WO2016090556A1 - 一种高温中性纤维素酶及其编码基因和应用 - Google Patents

一种高温中性纤维素酶及其编码基因和应用 Download PDF

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WO2016090556A1
WO2016090556A1 PCT/CN2014/093378 CN2014093378W WO2016090556A1 WO 2016090556 A1 WO2016090556 A1 WO 2016090556A1 CN 2014093378 W CN2014093378 W CN 2014093378W WO 2016090556 A1 WO2016090556 A1 WO 2016090556A1
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cellulase
cel45
seq
high temperature
gene
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French (fr)
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姚斌
石鹏君
杨虹
黄火清
罗会颖
杨培龙
王亚茹
苏小运
柏映国
师霞
马锐
孟昆
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中国农业科学院饲料研究所
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Priority to CN201480083938.2A priority Critical patent/CN107429240B/zh
Priority to US15/534,491 priority patent/US10767170B2/en
Priority to PCT/CN2014/093378 priority patent/WO2016090556A1/zh
Publication of WO2016090556A1 publication Critical patent/WO2016090556A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • the present invention relates to the field of genetic engineering, and in particular, to a high temperature neutral vitamin enzyme and its encoding gene and application.
  • Cellulose is widely found in the aleurone layer and endosperm cell wall of crops such as barley, wheat, corn, rice and sorghum. Cellulose accounts for about 40% of the dry weight of cells, and is a linear structural molecule composed of glucose linked by ⁇ -1,4-glycosidic bonds. Cellulases can be synthesized by a variety of microorganisms, including fungi, actinomycetes, myxobacteria, and eubacteria, and can also be synthesized by plants.
  • the hydrolysis of cellulose to glucose by microorganisms involves three main types of cellulases: (i) endoglucanases that randomly cleave ⁇ -1,4-glycosidic linkages in cellulose molecules (EC 3.2.1.4); (ii) cellobiohydrolase (EC 3.2.1.91) which releases cellobiose from the non-reducing end to digest cellulose; (iii) ⁇ -glucosidase which hydrolyzes cellobiose and low molecular weight cellodextrin to release glucose (EC 3.2.1.21).
  • endoglucanases that randomly cleave ⁇ -1,4-glycosidic linkages in cellulose molecules (EC 3.2.1.4);
  • cellobiohydrolase EC 3.2.1.91 which releases cellobiose from the non-reducing end to digest cellulose
  • ⁇ -glucosidase which hydrolyzes cellobiose and low molecular weight cellodextrin to release glucose
  • Cellulase has a variety of industrial applications, such as food, feed, beer, pharmaceuticals, textiles, bio-energy and other fields. Different industrial applications have different requirements for cellulase properties.
  • the feed industry requires acidophilic cellulase
  • the textile industry requires high temperature alkali-resistant cellulase.
  • the cellulase used in the industry is mainly from the genus Trichoderma. The optimum pH of these enzymes is about 5.0, and the optimum temperature is between 50-60 ° C, which cannot meet the industrial requirements of the cotton fabric washing industry and the paper pulping industry.
  • thermophilic and thermostable enzymes will be more advantageous because heat resistance can increase the reaction rate, lower the viscosity of the substrate, and also inhibit the contamination of the bacteria. Therefore, it is of great significance to obtain a new type of neutral cellulase resistant to alkali and high temperature.
  • the invention screens a 45 family cellulase produced by Thielavia arenaria XZ7 with an optimum pH of 5.5, an optimum temperature of 60 ° C, certain enzyme activity under alkaline conditions and good alkali resistance at 90 ° C. Under the treatment for 1h, the optimal enzyme condition was retained at about 70%. After treatment for 1 hour in boiling water, it still had the optimum 50% enzyme activity, and the thermal stability was very good, which can be well applied in textile, papermaking and other fields.
  • Another object of the present invention is to provide a gene cel45 encoding the above high temperature neutral cellulase.
  • Another object of the present invention is to provide a recombinant vector comprising the above gene.
  • Another object of the present invention is to provide a recombinant strain comprising the above gene.
  • Another object of the present invention is to provide a process for preparing the above high temperature neutral cellulase CEL45.
  • Another object of the present invention is to provide the use of the above high temperature neutral cellulase CEL45.
  • the present invention isolates a novel high temperature neutral cellulase CEL45 from the thermophilic fungus Thielavia arenaria, and constructs a recombinant yeast strain capable of efficiently expressing the cellulase.
  • the present invention provides a high temperature neutral cellulase CEL45 selected from the group consisting of:
  • the enzyme comprises 249 amino acids and a stop codon, and the N-terminal 20 amino acids are its predicted signal peptide sequence "mhlsllaplslllgpvfvsa" (SEQ ID NO. 3).
  • the mature high temperature neutral cellulase CEL45 has a theoretical molecular weight of 24.2 kDa and its amino acid sequence is shown in SEQ ID NO.
  • the high temperature neutral cellulase CEL45 of the present invention has characteristics such as high temperature resistance and high enzyme activity under neutral conditions.
  • the invention screens a 45th family cellulase produced by Thielavia arenaria, the optimum pH value is 5.5, the optimum temperature is 60 ° C, certain enzyme activity under alkaline conditions and alkali resistance is good, at 90 The enzyme activity of about 70% of the optimum conditions was retained after treatment at °C for 1 hour. After treatment in boiling water, the enzyme activity of 50% of the optimum conditions was obtained, and the thermal stability was very good.
  • the present invention provides a gene cel45 encoding the above high temperature neutral cellulase CEL45, in particular the gene:
  • the gene cel45 encoding the above high temperature neutral cellulase CEL45 according to the present invention is selected from the group consisting of:
  • genomic sequence of the gene is as shown in SEQ ID NO. 4:
  • the cellulase-derived gene cel45 is isolated and cloned by a PCR-based method, and the complete sequence analysis of the DNA indicates that the cDNA of the structural gene cel45 of the cellulase CEL45 is 750 bp in length.
