CN110982805A - α -L-arabinofuranosidase and related products - Google Patents
α -L-arabinofuranosidase and related products Download PDFInfo
- Publication number
- CN110982805A CN110982805A CN201911410865.4A CN201911410865A CN110982805A CN 110982805 A CN110982805 A CN 110982805A CN 201911410865 A CN201911410865 A CN 201911410865A CN 110982805 A CN110982805 A CN 110982805A
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- China
- Prior art keywords
- arabinofuranosidase
- recombinant
- enzyme
- encoding
- bacillus subtilis
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Abstract
The invention provides α -L-arabinofuranosidase and related products thereof, wherein recombinant α -L-arabinofuranosidase is obtained by secretion of bacillus subtilis, and the bacillus subtilis contains or integrates a target gene for coding α -L-arabinofuranosidase.
Description
Technical Field
The invention relates to the technical field of biology, in particular to α -L-arabinofuranosidase and a related product thereof.
Background
Plant cell walls are a semi-rigid complex structure composed primarily of carbohydrates, proteins, lignin, and water. The carbohydrates of the cell wall are mainly composed of cellulose, hemicellulose and pectin. Cellulose is a linear homopolymer of glucose, hemicellulose is a mixed heteropolysaccharide which is linked to each other and also to cellulose and pectin, and mainly consists of xylan, arabinoxylan, mannan, galactomannan, arabinogalactan, and the like. The biological effect of hemicellulose is to strengthen the cell wall by interactions, its content in wood is approximately between 20% and 40%, and the composition and content varies between the fruits, stems, branches, roots and bark of plants. Xylan in hemicellulose is second only to cellulose and is the second most abundant macromolecular substance on the earth, the main chain of the hemicellulose is xylose residue connected with beta- (1-4) bonds, and the main chain also contains a plurality of side chain substitutes, usually arabinose, ferulic acid, glucuronic acid, acetic acid, coumaric acid and the like.
In order to complete the complete degradation of hemicellulose and pectin in plants, it is critical to cleave α -L-arabinofuranose bond by means of arabinouronic acid enzymes, which act synergistically with other hemicellulases, pectinases, cellulases, etc., the catalytic reaction can be accelerated.
There are two types of a-L-arabinofuranosidases, endo (EC3.2.1.99) and exo (EC3.2.1.55). endo-arabinofuranosidases cleave the arabinofuranose linkages in α -1-5-arabinose, while exo-arabinofuranosidases cleave the linkages of α -1-2-, α -1-3-, and α -1-5-linked arabinose and side chain polysaccharides. arabinofuranosidases have important utility values, e.g., fruits are rich in carbohydrate polysaccharides such as pectin and arabinose, prokaryotic polysaccharides, proteins, and polyphenols tend to precipitate during storage and juice addition, arabinofuranosidases are useful in juice clarification in the juice industry. α -L-arabinofuranosidases are used in conjunction with pectinases to increase the solubility of components in juice, clarity and reducing sugar concentrations, and to increase juice yield. α -L-arabinofuranosidases are currently commercially produced in a wide variety of fungal-derived partial acid polypeptides which are more suitable for performing reactions in a neutral environment.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide α -L-arabinofuranosidase and related products, which is a bacterial neutral α -L-arabinofuranosidase product with high efficiency and good quality for the market.
In order to achieve the above objects and other related objects, the first aspect of the present invention provides a recombinant α -L-arabinofuranosidase having an amino acid sequence shown in SEQ ID NO. 7.
Further, the recombinant α -L-arabinofuranosidase is secreted from Bacillus subtilis, which contains or incorporates a gene of interest encoding α -L-arabinofuranosidase.
In a second aspect, the invention provides a recombinant α -L-arabinofuranosidase encoding polynucleotide encoding the recombinant α -L-arabinofuranosidase.
Further, the polynucleotide kinase sequence is a sequence shown in SEQ ID NO.1 or a sequence with more than 90% homology with the sequence shown in SEQ ID NO. 1.
In a third aspect, the invention provides a recombinant expression vector comprising said polynucleotide encoding α -L-arabinofuranosidase.
Further, the recombinant expression vector is pMK4-PxylA-abfA2, wherein abfA2 is the recombinant α -L-arabinofuranosidase encoding polynucleotide.
Furthermore, the sequence of the recombinant expression vector is shown as SEQ ID NO. 2.
The fourth aspect of the invention provides an engineering bacterium, which contains or integrates the recombinant expression vector.
Further, the engineering bacteria can adopt engineering bacteria commonly used in the field. For example, Bacillus subtilis 6051a can be used as the starting strain.
The fifth aspect of the invention provides a complex enzyme, which comprises the recombinant α -L-arabinofuranosidase as described above.
The sixth aspect of the invention provides a cellulosic ethanol complex enzyme, which comprises the recombinant α -L-arabinofuranosidase
Further, the fiber ethanol compound enzyme also comprises cellobiase and/or xylanase.
Furthermore, the fiber ethanol complex enzyme comprises the following components in parts by weight:
α -25-45 parts of L-arabinofuranosidase;
90-100 parts of cellobiase;
40-60 parts of xylanase.
The seventh aspect of the invention provides a method for producing arabinofuranosidase by fermenting recombinant bacillus subtilis, which comprises extracting bacillus subtilis secretion, wherein the bacillus subtilis contains or integrates a target gene for coding α -L-arabinofuranosidase.
Further, the method specifically comprises the steps of culturing the bacillus subtilis in a proper culture medium, adding xylose when the strain grows to a logarithmic phase, performing fermentation culture, and collecting the secretion of the strain.
Further, the culture conditions are: the temperature is 15-37 ℃, and the rotation speed is 100-300 rpm; to-be-treated bacterium OD600And (3) reaching 0.75-0.85, adding xylose to the final concentration of 1.0% (m/v), and continuing to culture.
Such suitable media can be formulated by the person skilled in the art themselves or obtained commercially.
Preferably, the collection of the obtained secretion requires further purification treatment.
Further, the specific method for collecting the secretion of the thallus is to take fermentation liquor, centrifuge and take supernatant.
An eighth aspect of the present invention provides a method for producing cellulosic ethanol, the method comprising: the cellulosic ethanol is obtained by carrying out enzymolysis on the raw materials by using the cellulosic ethanol complex enzyme.
