CN110423701A

CN110423701A - A kind of Aspergillus niger strain of high yield arabinofuranosidase

Info

Publication number: CN110423701A
Application number: CN201910512907.9A
Authority: CN
Inventors: 徐晓东; 刘文瑶; 朱倩倩
Original assignee: Qingdao Vland Biotech Group Co Ltd
Current assignee: Qingdao Vland Biotech Group Co Ltd
Priority date: 2019-06-14
Filing date: 2019-06-14
Publication date: 2019-11-08
Anticipated expiration: 2039-06-14
Also published as: CN110423701B

Abstract

The present invention provides Aspergillus niger strain and its applications that a plant height produces arabinofuranosidase.Applicant constructs first obtains the aspergillus niger engineering bacteria strain of recombinant expression arabinofuranosidase gene, then the mutant strain that a plant height produces arabinofuranosidase is further obtained by the screening of ultraviolet mutagenesis method, deposit number is CCTCC NO:M2019433.The mutant bacteria advantageously reduces the production cost of arabinofuranosidase glucosides, has a extensive future.

Description

A kind of Aspergillus niger strain of high yield arabinofuranosidase

Technical field

The invention belongs to gene engineering technology fields, and in particular to the black-koji mould of plant height production arabinofuranosidase Strain and its application.

Technical background

In nature, main component of the hemicellulose as plant cell wall is to be only second to the second largest of cellulose to enrich Polysaccharide, improve its biodegradability with far-reaching biological significance.Xylan is that the representativeness in hemicellulose is main Constituent component is wanted, structure is extremely complex, and main chain D- xylopyranose is formed by β-Isosorbide-5-Nitrae glucosides key connection, is also connected on side chain There are the different substituent groups such as arabinose, glucuronic acid, ferulic acid ester group, acetyl group, therefore, the degradable need of xylan Wanting the collective effect of multiple enzymes could complete.Wherein, zytase acts on the master of xylan as apoenzyme in a manner of inscribe Chain, arabinofuranosidase are then Side chain cleavage enzymes, and play important facilitation to the degradation of main chain.

Arabinofuranosidase belongs to glycosyl hydrolase family, according to the difference of substrate conformation, can be divided into β-L- I Primary furanoside enzyme (β-L-arabinofuranosidase) and α-l-arabfuranglycosidase (α-L- Arabinofuranosidase, Anabf A) two classes.β-L- arabinofuranosidase can be from the non-reducing end of a variety of substrates β-L-arabinose residue under degrading.Anabf A can be from araboxylan (arabinoxylans, AX), araban (Arabinan) and the hydrolysis of the non-reducing end of the polymers such as arabogalactan (arabinogalactans) generates one α-L-arabinose molecule.Existing arabinose residues are mainly α-L-arabinose residue in nature.

As one of hemicellulose degrading enzymes system, α-l-arabfuranglycosidase participates in the agricultural product residue of low price again It utilizes, generation, which can be used for fermenting, generates the monosaccharide and other byproducts of alcohol fuel, therefore it has in agricultural-industrial industry Good application prospect.In feedstuff industry, cellulose and hemicellulose are the main energy sources of ruminant animals, but by Only 40-60% low in its degradation rate, really being utilized by animal.Arabinofuranosidases are as a kind of feed addition Agent eliminates arabinose side chains on xylan, promotes the degradation of xylan, is easy to be digested and assimilated by animal.Arabinofuranosidase Glycosidase is also extensively used in food service industry.L-arabinose can replace traditional as a kind of sweetener of low intake Sucrose, is suitable for the elderly and hyperglycemic patients are edible.Meanwhile it can also block absorption of the human body to sucrose, losing weight and controlling There is good application prospect in terms of diabetes.Arabinofuranosidase also can increase the concentration of terpenol during wine brewing, mention Araban since which removes arabinose side chains, and can be hydrolyzed into the solable matter of short chain by the fragrance of high wine, be increased Add the clarity of fruit juice, and is applied to juice production industry.

In nature, the source of α-l-arabfuranglycosidase is than wide.Researcher is from fungi, bacterium With discovery and isolated α-l-arabfuranglycosidase gene in plant, and heterogenous expression is successfully carried out.But production at present The yield of α-l-arabfuranglycosidase is generally relatively low in strain, high so as to cause the production cost of the enzyme, limitation Its application.

Summary of the invention

The present invention be solve prior art problem, provide a plant height produce arabinofuranosidase Aspergillus niger strain and It is applied.Applicant construct first obtains recombinant expression arabinofuranosidase gene aspergillus niger engineering bacteria strain, then into One step obtains one plant of arabinofuranosidase superior strain by the screening of ultraviolet mutagenesis method, advantageously reduces the production of the enzyme Cost has a extensive future.

One aspect of the present invention provides a kind of recombinant plasmid, and the recombinant plasmid carries arabinofuranosidase base Cause.

