFLNRYILAMHRAMEEGTDIRGYFQWTLTDNFEWSCGYRDRFGLIYVDFGTQRRYRKDSSYWFEELIRTRGGNLMKEEPITFTEGPVEGEPRRLQGKLTLPGGGKLRVISGYATADSLSLIPGDTVRLPSRQVLLEGEMTYILEESL

[0054] β-glucosidase encoded by SEQ ID NO:8-MGYL-2

[0055] MKKTLIFTILSYALACTAMAQEKFELGKPGNDNYRYLDEYHALKEYIDREKYPNFKLGSGTTVSSYLNETTFRNMINKNFDETVAGNAMKMSSCVDGNGNMNFSTVTQYVNAATKAGLNVYGHTLAWHSQQPNGWLRKLIADKPAPDLTDGDVDVMVQKYSKDFRTNQSVGWTADKTQYGFSLSFDTTNGLRIHTTKKINSWEVQFVGADNIILETGKTYKMTMTIRGTAAGKMHTKLGDWSSGTTLDVPFTTEWKDVEVNYKAVMASSFLLLQCGDFVGDIYIKSIKFEEKKKGKTINEERRCIKAEATAKQAETWDNQAWFVLGSFPANAKYELKADVRADKPAYATTQIHTAPGTYVHYEAVGNINFTTEWKTISLSGTLSKAGESIALNLSEFADANNYYFDNVSFKINGVERIKNGDFEGTDVSSFRVKKNGSGVTTPTICTNLTYVYIPSTIPLSQKERHDTLVYAMDKWIKGMMQACDGKVKGWDLVNEAISGGGNDGSGNYTLQHSEGYTPGGTWDVGGDAFYWQDYMGDLEYVRQACRLARKYGPSDVKLFINDYNLESDWDSNKKLKSLINWIKKWEADGVTHIDGIGTQMHISFYKNSSTQTSKKNAITQMFKLMAATGKYVRVSEMDMGYVDASGKDVPTGSMTEAQHKEMADFYEWIIKQYLTLVPPAQQWGICQWCPTDAPSNSGWRANTPVGIWDINYYRKHVYAGFVRGLGGKLTAIDEVETETPNMDNGAFDIRGNRLPKGVSFNELPSGLYIINGKKVLKR

[0056] Nucleotide sequence of SEQ ID NO:9 - ML117-1 expressed in Escherichia coli

[0057] Nucleotide sequence of SEQ ID NO:10–ML117-1 expressed in Bacillus subtilis

[0058] Nucleotide sequence of SEQ ID NO:11–ML117-1 expressed in Pichia pastoris

[0059] SEQ ID NO:12 - Nucleotide sequence of ML-4 expressed in Escherichia coli

[0060] SEQ ID NO:13 - Nucleotide sequence of ML-4 expressed in Bacillus subtilis

[0061] SEQ ID NO:14 - Nucleotide sequence of ML-4 expressed in Pichia pastoris

[0062] SEQ ID NO:15 - Nucleotide sequence of EHL-18 expressed in Escherichia coli

[0063] SEQ ID NO:16 - Nucleotide sequence of EHL-18 expressed in Bacillus subtilis

[0064] SEQ ID NO:17 - Nucleotide sequence of EHL-18 expressed in Pichia pastoris

[0065] SEQ ID NO:18 - Nucleotide sequence of PY-3 expressed in Escherichia coli

[0066] SEQ ID NO:19 - Nucleotide sequence of PY-3 expressed in Bacillus subtilis

[0067] SEQ ID NO:20 - Nucleotide sequence of PY-3 expressed in Pichia pastoris SEQ IDNO:21 - Nucleotide sequence of MY2-2-K expressed in Escherichia coli

[0068] SEQ ID NO:22 - Nucleotide sequence of MY2-2-K expressed in Bacillus subtilis

[0069] SEQ ID NO:23 - Nucleotide sequence of MY2-2-K expressed in Pichia pastoris

[0070] SEQ ID NO:24 - Nucleotide sequence of N3F-4-K expressed in Escherichia coli

[0071] SEQ ID NO:25 - Nucleotide sequence of N3F-4-K expressed in Bacillus subtilis

[0072] Nucleotide sequence of SEQ ID NO:26–N3F-4-K expressed in Pichia pastoris

[0073] Nucleotide sequence of SEQ ID NO:27–T4F-1-K expressed in Escherichia coli

[0074] Nucleotide sequence of SEQ ID NO:28–T4F-1-K expressed in Bacillus subtilis

[0075] Nucleotide sequence of SEQ ID NO:29–T4F-1-K expressed in Pichia pastoris

[0076] Nucleotide sequence of SEQ ID NO:30–MGYL-2 expressed in Escherichia coli

[0077] Nucleotide sequence of SEQ ID NO:31–MGYL-2 expressed in Bacillus subtilis

[0078] Nucleotide sequence of SEQ ID NO:32–MGYL-2 expressed in Pichia pastoris Detailed implementation manners

[0079] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0080] The experimental methods used in the examples are all conventional methods unless otherwise specified.