  • the base sequence of the signal peptide is:
  • the gene sequence of the mature cellulase CEL45 is shown in SEQ ID NO.
  • the theoretical molecular weight of the mature protein was 24.2 kDa, and the cellulase gene cel45 sequence and its encoded amino acid sequence were BLAST aligned in GenBank to confirm that CEL45 is a new cellulase.
  • the invention also provides a derivative of the above protein which may be subjected to one or more of the polypeptide sequences set forth in SEQ ID NO. 1 or SEQ ID NO. 2 (eg, one or several, or such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of a value selected from 1 to 10, or a range intermediate the above values) Amino acid residue substitution, deletion and/or insertion obtained, and still having cellulase active.
  • a common strategy is to conservative amino acid substitutions by replacing amino acid residues with amino acid residues having similar side chains.
  • a family of amino acid residues having similar side chains is well defined in the art.
  • a fusion protein in order to construct a fusion protein, promote expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or Other suitable regions within the protein include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, such as 6His or Flag tag, or factor Xa or thrombin or enterokinase proteolytic enzyme sites.
  • suitable linker peptides signal peptides
  • leader peptides leader peptides
  • terminal extensions glutathione S-transferase (GST)
  • GST glutathione S-transferase
  • the cellulase-reactive protein of the present invention comprises a nucleoside hybridized under stringent conditions to the nucleotide sequence set forth in SEQ ID NO. 4 or SEQ ID NO. 5 of the Sequence Listing.
  • Acid sequence The encoded amino acid sequence.
  • hybridization under stringent conditions is used to describe hybridization and washing conditions in which nucleotide sequences that are typically at least 60% homologous to each other are still hybridizable to each other.
  • stringent conditions are those under which conditions having at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homology to each other will generally still hybridize to each other. .
  • stringent conditions are well known to those of ordinary skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions is hybridization in 6X SSC at about 45 °C followed by one or more washes in 0.2 x SSC, 0.1% SDS at 50-65 °C.
  • highly stringent conditions can be achieved by increasing the hybridization temperature, for example to 50 °C, 55 °C, 60 °C or 65 °C.
  • the present invention also encompasses a polypeptide having cellulase activity encoded by an allele or a natural variant of the polynucleotide molecule set forth in SEQ ID NO. 4 or SEQ ID NO.
  • the cellulase protein is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or at least about 70 %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, more preferably at least about 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, even more preferably at least about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% 99.7%, 99.8%, 99.9% or higher of the active protein homologous to the full-length amino acid
  • the invention provides a novel cellulase gene SEQ ID NO. 4 or SEQ ID NO.
  • the present invention further encompasses a nucleic acid molecule which differs from one of the nucleotide sequences of SEQ ID NO. 4 or SEQ ID NO. 5 of the present invention, and its encoded cellulase protein, due to the degeneracy of the genetic code.
  • an isolated nucleic acid molecule of the invention is a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO. 4 or SEQ ID NO. 5, preferably its allele or Natural Variants.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having the amino acid sequence set forth in SEQ ID NO. 1 or SEQ ID NO.
  • the nucleic acid molecule of the invention encodes a full length cellulase protein substantially identical to the amino acid sequence of SEQ ID NO. 1 or SEQ ID No. 2, for example, by substitution, deletion and/or insertion of one or more a protein (such as one or several, or a value selected from 1 to 10) amino acid residues derived from SEQ ID NO. 1 or SEQ ID NO. 2, or with SEQ ID NO. 1 or SEQ ID NO.
  • the nucleic acid molecule is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or at least about 70%, 71%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.7%, 97.8%, 97.9%, or at least about 98%, 98.1%, 98.2 %, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, even more preferably at least about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.
  • the present invention provides a recombinant vector comprising the above high temperature neutral cellulase gene cel45, selected as pPIC9-cel45.
  • the cellulase gene of the present invention is inserted between appropriate restriction sites of the expression vector such that the nucleotide sequence is operably linked to the expression control sequence.
  • the recombinant yeast expression plasmid pPIC9-cel45 was obtained.
  • the present invention also provides a recombinant strain comprising the above high temperature neutral cellulase gene cel45, preferably the strain is Escherichia coli, yeast, preferably recombinant strain GS115/cel45.
  • the invention also provides a method for preparing a high temperature neutral cellulase CEL45 comprising the following steps:
  • the host cell is a Pichia pastoris cell, a Saccharomyces cerevisiae cell or a Polymorpha cell, and the recombinant yeast expression plasmid is preferably transformed into Pichia pastoris GS115 to obtain a heavy weight.
  • the recombinant expression vectors of the invention can be designed to express cellulase proteins in prokaryotic or eukaryotic cells.
  • the cellulase gene can be in bacterial cells such as E. coli, yeast (such as Pichia, Aspergillus niger), insect cells (such as Sf9 cells or silkworm cells using a baculovirus expression vector) or plant cells (such as Pseudomonas).
  • the invention relates to a host cell, preferably Pichia pastoris, into which a recombinant expression vector of the invention has been introduced.
  • the host cell can be any prokaryotic or eukaryotic cell including, but not limited to, those host cells described above.
  • Pichia pastoris cells are preferred.
  • Pichia pastoris is a methanol yeast that can be metabolized with methanol as the sole carbon source. This system is known for its ability to express high levels of heterologous proteins.
  • As an efficient expression system many cellulase genes have been successfully expressed in Pichia pastoris. Also the novel cellulase genes provided by the present invention are also expressed in Pichia pastoris and have high expression levels. Therefore, mass production of cellulase by fermentation is very easy and the cost is lower than at any time.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in the Molecular Cloning Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY, 1989, Sambrook et al., and other laboratory manuals.
  • Host cells of the invention can be used to produce (i.e., express) cellulase proteins.