The amount of the enzyme to be used may be specifically set depending on the kind of the raw material.
Further, the feedstock may be an organic material containing cellulosic ethanol. Such as corn stover or other plant stover of around 80 mesh.
The ninth aspect of the invention provides the use of the recombinant α -L-arabinofuranosidase, the polynucleotide encoding α -L-arabinofuranosidase, the recombinant expression vector, the engineered bacterium, the complex enzyme, or the fibroethanol complex enzyme as described above for preparing fibroethanol.
As described above, the α -L-arabinofuranosidase of the present invention has the following advantageous effects:
1) the invention provides a bacterial gene which is found by extracting metagenome data from a fermented heap of dairy cow compost, and the neutral temperature-resistant α -L-arabinofuranosidase can be efficiently expressed and produced by utilizing a synthetic biology method through various biotechnological means such as gene synthesis, sequencing identification, plasmid construction, plasmid transformation, identification culture, fermentation culture and the like.
2) The invention performs fermentation expression on the produced recombinant bacillus subtilis, and the unit enzyme activity of the recombinant bacillus subtilis reaches more than 780U/mL.
3) The composite cellulose ethanol enzyme prepared from α -L-arabinofuranosidase disclosed by the invention can shorten the degradation time of cellulose straws, improve the yield of reducing sugar by 26-46%, finally improve the yield of cellulose ethanol and obviously reduce the production cost, and verifies the great prospect of α -L-arabinofuranosidase in industrial application.
Drawings
FIG. 1 is a schematic diagram of the structure of expression plasmid pMK4-PxylA-abfA 2.
FIG. 2 is a graph showing the variation of the enzyme activity secreted and expressed by α -L-arabinofuranosidase recombinant producing bacteria with fermentation time.
FIG. 3 is an evaluation of the effect of arabinofuranosidase in the biomass pretreatment in the mass production of cellulosic ethanol on the dissociation of reducing sugars.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, Vol.304, Chromatin (P.M. Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
EXAMPLE 1 construction of recombinant plasmid pMK4-PxylA-abfA2
The expression gene of the arabinofuranosidase is obtained by metagenomic analysis and sequence comparison, is obtained by gene synthesis after codon optimization, is named as abfA2, and the nucleic acid sequence of the expression gene is shown in SEQ ID NO. 1.
SEQ ID NO.1:
atgacagttaaacaagctaaaatgacaatcgataaagaatacaaagttggcgaagttgataaacgtctttacggctctttcatcgaacatcttggccgtgctgtttacgaaggcatctacgaacctgatcatcctgaagctgatgaatctggcttccgtaaagatgttatcaaacttgttaaagaattaaaggttccattcatccgttacccaggcggcaacttcgtttctggctacaactgggaagatggcgttggccctgttgaaaaacgtcctacacgtcttgatcttgcttgggctacaacagaacctaaccttgttggcacaaacgaattcatggattgggctaaacttgttggcgctgaagttaacatggctgttaaccttggcacacgtggcatcgatgctgctcgtaaccttgttgaatactgcaaccatccttctggctcttactactctgatcttcgtaaatctcatggctacaaagaacctcataaaatcaaaacatggtgccttggcaacgaaatggatggcccttggcaaatcggccataaaacagctgctgaatacggccgtatcgctgctgaagctgctaaagttatgaaatggacagatccttctatcgaacttgttgcttgcggctcttctggctctggcatgcaaacattcatcgattgggaaacaacagttcttgatcatacatacgatcatgttgaatacatctctcttcattcttactacggcaaccgtgataacgatcttcctaactaccttgctcgttctcttgatatggatcatttcatcaacacagtagttgctgtatgcgactacatgaaagctaaaaaacgttctaaaaaaacaatccatctttcttacgatgaatggaacgtttggtaccattctaacgaaaaagataaacttgttgaacgttgggaacgtgctcctcatcttcttgaagatatctacaacttcgaagatgctcttcttgttggctgcatgcttatcacaatgcttaaacatgctgatcgtgttaaaatcgcttgccttgctcaacttgttaacgttatcgctcctatcatgacagaaaaaggcggcgaagcttggcgtcaaacaatcttctaccctttcatgcatgcttctgtttacggccgtggcacagctcttcaaacagttgtttcttctcctaaatacgattctaaagatttcacagatgttccttaccttgaatctgtttctgttttcaacgaagaagctgaagaacttacaatcttcgctgttaaccgtgatacagaaggcggccttcaaatcgaagctgatgttcgttctttcgaaggctacgctgtttctgaacatatcgttcttgaacatgaagataacaaagctacaaacgaacaagatcgtaacaacgttgttcctcattctggcggcgatgctaaagtttgcgatggccgtcttacagctcatcttcctaaactttcttggaacgttatccgtcttaaaaaacgttaa
The invention adopts the expression of xylose promoter mediated abfA2, and the plasmid construction process is as follows:
firstly, obtaining an abfA2 fragment and a PxylA fragment by a PCR method respectively, wherein the PxylA fragment comprises a PxylA promoter and an xylR expression element, and taking synthetic DNA and a plasmid pAX01-comk as templates respectively, and the used primers are respectively as follows:
abfA2-F:ggaaatgggatccaaaggaggccataatatgacagttaaacaagctaaaatgacaatc(SEQ ID NO.3)
abfA2-R:gccagtgaattcccggggatccttaacgttttttaagacggataacg(SEQ ID NO.4)
pxylA-F:ttacgccaagcttggctgcagtcgcgatgattaattaattcagaacg(SEQ ID NO.5)
pxylA-R:gattgtcattttagcttgtttaactgtcatattatggcctcctttggatcccatttcc(SEQ ID NO.6)
after the PCR product was identified by agarose gel electrophoresis, the cleaning kit was used for cleaning and the DNA concentration was determined.
Then obtaining a PxylA-abfA2 fragment by an Overlap PCR method, which comprises the following specific operations:
a, first step reaction 20 μ L system, including 2 XPrimeStar 10 μ L, affA 250 ng, PxylA 50ng, add double distilled water to 20 μ L. The annealing temperature was set at 60 ℃, the extension time was 3min 15s, and the number of set cycles was 5. b, the second reaction step is 50 μ L, including 2 XPrimeStar 25 μ L, primer abfA 2-R2.5 μ L, primer pxyl A-F2.5 μ L, unclean product of the first reaction step 1 μ L, double distilled water 19 μ L. The annealing temperature was set at 55 ℃, the extension time was 3min 15s, and the number of set cycles was 30. After the PCR product was identified by agarose gel electrophoresis, the cleaning kit was used for cleaning and the DNA concentration was determined.