[0081] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be obtained through commercial channels.

[0082] Unless otherwise indicated, the terms used herein have the ordinary technical meanings understood by those skilled in the art. For definitions and terms in the art, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0083] As used herein, the term "comprising" or "including" is an open-ended description that includes all the specified components or steps described, as well as other specified components or steps that do not substantially affect; when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0084] As used herein, the term "and / or" includes all combinations of the items connected by this term, and each combination should be considered as having been separately listed herein. For example, "A and / or B" includes "A", "A and B", and "B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0085] As used herein, the term "substitution of an amino acid" refers to the replacement of one amino acid with another amino acid having similar or dissimilar properties, including conservative substitutions (i.e., having a relatively small impact on the structure or function of the protein) or non-conservative substitutions (i.e., having a relatively large impact on the structure or function of the protein). In the present invention, it is preferred that the amino acid substitution is a conservative substitution.

[0086] As used herein, the term "protein with the same function" refers to a protein having the same β-glucosidase activity, endoglucanase activity, filter paper enzyme activity, or hemicellulase activity.

[0087] As used herein, the term "hybridize with... under stringent conditions" refers to hybridizing and washing the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS in DNA or RNA hybridization experiments.

[0088] The present invention provides eight genes with β-glucosidase activity, which are named ML-4, ML117-1, EHL-18, PY-3, MY2-2-K, N3F-4-K, T4F-1-K, and MGYL-2, respectively.

[0089] The present invention also provides recombinant vectors containing the above genes. Further, the starting vector of the recombinant vector is a plasmid vector applicable to Escherichia coli, Bacillus subtilis, and yeast fungi.

[0090] The present invention also provides recombinant microbial cells, which are recombinant microbial cells carrying the above genes or the above recombinant vectors.

[0091] The present invention provides the application of the above genes, the above recombinant vectors, and the above recombinant microbial cells in the preparation of β-glucosidase.

[0092] The present invention also provides a method for producing β-glucosidase, which is to obtain β-glucosidase by fermenting the above recombinant microbial cells.

[0093] The application of the gene with β-glucosidase activity in bacterial or fungal expression can be achieved in the following manner:

[0094] 1) Express the eight genes with β-glucosidase activity to obtain corresponding recombinant engineering bacteria.

[0095] 2) Use filter paper, CMC-Na, and corncob xylan as substrates respectively, and use the DNS method to measure the filter paper enzyme activity, endoglucanase activity, and hemicellulase activity; use p-nitrophenyl-β-D-galactopyranoside (pNPG) as the substrate, and terminate the reaction with 1 mol / L Na2CO3 to measure the β-glucosidase activity;

[0096] 3) Measure the substrate specificity of the recombinant enzyme with the supernatant of the recombinant strain.

[0097] Furthermore, in the step 2), the protein encoded by the ML117-1 gene has a β-glucosidase activity of 27.089 U / mg - 21435.592 U / mg. The proteins encoded by the ML-4, EHL-18, PY-3, MY2-2-K, N3F-4-K, T4F-1-K, and MGYL-2 genes have a β-glucosidase activity of 672.317 - 829.896 U / ml. The proteins encoded by the ML-4 and MGYL-2 genes have an endoglucanase activity of 0.701 - 1.4 U / ml. The proteins encoded by the ML-4, EHL-18, PY-3, MY2-2-K, T4F-1-K, and MGYL-2 genes have a filter paper enzyme activity of 0.07 - 3.365 U / ml. The proteins encoded by the ML-4, EHL-18, PY-3, N3F-4-K, T4F-1-K, and MGYL-2 genes have a hemicellulase activity of 2.548 - 130.66 U / ml.

