WO2017144008A1 - α淀粉酶变体及其应用 - Google Patents

α淀粉酶变体及其应用 Download PDF

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WO2017144008A1
WO2017144008A1 PCT/CN2017/074724 CN2017074724W WO2017144008A1 WO 2017144008 A1 WO2017144008 A1 WO 2017144008A1 CN 2017074724 W CN2017074724 W CN 2017074724W WO 2017144008 A1 WO2017144008 A1 WO 2017144008A1
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alpha amylase
alpha
amylase variant
seq
amino acid
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French (fr)
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范岩
杜秀贞
郝名慧
孙艳
黄珂
李峰
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Nanjing Bestzyme Bioengineering Co Ltd
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Nanjing Bestzyme Bioengineering Co Ltd
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Priority to CN201780013526.5A priority Critical patent/CN108699544B/zh
Priority to US16/080,277 priority patent/US11104909B2/en
Priority to JP2018563750A priority patent/JP2019506186A/ja
Priority to EP17755837.6A priority patent/EP3421596A4/en
Priority to AU2017222025A priority patent/AU2017222025A1/en
Publication of WO2017144008A1 publication Critical patent/WO2017144008A1/zh
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    • 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
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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    • 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
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)

Definitions

  • the invention belongs to the field of enzyme engineering, and relates to an alpha amylase variant and application thereof.
  • alpha amylase In industry, the hydrolysis of starch is mainly started from alpha amylase. These synergistic applications of microbial alpha-amylase and other enzymes, such as pullulanase, glucoamylase and glucose isomerase, are effective. Decomposition of starch macromolecules, these small molecule polysaccharides or monosaccharides produced in food manufacturing, grain processing, beer processing, alcohol production and other industries have many applications, it is essential.
  • Alpha amylase is a kind of saccharification hydrolase. Its main structural feature is ( ⁇ / ⁇ ) 8 folding, which contains a special starch substrate binding site. The length is generally no more than 10 sugar monomers, but many When the binding sites of amylase work together, multi-site binding can be carried out to successfully cut starch macromolecules.
  • Alpha amylase can effectively cut the ⁇ -1,4 glycosidic bond in the starch substrate, thereby rapidly reducing the molecular weight and viscosity of the starch substrate, and the products are mainly dextrin of different lengths.
  • alpha amylases There are different types of alpha amylases, and the industrial application conditions of these species vary greatly depending on the nature of the desired product.
  • Alpha amylase ( ⁇ -1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) is effective in hydrolyzing ⁇ -1,4 glycosidic linkages in starch and other polysaccharides.
  • alpha-amylase which can support effective starch liquefaction in different application fields.
  • the focus of the enzyme-based technology to improve the enzyme species is mainly in heat resistance, acid-base tolerance performance improvement and liquefaction effect.
  • alpha-amylases have been found and defined from plants and microorganisms and have commercial value, mainly including B.licheniformis alpha amylase, B. amyloliquefaciens alpha amylase and G. stearothermophilus alpha amylase, in which B.licheniformis alpha-amylase is used as a template.
  • the number of variants is the largest and the most widely used.
  • alpha amylase variant obtained by mutating or deleting at least one amino acid residue in the amino acid sequence of the parent alpha amylase, while still maintaining the ability of the parent to hydrolyze the ⁇ -1,4 glycosidic linkage; Sequence homology is above 95%.
  • the parent alpha amylase is preferably a natural alpha amylase, ie a bacterial alpha amylase, further preferably an alpha amylase of any one of Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, G. stearothermophilus or Bacillus cereus, further An alpha amylase of B. licheniformis or G. stearothermophilus is preferred, and an alpha amylase of G. stearothermophilus is most preferred.
  • the full-length coding gene sequence of the alpha-amylase of G. stearothermophilus is shown in SEQ ID NO. 1; the corresponding amino acid sequence is shown in SEQ ID NO.
  • the alpha amylase variant is preferably selected from any one of the following or any combination of the following:
  • a parent alpha-amylase of G. stearothermophilus is deleted from its N-terminal first to fifth amino acid residues and replaced with VN or ANLN;
  • the alpha amylase variant is also preferably based on any of the above three conditions or any combination of the three conditions, the C-terminus of which increases the FAN three amino acid residues.
  • amino acid sequence of the alpha amylase variant is further preferably from any one of the following: SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO.
  • the nucleotide coding sequence of the alpha amylase variant is selected from any one of the following: SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. .
  • a gene encoding an alpha amylase variant of the invention is provided.
  • the gene is preferably selected from any one of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO.
  • An expression vector for expressing an alpha amylase variant of the invention comprising the gene encoding an alpha amylase variant of claim 8.
  • the expression vector comprises a natural or synthetic promoter sequence, a natural or synthetic ribosome binding site, a natural or synthetic terminator sequence, and an alpha amylase variant of the invention.
  • the gene sequences together form an expression component.
  • a recombinant cell for expressing an alpha amylase variant of the invention comprising one or more genes encoding an alpha amylase variant of the invention.
  • the host cell of the recombinant cell is preferably from a Bacillus strain, further preferably B. licheniformis or a Bacillus strain genetically engineered to inactivate some endogenous proteins; most preferably B.licheniformis genetically engineered to inactivate AprE and/or NprE .
  • a method for producing an alpha amylase variant of the present invention comprising culturing a recombinant cell containing a sequence encoding an alpha amylase variant under conditions suitable for expression of an alpha amylase variant, and from recombinant cells or An alpha amylase variant is obtained in the culture supernatant.
  • the high temperature is preferably from 80 ° C to 110 ° C, more preferably from 100 ° C to 110 ° C; and the low pH is preferably from pH 5.0 to 5.5.
  • the invention provides a series of alpha amylase variants which have higher catalytic activity under acidic conditions of pH 5.0 and high temperature conditions above 100 °C.
  • the acid and thermal stability of these alpha amylase variants is suitable for the liquefaction of starch.
  • Figure 1 shows the pYF-tsDE vector, including a temperature-sensitive element (replicating activity at 30 ° C), an erythromycin-determining gene (ErmC) - which can tolerate 300 ⁇ g/mL of erythromycin in E. coli.
  • ErmC erythromycin-determining gene
  • B.licheniformis can tolerate 5 ⁇ g/mL of erythromycin.
  • Recombinant host cells containing the nucleotide sequence encoding the alpha amylase variant were screened with erythromycin.
  • Figure 2 is a schematic representation of the pUC57-KS-erm vector from which the pYF-tsDE vector of the present invention can be obtained.
  • Figure 3 is a schematic representation of the pYF-tsINT-amy vector.
  • Figure 4 shows the protein flocculation and viscosity.
  • Figure 5 shows the results of liquefaction experiments under different starch slurry concentrations
  • the alpha amylase refers to an enzyme capable of hydrolyzing the ⁇ -1,4 glycosidic bond of the polysaccharide.
  • alpha amylase can hydrolyze starch to dextrin.
  • the parent alpha amylase refers to a natural alpha amylase.
  • Natural alpha amylases are bacterial alpha amylases, including but not limited to Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, G. stearothermophilus and Bacillus cereus.
  • the native alpha amylase is derived from a strain of Bacillus - especially B. licheniformis and G. stearothermophilus.
  • the full-length coding sequence of G. stearothermophilus is shown in SEQ ID NO. 1; the corresponding amino acid sequence is shown in SEQ ID NO.
  • alpha amylase variant means a non-naturally occurring mutation or deletion of one or several amino acid residues at the effective position of the amino acid sequence of the parent alpha amylase while still maintaining parental hydrolysis.
  • Alpha-amylase capable of alpha-1,4 glycosidic linkages.
  • “Liquidization” in the present invention generally refers to a process of decomposing carbohydrates into small molecule polysaccharides.
  • “liquefaction” refers specifically to the hydrolysis of the alpha-1,4 glycosidic linkage of the carbohydrate.
  • the " ⁇ -1,4 glycosidic bond” refers to a bond connecting C1 of the former glucose to C4 of the latter glucose, that is, an ⁇ -1,4 glycosidic bond.
  • the present invention relates to an "alpha amylase variant" obtained by sequence engineering a parent alpha amylase.
  • the parent alpha amylase is a natural alpha amylase, particularly a natural alpha amylase derived from bacteria.
  • the alpha amylase variant is the mutation or deletion of one or several amino acid residues at the effective position of the amino acid sequence of the parent alpha amylase.
  • the invention includes a series of alpha amylase variants.
  • the amino acid sequence of this series of alpha amylase variants has a homology of at least 95%, reaching 95%, 96%, 97%, 98%, 99% or 100%, respectively.
  • the alpha amylase variant is selected from any of the following:
  • the parent alpha-amylase of G. stearothermophilus is deleted from the N-terminal first to fifth amino acid residues and replaced with VN, and the C-terminal first to 27th amino acid residues are deleted, and the amino acid is obtained.