Meanwhile, plasmid pMK4 was treated with restriction enzymes Pst I and BamH I using a 50. mu.L system including pMK 41. mu.g, Pst I (Fast Digest) 1.5. mu.L, BamH I (Fast Digest) 1.5. mu.L, 10 Xfast Digest buffer 5. mu.L. After 1h of isothermal treatment at 37 ℃ the cleaning kit was used for cleaning and the DNA concentration was determined.
And finally, constructing plasmids by adopting an In-fusion kit and a method thereof, wherein the connected fragments are the DNA product of the Overlap PCR and pMK4 enzyme digestion linearized plasmid, the transformed host is Escherichia coli DH5 α, screening and culturing are carried out on an LB plate with 100 mu g/mL ampicillin, positive transformants are selected for culturing and plasmids are extracted, the plasmids are submitted to a sequencing company for sequencing, shown In SEQ ID NO.2, the plasmids which are identified and compared to be designed are pMK4-PxylA-abfA2, and the plasmids pMK4-PxylA-abfA2 are used for constructing subsequent strains.
SEQ ID NO.2:
gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttggctgcagtcgcgatgattaattaattcagaacgctcggttgccgccgggcgttttttatgcagcaatggcaagaacgtcccggggagctcctaacttataggggtaacacttaaaaaagaatcaataacgatagaaaccgctcctaaagcaggtgcattttttcctaacgaagaaggcaatagttcacatttattgtctaaatgagaatggactctagaagaaacttcgtttttaatcgtatttaaaacaatgggatgagattcaattatatgatttctcaagataacagcttctatatcaaatgtattaaggatattggttaatccaattccgatataaaagccaaagttttgaagtgcatttaacatttctacatcatttttatttgcgcgttccacaatctcttttcgagaaatattcttttcttctttagagagcgaagccagtaacgctttttcagaagcatataattcccaacagcctcgatttccacagctgcatttgggtccattaaaatctatcgtcatatgacccatttccccagaaaaaccctgaacacctttatacaattcgttgttaataacaagtccagttccaattccgatattaatactgatgtaaacgatgttttcatagttttttgtcataccaaatactttttcaccgtatgctcctgcattagcttcattttcaacaaaaaccggaacattaaactcactctcaattaaaaactgcaaatctttgatattccaatttaagttaggcatgaaaataatttgctgatgacgatctacaaggcctggaacacaaattcctattccgactagaccataaggggactcaggcatatgggttacaaaaccatgaataagtgcaaataaaatctcttttacttcactagcggaagaactagacaagtcagaagtcttctcgagaataatatttccttctaagtcggttagaattccgttaagatagtcgactcctatatcaataccaatcgagtagcctgcattcttattaaaaacaagcattacaggtcttctgccgcctctagattgccctgccccaatttcaaaaataaaatctttttcaagcagtgtatttacttgagaggagacagtagacttgtttaatcctgtaatctcagagagagttgccctggagacaggggagttcttcaaaatttcatctaatattaatttttgattcattttttttactaaagcttgatctgcaatttgaataataaccactcctttgtttatccaccgaactaagttggtgttttttgaagcttgaattagatatttaaaagtatcatatctaatattataactaaattttctaaaaaaaacattgaaataaacatttattttgtatatgatgagataaagttagtttattggataaacaaactaactcaattaagatagttgatggataaacttgttcacttaaatcaaagggggaaatgacaaatggtccaaactagtgatatctaaaaatcaaagggggaaatgggatccaaaggaggccataatatgacagttaaacaagctaaaatgacaatcgataaagaatacaaagttggcgaagttgataaacgtctttacggctctttcatcgaacatcttggccgtgctgtttacgaaggcatctacgaacctgatcatcctgaagctgatgaatctggcttccgtaaagatgttatcaaacttgttaaagaattaaaggttccattcatccgttacccaggcggcaacttcgtttctggctacaactgggaagatggcgttggccctgttgaaaaacgtcctacacgtcttgatcttgcttgggctacaacagaacctaaccttgttggcacaaacgaattcatggattgggctaaacttgttggcgctgaagttaacatggctgttaaccttggcacacgtggcatcgatgctgctcgtaaccttgttgaatactgcaaccatccttctggctcttactactctgatcttcgtaaatctcatggctacaaagaacctcataaaatcaaaacatggtgccttggcaacgaaatggatggcccttggcaaatcggccataaaacagctgctgaatacggccgtatcgctgctgaagctgctaaagttatgaaatggacagatccttctatcgaacttgttgcttgcggctcttctggctctggcatgcaaacattcatcgattgggaaacaacagttcttgatcatacatacgatcatgttgaatacatctctcttcattcttactacggcaaccgtgataacgatcttcctaactaccttgctcgttctcttgatatggatcatttcatcaacacagtagttgctgtatgcgactacatgaaagctaaaaaacgttctaaaaaaacaatccatctttcttacgatgaatggaacgtttggtaccattctaacgaaaaagataaacttgttgaacgttgggaacgtgctcctcatcttcttgaagatatctacaacttcgaagatgctcttcttgttggctgcatgcttatcacaatgcttaaacatgctgatcgtgttaaaatcgcttgccttgctcaacttgttaacgttatcgctcctatcatgacagaaaaaggcggcgaagcttggcgtcaaacaatcttctaccctttcatgcatgcttctgtttacggccgtggcacagctcttcaaacagttgtttcttctcctaaatacgattctaaagatttcacagatgttccttaccttgaatctgtttctgttttcaacgaagaagctgaagaacttacaatcttcgctgttaaccgtgatacagaaggcggccttcaaatcgaagctgatgttcgttctttcgaaggctacgctgtttctgaacatatcgttcttgaacatgaagataacaaagctacaaacgaacaagatcgtaacaacgttgttcctcattctggcggcgatgctaaagtttgcgatggccgtcttacagctcatcttcctaaactttcttggaacgttatccgtcttaaaaaacgttaaggatccccgggaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatccatatccttctttttctgaaccgacttctcctttttcgcttctttattccaattgctttattgacgttgagcctcggaacccttaacaatcccaaaacttgtcgaatggtcggcttaatagctcacgctatgccgacattcgtctgcaagtttagttaagggttcttctcaacgcacaataaattttctcggcataaatgcgtggtctaatttttatttttaataaccttgatagcaaaaaatgccattccaatacaaaaccacatacctataatcgataaccacataacagtcataaaaccactcctttttaacaaactttatcacaagaaatatttaaattttaaatgcctttattttgaattttaaggggcattttaaagatttaggggtaaatcatatagttttatgcctaaaaacctacagaagcttttaaaaagcaaatatgagccaaataaatatattctaattctacaaacaaaaatttgagcaaattcagtgtcgattttttaagacactgcccagttacatgcaaattaaaattttcatgattttttatagttcctaacagggttaaaatttgtataacgaaagtataatgtttatataacgttagtataataaagcattttaacattatacttttgataatcgtttatcgtcgtcatcacaataacttttaaaatactcgtgcataattcaacagctgacctcccaataactacatggtgttatcgggaggtcagctgttagcacttatattttgttattgttcttcctcgatttcgtctatcattttgtgattaatttctcttttttcttgttctgttaagtcataaagttcactagctaaatactctttttgtttccaaatataaaaaatttgatagatatattcggttggatcaatttcttttaagtaatctaaatccccattttttaatttctttttagcctctttaaataatcctgaataaactaatacctgtttacctttaagtgatttataaaatgcatcaaagactttttgatttattaaataatcactatctttaccagaatacttagccatttcatataattctttattattattttgtcttattttttgaacttgaacttgtgttatttctgaaatgcccgttacatcacgccataaatctaaccattcttgttggctaatataatatcttttatctgtgaaatacgatttatttactgcaattaacacatgaaaatgaggattataatcatctctttttttattatatgtaatctctaacttacgaacatatccctttataacactacctactttttttctctttataagttttctaaaagaattattataacgttttatttcattttctaattcatcactcattacattaggtgtagtcaaagttaaaaagataaactcctttttctcttgctgcttaatatattgcatcatcaaagataaacccaatgcatcttttctagcttttctccaagcacagacaggacaaaatcgatttttacaagaattagctttatataatttctgtttttctaaagttttatcagctacaaaagacagaaatgtattgcaatcttcaactaaatccatttgattctctccaatatgacgtttaataaatttctgaaatacttgatttctttgttttttctcagtatacttttccatgttataacacataaaaacaacttagttttcacaaactatgacaataaaaaaagttgctttttcccctttctatgtatgttttttactagtcatttaaaacgatacattaataggtacgaaaaagcaactttttttgcgcttaaaaccagtcataccaataacttaagggtaactagcctcgccggcaatagttacccttattatcaagataagaaagaaaaggatttttcgctacgctcaaatcctttaaaaaaacacaaaagaccacattttttaatgtggtcttttattcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatatttttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattgggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtactttttacagtcggttttctaatgtcactaacctgccccgttagttgaagaaggtttttatattacagctccagatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagaagcggaaga
Example 2 molecular biological construction of α -L-arabinofuranosidase recombinant Bacillus subtilis
The invention selects Bacillus subtilis 6051a as the starting strain to construct the production strain of the arabinofuranosidase. First, the method of preparing the competence of Bacillus subtilis by using a hypertonic solution as reported by Yoshito Sadaie et al in 1983 (Formation of component Tail)lus subtilis cells, J Bacteriol.1983 Feb; 153(2) 813-821) competent cells of Bacillus subtilis 6051a were prepared as follows: culturing bacillus subtilis 6051a in LB culture medium overnight under the conditions of 37 ℃ and 200 rpm; then transferring the overnight culture into 40mL of thallus enrichment medium (LB +90g/L sorbitol), wherein the inoculum size is 1%, and continuing to culture at 37 ℃ and 200 rpm; cultured to OD600After the concentration is about 0.9, placing the thalli on ice to be cooled for 10-20 min, and then collecting the thalli on a freezing centrifugal machine, wherein the centrifugal conditions are set to be 6000rpm, 10min and 4 ℃; the bacillus subtilis competence is prepared by washing the thalli twice by using hypertonic solution (90g/L sorbitol, 92.5g/L mannitol and 10g/L glycerol) and resuspending the thalli.
Bacillus subtilis 6051a was then transformed with plasmid pMK4-PxylA-afbA2 by electroporation. The specific operation is as follows: mu.L of competent cells were taken, 1. mu.g of pMK4-PxylA-abfA2 was added, mixed well, charged into a 2mm electric rotor, and subjected to electric shock at 2.5 kV. After the electric shock, the electric rotor was taken out and 1mL of a recovery medium (LB +90g/L sorbitol +70g/L mannitol) was immediately added thereto, incubated at 37 ℃ for 3 hours at 200rpm, and then coated with a 10. mu.g/mL chloramphenicol-resistant plate for overnight selection. The grown transformant is identified to be a positive transformant through quality-improved particle identification, namely the production strain ABF1 of the arabinofuranosidase.
EXAMPLE 3 fermentative production of α -L-arabinofuranosidase by recombinant Bacillus subtilis Strain
Taking ABF1 as a production strain of α -L-arabinofuranosidase and LB as a fermentation medium for culture as an example, the method specifically comprises the following steps:
selecting single ABF1 colonies, inoculating the single ABF1 colonies into a test tube filled with 3mL of liquid LB culture medium, and culturing the test tube in a constant-temperature shaking incubator at 37 ℃ and 200rpm for 10-12 h; transferring the bacterial liquid into a 250 mL-specification shake flask filled with 30mL of liquid LB, wherein the inoculation amount is 1%. Culturing at 37 deg.C (or 25 deg.C, 16 deg.C) and 200rpm for 4 hr to obtain OD600Reaching about 0.8, adding xylose to the final concentration of 1% (m/v) for induction expression, and then continuing culture and fermentation. Taking the fermentation liquor, centrifuging for 10min at 4 ℃ and 12000rpm, and taking the supernatant after centrifugation for enzyme activity determination. Determination of enzyme ActivitypNPA (4-nitrophenyl α -L-arabinofuranoside) is used as a substrate, the determination conditions are pH 6.5 and the temperature is 45 ℃, and the enzyme amount required for decomposing the pNPA to release 1 mu mol of p-nitrophenol per minute is defined as one enzyme activity unit.