[0098] The present invention provides eight novel β-glucosidase sequences, which can be heterologously expressed in Escherichia coli, Bacillus subtilis, and yeast and have cleavage activity on lignocellulose. The optimal working temperature of the β-glucosidase encoded by the ML117-1 gene is 46°C, and it can maintain more than 80% of its enzyme activity in the range of 37°C - 80°C; the optimal pH of this β-glucosidase is 9.0, and in the range of pH 5.0 - 10.0, its enzyme activity remains above 60%. The proteins encoded by the ML-4, EHL-18, PY-3, MY2-2-K, N3F-4-K, T4F-1-K, and MGYL-2 genes have a β-glucosidase activity of 672.317 - 829.896 U / ml. The proteins encoded by the ML-4 and MGYL-2 genes have an endoglucanase activity of 0.701 - 1.4 U / ml. The proteins encoded by the ML-4, EHL-18, PY-3, MY2-2-K, T4F-1-K, and MGYL-2 genes have a filter paper enzyme activity of 0.07 - 3.365 U / ml. The proteins encoded by the ML-4, EHL-18, PY-3, N3F-4-K, T4F-1-K, and MGYL-2 genes have a hemicellulase activity of 2.548 - 130.66 U / ml. The proteins encoded by the genes involved in the present invention have significant advantages in the degradation of cellulose and hemicellulose and can provide potential utilization value for multiple fields such as food, feed, medicine, and bioenergy.

[0099] All patents and publications mentioned in this application are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain changes can be made to the present invention without departing from the spirit or scope of the invention. The following examples further illustrate the present invention and should not be construed as limiting the invention or the scope of the specific methods described herein.

[0100] Example 1 Obtaining the β-glucosidase gene

[0101] 1. Test materials and reagents

[0102] Luria-Bertani medium (LB medium): 10 g of tryptone, 10 g of NaCl, 5 g of yeast extract, add distilled water to 1000 mL.

[0103] LB solid medium: Add 1.8 - 2 g of agar to 100 mL of LB liquid medium, autoclave at 121 °C for 20 min.

[0104] Whatman No. 1 filter paper, purchased from whatman company, product number: 1001 - 047.

[0105] Sodium carboxymethyl cellulose (CMC-Na), purchased from Solarbio company, product number: C8621.

[0106] Corncob xylan, purchased from Solarbio company, product number: X8163.

[0107] pNPG (p-nitrophenyl-β-D-glucopyranoside), purchased from Solarbio company, product number: N8700.

[0108] DNS (Ghose method) reagent, purchased from Shanghai Yuanye Bio-Technology Co., Ltd., product number: R30591.

[0109] 0.1 moL / L pH 4.8 citrate-sodium citrate buffer: Mix 460 mL of 0.1 moL / L citric acid solution with 540 mL of 0.1 moL / L sodium citrate solution evenly, filter sterilize, and store in a 4 °C refrigerator.

[0110] 0.1 mol / L pH 5.5 acetic acid-sodium acetate buffer: Dissolve 46.76 g of sodium acetate and 5.7 mL of acetic acid to 1 L, filter sterilize, and store in a 4 °C refrigerator.

[0111] pH 5.5 citrate-disodium hydrogen phosphate buffer: Mix 425 mL of 0.1 moL / L citric acid solution with 575 mL of 0.2 moL / L disodium hydrogen phosphate solution evenly, filter sterilize, and store in a 4 °C refrigerator.

[0112] 1 mol / L Na2CO3 solution: 106 g of Na2CO3 was made up to 1 L.

[0113] 1% (W / V) carboxymethyl cellulose suspension: 1 g of carboxymethyl cellulose was dissolved in 100 mL of acetic acid - sodium acetate buffer at pH 5.5, mixed well, and stored in a refrigerator at 4°C for later use.

[0114] 1% (W / V) corncob xylan solution: 1 g of corncob xylan was dissolved in 100 mL of acetic acid - sodium acetate buffer at pH 5.5, mixed well, and stored in a refrigerator at 4°C for later use.

[0115] 5 mmol / L pNPG solution: 0.1506 g of pNPG was dissolved in 100 mL of citric acid - disodium hydrogen phosphate buffer at pH 5.5, mixed well, and stored in a refrigerator at 4°C for later use.