  • the invention also provides methods of making cellulase proteins using the host cells of the invention.
  • the method comprises culturing a host cell of the invention in a suitable culture medium into which a recombinant expression vector encoding a cellulase protein has been introduced, or a gene encoding wild type or altered protein has been introduced into the genome The gene of the enzyme protein) until the cellulase protein is produced.
  • the method further comprises isolating the cellulase protein from the culture medium or host cell.
  • a further aspect of the invention is a cellulase expressed in Pichia pastoris.
  • cellulases are purified by simple methods such as ammonium sulfate precipitation, dialysis, ultrafiltration and chromatography. After simple purification, the purity of the cellulase is sufficient to study its enzymatic properties.
  • the invention also provides the use of the above high temperature neutral cellulase CEL45, including industrial applications such as textiles and paper.
  • the first technical problem to be solved by the present invention is that the existing microbial-derived cellulase has few neutral and alkali-resistant properties and poor thermal stability, thereby providing a new and successfully applied to the textile and paper industry.
  • High temperature neutral cellulase The cellulase provided by the invention has an optimum pH value of 5.5 and an optimum temperature of 60 ° C. It has certain enzyme activity under alkaline conditions and has good alkali resistance at 90 ° C. After 1 h of treatment, the optimum enzyme activity of about 70% was retained, and the enzyme activity of about 50% of the optimum conditions was obtained in boiling water for 1 hour. The thermal stability was very good, and it can be well applied in the fields of textiles and papermaking.
  • the present invention isolated a novel high temperature neutral cellulase CEL45 from the thermophilic fungus Thielavia arenaria XZ7.
  • Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.
  • Endonuclease was purchased from TaKaRa, and ligase was purchased from Invitrogen.
  • Barley dextran, sodium carboxymethyl cellulose (CMC-Na) was purchased from Sigma, and all others were domestically produced reagents (all available from the general biochemical reagent company).
  • Thermophilic fungus Thielavia arenaria XZ7 medium is potato juice medium: 1000 mL potato juice, 10 g glucose, 25 g agar, pH is natural.
  • E. coli medium LB 1% peptone, 0.5% yeast extract, 1% NaCl, pH natural.
  • BMGY medium 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V).
  • BMMY medium Divided by 0.5% methanol instead of glycerol, the rest of the ingredients are the same as BMGY, and the pH is natural.
  • the mycelium cultured in liquid for 3 days was filtered into a mortar with a sterile filter paper, 2 mL of the extract was added, and the mixture was ground for 5 minutes. Then, the slurry was placed in a 50 mL centrifuge tube, and lysed in a 65 ° C water bath for 120 minutes, mixed every 20 minutes. Once again, centrifuge at 13000 rpm for 10 min at 4 °C. The supernatant was extracted from phenol/chloroform to remove the heteroprotein, and the supernatant was added to an equal volume of isopropanol, and allowed to stand at -20 ° C for 30 min, and then centrifuged at 13,000 rpm for 10 min at 4 ° C. The supernatant was discarded, and the precipitate was washed twice with 70% ethanol, dried under vacuum, dissolved in an appropriate amount of TE, and placed at -20 ° C until use.
  • Degenerate primers P1, P2 were designed based on the conserved regions GXTTRYWDC and QFDXXIPGG of the 45th family cellulase gene.
  • P1 5'-GGYAMVACCACYCGYTAYTGGGAYTGYT-3';
  • PCR amplification was carried out using the total DNA of Thielavia arenaria XZ7 as a template.
  • the PCR reaction parameters were: denaturation at 94 ° C for 5 min; then denaturation at 94 ° C for 30 sec, annealing at 45 ° C for 30 sec, extension at 72 ° C for 1 min, and incubation at 72 ° C for 10 min after 30 cycles.
  • a fragment of about 292 bp was obtained, and the fragment was recovered and ligated to the pEASY-T3 vector and sent to Sanbo Biotechnology Co., Ltd. for sequencing.
  • TAIL-PCR specific primers were designed upstream and downstream: the design direction was the direction of the unknown region that needs to be amplified, and the position of sp2 was designed inside the sp1.
  • the distance between each two primers is not strictly defined, the primer length is generally 22 to 30 nt, and the annealing temperature is 60 to 65 °C.
  • usp upstream specific primer
  • dsp downstream specific primer
  • the flanking sequence of the known gene sequence was obtained by reverse TAIL-PCR, and the amplified product was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing.
  • the cellulase CEL45 gene has a full length of 938 bp and contains two intron regions.
  • the mature gene sequence is 750 bp in length and encodes 249 amino acids. A stop codon.
  • Analysis with SignalP http://www.cbs.dtu.dk/services/SignalP) indicated that the N-terminal 20 amino acids were the predicted signal peptides.
  • the predicted molecular weight of the mature protein encoded by this gene is predicted to be 24.2 kDa.
  • the expression vector pPIC9 was digested (EcoR I+Not I), and the gene encoding cellulase CEL45 was cleavage (EcoR I+Not I), and the gene fragment encoding mature cellulase was excised (excluding The signal peptide sequence was ligated to the expression vector pPIC9 to obtain a recombinant plasmid pPIC9-cel45 containing the Thielavia arenaria XZ7 cellulase gene cel45 and transformed into Pichia pastoris GS115 to obtain recombinant Pichia pastoris strain GS115/cel45.
  • a recombinant expression vector comprising a signal peptide sequence was constructed in the same manner, and transformed and obtained a recombinant Pichia strain.
  • the GS115 strain containing the recombinant plasmid was inoculated into 400 mL of BMGY culture medium, and cultured at 30 ° C for 250 hours with shaking at 250 rpm, and then the cells were collected by centrifugation. Then, it was resuspended in 200 mL of BMMY medium, and cultured at 30 ° C with shaking at 250 rpm. After induction for 48 h, the supernatant was collected by centrifugation. The viability of the cellulase was measured. SDS-PAGE results showed that recombinant cellulase was expressed in Pichia pastoris.