FIG. 2 is a statistical graph of enzyme activity of shake flask fermentation of recombinant Bacillus subtilis ABF1, wherein the enzyme activity of the produced arabinofuranosidase increases with the change of fermentation time, the enzyme activity reaches the highest point after fermentation for 48h, and the highest specific enzyme activity is about 780U/mL. The sequence of the recombinase polypeptide is sequenced, and the result is shown in SEQ ID NO.3 (containing secretion peptide), which is consistent with the expected target object.
SEQ ID NO.7:
GFRKDVIKLVKELKVPFIRYPGGNFVSGYNWEDGVGPVEKRPTRLDLAWA 100
TTEPNLVGTNEFMDWAKLVGAEVNMAVNLGTRGIDAARNLVEYCNHPSGS 150
MKWTDPSIELVACGSSGSGMQTFIDWETTVLDHTYDHVEYISLHSYYGNR 250
DNDLPNYLARSLDMDHFINTVVAVCDYMKAKKRSKKTIHLSYDEWNVWYH 300
SNEKDKLVERWERAPHLLEDIYNFEDALLVGCMLITMLKHADRVKIACLA 350
DFTDVPYLESVSVFNEEAEELTIFAVNRDTEGGLQIEADVRSFEGYAVSE 450
HIVLEHEDNKATNEQDRNNVVPHSGGDAKVCDGRLTAHLPKLSWNVIRLK 500
KR
Example 4 application of arabinofuranosidase in the Mass production of cellulosic ethanol
(1) Preparing 5 groups of compound enzymes A, B, C, D, E, wherein each group of compound enzymes contains 100 g/kg cellobiase (containing about 10 ten thousand units of enzyme activity per kilogram of compound enzyme), 50 g/kg xylanase (containing about 20 ten thousand units of enzyme activity per kilogram of compound enzyme), 25 g/kg α -L-arabinofuranosidase (A group), 30 g/kg (B group), 35 g/kg (C group), 40 g/kg (D group) and 45 g/kg (E group), respectively treating crushed corn straws (about 80 meshes) with the 5 groups of enzymes, adding the crushed corn straws into the straws according to a liquid-solid ratio of 9:1, controlling the reaction temperature at 50 ℃ and the pH value at 4.8-5.0, adding 0.05g of penicillin potassium after the pH value is adjusted, pouring the mixed sample into a stainless steel tank, adjusting the temperature in a constant-temperature water bath fermentation tank to 50 ℃, and testing for 3 days (taking at least one sample per day for testing).
The result is shown in figure 3, which shows that the yield of reducing sugar is increased remarkably with the increase of enzyme amount, and the calculation result shows that the catalytic activity of the cellulose complex enzyme is increased with the increase of the arabinofuranosidase, so that the degradation efficiency of cellulose can be improved remarkably, and therefore, the α -L-arabinofuranosidase is an important component of the cellulose alcohol complex enzyme.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
<110> Hunan Lierkang Bio-Ltd
<120> α -L-arabinofuranosidase and related products
<160>7
<170>SIPOSequenceListing 1.0
<210>1
<211>1509
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
atgacagtta aacaagctaa aatgacaatc gataaagaat acaaagttgg cgaagttgat 60
aaacgtcttt acggctcttt catcgaacat cttggccgtg ctgtttacga aggcatctac 120
gaacctgatc atcctgaagc tgatgaatct ggcttccgta aagatgttat caaacttgtt 180
aaagaattaa aggttccatt catccgttac ccaggcggca acttcgtttc tggctacaac 240
tgggaagatg gcgttggccc tgttgaaaaa cgtcctacac gtcttgatct tgcttgggct 300
acaacagaac ctaaccttgt tggcacaaac gaattcatgg attgggctaa acttgttggc 360
gctgaagtta acatggctgt taaccttggc acacgtggca tcgatgctgc tcgtaacctt 420
gttgaatact gcaaccatcc ttctggctct tactactctg atcttcgtaa atctcatggc 480
tacaaagaac ctcataaaat caaaacatgg tgccttggca acgaaatgga tggcccttgg 540
caaatcggcc ataaaacagc tgctgaatac ggccgtatcg ctgctgaagc tgctaaagtt 600
atgaaatgga cagatccttc tatcgaactt gttgcttgcg gctcttctgg ctctggcatg 660
caaacattca tcgattggga aacaacagtt cttgatcata catacgatca tgttgaatac 720
atctctcttc attcttacta cggcaaccgt gataacgatc ttcctaacta ccttgctcgt 780
tctcttgata tggatcattt catcaacaca gtagttgctg tatgcgacta catgaaagct 840
aaaaaacgtt ctaaaaaaac aatccatctt tcttacgatg aatggaacgt ttggtaccat 900
tctaacgaaa aagataaact tgttgaacgt tgggaacgtg ctcctcatct tcttgaagat 960
atctacaact tcgaagatgc tcttcttgtt ggctgcatgc ttatcacaat gcttaaacat 1020
gctgatcgtg ttaaaatcgc ttgccttgct caacttgtta acgttatcgc tcctatcatg 1080
acagaaaaag gcggcgaagc ttggcgtcaa acaatcttct accctttcat gcatgcttct 1140
gtttacggcc gtggcacagc tcttcaaaca gttgtttctt ctcctaaata cgattctaaa 1200
gatttcacag atgttcctta ccttgaatct gtttctgttt tcaacgaaga agctgaagaa 1260
cttacaatct tcgctgttaa ccgtgataca gaaggcggcc ttcaaatcga agctgatgtt 1320
cgttctttcg aaggctacgc tgtttctgaa catatcgttc ttgaacatga agataacaaa 1380
gctacaaacg aacaagatcg taacaacgtt gttcctcatt ctggcggcga tgctaaagtt 1440
tgcgatggcc gtcttacagc tcatcttcct aaactttctt ggaacgttat ccgtcttaaa 1500
aaacgttaa 1509
<210>2
<211>8635
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccaggctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgg 240
ctgcagtcgc gatgattaat taattcagaa cgctcggttg ccgccgggcg ttttttatgc 300
agcaatggca agaacgtccc ggggagctcc taacttatag gggtaacact taaaaaagaa 360
tcaataacga tagaaaccgc tcctaaagca ggtgcatttt ttcctaacga agaaggcaat 420
agttcacatt tattgtctaa atgagaatgg actctagaag aaacttcgtt tttaatcgta 480
tttaaaacaa tgggatgaga ttcaattata tgatttctca agataacagc ttctatatca 540