[0116] 2. Extraction of DNA and gene synthesis

[0117] Taking the rumen microorganisms of Tarim red deer, goitered gazelle, argali, sheep, goat and camel, and the monogastric microorganisms of Mongolian wild ass as experimental samples, genomic DNA was extracted using a soil DNA extraction kit (brand: Yeasen, product number: 18815ES08), and the operation was carried out according to the instruction manual provided by the merchant. Then it was sent to a sequencing company for metagenomic sequencing and annotation. Through data analysis, functional genes of β-glucosidase were screened out, and eight functional genes with β-glucosidase were obtained and named ML117-1 (SEQ ID NO:9), ML-4 (SEQ ID NO:12), EHL-18 (SEQ ID NO:15), PY-3 (SEQ ID NO:18), MY2-2-K (SEQ ID NO:21), N3F-4-K (SEQ ID NO:24), T4F-1-K (SEQ ID NO:27) and MGYL-2 (SEQ ID NO:30), and the amino acid sequences encoded by them are shown in SEQ ID NOs: 1 to 8 respectively.

[0118] PCR amplification of its functional genes was carried out using a high-fidelity enzyme gold medal Mix (Beijing Tsingke Biotechnology Co., Ltd.). The amplification conditions were: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s. After 32 cycles, the final extension was at 72°C for 10 min. The PCR products were identified by 1.0% agarose gel electrophoresis at 100 V for 90 min, observed under ultraviolet light, and the correct fragments were purified using a Sangon gel recovery and purification kit.

[0119] The above PCR gel recovery product and vector pET28a(+) were double digested with restriction endonucleases (NcoI, XhoI) according to the instructions, and the digested products were recovered. The digested PCR product and vector were ligated by T4 ligase, and the ligation product was introduced into Escherichia coli BL21 competent cells by heat shock method. Positive clones were screened by colony PCR, and finally further verified by sequencing to obtain the recombinant vector. The recombinant vector containing the ML117-1 gene is as Figure 1 shown.

[0120] Example 2 Study on the properties of recombinant protein

[0121] 1. Preparation of crude enzyme solution

[0122] The above recombinant strain was cultured overnight, and then cultured to an OD 600 of 0.6 - 0.8 at an inoculation amount of 1%, and induced with IPTG (isopropyl-β-D-thiogalactoside) at a final concentration of 0.2 mmol / L for 6 h. The bacterial solution was centrifuged at 4500 / min. Lysozyme solution at 1 mg / mL was added to the precipitate, and it was repeatedly frozen and thawed 3 times, sonicated for 30 min, and centrifuged at 4500 rpm. The supernatant obtained was the crude enzyme solution.

[0123] 2. Study on the properties of recombinant protein

[0124] (1) Study on the properties of multifunctional enzyme activity

[0125] The enzyme activities of the recombinant protein on four model substrates of filter paper, CMC-Na, corncob xylan, and pNPG.

[0126] The detection method is as follows:

[0127] The internationally common filter paper enzyme activity assay method was used, mainly referring to the standard method NREL / TP-510-42628 formulated by the National Renewable Energy Laboratory of the United States. Approximately 25 mg of Whatman No. 1 filter paper (1 cm × 3 cm) was put into a test tube, 0.45 mL of pH 4.8 citrate-sodium citrate buffer and 50 μL of crude enzyme solution were added, and it was shaken well. It was incubated at 50 °C for 60 min (in the control group, 50 μL of crude enzyme solution was first boiled for 10 min to inactivate). Then, 1.5 mL of DNS solution was immediately added to terminate the reaction, and the test tube was heated in a boiling water bath for 5 min for color development, then quickly cooled, and made up to 5 ml, and the absorbance value was measured at 540 nm. The content of glucose in the system was calculated according to the glucose standard curve.

[0128] The activity of endoglucanase was determined by DNS method. Using sodium acetate buffer (pH 5.5) as the solvent, 1% CMC-Na was prepared as the substrate. 50 μL of crude enzyme solution was incubated with 450 μL of substrate at 50 °C for 30 min (in the control group, 50 μL of crude enzyme solution was first boiled for 10 min to inactivate). Then 1.5 mL of DNS solution was added to terminate the enzymatic reaction. The mixture was boiled at 100 °C for 5 min, and distilled water was added to make the volume up to 5 mL. The OD 540 value was measured with an enzyme-labeling instrument to determine the amount of reducing sugar formed during the incubation process. The content of glucose in the system was calculated according to the glucose standard curve.

[0129] Using 5 mmol / L pNPG as the substrate (pH 5.5), 50 μL of crude enzyme solution was incubated with 450 μL of substrate in a water bath at 45 °C for 20 min (in the control group, 50 μL of crude enzyme solution was first boiled for 10 min to inactivate). Then 1 mL of 1 mol / L Na2CO3 was added to terminate the reaction, and distilled water was added to make the volume up to 5 mL. 200 μL of the supernatant was taken and added to an enzyme-labeling plate, and the absorbance at 410 nm was measured. The amount of enzyme required to produce 1 μmol of p-nitrophenol per minute was defined as one unit of β-glucosidase activity.