  • DNS method The specific method is as follows: at pH 6.0 (0.1M sodium dihydrogen phosphate-citric acid), at 65 ° C, 1 mL of the reaction system includes 100 ⁇ L of appropriate diluted enzyme solution and 900 ⁇ L (1%, w/v) substrate. The reaction was carried out for 10 min, and the reaction was terminated by adding 1.5 mL of DNS, and boiled in water for 5 min. The OD value was measured at 540 nm after cooling.
  • pH 6.0 0.1M sodium dihydrogen phosphate-citric acid
  • 1 mL of the reaction system includes 100 ⁇ L of appropriate diluted enzyme solution and 900 ⁇ L (1%, w/v) substrate.
  • the reaction was carried out for 10 min, and the reaction was terminated by adding 1.5 mL of DNS, and boiled in water for 5 min. The OD value was measured at 540 nm after cooling.
  • One enzyme unit (U) is defined as the amount of enzyme that releases 1 ⁇ mol of reducing sugar per minute under given conditions.
  • Example 2 The properties of the recombinant cellulase obtained in Example 2 were determined.
  • the recombinant cellulase CEL45 purified in Example 2 was subjected to an enzymatic reaction at different pH to determine its optimum pH.
  • Substrate cellulase with different pH buffers 0.1M Gly-HCl pH 1.0-3.0; 0.1M lemon Acid-dihydrogen phosphate disodium pH 3.0-8.0; 0.1 M Tris-HCl pH 8.0-9.0; 0.1 M Gly-NaOH pH 9.0-12.0
  • Enzyme activity assay was carried out at 60 °C. As a result (Fig.
  • the cellulase CEL45 had an optimum pH of 5.5, maintained 90% or more of the enzyme activity in the range of pH 5.0 to 7.0, and had an optimum condition of about 25% at pH 9.0.
  • the cellulase was treated at 37 ° C for 60 min in the various pH buffers described above, and the enzyme activity was measured at 60 ° C in a pH 5.5 buffer system to study the pH stability of the enzyme.
  • the results showed that the cellulase CEL45 was stable between pH 2.0 and 10.0.
  • the residual enzyme activity was above 80% after 60 min treatment in this pH range, indicating that the enzyme has good pH stability.
  • the optimum temperature for cellulase is determined by enzymatic reaction in a citric acid-disodium hydrogen phosphate buffer system (pH 5.5) and at different temperatures. The heat resistance was measured after the cellulase was treated at different temperatures for 2, 5, 10, 20, 30, and 60 minutes, and the enzyme activity was measured at an optimum temperature.
  • the optimum temperature measurement result of the enzyme reaction indicates that the optimum temperature is 60 °C.
  • the thermostability experiment of the enzyme showed (Fig. 4) that the cellulase CEL45 has good thermal stability. When incubated at 90 °C for 1 h, it can maintain more than 70% of enzyme activity, and it is still optimal after treatment for 1 h in boiling water. About 50% of the enzyme activity under the conditions.