  • the sequence is shown in SEQ ID NO. 4;
  • the parent alpha-amylase of G. stearothermophilus lacks the N-terminal 1st to 5th amino acid residues and is substituted with ANLN, deleting the N-terminal 180th and 181th amino acid residues, and is deleted.
  • the first to the 27th amino acid residues of the C-terminus are obtained, and the amino acid sequence is as shown in SEQ ID NO.
  • the parent alpha-amylase of G. stearothermophilus lacks the N-terminal 1st to 5th amino acid residues and is substituted with ANLN, deleting the N-terminal 180th and 181th amino acid residues, and is deleted.
  • the first to the 32th amino acid residues of the C-terminus are obtained, and the amino acid sequence is as shown in SEQ ID NO.
  • the parent alpha-amylase of G. stearothermophilus lacks the N-terminal 1st to 5th amino acid residues and is replaced by ANLN, deleting the N-terminal 180th and 181th amino acid residues, and deleting C - the first to the 27th amino acid residues at the end, and the FAN three amino acid residues are added at the C-terminus, the amino acid sequence is shown in SEQ ID NO.
  • amino acid sequence is selected from any one of SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO.
  • the alpha amylase variants of the invention retain the ability to hydrolyze alpha-1,4 glycosidic linkages.
  • the performance of these alpha amylases is consistent with industrial production requirements, such as increased liquefaction efficiency, and stable catalytic activity under acidic pH or high temperature conditions.
  • an alpha amylase variant has stable catalytic activity under acidic conditions of pH 5.0 or conditions above 100 ° C (especially between 100 ° C and 110 ° C). These enhanced properties of alpha amylase variants are more responsive to the liquefaction of the starch industry. Because the liquefaction process is often carried out under low pH and high temperature conditions in the starch industry.
  • alpha amylase variants of the invention can be used in liquefaction reactions.
  • the alpha amylase variant is derived from a parent alpha amylase, particularly an alpha amylase derived from the G. stearothermophilus parent.
  • the alpha amylase variant amino acid sequence is set forth in SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO.
  • any carbohydrate containing an ⁇ -1,4 glycosidic bond can be used for the liquefaction reaction.
  • Carbohydrates containing one or more alpha-1,4 glycosidic linkages include, but are not limited to, starch, amylopectin, amylose, and dextran.
  • alpha-1,6-glycosidic linkages and alpha-1,4-glycosidic linkages such as amylopectin.
  • the " ⁇ -1,4-glycosidic bond" refers to a bond in which C1 of the former glucose is linked to C4 of the latter glucose, that is, an ⁇ -1,4 glycosidic bond. Therefore, the alpha amylase variant of the present invention can be used in combination with a pullulanase capable of hydrolyzing alpha-1,6 glycosidic linkages during saccharification.
  • Enzymes capable of hydrolyzing alpha-1,4 glycosidic linkages include, but are not limited to, alpha amylases.
  • the enzyme that catalyzes the hydrolysis of the alpha-1,4 glycosidic linkage is an alpha amylase.
  • a method of further catalyzing the efficiency of the saccharification reaction is to use a pullulanase in combination.
  • "Pluronic enzyme” means a hydrolase capable of hydrolyzing an ⁇ -1,6 glycosidic bond.
  • the use of the alpha amylase of the present invention in combination with pullulanase in the saccharification of starch can increase the purity of glucose and maltose.
  • the use of the above complex enzyme in the saccharification reaction can effectively reduce the substrate concentration, increase the conversion efficiency, and can also have higher catalytic activity at acidic pH or higher, and can better adapt to the conditions of industrial hydrolysis of starch.
  • the present invention provides a method in which an alpha amylase variant is capable of hydrolyzing alpha-1,4 glycosidic linkages for saccharification at any temperature and pH suitable for industrial production.
  • the liquefaction reaction can be carried out at a high temperature of from 80 ° C to 110 ° C, such as 80 ° C, 90 ° C, 100 ° C, 105 ° C and 110 ° C.
  • the saccharification reaction can also be carried out under acidic pH conditions of pH 5.0 to pH 5.5, such as pH 5.0, 5.1, 5.2, 5.3, 5.4 and 5.5.
  • the liquefaction reaction catalyzed by the alpha amylase variant is stable at acidic pH and at a temperature above 100 °C.
  • the expression vector of the present invention comprises a synthetic nucleotide sequence encoding an alpha amylase variant
  • the recombinant host cell comprises the above expression vector.
  • the expression vector contains a series of synthetic nucleotide sequences encoding different alpha amylase variants.
  • the expression vector is capable of integrating into the genome of the host cell.
  • the expression vector contains the synthetic nucleotide sequence SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO.
  • the expression vector of the present invention preferably comprises a natural or synthetic promoter sequence, a natural or synthetic ribosome binding site, a natural or synthetic terminator sequence.
  • These genetic elements together with the synthetic alpha amylase variant coding sequence constitute an expression component, and the expression component and the vector backbone constitute an expression vector.
  • the expression vector includes a The expression module, and the expression module comprises the following elements: a promoter sequence, a synthetic ribosome binding site, a synthetic nucleotide sequence encoding an alpha amylase variant of the invention, and a terminator sequence.
  • the signal sequence is capable of directing the secretion of the alpha amylase variant, and introducing the signal sequence into the expression vector or expression component, particularly by introducing the signal sequence upstream of the initiation codon is more favorable for secretion of the alpha amylase variant.
  • the expression vector is suitably expressed in bacteria, in particular a Bacillus strain, more preferably expressed in B. licheniformis.
  • the expression vector is capable of integration into the genome of Bacillus, particularly the genome of B. licheniformis.
  • An expression vector for a host cell that can be used to integrate a polynucleotide sequence into a chromosome, and a method of constructing such an expression vector, are a common skill well known in the field of contemporary biology.
  • a recombinant host cell can genetically engineer a nucleic acid sequence comprising one or more alpha amylase variant gene expression. Any technique can be used to genetically engineer a host cell comprising one or more alpha amylase variants of the invention encoding a synthetic nucleic acid sequence, eg, chromosomal integration.
  • Vectors containing temperature sensitive origin and resistance screening markers can be used in the integration step. These vectors are integrated with specific regions of the genome by the Campbell mechanism, and recombinant strains are obtained by resistance screening, and the recombinant bacteria remove the resistance screening markers by homologous recombination in the subsequent culture.
  • the recombinant host cell is genetically engineered to inactivate some endogenous proteins.
  • Endogenous properties that can be inactivated include, but are not limited to, extracellular proteases.
  • the recombinant host cell inactivates some endogenous protein before or after the transformation of the nucleic acid sequence containing the alpha amylase variant expression gene.
  • a more preferred method is to inactivate the exogenous secreted protease of the host bacteria prior to transfer to the vector of the alpha amylase variant expressing gene.
  • B.licheniformis has been engineered to have inactivated some exogenous protease genes.
  • the B. licheniformis strain can inactivate some extracellular proteases, such as subtilisin (AprE), glutamic acid-specific protease (Blase).
  • SorE subtilisin
  • Blase glutamic acid-specific protease
  • the present invention provides a method of producing an alpha amylase variant.
  • the method comprises culturing a recombinant host cell comprising a nucleotide sequence encoding an alpha amylase variant under conditions suitable for expression of an alpha amylase variant, and from the recombinant host cell or supernatant thereof An alpha amylase variant is obtained.
  • All recombinant host cells of the invention are capable of producing alpha amylase variants.
  • the recombinant host cell comprises at least one copy of a nucleotide sequence encoding an alpha amylase variant.
  • These nucleotide sequences encoding alpha amylase variants are capable of expressing alpha amylase variants under suitable conditions.
  • Alpha amylase variants secreted from recombinant host cells can be harvested from recombinant cells or supernatants. Methods of collection include, but are not limited to, filtration, centrifugation, and the like.
  • B. licheniformis is genetically engineered to produce high alpha amylase variants by fermentation.
  • B. licheniformis introduced a nucleotide sequence encoding an alpha amylase variant by genetic engineering. More preferably, the B. licheniformis of the present invention has removed the resistance screening gene, is environmentally friendly and produces alpha amylase variants more suitable for use in the food industry.
  • pYF-tsDE (Fig. 1) is a temperature sensitive E. coli/B.licheniformis shuttle plasmid.
  • the plasmid consists of a temperature-sensitive replication origin (active at 30 ° C) and an erythromycin resistance gene (ErmC), the resistance of which is 300 ug/ml in E. coli, in B.
  • ErmC erythromycin resistance gene
  • the resistance in licheniformis is 5 ug/ml.
  • the origin of replication on the plasmid was inactivated and the plasmid was integrated into the designated site of the host genome and screened with ErmC.