aatgtattaa ggatattggt taatccaatt ccgatataaa agccaaagtt ttgaagtgca 600
tttaacattt ctacatcatt tttatttgcg cgttccacaa tctcttttcg agaaatattc 660
ttttcttctt tagagagcga agccagtaac gctttttcag aagcatataa ttcccaacag 720
cctcgatttc cacagctgca tttgggtcca ttaaaatcta tcgtcatatg acccatttcc 780
ccagaaaaac cctgaacacc tttatacaat tcgttgttaa taacaagtcc agttccaatt 840
ccgatattaa tactgatgta aacgatgttt tcatagtttt ttgtcatacc aaatactttt 900
tcaccgtatg ctcctgcatt agcttcattt tcaacaaaaa ccggaacatt aaactcactc 960
tcaattaaaa actgcaaatc tttgatattc caatttaagt taggcatgaa aataatttgc 1020
tgatgacgat ctacaaggcc tggaacacaa attcctattc cgactagacc ataaggggac 1080
tcaggcatat gggttacaaa accatgaata agtgcaaata aaatctcttt tacttcacta 1140
gcggaagaac tagacaagtc agaagtcttc tcgagaataa tatttccttc taagtcggtt 1200
agaattccgt taagatagtc gactcctata tcaataccaa tcgagtagcc tgcattctta 1260
ttaaaaacaa gcattacagg tcttctgccg cctctagatt gccctgcccc aatttcaaaa 1320
ataaaatctt tttcaagcag tgtatttact tgagaggaga cagtagactt gtttaatcct 1380
gtaatctcag agagagttgc cctggagaca ggggagttct tcaaaatttc atctaatatt 1440
aatttttgat tcattttttt tactaaagct tgatctgcaa tttgaataat aaccactcct 1500
ttgtttatcc accgaactaa gttggtgttt tttgaagctt gaattagata tttaaaagta 1560
tcatatctaa tattataact aaattttcta aaaaaaacat tgaaataaac atttattttg 1620
tatatgatga gataaagtta gtttattgga taaacaaact aactcaatta agatagttga 1680
tggataaact tgttcactta aatcaaaggg ggaaatgaca aatggtccaa actagtgata 1740
tctaaaaatc aaagggggaa atgggatcca aaggaggcca taatatgaca gttaaacaag 1800
ctaaaatgac aatcgataaa gaatacaaag ttggcgaagt tgataaacgt ctttacggct 1860
ctttcatcga acatcttggc cgtgctgttt acgaaggcat ctacgaacct gatcatcctg 1920
aagctgatga atctggcttc cgtaaagatg ttatcaaact tgttaaagaa ttaaaggttc 1980
cattcatccg ttacccaggc ggcaacttcg tttctggcta caactgggaa gatggcgttg 2040
gccctgttga aaaacgtcct acacgtcttg atcttgcttg ggctacaaca gaacctaacc 2100
ttgttggcac aaacgaattc atggattggg ctaaacttgt tggcgctgaa gttaacatgg 2160
ctgttaacct tggcacacgt ggcatcgatg ctgctcgtaa ccttgttgaa tactgcaacc 2220
atccttctgg ctcttactac tctgatcttc gtaaatctca tggctacaaa gaacctcata 2280
aaatcaaaac atggtgcctt ggcaacgaaa tggatggccc ttggcaaatc ggccataaaa 2340
cagctgctga atacggccgt atcgctgctg aagctgctaa agttatgaaa tggacagatc 2400
cttctatcga acttgttgct tgcggctctt ctggctctgg catgcaaaca ttcatcgatt 2460
gggaaacaac agttcttgat catacatacg atcatgttga atacatctct cttcattctt 2520
actacggcaa ccgtgataac gatcttccta actaccttgc tcgttctctt gatatggatc 2580
atttcatcaa cacagtagtt gctgtatgcg actacatgaa agctaaaaaa cgttctaaaa 2640
aaacaatcca tctttcttac gatgaatgga acgtttggta ccattctaac gaaaaagata 2700
aacttgttga acgttgggaa cgtgctcctc atcttcttga agatatctac aacttcgaag 2760
atgctcttct tgttggctgc atgcttatca caatgcttaa acatgctgat cgtgttaaaa 2820
tcgcttgcct tgctcaactt gttaacgtta tcgctcctat catgacagaa aaaggcggcg 2880
aagcttggcg tcaaacaatc ttctaccctt tcatgcatgc ttctgtttac ggccgtggca 2940
cagctcttca aacagttgtt tcttctccta aatacgattc taaagatttc acagatgttc 3000
cttaccttga atctgtttct gttttcaacg aagaagctga agaacttaca atcttcgctg 3060
ttaaccgtga tacagaaggc ggccttcaaa tcgaagctga tgttcgttct ttcgaaggct 3120
acgctgtttc tgaacatatc gttcttgaac atgaagataa caaagctaca aacgaacaag 3180
atcgtaacaa cgttgttcct cattctggcg gcgatgctaa agtttgcgat ggccgtctta 3240
cagctcatct tcctaaactt tcttggaacg ttatccgtct taaaaaacgt taaggatccc 3300
cgggaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 3360
acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 3420
caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta 3480
ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat 3540
ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 3600
ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 3660
ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 3720
gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 3780
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 3840
tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 3900
gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 3960
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 4020
tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 4080
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 4140
atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 4200
cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 4260
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 4320
gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 4380
ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 4440
gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 4500
agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 4560
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 4620
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 4680
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 4740
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 4800
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccata tccttctttt 4860
tctgaaccga cttctccttt ttcgcttctt tattccaatt gctttattga cgttgagcct 4920
cggaaccctt aacaatccca aaacttgtcg aatggtcggc ttaatagctc acgctatgcc 4980