  • the enzyme activity was measured in a citric acid-dibasic sodium phosphate buffer system (pH 5.5) at 60 ° C, and its K m value at 60 ° C was calculated.
  • the K m value at the time of using dextran as a substrate was determined to be 11.28 mg/ml, and the maximum reaction rate V max was 1256.44 ⁇ mol/min ⁇ mg.
  • the K m value when CMC-Na was used as a substrate was 10.79 mg/ml, and the maximum reaction rate V max was 1177.44 ⁇ mol/min ⁇ mg.

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Abstract

提供了一种真菌来源的高温中性45家族纤维素酶及其编码基因和应用。所述纤维素酶最适pH值为5.5，最适温度为60℃，在碱性条件下具有一定酶活力且耐碱性好，在90℃下处理1h保留最适条件70％左右的酶活力，沸水里处理1h仍具有最适条件50％左右的酶活力，可很好的应用于纺织和造纸等领域。

Description

一种高温中性纤维素酶及其编码基因和应用 技术领域
本发明涉及基因工程领域，具体地，本发明涉及一种高温中性维素酶及其编码基因和应用。
背景技术
纤维素广泛存在于大麦、小麦、玉米，水稻和高粱等作物的糊粉层和胚乳细胞壁中。纤维素大约占细胞干重的40％左右，是由葡萄糖通过β-1,4-糖苷键连接而成的线状结构分子。纤维素酶可由多种微生物合成，包括真菌、放线菌、粘细菌和真细菌，也可以由植物合成。微生物将纤维素水解为葡萄糖涉及以下三类主要的纤维素酶：(i)在纤维素分子中随机切割β-1,4-糖苷键的内切葡聚糖酶(EC3.2.1.4)；(ii)从非还原末端消化纤维素而释放纤维二糖的纤维二糖水解酶(EC 3.2.1.91)；(iii)水解纤维二糖和低分子量纤维糊精以释放葡萄糖的β-葡萄糖苷酶(EC3.2.1.21)。目前研究最多的主要为微生物来源的纤维素酶，其中真菌由于其分泌量大，且分泌到胞外而成为研究的热点。
纤维素酶有多种工业应用，如在食品、饲料、啤酒、医药、纺织、生物能源等领域有着广泛的应用。而不同的工业应用，对纤维素酶性质的要求是不同的，例如，饲料工业需要嗜酸的纤维素酶，纺织工业需要高温耐碱的纤维素酶。现在工业中应用的纤维素酶主要来自木霉属，这些酶的最适pH在5.0左右，最适温度在50-60℃之间，不能满足棉织品水洗整理工业和造纸制浆业的工业要求。因为棉纤维耐碱不耐酸，强酸性处理后易损坏(许爱国等，中性纤维素酶在棉织物整理中的应用，印染，2006，21，11-12)。而且嗜热和热稳定性好的酶将更具优势，因为耐热可以提高反应速率，降低底物的粘度，同时还可以抑制杂菌的污染。因此，获得新型且耐碱高温的中性纤维素酶的研究具有重大意义
本发明筛选到Thielavia arenaria XZ7产生的一种45家族纤维素酶，其最适pH值为5.5，最适温度为60℃，在碱性条件下具有一定酶活力且耐碱性好，在90℃下处理1h保留最适条件70％左右的酶活力，沸水里处理1h后仍具有最适条件50％的酶活力，热稳定性非常好，可很好的应用于纺织、造纸等领域。
发明内容
本发明的目的是提供一种真菌来源的高温中性纤维素酶CEL45。
本发明的另一目的是提供编码上述高温中性纤维素酶的基因cel45。
本发明的另一目的是提供包含上述基因的重组载体。
本发明的另一目的是提供包含上述基因的重组菌株。
本发明的另一目的是提供一种制备上述高温中性纤维素酶CEL45的方法。
本发明的另一目的是提供上述高温中性纤维素酶CEL45的应用。本发明从嗜热真菌Thielavia arenaria中分离得到一种新的高温中性纤维素酶CEL45，并构建了能够高效表达此纤维素酶的重组酵母菌株。
本发明提供了一种高温中性纤维素酶CEL45，其选自：
(a)包含SEQ ID NO.1或SEQ ID NO.2所示氨基酸序列的多肽；或者
(b)SEQ ID NO.1或SEQ ID NO.2所示氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有纤维素酶活性的由(a)衍生的多肽。
Figure PCTCN2014093378-appb-000001
其中，该酶包括249个氨基酸和一个终止密码子，N端20个氨基酸为其预测的信号肽序列“mhlsllaplslllgpvfvsa”(SEQ ID NO.3)。
因此，成熟的高温中性纤维素酶CEL45的理论分子量为24.2kDa，其氨基酸序列如SEQ ID NO.2所示：
Figure PCTCN2014093378-appb-000002
本发明的高温中性纤维素酶CEL45具有耐高温、中性条件下有较高酶活性等特性。
本发明筛选到Thielavia arenaria所产生的一种第45家族纤维素酶，其最适pH值为5.5，最适温度为60℃，在碱性条件下具有一定酶活力且耐碱性好，在90℃下处理1h保留最适条件70％左右的酶活力，沸水里处理1后仍具有最适条件50％的酶活力，热稳定性非常好。
本发明提供了编码上述高温中性纤维素酶CEL45的基因cel45，具体地该基因：
(a)编码SEQ ID NO.1或SEQ ID NO.2所示氨基酸序列的多肽；
(b)编码SEQ ID NO.1或SEQ ID NO.2所示氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有纤维素酶活性的由(a)衍生的多肽；
优选地，根据本发明的编码上述高温中性纤维素酶CEL45的基因cel45，所述基因选自：
(a)包含SEQ ID NO.4或SEQ ID NO.5所示核酸分子的DNA；或
(b)在严谨条件下与SEQ ID NO.4或SEQ ID NO.5所示DNA序列杂交、且编码具有纤维素酶活性的多肽的DNA分子。
具体地，该基因的基因组序列如SEQ ID NO.4所示：
Figure PCTCN2014093378-appb-000003