  • the pYF-tsDE plasmid was constructed by double-digesting the plasmid pUC57-KS-erm (combined with Genscript, sequence shown in CN104073458A, Figure 2) with BglII, and recovering the purified 3.8 kbp fragment using T4 ligase (New England Biolabs). Self-ligated, the cloned plasmid is pYF-tsDE. Transformants were propagated in E. coli TOP10 and served as a backbone for all of the following genes.
  • Example 2 Construction of a protease deficient B. licheniformis strain
  • a single alpha amylase variant expression cassette comprises a natural or synthetic promoter sequence, a signal peptide sequence screened from Bacillus, a synthetic ribosome binding site, and one from G.
  • the ⁇ -amylase variant encoding gene of .stearothermophilus and a transcription terminator.
  • This design will be greatly enhanced The expression level of the gene in the host strain and the amount of secretion of the alpha amylase variant.
  • Replacing the alpha amylase variant encoding gene with a specific site on the B. licheniformis cell genome is achieved by plasmid-mediated single cross-homologous recombination.
  • B. licheniformis the activity of extracellular proteases is detrimental to the secretion of heterologous enzymes.
  • Two major extracellular proteases have been identified: subtilisin (AprE), glutamic acid-specific protease (Blase), and large steps of extracellular protease activity in B. licheniformis are derived from these two proteases.
  • 2.1pYF-tsDE was digested with BglII and treated with CIP to inhibit self-ligation;
  • genomic DNA of licheniformis (CICC 22794, purchased from Chinese microbial strain library) was used as a template to amplify about 500 bp homology from both sides of the gene to be deleted by PCR. sequence.
  • the monoclonal clone of licheniformis can be used directly as a genomic DNA template in a PCR reaction after 98 minutes of pre-denaturation at 98 °C.
  • the primers used in the PCR reaction were synthesized by Genscript.
  • the primer sequences are as follows:
  • the primers that amplify the upstream sequence of the Apr gene are:
  • lichApr_F1 TTATTGAGCGGCAGCTTCGACATTGATCAGACCTT
  • lichApr_R1 CCTTACGGCATTCCTCTCAACAGCGGATCTTCAG
  • lichApr_F2 CCTGAAGATCCGCTGTTGAGAGGAATGCCGTAAGG
  • the primers that amplify the downstream sequence of the Blase gene are:
  • the PCR amplification system is 50ul, and the reaction procedure is as follows:
  • Bacillus subtilis B. licheniformis 14580 monoclonal pre-denaturation at 98 ° C, 8 minutes;
  • the PCR product was detected by electrophoresis on a 0.8% agarose gel and purified using an EST kit.
  • the internal deletion fragment of the gene was obtained by overlap extension PCR (SOE), and the specific operations are as follows:
  • PCR was performed using primers XX ⁇ CZ ⁇ F1 and XX ⁇ CZ ⁇ R2 (“XX” for Apr or Blase) by mixing 1:1 molar ratio of the upstream and downstream homologous sequences of each gene as a template.
  • XX for Apr or Blase
  • the above fragment was subsequently recombined into the BglII linearized pYF-tsDE vector using the Clone-EZ cloning kit (provided by Genscript), and the obtained recombinant plasmids were named pYF-tsDE-Apr and pYF-tsDE-Blase, respectively.
  • These recombinant plasmids are temperature-sensitive plasmids in which the contained Apr gene or Blase gene lacks an internal sequence of about 400-500 bp relative to the entire gene.
  • the replacement of different alleles can be achieved by homologous recombination.
  • homologous recombination see CN102124112A, and other well-known methods of homologous recombination in the art can also be used.
  • the method and screening process for transforming the knockout plasmid into the competent cells of licheniformis in this experiment are as follows:
  • the positive clone strain was further transferred to a condition of 37 ° C to allow the temperature sensitive plasmid to be fused to the host genome.
  • several clones were selected and inoculated in 2 ⁇ YT medium for 24 hours and then subcultured. The whole process was repeated 4-5 times (generally 5-7 days) ).
  • the construction of the integration plasmid was carried out in the same manner as the above pYF-tsDE plasmid.
  • a homologous region of about 800 bp was designed upstream and downstream of the AmyE site on the genome, ligated to the side of an alpha amylase variant expression cassette.
  • some bacterial chromosomal DNA fragments and functional synthetic sequences naturally selected from beginning to end are assembled, which are necessary for controlling the expression of the alpha amylase variant gene.
  • a typical amylase expression cassette consists of the following components:
  • a typical alpha amylase variant expression cassette consists of the following elements: a natural or synthetic promoter sequence (SEQ ID NO. 13), a synthetic ribose The body binding site aaaggagg, an alpha amylase variant encoding gene derived from G. stearothermophilus (SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID, respectively) NO. 11) and a synthetic termination sequence (SEQ ID NO. 14).
  • a strong native signal sequence SEQ ID: NO.
  • the complete alpha amylase variant expression cassette was inserted into the BglII site of the linearized pYF-tsDE using the Clone-EZ cloning kit (Genscript), and the resulting temperature-sensitive integrated plasmid was named pYF-tsINT-amy ( image 3).
  • Genscript Clone-EZ cloning kit
  • the synthesis of the above sequence was carried out by Genscript, and the above sequences were sequentially serially connected in series to obtain an ⁇ -amylase enzyme expression cassette.
  • the signal peptide sequence in this framework is selected from Bacillus subtilis and can effectively increase the secretion of alpha amylase.
  • the entire ⁇ -amylase expression cassette (including the upstream and downstream homologous fragments of the amyE gene) was cyclized with BglII linearized pYF-tsDE plasmid (recombinant kit provided by Genscript) using a recombinant technique, and the constructed temperature-sensitive plasmid was named as pYF-tsINT-amy.
  • This plasmid was used for transformation into Bacillus licheniformis with the ApE and Blase protease gene deletion (CICC22794, purchased from the Chinese microbial strain bank), and the AmyE was replaced by the resistance-labeled alpha amylase variant expression cassette.
  • B. licheniformis engineered strains producing alpha amylase variants were stored at -80 °C.
  • Example 4 Shake flask fermentation of alpha amylase variant production
  • the alpha amylase variant has a molecular weight of approximately 53 kD.
  • the alpha amylase variant activity was determined in the same manner as in Example 6.
  • Example 5 ⁇ -amylase variant step-wise feeding fermentation process
  • the genetically engineered B. licheniformis strain cryopreserved at -80 °C obtained in Example 3 was streaked onto agar slants, and cultured at 37 ° C overnight.
  • the agar slant formula was as follows: peptone 1%, yeast extract 0.5%, NaCl 1%, agar powder 2%.
  • seed shake flask formula: maltose syrup 4.0%, peptone 2.0%, yeast extract 0.1%, KH 2 PO 4 0.6%. After 16 hours, all of the seed broth was transferred to a 7 L stainless steel fermentor containing 4 L of medium at 37 ° C, stirring at 350 rpm, and aeration rate of 650 L/H for 12 hours. Fermentor formula: maltose syrup 6.0%, peptone 1.0%, yeast extract 1%, KH 2 PO 4 0.2%, MnCl 2 0.003%.
  • the fermentation pH was then controlled at 5.7 ⁇ 0.2 with 5% phosphoric acid and continuously fed to the fermentor at a rate of 0.5 L/18 hrs at a rate of 1 L/18 hrs for the first 18 hours.
  • the feeding formula was as follows: maltose syrup 48%, peptone 6%, yeast extract 8%.
  • the entire fermentation process lasts for 140-150 hours. All media in the fermentor were collected and centrifuged at 1010 krpm for 30 minutes at 4 ° C. The supernatant after centrifugation was used for alpha amylase variant enzyme activity analysis.
  • the amylase activity was determined using the Bassella amylase activity (BAU). Definition of 1 BAU unit: The amount of enzyme required to liquefy 1 mg of soluble starch in 1 minute at pH 6.0 and 70 °C.
  • the enzyme activity was determined as follows: 20 ml of 20 g/L soluble starch solution was mixed with 5 ml of pH 6.0 phosphate buffer, preheated at 70 ° C for 8 min, and 1.0 ml of diluted enzyme solution was added to accurately react for 5 minutes. Take 1 ml of the reaction solution, add a test tube containing 0.5 ml of 0.1 mol/L hydrochloric acid solution and 5 ml of dilute iodine solution, shake well, and add 0.5 ml 0.1 mol/L. The hydrochloric acid solution and 5 ml of dilute iodine solution were blank, and the absorbance was quickly measured at a wavelength of 660 nm, and the enzyme activity of the test sample was obtained according to the absorbance table.
  • 1BAU amylase activity was determined using the Bassell amylase unit (BAU).
  • BAU Bassell amylase unit
  • One BAU is defined as the amount of enzyme required to liquefy 1 mg of soluble starch in 1 minute at pH 6.0 and 70 °C.
  • tDS dry matter per ton.