gacattcgtc tgcaagttta gttaagggtt cttctcaacg cacaataaat tttctcggca 5040
taaatgcgtg gtctaatttt tatttttaat aaccttgata gcaaaaaatg ccattccaat 5100
acaaaaccac atacctataa tcgataacca cataacagtc ataaaaccac tcctttttaa 5160
caaactttat cacaagaaat atttaaattt taaatgcctt tattttgaat tttaaggggc 5220
attttaaaga tttaggggta aatcatatag ttttatgcct aaaaacctac agaagctttt 5280
aaaaagcaaa tatgagccaa ataaatatat tctaattcta caaacaaaaa tttgagcaaa 5340
ttcagtgtcg attttttaag acactgccca gttacatgca aattaaaatt ttcatgattt 5400
tttatagttc ctaacagggt taaaatttgt ataacgaaag tataatgttt atataacgtt 5460
agtataataa agcattttaa cattatactt ttgataatcg tttatcgtcg tcatcacaat 5520
aacttttaaa atactcgtgc ataattcaac agctgacctc ccaataacta catggtgtta 5580
tcgggaggtc agctgttagc acttatattt tgttattgtt cttcctcgat ttcgtctatc 5640
attttgtgat taatttctct tttttcttgt tctgttaagt cataaagttc actagctaaa 5700
tactcttttt gtttccaaat ataaaaaatt tgatagatat attcggttgg atcaatttct 5760
tttaagtaat ctaaatcccc attttttaat ttctttttag cctctttaaa taatcctgaa 5820
taaactaata cctgtttacc tttaagtgat ttataaaatg catcaaagac tttttgattt 5880
attaaataat cactatcttt accagaatac ttagccattt catataattc tttattatta 5940
ttttgtctta ttttttgaac ttgaacttgt gttatttctg aaatgcccgt tacatcacgc 6000
cataaatcta accattcttg ttggctaata taatatcttt tatctgtgaa atacgattta 6060
tttactgcaa ttaacacatg aaaatgagga ttataatcat ctcttttttt attatatgta 6120
atctctaact tacgaacata tccctttata acactaccta ctttttttct ctttataagt 6180
tttctaaaag aattattata acgttttatt tcattttcta attcatcact cattacatta 6240
ggtgtagtca aagttaaaaa gataaactcc tttttctctt gctgcttaat atattgcatc 6300
atcaaagata aacccaatgc atcttttcta gcttttctcc aagcacagac aggacaaaat 6360
cgatttttac aagaattagc tttatataat ttctgttttt ctaaagtttt atcagctaca 6420
aaagacagaa atgtattgca atcttcaact aaatccattt gattctctcc aatatgacgt 6480
ttaataaatt tctgaaatac ttgatttctt tgttttttct cagtatactt ttccatgtta 6540
taacacataa aaacaactta gttttcacaa actatgacaa taaaaaaagt tgctttttcc 6600
cctttctatg tatgtttttt actagtcatt taaaacgata cattaatagg tacgaaaaag 6660
caactttttt tgcgcttaaa accagtcata ccaataactt aagggtaact agcctcgccg 6720
gcaatagtta cccttattat caagataaga aagaaaagga tttttcgcta cgctcaaatc 6780
ctttaaaaaa acacaaaaga ccacattttt taatgtggtc ttttattctt caactaaagc 6840
acccattagt tcaacaaacg aaaattggat aaagtgggat atttttaaaa tatatattta 6900
tgttacagta atattgactt ttaaaaaagg attgattcta atgaagaaag cagacaagta 6960
agcctcctaa attcacttta gataaaaatt taggaggcat atcaaatgaa ctttaataaa 7020
attgatttag acaattggaa gagaaaagag atatttaatc attatttgaa ccaacaaacg 7080
acttttagta taaccacaga aattgatatt agtgttttat accgaaacat aaaacaagaa 7140
ggatataaat tttaccctgc atttattttc ttagtgacaa gggtgataaa ctcaaataca 7200
gcttttagaa ctggttacaa tagcgacgga gagttaggtt attgggataa gttagagcca 7260
ctttatacaa tttttgatgg tgtatctaaa acattctctg gtatttggac tcctgtaaag 7320
aatgacttca aagagtttta tgatttatac ctttctgatg tagagaaata taatggttcg 7380
gggaaattgt ttcccaaaac acctatacct gaaaatgctt tttctctttc tattattcca 7440
tggacttcat ttactgggtt taacttaaat atcaataata atagtaatta ccttctaccc 7500
attattacag caggaaaatt cattaataaa ggtaattcaa tatatttacc gctatcttta 7560
caggtacatc attctgtttg tgatggttat catgcaggat tgtttatgaa ctctattcag 7620
gaattgtcag ataggcctaa tgactggctt ttataatatg agataatgcc gactgtactt 7680
tttacagtcg gttttctaat gtcactaacc tgccccgtta gttgaagaag gtttttatat 7740
tacagctcca gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 7800
gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 7860
atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 7920
tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 7980
gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 8040
actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 8100
gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 8160
agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 8220
ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 8280
aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 8340
cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 8400
gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 8460
cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 8520
cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 8580
gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagaagcg gaaga 8635
<210>4
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ggaaatggga tccaaaggag gccataatat gacagttaaa caagctaaaa tgacaatc 58
<210>5
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
gccagtgaat tcccggggat ccttaacgtt ttttaagacg gataacg 47
<210>6
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
ttacgccaag cttggctgca gtcgcgatga ttaattaatt cagaacg 47
<210>7
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gattgtcatt ttagcttgtt taactgtcat attatggcct cctttggatc ccatttcc 58
<210>4
<211>502
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Met Thr Val Lys Gln Ala Lys Met Thr Ile Asp Lys Glu Tyr Lys Val
1 5 10 15
Gly Glu Val Asp Lys Arg Leu Tyr Gly Ser Phe Ile Glu His Leu Gly
20 25 30
Arg Ala Val Tyr Glu Gly Ile Tyr Glu Pro Asp His Pro Glu Ala Asp