根据本发明的具体实施方式，基于PCR的方法分离克隆了纤维素酶的基因cel45，DNA全序列分析结果表明，纤维素酶CEL45的结构基因cel45的cDNA全长750bp。其中，信号肽的碱基序列为：
Figure PCTCN2014093378-appb-000004
成熟的纤维素酶CEL45的基因序列如SEQ ID NO.5所示。
Figure PCTCN2014093378-appb-000005
成熟蛋白理论分子量为24.2kDa，将纤维素酶基因cel45序列及其编码的氨基酸序列在GenBank中进行BLAST比对，确定CEL45是一种新的纤维素酶。
本发明也提供了上述蛋白的衍生物，其可由SEQ ID NO.1或SEQ ID NO.2所示的多肽序列经过一个或多个(例如，一个或几个，或者如1、2、3、4、5、6、7、8、9或10的选自1～10的值，或者介于上述值中间的范围)氨基酸残基的取代、缺失和/或插入获得，并仍然具有纤维素酶活性。例如，一个常见的策略是保守氨基酸取代，即将氨基酸残基用具有相似侧链的氨基酸残基替换。具有相似侧链的氨基酸残基的家族在本领域已有明确定义。因此，在纤维素酶蛋白中用来自同一侧链的另一氨基酸残基替换一个或几个位点，将不会在实质上影响其酶活性。此外，本领域技术人员公知，在基因的克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的蛋白末端引入了一个或多个不相干的残基，而这并不影响目的蛋白的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、如6His或Flag的标签，或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。
又在一实施例中，本发明的具有纤维素酶活性的蛋白包括由在严谨条件下与序列表中的SEQ ID NO.4或SEQ ID NO.5所示的核苷酸序列杂交的核苷酸序列 编码的氨基酸序列。如此处所用，术语“在严谨条件下杂交”是用来描述典型地相互间至少60％同源的核苷酸序列仍可相互杂交的杂交和清洗条件。优选地，严谨条件为这样的条件，在此条件下相互间具有至少约65％、更优选地至少约70％、且甚至更优选地至少约75％或更高同源性的序列一般仍可相互杂交。此严谨条件为本领域普通技术人员所公知，可在Current Protocols in Molecular Biology,John Wiley&Sons,N.Y.(1989),6.3.1-6.3.6中找到。严谨杂交条件的一个优选、非限制性实例为：在6×SSC中于约45℃杂交，然后在0.2×SSC、0.1％SDS中于50-65℃洗涤一次或多次。本领域技术人员能够理解，高度严谨条件可通过提高杂交温度，例如至50℃、55℃、60℃或65℃来实现。
另外，本领域普通技术人员将会理解：由于自然变异所致的遗传多态性可在群体中的个体间存在。此类自然变异一般可在纤维素酶基因的核苷酸序列中导致1-5％的差异。纤维素酶中任何以及所有这种自然变异产生的、并且不改变纤维素酶功能活性的核苷酸突变和所得氨基酸多态性也在本发明的范围之内。因此，本发明也包括由SEQ ID NO.4或SEQ ID NO.5所示多聚核苷酸分子的等位基因或天然变体所编码的具有纤维素酶活性的多肽。
在另一优选的实施方案中，纤维素酶蛋白为至少约60％、61％、62％、63％、64％、65％、66％、67％、68％、69％，或至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％，或至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％，或者至少约90％、91％、92％、93％、94％、95％、96％、97％、98％，更优选地至少约98.1％、98.2％、98.3％、98.4％、98.5％、98.6％、98.7％、98.8％、98.9％，甚至更优选地至少约99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％或更高地同源于本发明的SEQ ID NO.1或SEQ ID NO.2所示的全长氨基酸序列的活性蛋白。介于上述值中间的范围和同一性值(例如，69-90％同源性或98.1-99.9％一致性)也包括在本发明中。
另一方面，本发明提供一种新的纤维素酶基因SEQ ID NO.4或SEQ ID NO.5。本发明进一步包括由于遗传密码的简并性而不同于本发明的SEQ ID NO.4或SEQ ID NO.5所述核苷酸序列之一的核酸分子、及其编码的纤维素酶蛋白。在一个实施方案中，本发明的分离的核酸分子为如在严谨条件下与SEQ ID NO.4或SEQ ID NO.5所示核苷酸序列杂交的核苷酸序列，优选其等位基因或天然突 变体。在另一实施例中，本发明的分离核酸分子具有编码具SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列的蛋白质的核苷酸序列。在一实施例中，本发明的核酸分子编码与SEQ ID NO.1或SEQ ID No.2的氨基酸序列基本一致的全长纤维素酶蛋白质，例如，通过取代、删除和/或插入一个或多个(如一个或几个，或者选自1～10的值)氨基酸残基而源自SEQ ID NO.1或SEQ ID NO.2的蛋白，或者，与SEQ ID NO.1或SEQ ID NO.2的氨基酸序列至少99％同源的蛋白。该核酸分子优选为至少约60％、61％、62％、63％、64％、65％、66％、67％、68％、69％,或至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％，或至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％，更优选至少约90％、91％、92％、93％、94％、95％、96％、97％、97.7％、97.8％、97.9％，或至少约98％、98.1％、98.2％、98.3％、98.4％、98.5％、98.6％、98.7％、98.8％、98.9％,甚至更优选地至少约99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％或更高地同源于SEQ ID NO.4或SEQ ID NO.5的核苷酸序列，介于上述值的范围或同一性值(如76～97％同源或97.8～99.9％相同)也包括在本发明中。