  • the amylase expressed and isolated from B. licheniformis cells was first tested with corn starch for the first round of liquefaction. Test conditions: 18 Baume (°Bé), thoroughly mixed, pH adjusted to 5.2 with hydrochloric acid. 0.22 kg/tDS of amylase was added, and the injection temperature was 100 ° C, 105 ° C, 108 ° C, 110 ° C, and 115 ° C, respectively. After 5-8 min, the mixture was flashed and maintained at 95 ° C for 120 min. After liquefaction, the DE and iodine test were carried out, and the protein flocculation and viscosity were observed. The results are shown in Table 1 and Figure 4.
  • the amylase variant can still be liquefied normally under the condition of starch slurry concentration up to 22 ° Bé, indicating that the alpha amylase variant can be liquefied by thick slurry, which effectively saves Factory cost.
  • the ⁇ -amylase variant can still be liquefied normally under the condition of low pH and 0.15-0.3 kg/tDS addition, indicating that the ⁇ -amylase variant has strong tolerance to low pH.
  • the ⁇ -amylase variant can still be liquefied normally, which can effectively reduce the enzyme cost of the factory.
  • the ⁇ -amylase variant liquefaction solution and the Liquozyme Supra liquefaction solution were used, and the saccharification effects of the two were completely the same, indicating that the ⁇ -amylase variant can be applied to the starch sugar industry.
  • Amylase DE (%) Viscosity (mPas)
  • the alpha amylase variant 10.02 11650 Liquozyme SC 10.05 11710
  • the alpha amylase variant and Liquzoyme SC can achieve the same application effect.
  • the series of ⁇ -amylase variants have good heat resistance and pH tolerance, and can be applied to high-concentration starch slurry liquefaction, and thus can be applied to the starch sugar industry and Alcohol industry.

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Abstract

提供了一种α淀粉酶变体，由亲代α淀粉酶氨基酸序列中至少有一个氨基酸残基突变或删除得到，同时仍然保持了亲代水解α-1,4糖苷键的能力的α淀粉酶；二者氨基酸序列同源性达到95％以上。提供的一系列α淀粉酶变体，在pH5.0酸性条件下和100℃以上高温条件下有较高的催化活度。

Description

α淀粉酶变体及其应用 技术领域
本发明属于酶工程领域，涉及α淀粉酶变体及其应用。
背景技术
在工业界，淀粉的水解主要是从α淀粉酶来开始的，这些来源于微生物的α淀粉酶和其他酶种的协同应用，比如说普鲁兰酶，糖化酶及葡萄糖异构酶，可有效分解淀粉大分子，这些产生的小分子多糖或单糖在食品制造、谷物处理、啤酒加工、酒精生产等行业有很多应用，至关重要。α淀粉酶隶属于糖化水解酶的一种，它的主要结构特征是(α/β)8折叠，其中含有特殊的淀粉底物结合位点，长度一般为不超过10个糖单体，但是很多淀粉酶的结合位点在一起作用，就可以实行多点位结合，从而成功剪切淀粉大分子。
α淀粉酶可以有效剪切淀粉底物中的α-1，4糖苷键，从而快速降低淀粉底物的分子量和粘度，产物主要是不同长度的糊精。α淀粉酶有不同的种类，这些种类在工业上的应用条件根据所需产品的性征有很大的不同。
α淀粉酶(α-1,4-glucan-4-glucanohydrolases,E.C.3.2.1.1)能有效水解淀粉和其他的多糖中的α-1，4糖苷键。鉴于在淀粉在水解过程中对酶效率的提高和降低生产成本的需求，寻求在不同应用领域能支持有效淀粉液化的α淀粉酶已成为学术界和工业界的一个重要研发方向。目前利用酶工程的技术改良该酶种的焦点主要在耐热性，酸碱耐受性能的改善和液化效果的提升。
已经从植物和微生物中发现和定义了很多α淀粉酶具有商业价值，主要包括B.licheniformisα淀粉酶，B.amyloliquefaciensα淀粉酶和G.stearothermophilusα淀粉酶，其中以B.licheniformisα-amylase为模板有衍生的变体数量为最多，应用也最广泛。
在本发明中，为适应工业化生产的需要，我们利用G.stearothermophilusα淀粉酶为模板构建了一系列α淀粉酶变体，提高了该酶种的应用效率，特别是在低pH以及减少添加量的情况下液化效率同市场主流产品可相匹配。
发明内容
本发明的目的是提供一系列G.stearothermophilusα淀粉酶变体，该系列变体可以提高液化效率，并能适应工业化生产的需求。特别是在100℃以上的温度和pH为5.0的条件下，本发明的α淀粉酶变体的酶活及其他性质可同市场主流产品相匹配。
本发明的目的是提供编码该α淀粉酶变体的基因。
本发明的又一目的是提供该α淀粉酶变体的生产方法和应用。
本发明的目的可通过以下技术方案实现：
一种α淀粉酶变体，由亲代α淀粉酶氨基酸序列中至少有一个氨基酸残基突变或删除得到，同时仍然保持了亲代水解α-1,4糖苷键的能力的α淀粉酶；二者氨基酸序列同源性达到95％以上。
所述的亲代α淀粉酶优选天然α淀粉酶，即细菌α淀粉酶，进一步优选Bacillus subtilis,B.licheniformis，B.amyloliquefaciens，G.stearothermophilus或Bacillus cereus中的任意一种的α淀粉酶，更进一步优选B.licheniformis或G.stearothermophilus的α淀粉酶，最优选G.stearothermophilus的α淀粉酶。
所述的G.stearothermophilus的α淀粉酶的全长编码基因序列见SEQ ID NO.1；对应的氨基酸序列见SEQ ID NO.2。
所述的α淀粉酶变体优选自以下任意一种或以下三种情况的任意结合：
(1)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第1个至第5个氨基酸残基并替换成VN或ANLN得到；
(2)由G.stearothermophilus的亲代α淀粉酶缺失其C-末端27个至32个氨基酸残基得到；如缺失其C-末端第1～个至第27个氨基酸残基，或缺失其C-末端第1～个至第29个氨基酸残基，或缺失其C-末端第1～个至第32个氨基酸残基；
(3)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第180个和第181个氨基酸残基而获得。
所述的α淀粉酶变体还优选基于上述三种情况或三种情况的任意结合的基础上，其C-末端增加FAN三个氨基酸残基。
所述的α淀粉酶变体的氨基酸序列进一步优选自以下任意一种：SEQ ID NO.4，SEQ ID NO.6，SEQ ID NO.8，SEQ ID NO.10和SEQ ID NO.12。
所述的α淀粉酶变体的核苷酸编码序列选自以下任意一种：SEQ ID NO.3，SEQ ID NO.5，SEQ ID NO.7，SEQ ID NO.9和SEQ ID NO.11。
编码本发明所述的α淀粉酶变体的基因。