35 40 45
Glu Ser Gly Phe Arg Lys Asp Val Ile Lys Leu Val Lys Glu Leu Lys
50 55 60
Val Pro Phe Ile Arg Tyr Pro Gly Gly Asn Phe Val Ser Gly Tyr Asn
65 70 75 80
Trp Glu Asp Gly Val Gly Pro Val Glu Lys Arg Pro Thr Arg Leu Asp
85 90 95
Leu Ala Trp Ala Thr Thr Glu Pro Asn Leu Val Gly Thr Asn Glu Phe
100 105 110
Met Asp Trp Ala Lys Leu Val Gly Ala Glu Val Asn Met Ala Val Asn
115 120 125
Leu Gly Thr Arg Gly Ile Asp Ala Ala Arg Asn Leu Val Glu Tyr Cys
130 135 140
Asn His Pro Ser Gly Ser Tyr Tyr Ser Asp Leu Arg Lys Ser His Gly
145 150 155 160
Tyr Lys Glu Pro His Lys Ile Lys Thr Trp Cys Leu Gly Asn Glu Met
165 170 175
Asp Gly Pro Trp Gln Ile Gly His Lys Thr Ala Ala Glu Tyr Gly Arg
180 185 190
Ile Ala Ala Glu Ala Ala Lys Val Met Lys Trp Thr Asp Pro Ser Ile
195 200 205
Glu Leu Val Ala Cys Gly Ser Ser Gly Ser Gly Met Gln Thr Phe Ile
210 215 220
Asp Trp Glu Thr Thr Val Leu Asp His Thr Tyr Asp His Val Glu Tyr
225 230 235 240
Ile Ser Leu His Ser Tyr Tyr Gly Asn Arg Asp Asn Asp Leu Pro Asn
245 250 255
Tyr Leu Ala Arg Ser Leu Asp Met Asp His Phe Ile Asn Thr Val Val
260 265 270
Ala Val Cys Asp Tyr Met Lys Ala Lys Lys Arg Ser Lys Lys Thr Ile
275 280 285
His Leu Ser Tyr Asp Glu Trp Asn Val Trp Tyr His Ser Asn Glu Lys
290 295 300
Asp Lys Leu Val Glu Arg Trp Glu Arg Ala Pro His Leu Leu Glu Asp
305 310 315 320
Ile Tyr Asn Phe Glu Asp Ala Leu Leu Val Gly Cys Met Leu Ile Thr
325 330 335
Met Leu Lys His Ala Asp Arg Val Lys Ile Ala Cys Leu Ala Gln Leu
340 345 350
Val Asn Val Ile Ala Pro Ile Met Thr Glu Lys Gly Gly Glu Ala Trp
355 360 365
Arg Gln Thr Ile Phe Tyr Pro Phe Met His Ala Ser Val Tyr Gly Arg
370 375 380
Gly Thr Ala Leu Gln Thr Val Val Ser Ser Pro Lys Tyr Asp Ser Lys
385 390 395 400
Asp Phe Thr Asp Val Pro Tyr Leu Glu Ser Val Ser Val Phe Asn Glu
405 410 415
Glu Ala Glu Glu Leu Thr Ile Phe Ala Val Asn Arg Asp Thr Glu Gly
420 425 430
Gly Leu Gln Ile Glu Ala Asp Val Arg Ser Phe Glu Gly Tyr Ala Val
435 440 445
Ser Glu His Ile Val Leu Glu His Glu Asp Asn Lys Ala Thr Asn Glu
450 455 460
Gln Asp Arg Asn Asn Val Val Pro His Ser Gly Gly Asp Ala Lys Val
465 470 475 480
Cys Asp Gly Arg Leu Thr Ala His Leu Pro Lys Leu Ser Trp Asn Val
485 490 495
Ile Arg Leu Lys Lys Arg
500
Claims (15)
1. A recombinant α -L-arabinofuranosidase has an amino acid sequence shown in SEQ ID NO. 7.
2. The recombinant α -L-arabinofuranosidase according to claim 1, wherein the recombinant α -L-arabinofuranosidase is secreted from Bacillus subtilis that contains or incorporates a gene of interest encoding α -L-arabinofuranosidase.
3. A recombinant α -L-arabinofuranosidase encoding the recombinant α -L-arabinofuranosidase of claim 1 or 2.
4. The recombinant α -L-arabinofuranosidase-encoding polynucleotide of claim 3 having the sequence shown in SEQ ID No.1 or a sequence with more than 90% homology to the sequence shown in SEQ ID No. 1.
5. A recombinant expression vector comprising the polynucleotide encoding α -L-arabinofuranosidase according to claim 3 or 4.
6. An engineered bacterium containing or incorporating the recombinant expression vector of claim 5.
7. A complex enzyme comprising the recombinant α -L-arabinofuranosidase according to claim 1 or 2.
8. A fibroethanol complex enzyme, which comprises α -L-arabinofuranosidase according to claim 1 or 2.
9. The complex cellosolve enzyme as claimed in claim 8, wherein the complex cellosolve enzyme further comprises cellobiase and/or xylanase.
10. A process for producing the recombinant α -L-arabinofuranosidase of claim 1 or 2, which comprises extracting a secretion from Bacillus subtilis harboring or incorporating a gene of interest encoding α -L-arabinofuranosidase.
11. The method as claimed in claim 10, wherein the bacillus subtilis is cultured in a suitable medium, xylose is added after the strain grows to logarithmic phase, fermentation culture is carried out, and the secretion of the strain is collected.
12. The method of claim 11, wherein the culture conditions are: the temperature is 15-37 ℃, and the rotation speed is 100-300 rpm; to-be-treated bacterium OD600And (3) reaching 0.75-0.85, adding xylose to the final concentration of 1.0% (m/v), and continuing to culture.
13. The method according to claim 12, wherein the secretion of the microorganism is collected by taking a fermentation broth, centrifuging the fermentation broth, and taking a supernatant.
14. A method of making cellulosic ethanol, the method comprising: the cellulosic ethanol is obtained by carrying out enzymolysis on the raw material by using the cellulosic ethanol complex enzyme as claimed in any one of claims 8 or 9.
15. Use of the recombinant α -L-arabinofuranosidase of claim 1 or 2, the recombinant α -L-arabinofuranosidase-encoding polynucleotide of claim 3 or 4, the recombinant expression vector of claim 5, the recombinant engineered bacterium of claim 6, the complex enzyme of claim 7, or the fibroethanol complex enzyme of claim 8 or 9 for the preparation of fibroethanol.
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