本发明提供了包含上述高温中性纤维素酶基因cel45的重组载体，选为pPIC9-cel45。将本发明的纤维素酶基因插入到表达载体合适的限制性酶切位点之间，使其核苷酸序列可操作的与表达调控序列相连接。作为本发明的一个最优选的实施方案，优选为将本发明的纤维素酶基因插入到质粒pPIC9上的EcoR I和Not I限制性酶切位点之间，使该核苷酸序列位于AOX1启动子的下游并受其调控，得到重组酵母表达质粒pPIC9-cel45。
本发明还提供了包含上述高温中性纤维素酶基因cel45的重组菌株，优选所述菌株为大肠杆菌、酵母菌，优选为重组菌株GS115/cel45。
本发明还提供了一种制备高温中性纤维素酶CEL45的方法，包括以下步骤：
1)用上述的重组载体转化宿主细胞，得重组菌株；
2)培养重组菌株，诱导重组纤维素酶表达；
3)回收并纯化所表达的纤维素酶CEL45蛋白。
其中，优选所述宿主细胞为毕赤酵母细胞、啤酒酵母细胞或多型逊酵母细胞，优选将重组酵母表达质粒转化毕赤酵母细胞(Pichia pastoris)GS115，得到重 组菌株GS115/cel45。
本发明的重组表达载体可设计用于在原核或真核细胞中表达纤维素酶蛋白。例如，纤维素酶基因可在如大肠杆菌的细菌细胞、酵母(如毕赤酵母、黑曲霉)、昆虫细胞(如使用杆状病毒表达载体的Sf9细胞或家蚕细胞)或植物细胞(如脓杆菌介导的拟南芥、烟草、玉米等)中表达。从而，本发明涉及已导入本发明的重组表达载体的宿主细胞、优选毕赤酵母。宿主细胞可为任何原核或真核细胞，其包括但不限于上述的那些宿主细胞。优选毕赤酵母细胞。巴斯德毕赤酵母(Pichia pastoris)是一种甲醇酵母，能够以甲醇作为唯一碳源进行代谢。这个系统因为其表达高水平的异源蛋白的能力而闻名。作为有效的表达系统，许多纤维素酶基因已成功地在毕赤酵母中表达。同样本发明提供的新的纤维素酶基因也在毕赤酵母中表达，并具有高表达水平。因此，通过发酵大规模生产纤维素酶非常容易，并且成本比任何时候都低。
载体DNA可通过常规转化或转染技术导入原核或真核细胞中。转化或转染宿主细胞的合适方法可在《分子克隆》实验室手册第二版，冷泉港实验室出版社,NY,1989，Sambrook等人，和其它实验室手册中找到。
本发明的宿主细胞(如培养的原核或真核宿主细胞)可用于产生(即表达)纤维素酶蛋白。因此，本发明还提供了使用本发明的宿主细胞制备纤维素酶蛋白的方法。在一个实施例中，该方法包括在合适的培养基中培养本发明的宿主细胞(其中已导入了编码纤维素酶蛋白的重组表达载体，或其基因组中已导入了编码野生型或改变的纤维素酶蛋白的基因)，直至产生纤维素酶蛋白。在另一实施方案中，该方法还包括从培养基或宿主细胞分离纤维素酶蛋白。
本发明的再一方面为在毕赤酵母中表达的纤维素酶。为了测定纤维素酶的性质，通过如硫酸铵沉淀，透析、超滤和层析的简单方法纯化纤维素酶。经过简单的净化，纤维素酶的纯度足以研究其酶学性质。
本发明还提供了上述高温中性纤维素酶CEL45的应用，包括纺织和造纸等工业方面的应用。
本发明首先所要解决的技术问题是现有微生物来源的纤维素酶很少具有中性且耐碱的性质，且热稳定性差的问题，从而提供一种新的能够成功应用于纺织和造纸工业的高温中性纤维素酶。本发明所提供的纤维素酶最适pH值为5.5，最适温度为60℃，在碱性条件下具有一定酶活力且耐碱性好，在90℃下 处理1h保留最适条件70％左右的酶活力，沸水里处理1h仍具有最适条件50％左右的酶活力，热稳定性非常好，可很好的应用于纺织和造纸等领域。
附图说明
图1重组纤维素酶CEL45的最适pH。
图2重组纤维素酶CEL45的pH稳定性。
图3重组纤维素酶CEL45的最适温度。
图4重组纤维素酶CEL45的热稳定性。
具体实施方式
试验材料和试剂
1、菌株及载体：本发明从嗜热真菌Thielavia arenaria XZ7中分离得到一种新的高温中性纤维素酶CEL45。毕赤酵母表达载体pPIC9及菌株GS115购自于Invitrogen公司。
2、酶类及其它生化试剂：内切酶购自TaKaRa公司，连接酶购自Invitrogen公司。大麦葡聚糖、羧甲基纤维素钠(CMC-Na)购自Sigma公司，其它都为国产试剂(均可从普通生化试剂公司购买得到)。
3、培养基：
(1)嗜热真菌Thielavia arenaria XZ7培养基为马铃薯汁培养基：1000mL马铃薯汁，10g葡萄糖，25g琼脂，pH自然。
(2)大肠杆菌培养基LB(1％蛋白胨、0.5％酵母提取物、1％NaCl,pH自然)。
(3)BMGY培养基：1％酵母提取物，2％蛋白胨，1.34％YNB，0.00004％Biotin，1％甘油(V/V)。
(4)BMMY培养基：除以0.5％甲醇代替甘油,其余成份均与BMGY相同，pH自然。
说明：以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1梭孢菌Thielavia arenaria XZ7中纤维素酶编码基因cel45的克隆
提取嗜热梭孢菌(Thielavia arenaria XZ7)基因组DNA：
将液体培养3天的菌丝体用无菌滤纸过滤放入研钵中,加入2mL提取液，研磨5min,然后将研磨液置于50mL离心管中，65℃水浴锅裂解120min，每隔20min混匀一次,在4℃下13000rpm离心10min。取上清于酚/氯仿中抽提除去杂蛋白,再取上清加入等体积异丙醇,于-20℃静置30min后,4℃下13000rpm离心10min。弃上清,沉淀用70％的乙醇洗涤两次,真空干燥,加入适量TE溶解,置于-20℃备用。
根据第45家族纤维素酶基因的保守区GXTTRYWDC和QFDXXIPGG序列设计合成了简并引物P1，P2
P1:5'-GGYAMVACCACYCGYTAYTGGGAYTGYT-3'；
P2:5'-WCCBCCKGGRATSRMSADRTCRAAYTG-3'
以梭孢菌(Thielavia arenaria XZ7)总DNA为模板进行PCR扩增。PCR反应参数为：94℃变性5min；然后94℃变性30sec，45℃退火30sec，72℃延伸1min，30个循环后72℃保温10min。得到一约292bp片段，将该片段回收后与pEASY-T3载体相连送三博生物技术有限公司测序。
根据测序得到的核苷酸序列，设计上游和下游各2条TAIL-PCR特异性引物：设计方向为需要扩增的未知区域方向，sp2的位置设计在sp1的内侧。每两个引物之间的距离没有严格规定，引物长度一般22～30nt，退火温度在60～65℃。并将它们分别命名为usp(上游特异性引物),dsp(下游特异性引物)见表1。
表1.纤维素酶CEL45TAIL-PCR特异性引物
Figure PCTCN2014093378-appb-000006