所述的基因优选自SEQ ID NO.3，SEQ ID NO.5，SEQ ID NO.7，SEQ ID NO.9和SEQ ID NO.11中的任意一种。
用于表达本发明所述的α淀粉酶变体的表达载体，其中含有权利要求8所述的编码α淀粉酶变体的基因。
所述的表达载体包括主要由一个天然或者合成的启动子序列，一个天然或合成的核糖体结合位点，一个天然或合成的终止子序列，以及本发明所述的编码α淀粉酶变体的基因序列一同组成了一个表达组件。
一种用于表达本发明所述的α淀粉酶变体的重组细胞，其中包含一个或多个本发明所述的编码α淀粉酶变体的基因。
重组细胞的宿主细胞优选自Bacillus菌株,进一步优选B.licheniformis或经过基因工程改造失活了一些内源性蛋白的Bacillus菌株；最优选经过基因工程改造失活了AprE和/或NprE的B.licheniformis。
一种本发明所述的α淀粉酶变体的生产方法，包括在适宜α淀粉酶变体表达的条件下对含有编码α淀粉酶变体基因序列的重组细胞进行培养，并从重组细胞或者其培养上清液中获得α淀粉酶变体。
本发明所述的α淀粉酶变体在水解多糖的α-1,4糖苷键中的应用；优选在高温和/或低pH条件下水解多糖的α-1,4糖苷键中的应用；所述的高温优选80℃～110℃，进一步优选100℃～110℃；所述的低pH优选pH值为5.0～5.5。
有益效果
本发明提供的一系列α淀粉酶变体，在pH 5.0酸性条件下和100℃以上高温条件下有较高的催化活度。这些α淀粉酶变体的耐酸和热稳定性，适用于淀粉液化。
附图说明
图1为pYF-tsDE载体，包括一个温度敏感原件(30℃时有复制活性)，一个红霉素决定基因(ErmC)--在E.coli中可以耐受300μg/mL的红霉素而在B.licheniformis中可以耐受5μg/mL的红霉素。用红霉素筛选出含有编码α淀粉酶变体核苷酸序列的重组宿主细胞。
图2为pUC57-KS-erm载体的示意图，从该载体中可以获得本发明中的pYF-tsDE载体。
图3为pYF-tsINT-amy载体的示意图。
图4蛋白絮凝和粘度情况。
图5不同淀粉浆浓度条件下的液化实验结果图
图6α淀粉酶变体的耐酸性实验结果
本发明的详细描述
除非另有规定，所有技术和科学术语在本发明中的具有相同的含义，通常理解为一般的专业技术。在此应用中，某些术语意义同规范所述。必须指出的是，本文所用的和所附的权利要求，单数形式“一个”“这个”包括复数形式，除非上下文中另有明确规定。
本发明中，α淀粉酶指的是能够水解多糖的α-1,4糖苷键的酶。例如，α淀粉酶能将淀粉水解成糊精。
本发明中，亲代α淀粉酶是指天然的α淀粉酶。天然的α淀粉酶是细菌α淀粉酶，来源包括但不局限于Bacillus subtilis,B.licheniformis，B.amyloliquefaciens，G.stearothermophilus和Bacillus cereus。
根据本发明的优选实施例，天然α淀粉酶是来源于芽孢杆菌菌株----尤其是B.licheniformis和G.stearothermophilus。G.stearothermophilus的全长编码序列见SEQ ID NO.1；对应的氨基酸序列见SEQ ID NO.2。
本发明中，术语“α淀粉酶变体”是指非天然存在的，在亲代的α淀粉酶氨基酸序列的有效点位进行一个或数个氨基酸残基的突变或删除，同时仍然保持了亲代水解α-1,4糖苷键的能力的α淀粉酶。
本发明中“液化”一般是指将碳水化合物分解为小分子的多糖的过程。当加入α淀粉酶或者α淀粉酶变体时，“液化”特指水解碳水化合物的α-1,4糖苷键。
本发明中，“α-1,4糖苷键”指将前一个葡萄糖的C1与后一个葡萄糖的C4连接起来的键，即为α-1,4糖苷键。
本发明涉及将亲代α淀粉酶做序列改造获得的“α淀粉酶变体”。亲代α淀粉酶是天然的α淀粉酶，特别来源于细菌的天然α淀粉酶。根据本发明的实施例，α淀粉酶变体是将亲代的α淀粉酶的氨基酸序列的有效点位进行一个或数个氨基酸残基的突变或删除。
本发明包括一系列的α淀粉酶变体。根据本发明的实施例，这一系列的α淀粉酶变体的氨基酸序列的同源性至少达到95％，分别达到95％，96％，97％，98％，99％或者100％。
作为说明性和非限制性的本发明示例，α淀粉酶变体选自以下任意一种：
(1)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第1个至第5个氨基酸残基并替换成VN，且缺失C-末端第1个至第27个氨基酸残基得到，氨基酸序列如SEQ ID NO.4所示；
(2)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第1个至第5个氨基酸残基并替换成ANLN，缺失其N-末端第180个和第181个氨基酸残基，且缺失C-末端第1个至第27个氨基酸残基得到，氨基酸序列如SEQ ID NO.6所示；
(3)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第1个至第5个氨基酸残基并替换成ANLN，缺失其N-末端第180个和第181个氨基酸残基，且缺失C-末端第1个至第29个氨基酸残基得到，氨基酸序列如SEQ ID NO.8所示；
(4)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第1个至第5个氨基酸残基并替换成ANLN，缺失其N-末端第180个和第181个氨基酸残基，且缺失C-末端第1个至第32个氨基酸残基得到，氨基酸序列如SEQ ID NO.10所示；
(5)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第1个至第5个氨基酸残基并替换成ANLN，缺失其N-末端第180个和第181个氨基酸残基，缺失C-末端第1个至第27个氨基酸残基，并在C-末端增加FAN三个氨基酸残基得到，氨基酸序列如SEQ ID NO.12所示；
的氨基酸序列选自，SEQ ID NO.6，SEQ ID NO.8，SEQ ID NO.10和SEQ ID NO.12中的任意一种。
本发明中的α淀粉酶变体保持了水解α-1,4糖苷键的能力。另外，这些α淀粉酶的性能使用上符合工业生产要求，比如，液化效率的提高，酸性pH或高温条件下催化活性稳定。
根据本发明的实施例，一个α淀粉酶变体在pH 5.0的酸性条件或者温度高于100℃的条件(尤其是温度在100℃-110℃之间)下，催化活性稳定。α淀粉酶变体这些提高的性能更适应淀粉工业的液化反应。因为在淀粉工业上液化工艺常常在低pH和高温条件下进行。
本发明的所有α淀粉酶变体都可以用于液化反应。在优选的实施例中，α淀粉酶变体来源于亲代α淀粉酶，尤其是来源于G.stearothermophilus亲代的α淀粉酶。在特定的优选实施例中，α淀粉酶变体氨基酸序列见SEQ ID NO.4，SEQ ID NO.6，SEQ ID NO.8，SEQ ID NO.10和SEQ ID NO.12。
根据本发明，任何含有α-1,4糖苷键的碳水化合物都可以用于液化反应。含有一个或多个α-1,4糖苷键的碳水化合物包括但不仅限于淀粉，支链淀粉，直链淀粉和葡萄聚糖。
很多碳水化合物含有α-1,6-糖苷键和α-1,4-糖苷键，比如，支链淀粉。“α-1,4-糖苷键”是指前一个葡萄糖的C1与后一个葡萄糖的C4连接起来的键，即为α-1,4糖苷键。因此，本发明的α淀粉酶变体可以与能够水解α-1,6糖苷键的普鲁兰酶在糖化过程中配合使用。能够水解α-1,4糖苷键的酶包括但不仅限于α淀粉酶。在本发明的优选实施例中，催化水解α-1,4糖苷键的酶为α淀粉酶。
因此，根据本发明的实施例，进一步催化糖化反应提高效率的方法是配合使用普鲁兰酶。本发明中，“普鲁兰酶”是指能够水解α-1,6糖苷键的水解酶。
本发明中的α淀粉酶与普鲁兰酶配合在淀粉的糖化过程中使用能够提高葡萄糖和麦芽糖的纯度。此外，在糖化反应使用上述复合酶能有效减少底物浓度，提高转化效率，在酸性pH或更高的温度下也能有更高的催化活性，更能适应工业上水解淀粉的条件。
本发明提供了一种α淀粉酶变体能够在任何适合工业化生产的温度和pH条件下水解α-1，4糖苷键进行糖化反应的方法。根据本发明，液化反应可以在80℃至110℃的高温下反应，比如80℃，90℃，100℃，105℃和110℃。糖化反应也能在pH5.0至pH5.5的酸性pH条件下进行，比如pH5.0，5.1,5.2,5.3,5.4和5.5。
根据本发明的实施例，α淀粉酶变体催化的液化反应在酸性pH和100℃以上温度条件下催化活性稳定。
另一方面，本发明中的表达载体包含一个合成的编码α淀粉酶变体的核苷酸序列，而重组宿主细胞包含了上述表达载体。表达载体含有一系列合成的编码不同α淀粉酶变体的核苷酸序列。表达载体能够整合至宿主细胞的基因组上。例如，表达载体含有合成的核苷酸序列SEQ ID NO.3，SEQ ID NO.5，SEQ ID NO.7，SEQ ID NO.9和SEQ ID NO.11。
本发明中的表达载体优选包括一个天然或者合成的启动子序列，一个天然或合成的核糖体结合位点，一个天然或合成的终止子序列。这些遗传原件与合成的α淀粉酶变体编码序列一同组成了一个表达组件，表达组件与载体骨架构成了表达载体。例如，表达载体包括一个 表达组件，而表达组件包括以下的元件：一个启动子序列，一个合成的核糖体结合位点，一个合成的编码本发明中α淀粉酶变体的核苷酸序列和一个终止子序列。信号序列能够指导α淀粉酶变体的分泌，将信号序列引入表达载体或者表达组件，尤其是将信号序列引入起始密码子的上游更有利于α淀粉酶变体的分泌。