[0130] Hemicellulose-degrading enzymes are mainly represented by xylanase. The reaction substrate was 1% corncob xylan, prepared with 0.1 mol / L acetic acid-sodium acetate buffer at pH 5.5. 50 μL of crude enzyme solution was incubated with 450 μL of substrate at 45 °C for 30 min (in the control group, 50 μL of crude enzyme solution was first boiled for 10 min to inactivate). Subsequently, 1.5 mL of DNS color-developing solution was added, and after mixing, it was heated in boiling water for 5 min for color development. After rapid cooling, distilled water was added to make the volume up to 5 mL, and the absorbance was measured at 540 nm. The content of xylose in the system was calculated according to the xylose standard curve.

[0131] According to the above method, the specific activity of the glucosidase encoded by the ML117-1 gene was 12657.51 ± 113.17 U / mg.

[0132] (2) Determination of the optimal temperature and temperature stability of the glucosidase encoded by the ML117-1 gene

[0133] The electrophoretogram of the purified β-glucosidase encoded by the ML117-1 gene is as Figure 2 shown.

[0134] The His-tag gravity column protein purification steps are as follows: First, perform column pretreatment. Rinse the column with deionized water to remove the preservation solution ethanol. Equilibrate the column with the equilibration buffer (containing 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) for 15 - 20 min. Slowly add the protein supernatant after cell disruption (dissolved in the binding buffer, 2 - 4 mM imidazole) to the column and bind for 2 h. Elute the unbound miscellaneous proteins with the washing buffer (containing 20 - 50 mM imidazole), and elute the target protein with the elution buffer (containing 50 - 500 mM imidazole). Collect the eluate (collect in multiple tubes to avoid dilution), and verify the protein purity by SDS-PAGE.

[0135] For the purified β-glucosidase, its enzyme activity was measured at different temperatures from 37 - 80 °C under the condition of pH 5.5. The experimental results ( Figure 3 ) showed that the optimal temperature range of this enzyme was 40 - 50 °C, and within this temperature range, the enzyme activity presented an obvious peak region. Further narrowing the temperature range for research found that within the temperature range of 41 - 46 °C, the enzyme activity was in the gradually increasing range, and at 46 °C, the enzyme activity was in the range of 13714.09 - 14057.03 U / mg ( Figure 4 ).

[0136] The enzyme activity of this enzyme and commercial cellulase (Novozymes celluclast) was measured simultaneously within the same temperature range, and the specific enzyme activity of the two enzymes was measured respectively. The results are as Figure 3 shown. Within the same temperature range, the specific enzyme activity of commercial cellulase was much lower than that of this enzyme.

[0137] (3) Determination of the optimal pH and pH stability of the β-glucosidase encoded by the ML117-1 gene

[0138] Enzymatic reactions of the purified and expressed β-glucosidase were carried out under different pH conditions to determine its optimal pH. The purified β-glucosidase was placed in buffer systems with different pH values, and its pH suitability was measured at 46 °C. The results ( Figure 5 ) showed that the optimal pH range of this β-glucosidase was around 9.0, and at this time, the specific activity was in the relatively high range of 21254.56 - 21616.62 U / mg.

[0139] Within the range of pH 5.0 - 10.0, the enzyme activity of this β-glucosidase remained above 60%.

[0140] The enzyme activity of this enzyme and commercial cellulase was measured simultaneously within the same pH range, and the specific enzyme activity of the two enzymes was measured respectively. The results are as Figure 5 shown. Within the same pH range, the specific enzyme activity of commercial cellulase was much lower than that of this enzyme.

[0141] (4) Determination of the effects of different metal ions on the activity of β-glucosidase encoded by the ML117-1 gene

[0142] To determine the effects of metal ions and organic reagents on β-glucosidase, different metal ions (Mn 2+ , Ca 2+ , Zn 2+ , Mg 2+ , Cu 2+ , Fe 2+ , K + ) (200 mM) were added to the enzyme solution at 46 °C and pH 9 respectively, and the measurement was carried out after 30 min of treatment. The measurement results are as Figure 6 shown. The results show that: the enzyme completely loses its activity in the metal ion environments of Mn 2+ and Fe 2+ , and no significant inhibitory effects are shown by other ions and reagents. The commercial enzyme only shows low activity in Mg 2+ , Cu 2+ , K + and is completely inactivated in the remaining environments. The β-glucosidase of ML117-1 provided by the present invention has superior thermal stability and pH tolerance compared with the commercial enzyme, and has extremely high enzyme activity. This enzyme can be applied to environments such as high temperature, strong acid and strong alkali, and has potential value in the food, health care, feed and medical industries.