通过反向TAIL-PCR得到已知基因序列的侧翼序列，扩增得到产物回收后送三博生物技术有限公司测序。拼接后纤维素酶CEL45基因DNA全长938bp，包含2个内含子区域，其成熟基因序列全长750bp，编码249个氨基酸和 一个终止密码子。用SignalP(http://www.cbs.dtu.dk/services/SignalP)进行分析表明N端20个氨基酸为预测的信号肽。预测该基因所编码的成熟蛋白的理论分子量为24.2kDa。
实施例2重组纤维素酶的制备
将表达载体pPIC9进行双酶切(EcoR I+Not I)，同时将编码纤维素酶CEL45的基因cel45双酶切(EcoR I+Not I)，切出编码成熟纤维素酶的基因片段(不包含信号肽序列)与表达载体pPIC9连接，获得含有Thielavia arenaria XZ7纤维素酶基因cel45的重组质粒pPIC9-cel45并转化毕赤酵母GS115，获得重组毕赤酵母菌株GS115/cel45。
以同样的方法构建包含信号肽序列的重组表达载体，转化、获得重组毕赤酵母菌株。
取含有重组质粒的GS115菌株，接种于400mL BMGY培养液中，30℃250rpm振荡培养48h后，离心收集菌体。然后于200mL BMMY培养基重悬，30℃250rpm振荡培养。诱导48h后，离心收集上清。测定纤维素酶的活力。SDS-PAGE结果表明，重组纤维素酶在毕赤酵母中得到了表达。
实施例3重组纤维素酶的活性分析
纤维素酶活性的测定：在540nm下测定酶水解底物纤维素酶和羧甲基纤维素钠所生成的产物葡萄糖的量。
DNS法：具体方法如下：在pH 6.0(0.1M磷酸二氢钠-柠檬酸)，65℃条件下，1mL的反应体系包括100μL适当的稀释酶液和900μL(1％，w/v)底物，反应10min，加入1.5mL DNS终止反应，沸水煮5min。冷却后540nm测定OD值。
1个酶活单位(U)定义为在给定的条件下每分钟释放出1μmol还原糖的酶量。
测定实施例2获得的重组纤维素酶的性质
1、重组纤维素酶CEL45的最适pH和pH稳定性的测定方法如下：
将实施例2纯化的重组纤维素酶CEL45在不同的pH下进行酶促反应以测定其最适pH。底物纤维素酶用不同pH的缓冲液(0.1M Gly-HCl pH 1.0-3.0；0.1M柠檬 酸-磷酸氢二钠pH 3.0-8.0；0.1M Tris-HCl pH 8.0-9.0；0.1M Gly-NaOH pH 9.0-12.0)在60℃下进行酶活力测定。结果(图1)表明，纤维素酶CEL45的最适pH为5.5，在pH5.0～7.0的范围内维持90％以上的酶活性，在pH 9.0时也具有最适条件25％左右的活性。纤维素酶于上述各种不同pH的缓冲液中37℃处理60min，再在pH 5.5缓冲液体系中60℃下测定酶活性，以研究酶的pH稳定性。结果(图2)表明纤维素酶CEL45在pH 2.0～10.0之间均很稳定，在此pH范围内处理60min后剩余酶活性在80％以上，这说明此酶具有较好的pH稳定性。
2、纤维素酶CEL45的最适温度及热稳定性测定方法如下：
纤维素酶的最适温度的测定为在柠檬酸-磷酸氢二钠缓冲液体系(pH5.5)及不同温度下进行酶促反应。耐热性测定为纤维素酶在不同温度下分别处理2、5、10、20、30、60min后，于最适温度下进行酶活性测定。酶反应最适温度测定结果(图3)表明其最适温度为60℃。酶的热稳定性实验表明(图4)，纤维素酶CEL45有良好的热稳定性，在90℃下温育1h，能保持70％以上的酶活力，在沸水里处理1h后仍具有最适条件下50％左右的酶活力。
3、纤维素酶CEL45的动力学测定方法如下：
用不同浓度的葡聚糖为底物，在柠檬酸-磷酸氢二钠缓冲液体系中(pH5.5)，60℃下测定酶活性，计算出其在60℃下的Km值。经测定，以葡聚糖为底物时的Km值为11.28mg/ml，最大反应速度Vmax为1256.44μmol/min·mg。以CMC-Na为底物时的Km值为10.79mg/ml，最大反应速度Vmax为1177.44μmol/min·mg。
4、不同金属离子化学试剂对纤维素酶CEL45活性的影响测定如下：
在酶促反应体系中加入不同浓度的不同金属离子及化学试剂，研究其对酶活性的影响，各种物质终浓度为5mmol/L。在60℃、pH5.5条件下测定酶活性。结果表明，大多数离子和化学试剂对重组纤维素酶的活力没有影响，Ag+和SDS对其有抑制作用，Ca2+和Co2+对其有微弱的激活作用，β-巯基乙醇对其有明显的激活作用。

Claims (10)

  1. 一种高温中性纤维素酶CEL45，其特征在于，其选自：
    (a)包含SEQ ID NO.1或SEQ ID NO.2所示氨基酸序列的多肽；或者
    (b)SEQ ID NO.1或SEQ ID NO.2所示氨基酸序列经过取代、缺失或添加一个或几个氨基酸且不改变纤维素酶活性的由(a)衍生的多肽。
  2. 高温中性纤维素酶基因cel45，其特征在于，所述基因：
    (a)编码SEQ ID NO.1或SEQ ID NO.2所示氨基酸序列的多肽；
    (b)编码SEQ ID NO.1或SEQ ID NO.2所示氨基酸序列经过取代、缺失或添加一个或几个氨基酸且不改变纤维素酶活性的由(a)衍生的多肽。
  3. 根据权利要求2所述的高温中性纤维素酶基因cel45，其特征在于，所述基因选自：
    (a)包含SEQ ID NO.4或SEQ ID NO.5所示核酸分子的DNA；或
    (b)在严谨条件下与SEQ ID NO.4或SEQ ID NO.5所示DNA序列杂交、且编码且具有纤维素酶活性的多肽的DNA分子。
  4. 包含权利要求2所述的高温中性纤维素酶基因cel45的重组载体。
  5. 包含权利要求2所述的高温中性纤维素酶基因cel45的重组载体pPIC9-cel45。
  6. 包含权利要求2所述的高温中性纤维素酶基因cel45的重组菌株。
  7. 包含权利要求2所述的高温中性纤维素酶基因cel45的重组菌株GS115/cel45。
  8. 一种制备高温中性纤维素酶CEL45的方法，其特征在于，包括以下步骤：
    1)用权利要求4的重组载体转化宿主细胞，得重组菌株；
    2)培养重组菌株，诱导重组纤维素酶CEL45表达；
    3)回收并纯化所表达的纤维素酶CEL45。
  9. 权利要求1所述高温中性纤维素酶CEL45的应用。
  10. 权利要求1所述高温中性纤维素酶CEL45在纺织和造纸领域的应用。
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