根据本发明优选实施例，表达载体适宜在细菌中表达，尤其是Bacillus菌株,更适宜在B.licheniformis中表达。在特别优选实施例中，表达载体能够整合到Bacillus的基因组上，尤其是B.licheniformis的基因组上。可用于整合到染色体中的多核苷酸序列的宿主细胞的表达载体，以及这种表达载体的构建方法，是当代生物学领域众所周知的一个普通技能。
根据本发明实施例，重组宿主细胞可以基因工程改造包含一个或多个α淀粉酶变体基因表达的核酸序列。任何技术均可用于基因工程改造宿主细胞包含一个或多个本发明中的α淀粉酶变体编码合成的核酸序列，例如，染色体整合。含有温度敏感起源和抗性筛选标记的载体可以用于整合步骤。这些载体通过坎贝尔机制与基因组的特定区域整合，通过抗性筛选得到重组菌，重组菌在随后的培养过程中通过同源重组去掉抗性筛选标记。
根据本发明实施例，重组宿主细胞经过基因工程改造失活了一些内源性蛋白。能被失活的内源性包括但不限于胞外的蛋白酶。重组的宿主细胞转化含有α淀粉酶变体表达基因的核酸序列之前或者之后要失活一些内源性蛋白。更适宜的方法是，在转入α淀粉酶变体表达基因的载体之前进行宿主菌外源分泌蛋白酶的失活。
首先，B.licheniformis经过改造已经失活了一些外源性蛋白酶基因。特别是B.licheniformis菌株可以失活一些胞外蛋白酶，例如subtilisin(AprE)，glutamic acid-specific protease(Blase)。这些基因工程改造使得B.licheniformis菌株更适宜α淀粉酶变体的表达和分泌。
本发明提供了一种α淀粉酶变体的生产方法。根据本发明的实施例，该方法包括在适宜α淀粉酶变体表达的条件下对含有编码α淀粉酶变体核苷酸序列的重组宿主细胞进行培养，并从重组宿主细胞或者其上清液中获得α淀粉酶变体。
本发明的所有重组宿主细胞都能够生产α淀粉酶变体。重组宿主细胞包含了至少一个拷贝的编码α淀粉酶变体的核苷酸序列。这些编码α淀粉酶变体的核苷酸序列能够在适宜的条件下表达α淀粉酶变体。从重组宿主细胞中分泌的α淀粉酶变体能够从重组细胞或上清液中收集到。收集的方法包括但不仅限于过滤，离心等。
根据本发明的实施例，通过发酵经过基因工程改造B.licheniformis能够高产α淀粉酶变体。B.licheniformis通过基因工程改造导入了编码α淀粉酶变体的核苷酸序列。更好的是，本发明中的B.licheniformis已经去掉了抗性筛选基因，对环境无害并且生产的α淀粉酶变体更适合用于食品工业。
本发明中下列的例子进一步阐述了本发明的本质。应当理解，下面的例子不限制本发明，本发明的范围是由所附的权利要求确定。
具体实施方式
实施例1:pYF-tsDE质粒的构建
pYF-tsDE(图1)是一个温敏型的E.coli/B.licheniformis穿梭质粒。该质粒由一个温度敏感型的复制起点(在30℃有活性)和一个红霉素抗性基因(ErmC)组成，该抗性基因在E.coli中的抗性是300ug/ml，在B.licheniformis中的抗性是5ug/ml。在37℃时，质粒上的复制起点失活，质粒被整合到宿主菌基因组的指定位点，用ErmC进行筛选。
pYF-tsDE质粒的构建过程为：将质粒pUC57-KS-erm(委托Genscript合成,序列见CN104073458A，图2)用BglII双酶切，回收纯化3.8kbp的片段，用T4连接酶(New England Biolabs)自连，克隆好的质粒就是pYF-tsDE。转化子在E.coli TOP10中进行繁殖，并且作为以下所有基因操作的骨架。
实施例2:蛋白酶缺陷型B.licheniformis菌株的构建
作为重组酶产品宿主细胞的遗传工程菌株已经有文献报道(Widner et al.,Journal of Industrial Microbiology&Biotechnology,25,204-212,2000)。这些重组的宿主细胞一般包含一个或多个编码目标序列的核酸结构用以酶的表达。在本发明中，B.licheniformis被用作基因操作的受体菌。Bacillus的转化目前可以通过非常成熟的手段来达到，如感受态细胞转化，电转化和原生质体转化(Young et al.,J Bacteriology,81,823-829,1961；Shigekawa et al.,Biotechniques,6,742-751,1988；Chang et al.,Molecular General Genetics,168,111-115,1979)。
在本发明中，一个单独的α淀粉酶变体表达框，包含自然的或合成的启动子序列，一个从杆菌中筛选到的信号肽序列，一个合成的核糖体结合位点，一个来自于G.stearothermophilus的α淀粉酶变体编码基因，和一个转录终止子。这样的设计将大大增强 宿主菌株中基因的表达水平和α淀粉酶变体的分泌量。将α淀粉酶变体编码基因替换掉B.licheniformis细胞基因组上的特定位点是通过质粒介导的单交叉同源重组来实现的。
在B.licheniformis中，胞外蛋白酶的活性对异种酶的分泌是不利的。现已证实有2种主要的胞外蛋白酶：subtilisin(AprE)，glutamic acid-specific protease(Blase)，在B.licheniformis中大步伐的胞外蛋白酶活性都是源自于这两种蛋白酶。
在本发明中，为了获得α淀粉酶变体基因表达的结构整体性，上述两个基因被灭活，采用的是连续性方式单交叉坎贝尔型机制。具体操作如下：
2.1pYF‐tsDE经由BglII酶切后用CIP处理来抑制自连；
2.2基因敲除
(1)为了能够获得每个基因缺失片段，以地衣杆菌(CICC 22794，中国微生物菌种库购得)基因组DNA为模板用PCR的方法从要缺失的基因两侧各扩增大约500bp的同源序列。地衣杆菌的单克隆经过98℃,5分钟预变性后可以作为基因组DNA模板直接在PCR反应中使用。
用于PCR反应的引物是由Genscript公司合成的。引物序列如下：
扩增Apr基因上游序列的引物为：
lichApr_F1    TTATTGAGCGGCAGCTTCGACATTGATCAGACCTT
lichApr_R1    CCTTACGGCATTCCTCTCAACAGCGGATCTTCAG
扩增Apr基因下游序列的引物为：
lichApr_F2    CCTGAAGATCCGCTGTTGAGAGGAATGCCGTAAGG
lichApr_R2    ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGAC
扩增Blase基因上游序列的引物为：
blalich_F1    TTATTGTGCGCTGTTTTTCCAGTTGGTCAAATTGTCG
blalich_cR1   CGGACAAGGGTCACCAACGGGACAACTGTTACCATC
扩增Blase基因下游序列的引物为：
blalich_cF2   GATGGTAACAGTTGTCCCGTTGGTGACCCTTGTCC
blalich_R2    CGGCGTTGGTTAGTAAAAAGAGTGTTAAACGAGGTTTGAT
PCR扩增体系为50ul，反应程序如下：
(1)枯草芽孢杆菌B.licheniformis 14580单克隆预变性98℃，8分钟；
(2)96℃，15秒；
(3)58℃，15秒；
(4)72℃，30秒；重复2‐4步骤25‐30次；
(5)终延伸72℃，2分钟。
PCR产物用0.8％琼脂糖胶电泳检测后用爱思进试剂盒纯化。
2.3重叠PCR方法扩增内部大约400‐500bp序列缺失的目的基因
基因内部缺失片段是用重叠PCR方法(overlap extension PCR，SOE)获得的，具体操作如下：
(1)分别回收2.2中各基因上游、下游PCR片段并纯化；
(2)以各目的基因的上、下游同源序列片段1:1摩尔比混合后作为模板，用引物XX‐CZ‐F1和XX‐CZ‐R2(“XX”代表Apr或Blase)PCR扩增获得内部缺失片段的AprE基因或Blase基因。
上述片段随后用Clone‐EZ克隆试剂盒(Genscript公司提供)重组进经过BglII线性化的pYF‐tsDE载体中，获得的重组质粒分别命名为：pYF‐tsDE‐Apr和pYF‐tsDE‐Blase。这些重组质粒为温敏型质粒，其中包含的Apr基因或Blase基因相对于完整基因来说缺失了内部大约400‐500bp的序列。
不同等位基因的替换可通过同源重组来实现。方法参见CN102124112A，也可使用本领域的其他公知的同源重组的方法。
2.4质粒转化
本实验采用将敲除质粒转化到地衣杆菌感受态细胞中方法及筛选过程如下：
(1)将温敏型质粒pYF‐tsDE‐Apr或pYF‐tsDE‐Sig转化地衣杆菌(CICC 22794，中国微生物菌种库购得)感受态细胞；
(2)在30℃的条件下，在LB(每升含蛋白胨10g，酵母膏5g，氯化钠10g)培养基上用红霉素(5ug/ml)抗性来筛选阳性克隆菌株；