[0143] (5) Analysis of the enzymatic activity characteristics of the gene expression product

[0144] As Figure 7 shown, the protein expression of the gene expression products of MGYL-2, EHL-18, MY2-2-K, PY-3, T4F-1-K, N3F-4-K, and ML-4 was detected, and corresponding bands appeared at the corresponding sizes, indicating that the protein was successfully expressed.

[0145] The gel running order is as follows: Lane M: maker (10 - 200 KDa), Lane 1: before induction of T4F-1-K, Lane 2: after induction of T4F-1-K, Lane 3: before induction of PY-3, Lane 4: after induction of PY-3, Lane 5: before induction of EHL-18, Lane 6: after induction of EHL-18, Lane 7: before induction of MGYL-2, Lane 8: after induction of MGYL-2, Lane 9: before induction of N3F-4-K, Lane 10: after induction of N3F-4-K, Lane 11: before induction of ML-4, Lane 12: after induction of ML-4, Lane 13: before induction of MY2-2-K, Lane 14: after induction of MY2-2-K.

[0146] As Figure 8As shown, the enzyme activity assays of the gene expression products of MGYL-2, EHL-18, MY2-2-K, PY-3, T4F-1-K, N3F-4-K, and ML-4 showed that there were differences in the β-glucosidase activities of the gene expression products, among which the β-glucosidase activity of the gene expression product of T4F-1-K was prominent; in terms of hemicellulase activity, the activity of the gene expression product of N3F-4-K was relatively significant; the filter paper enzyme activity was generally low among the gene expression products; and in terms of endoglucanase activity, the gene expression product of ML-4 showed an obvious advantage. This result clearly demonstrated the enzyme activity characteristics of the different gene expression products of the present invention, provided key activity data support for their application in bioengineering fields such as cellulose degradation, and further highlighted the application value of the gene resources of the present invention.

[0147] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0148] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0149] Furthermore, any combination can be made between the various different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. β-glucosidase, which is selected from: (1) a protein having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; (2) a protein having the same function, which is obtained by substitution and / or deletion and / or addition of one or more amino acids in the amino acid sequence of the protein in (1); and (3) a protein having an amino acid sequence with at least 80% homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 and having the same function.

2. The β-glucosidase according to claim 1, which is selected from a protein having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, preferably a protein having an amino acid sequence as shown in SEQ ID NO:

1.

3. The β-glucosidase according to claim 1 or 2, which further has one or more of the following activities: endoglucanase activity, filter paper enzyme activity and hemicellulase activity, wherein the protein encoded by SEQ ID NO:1 has β-glucosidase activity: 27.089 U / mg - 21435.592 U / mg; the proteins encoded by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 have β-glucosidase activity: 672.317 - 829.896 U / ml; the proteins encoded by SEQ ID NO:2 and SEQ ID NO:8 have endoglucanase activity: 0.701 - 1.4 U / ml; the proteins encoded by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8 have filter paper enzyme activity: 0.07 - 3.365 U / ml; and / or the proteins encoded by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 genes have hemicellulase activity: 2.548 - 130.66 U / ml.

4. A gene encoding the β-glucosidase according to any one of claims 1 to 3.

5. The gene according to claim 4, which is selected from: (1) DNA with a nucleotide sequence as shown in any one of SEQ ID NOs: 9-32, preferably DNA as shown in SEQ ID NO: 9; (2) A DNA molecule that hybridizes with the DNA described in (1) under stringent conditions; and (3) A DNA molecule having at least 80% or more homology with the DNA described in (1).

6. A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium containing the gene according to claim 4 or 5.

7. The recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium according to claim 6, wherein the cell line is a eukaryotic cell line or a prokaryotic cell line, and / or the recombinant bacterium is a bacterium or a fungus.

8. Use of the gene according to claim 4 or 5 or the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium according to claim 6 or 7 in the production of β-glucosidase.

9. The use according to claim 8, wherein the β-glucosidase is used in the fields of food, feed, textile, medical treatment or bioenergy.

10. Use of the β-glucosidase according to any one of claims 1 to 3, the gene according to claim 4 or 5 or the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium according to claim 6 or 7 in the degradation of cellulose or hemicellulose.

Images