CN110982805A - α -L-arabinofuranosidase and related products - Google Patents

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

α -L-arabinofuranosidase and related products

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：

MTVKQAKMTIDKEYKVGEVDKRLYGSFIEHLGRAVYEGIYEPDHPEADES 50

GFRKDVIKLVKELKVPFIRYPGGNFVSGYNWEDGVGPVEKRPTRLDLAWA 100

TTEPNLVGTNEFMDWAKLVGAEVNMAVNLGTRGIDAARNLVEYCNHPSGS 150

YYSDLRKSHGYKEPHKIKTWCLGNEMDGPWQIGHKTAAEYGRIAAEAAKV 200

MKWTDPSIELVACGSSGSGMQTFIDWETTVLDHTYDHVEYISLHSYYGNR 250

DNDLPNYLARSLDMDHFINTVVAVCDYMKAKKRSKKTIHLSYDEWNVWYH 300

SNEKDKLVERWERAPHLLEDIYNFEDALLVGCMLITMLKHADRVKIACLA 350

QLVNVIAPIMTEKGGEAWRQTIFYPFMHASVYGRGTALQTVVSSPKYDSK 400

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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