(2)再将阳性克隆菌株转移到37℃的条件下培养，使该温敏质粒能够融合到宿主基因组上。为了使基因在设定的位点发生替换，挑选几个克隆同时接种于2×YT培养基中连续培养24小时后重新继代一次，整个过程继代4‐5次(一般需要5‐7天)。
(3)筛选红霉素敏感的枯草杆菌细胞进行PCR鉴定.可同时用1％的脱脂牛奶LB平板观察透明水解圈，敲除后的菌种应显示显著缩小的水解圈。
鉴定所用PCR引物：
AprE：Apr‐seqF1/Apr‐seqR3
Blase：Blase‐seqF1/Blase‐seqR3
Apr-seqF1：GCCAGGTTGAAGCGGTCTATTCAT
Apr‐seqR3：TACGGCCATCCGACCATAATGGAAC
Blase‐seqF1：GAAGAGCCGGTCACAATTGC
Blase‐seqR3：GGCCGTTAGATGTGACAGCC
实施例3:α淀粉酶变体菌株的整合构建
3.1淀粉酶表达框架构建
整合质粒的构建采用上述pYF-tsDE质粒同样的方法。为了将表达框整合到设计的基因组上的AmyE位点，在基因组上的AmyE位点的上下游设计800bp左右的同源区域，连接在一个α淀粉酶变体表达框两侧。同时组装了一些从头至尾自然选择的细菌染色体DNA片段和功能的合成序列，这些都是控制表达α淀粉酶变体基因所必须的。
一个典型的淀粉酶表达框有以下几个部分构成：一个典型的α淀粉酶变体表达框由以下元件组成：一个天然的或合成的启动子序列(SEQ ID NO.13)，一个合成的核糖体结合位点aaaggagg，一个源自于G.stearothermophilus的α淀粉酶变体编码基因(分别为SEQ ID NO.3，SEQ ID NO.5，SEQ ID NO.7，SEQ ID NO.9或SEQ ID NO.11)和一个合成的终止序列(SEQ ID NO.14)。一个从枯草芽孢杆菌中筛选的强的天然信号序列(SEQ ID:NO.15)被插入到α淀粉酶变体编码基因启动子的上游，用以增强表达酶的分泌效率。用Clone-EZ克隆试剂盒(Genscript)将完整的α淀粉酶变体表达框插入线性化的pYF-tsDE中的BglII位点，最终得到的温敏型整合质粒被命名为pYF-tsINT-amy(图3)。上述序列的合成由Genscript公司来完成，将上述序列依次无缝串联得到α淀粉酶酶表达框。本框架中信号肽序列是从枯草芽孢杆菌中筛选出的，能够有效的提高α淀粉酶的分泌。
3.2质粒转化
将上述整个α淀粉酶表达框(包含amyE基因上下游同源片段)利用重组技术环化BglII线性化的pYF-tsDE质粒(重组试剂盒由Genscript公司提供)，构建好的温敏型质粒命名为pYF-tsINT-amy。该质粒用于转化至AprE及Blase蛋白酶基因缺失的地衣芽孢杆菌中(CICC22794，中国微生物菌种库购得)，无抗性标记的α淀粉酶变体表达框将替换AmyE。采用上述的方法，成功整合α淀粉酶变体编码基因至B.licheniformis染色体上的菌株在蓝色淀粉平板上产生透明圈，PCR进一步验证表达框被整合在受体菌株的AmyE位点。
产α淀粉酶变体的B.licheniformis工程菌株在-80℃保存。
实施例4:α淀粉酶变体生产的摇瓶发酵
取一个活化的细菌单克隆(含有α淀粉酶变体表达框)，接种到20ml培养基中(含有麦芽糖浆4.0％，蛋白胨2.0％，酵母粉0.1％KH2PO4 0.6％以及相应抗生素)培养到对数期。取1.2ml培养液接种到30ml培养基中(含有麦芽糖浆12.0％，蛋白胨1.0％，酵母粉1％KH2PO4 0.2％，MnCl2 0.003％)，在往复摇床中120rpm振荡培养3天。分别在24小时，48小时和72小时取样1ml，1000rpm离心1min。将上清液保存，做SDS-PAGE分析。α淀粉酶变体分子量约为53kD。
测定α淀粉酶变体活性，方法同实施例6。
实施例5:α淀粉酶变体分步补料发酵工艺
将实施例3中得到的-80℃冷冻保存的基因工程B.licheniformis菌株划线与琼脂斜面上，37℃过夜恢复培养。琼脂斜面配方如下：蛋白胨1％，酵母提取物0.5％，NaCl 1％，琼脂粉2％。
首先，选取数个新鲜克隆在盛有50ml培养基的种子摇瓶中于37℃培养16小时。种子摇瓶配方：麦芽糖浆4.0％，蛋白胨2.0％，酵母提取物0.1％，KH2PO4 0.6％。16小时后，将所有的种子发酵液全部转移至盛有4L培养基的7L不锈钢发酵罐中于37℃，搅拌速度350rpm，通气速率为650L/H的条件下持续发酵12小时。发酵罐配方：麦芽糖浆6.0％，蛋白胨1.0％，酵母提取物1％，KH2PO4 0.2％，MnCl2 0.003％。然后用5％磷酸控制发酵pH在5.7±0.2左右，并在前18小时以速率1L/18hrs后110小时以速率0.5L/18hrs向发酵罐中不断补料。补料配方如下：麦芽糖浆48％，蛋白胨6％，酵母提取物8％。整个发酵过程持续140-150小时。收集并在4℃，1010krpm，30分钟离心发酵罐中所有培养基，离心后的上清用于α淀粉酶变体酶活分析。
实施例6:淀粉酶活力测定
淀粉酶活力测定用的是百斯杰淀粉酶活力(BAU)。1个BAU单位的定义：在pH6.0、70℃条件下，1分钟液化1mg可溶性淀粉所需要的酶量。
简单的说，酶活测定如下操作：20ml 20g/L可溶性淀粉溶液与5ml pH6.0磷酸缓冲液混合，先在70℃预热8min，加入1.0ml稀释后的酶液，准确反应5分钟，然后取1ml反应液，加入预先盛有0.5ml 0.1mol/L盐酸溶液和5ml稀碘液的试管中，摇匀，并以0.5ml 0.1mol/L 盐酸溶液和5ml稀碘液为空白，于660nm波长下，迅速测定其吸光值，根据吸光度查表，获得测试样品的酶活。
实施例7:淀粉酶的应用
以下所有结果都是基于α淀粉酶变体的序列SEQ ID NO.8。
除另外说明的，1BAU:淀粉酶的活力测定用的是百斯杰淀粉酶单位(BAU)。一个BAU被定义为在pH6.0、70℃条件下，1分钟液化1mg可溶性淀粉所需要的酶量。
tDS:每吨干物质。
从B.licheniformis细胞中表达并分离获得的淀粉酶首先用玉米淀粉进行第一轮液化测试。测试条件：18波美度(°Bé)、充分混匀、pH用盐酸调节至5.2。加入0.22kg/tDS的淀粉酶，分别采用喷射温度100℃、105℃、108℃、110℃、115℃，维持5-8min后闪蒸，95℃维持120min。液化后进行DE和碘试测试，同时注意观察蛋白絮凝和粘度情况，结果见表格1和图4。
表1:不同喷射温度条件下，淀粉酶液化应用比较
温度(℃) DE(％)
100 17.64
105 14.91
108 10.62
110 10.21
115 3.06
结果显示，在不同喷射温度情况下，100℃液化过头，105℃液化正合适，而且蛋白絮凝好，而在108℃和110℃，液化效果仍较好，蛋白絮凝正常，说明该α淀粉酶变体具有较好的耐热性，115℃液化很差，说明该α淀粉酶变体无法耐受115℃高温。
其次，我们通过在不同淀粉浆浓度条件下的液化实验，测定了该α淀粉酶变体对高底物浓度的耐受性。液化反应条件同上所述，喷射温度为108℃。结果见表2和图5。
表2:不同淀粉浆浓度下，淀粉酶液化应用比较
波美度(°Bé) DE(％)
15 8.58
18 10.73
20 12.10
22 14.29
如表2所示，在不同淀粉浆浓度条件下，淀粉浆浓度高达22°Bé条件下，该α淀粉酶变体仍能正常液化，说明该α淀粉酶变体可以进行浓浆液化，有效节约工厂成本。
然后，我们测定了该α淀粉酶变体的耐酸性，同时进行不同加酶量条件下的液化性能。液化反应条件同上所述，pH为5.0，加酶量分别为0.05、0.1、0.15、0.2、0.22、0.25、0.3kg/tDS。结果见表3和图6。
表3:在pH5.0下，该α淀粉酶变体不同加酶量液化应用比较
加酶量(kg/tDS) DE(％)
0.05 3.41
0.1 6.18
0.15 8.04
0.2 8.12
0.22 9.81
0.25 10.41
0.3 11.77
如表3所示，在低pH、0.15-0.3kg/tDS添加量条件下，该α淀粉酶变体仍能正常液化，说明该α淀粉酶变体对低pH具有较强的耐受性，同时在0.15kg/tDS低加酶量条件下，该α淀粉酶变体仍能正常液化，这可有效降低工厂用酶成本。
另外，我们进行了该α淀粉酶变体对糖化的影响测试，同时以Liquozyme Supra(购自Novozymes)液化制得的液化液进行对比，测试条件：32％干物质(DS)、充分混匀、pH用盐酸调节至4.3。加入0.45kg/tDS复合糖化酶，200ml的反应在60℃分别反应24和48小时。样品经0.22um膜过滤及100℃灭活后用于HPLC分析。结果见表格4。
表4:该α淀粉酶变体对糖化的影响
Figure PCTCN2017074724-appb-000001
Figure PCTCN2017074724-appb-000002
如表4所示，使用该α淀粉酶变体液化液和Liquozyme Supra液化液，两者糖化效果完全一样，说明该α淀粉酶变体可以应用于淀粉糖行业。
最后，因为α淀粉酶在酒精工业生产上有重要应用，我们还测试了该α淀粉酶变体在酒精生产上的液化效果，配制1：2.5料水比的玉米粉(40目)，用盐酸调节pH至5.8，加入0.145kg/tDS该α淀粉酶变体。在95℃蒸煮液化120min。反应结束后，测定样品DE和粘度，同时与Liquzoyme SC(购自Novozymes)进行对比试验，。结果见表5.
表5:该α淀粉酶变体在玉米酒精液化上的应用比较
淀粉酶 DE(％) 粘度(mPas)
该α淀粉酶变体 10.02 11650
Liquozyme SC 10.05 11710
如表5所示，该α淀粉酶变体与Liquzoyme SC可以达到同样的应用效果。说明其可以应用于玉米酒精工业。
综上所述，依照本发明中的实验结果，该系列α淀粉酶变体有较好的耐热性、pH耐受性，能应用于高浓度淀粉浆液化，因此能应用于淀粉糖行业和酒精行业。
本发明的实施例除了应用了本领域内的技术方法外，更离不开发明主旨的指导。因此，本发明不仅限于所公开的具体实施例，更要覆盖本发明精神和范围内的修订条款，详见权利要求。

Claims (18)

  1. 一种α淀粉酶变体，其特征在于由亲代α淀粉酶氨基酸序列中至少有一个氨基酸残基突变或删除得到，同时仍然保持了亲代水解α-1,4糖苷键的能力的α淀粉酶；二者氨基酸序列同源性达到95％以上。
  2. 根据权利要求1所述的α淀粉酶变体，其特征在于所述的亲代α淀粉酶为细菌α淀粉酶，选自Bacillus subtilis,B.licheniformis，B.amyloliquefaciens，G.stearothermophilus或Bacillus cereus中的任意一种的α淀粉酶。
  3. 根据权利要求2所述的α淀粉酶变体，其特征在于所述的亲代α淀粉酶为B.licheniformis或G.stearothermophilus的α淀粉酶，优选G.stearothermophilus的α淀粉酶。
  4. 根据权利要求3所述的α淀粉酶变体，其特征在于所述的G.stearothermophilus的α淀粉酶的全长编码基因序列见SEQ ID NO.1；对应的氨基酸序列见SEQ ID NO.2。
  5. 根据权利要求4所述的α淀粉酶变体，其特征在于所述的α淀粉酶变体选自以下任意一种或以下三种情况的任意结合：
    (1)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第1个至第5个氨基酸残基并替换成VN或ANLN得到；
    (2)由G.stearothermophilus的亲代α淀粉酶缺失其C-末端27个至32个氨基酸残基得到；
    (3)由G.stearothermophilus的亲代α淀粉酶缺失其N-末端第180个和第181个氨基酸残基而获得。
  6. 根据权利要求5所述的α淀粉酶变体，其特征在于所述的α淀粉酶变体的C-末端增加FAN三个氨基酸残基。
  7. 根据权利要求5或6所述的α淀粉酶变体，其特征在于所述的α淀粉酶变体的氨基酸序列选自以下任意一种：SEQ ID NO.4，SEQ ID NO.6，SEQ ID NO.8，SEQ ID NO.10和SEQ ID NO.12。
  8. 根据权利要求7所述的α淀粉酶变体，其特征在于所述的α淀粉酶变体的核苷酸编码序列选自以下任意一种：SEQ ID NO.3，SEQ ID NO.5，SEQ ID NO.7，SEQ ID NO.9和SEQ ID NO.11。
  9. 编码权利要求1～6中任一项所述的α淀粉酶变体的基因。
  10. 根据权利要求9所述的基因，其特征在于选自SEQ ID NO.3，SEQ ID NO.5，SEQ ID NO.7，SEQ ID NO.9和SEQ ID NO.11中的任意一种。
  11. 用于表达权利要求1～6中任一项所述的α淀粉酶变体的表达载体，其特征在于含有权利要求8所述的编码α淀粉酶变体的基因。
  12. 根据权利要求11所述的表达载体，其特征在于所述的表达载体包括主要由一个天然或者合成的启动子序列，一个天然或合成的核糖体结合位点，一个天然或合成的终止子序列，以及权利要求8所述的编码α淀粉酶变体的基因序列一同组成了一个表达组件。
  13. 一种用于表达权利要求1～6中任一项所述的α淀粉酶变体的重组细胞，其特征在于包含一个或多个权利要求9所述的编码α淀粉酶变体的基因。
  14. 根据权利要求13所述的重组细胞，其特征在于重组细胞的宿主细胞选自Bacillus菌株,优选B.licheniformis或经过基因工程改造失活了一些内源性蛋白的Bacillus菌株。
  15. 根据权利要求14所述的重组细胞，其特征在于重组细胞的宿主细胞选自经过基因工程改造失活了AprE和/或NprE的B.licheniformis。
  16. 一种权利要求1～6中任一项所述的α淀粉酶变体的生产方法，其特征在于包括在适宜α淀粉酶变体表达的条件下对含有编码α淀粉酶变体基因序列的重组细胞进行培养，并从重组细胞或者其培养上清液中获得α淀粉酶变体。
  17. 权利要求1～6中任一项所述的α淀粉酶变体在水解多糖的α-1,4糖苷键中的应用。
  18. 根据权利要求17所述的应用，其特征在于所述的α淀粉酶变体在高温和/或低pH条件下水解多糖的α-1,4糖苷键中的应用；所述的高温优选80℃～110℃，进一步优选100℃～110℃；所述的低pH优选pH值为5.0～5.5。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108699544B (zh) * 2016-02-26 2021-10-26 南京百斯杰生物工程有限公司 α淀粉酶变体及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102388132A (zh) * 2009-04-01 2012-03-21 丹尼斯科美国公司 包含α淀粉酶和蛋白酶的清洁系统
WO2013184577A1 (en) * 2012-06-08 2013-12-12 Danisco Us Inc. Alpha-amylase variants derived from the alpha amylase of cytophaga sp.amylase|(cspamy2).
CN103781910A (zh) * 2011-07-06 2014-05-07 诺维信公司 α淀粉酶变体及其编码多核苷酸

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR000862A1 (es) * 1995-02-03 1997-08-06 Novozymes As Variantes de una ó-amilasa madre, un metodo para producir la misma, una estructura de adn y un vector de expresion, una celula transformada por dichaestructura de adn y vector, un aditivo para detergente, composicion detergente, una composicion para lavado de ropa y una composicion para la eliminacion del
WO2005111203A2 (en) * 2004-04-08 2005-11-24 Genencor International, Inc. MUTANT α ΑMYLASES
RU2426775C2 (ru) * 2006-04-04 2011-08-20 Новозимс А/С Способ получения сусла
EP2215201A2 (en) * 2007-11-05 2010-08-11 Danisco US Inc. Alpha-amylase variants with altered properties
MX2010008358A (es) * 2008-02-04 2010-08-30 Danisco Us Inc Variantes de alfa-amilasa de bacillus stearothermophilus y usos de las mismas.
CN102057040A (zh) * 2008-06-06 2011-05-11 丹尼斯科美国公司 具有改良特性的嗜热脂肪土芽孢杆菌α-淀粉酶(AMYS)变体
CN108699544B (zh) * 2016-02-26 2021-10-26 南京百斯杰生物工程有限公司 α淀粉酶变体及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102388132A (zh) * 2009-04-01 2012-03-21 丹尼斯科美国公司 包含α淀粉酶和蛋白酶的清洁系统
CN103781910A (zh) * 2011-07-06 2014-05-07 诺维信公司 α淀粉酶变体及其编码多核苷酸
WO2013184577A1 (en) * 2012-06-08 2013-12-12 Danisco Us Inc. Alpha-amylase variants derived from the alpha amylase of cytophaga sp.amylase|(cspamy2).

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP3421596A4 *
WENDY A. OFFEN ET AL.: "Three-dimensional structure of a variant 'Termamyl-like' Geobacillus stearothermophilus a-amylase at 1. 9A° resolution", ACTA CRYSTALLOGRAPHICA SECTION F STRUCTURAL BIOLOGY COMMUNICATIONS, 1 January 2015 (2015-01-01), pages 66 - 70, XP055412007, ISSN: 2053-230X *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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