The amino acid sequence of the arabinofuranosidase is SEQ ID NO:2, coding nucleotide sequence SEQ ID NO:3。

One aspect of the present invention provides a kind of aspergillus niger engineering bacteria strain, and the bacterial strain carries above-mentioned recombinant plasmid.

The present invention also provides a kind of mutant strain aspergillus niger Su12-9969B(Aspergillus niger Su12- It 9969B), is to be obtained using above-mentioned aspergillus niger engineering bacteria as starting strain by ultraviolet mutagenesis.

The Chinese Typical Representative culture that the mutant strain has been preserved in Wuhan, China Wuhan University on June 5th, 2019 is protected Administrative center is hidden, deposit number is CCTCC NO:M2019433.

The present invention also provides application of the aspergillus niger mutant strain in production arabinofuranosidase.

The present invention will from microorganism Aspergillus aculeatus (Aspergillus aculeatus) arabinofuranosidase gene exist It is overexpressed in aspergillus niger host, building obtains aspergillus niger Su12-9969A.20L tank ferments after 160h, the strain fermentation supernatant Middle arabinofuranosidase enzyme activity reaches 751U/ml.

In order to improve the yield of arabinofuranosidase, applicant using aspergillus niger Su12-9969A as starting strain, into One step obtains a plant mutant bacterial strain aspergillus niger Su12-9969B by the method screening of ultraviolet mutagenesis, can increase substantially Arab The yield of furanoside enzyme.20L tank ferments after 160h, and arabinofuranosidase enzyme activity reaches in the mutant strain fermented supernatant fluid To 1281U/ml, 70.6% is improved than going out bacterium germination, unexpected technical results have been achieved.The mutant strain can answer extensively For the production of arabinofuranosidase, to advantageously reduce the production cost of arabinofuranosidase, promote its Popularization and application in the industrial circles such as feed, food.

Detailed description of the invention

Fig. 1 is plasmid pSU map；

Fig. 2 is 20L tank fermentation diagram；

Fig. 3 is SDS-PAGE protein electrophoresis figure: wherein: M is molecular weight of albumen Marker, and swimming lane 1,2 is respectively the black song of bacterium germination Mould Su12-9969A, mutant bacteria aspergillus niger Su12-9969B fermented supernatant fluid.

Specific embodiment

The routine techniques and method that the present invention has used genetic engineering and molecular biology field uses, such as MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT Documented method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These generality are with reference to text It offers and provides definition well known by persons skilled in the art and method.But those skilled in the art can be remembered in the present invention On the basis of the technical solution of load, using the other conventional methods in this field, experimental program and reagent, and it is not limited to tool of the present invention The restriction of body embodiment.

The present invention will be described in detail With reference to embodiment.

The clone of 1 arabinofuranosidase gene of embodiment

With microorganism Aspergillus aculeatus (Aspergillus aculeatus) genome be template, amplify me using primer 1 and primer 2 Primary furanoside enzyme gene segment, nucleotides sequence are classified as SEQ ID NO:2, and the amino acid sequence of coding is SEQ ID NO: 1。

PCR primer and reaction condition are as follows:

Primer 1(F): ATGCCTTCACGACGAACCCTC

Primer 2 (R): CTACGCGGACGCAAAGCCCGA

Reaction condition are as follows: 94 DEG C of denaturation 5min；Then 94 DEG C of denaturation 30s, 58 DEG C of renaturation 30s, 72 DEG C of extension 90s, 30 recycle Afterwards, 72 DEG C of heat preservation 10min.Agarose electrophoresis is the results show that the arabinofuranosidase gene size that amplification obtains is 1506bp。

The building of 2 recombinant vector of embodiment

The above-mentioned arabinofuranosidase gene of PCR amplification, primer both ends introduce the site XbaI.Primer sequence is as follows:

Primer 3(F): GTATCTAGA ATGCCTTCACGACGAACCCTC

Primer 4(R): GACTCTAGA CTACGCGGACGCAAAGCCCGA

PCR reaction condition are as follows: 94 DEG C of denaturation 5min；Then 94 DEG C of denaturation 30s, 58 DEG C of renaturation 30s, 72 DEG C of extension 90s, 30 are followed After ring, 72 DEG C of heat preservation 10min.Agarose gel electrophoresis results show that arabinofuranosidase gene is size 1506bp's Segment.

The arabinofuranosidase genetic fragment of above-mentioned acquisition and expression vector pSU are subjected to restriction enzyme respectively XbaI single endonuclease digestion, digestion condition are as follows:

PCR fragment digestion system (50ul)	Plasmid pSU digestion system (50ul)
		PCR fragment 20ul	PTG plasmid 20ul
10*M 5ul	10*M 5ul
		BSA 5ul	BSA 5ul
XbaI 2ul	XbaI 2ul
		ddH₂O 18ul	ddH₂O 18ul

37 DEG C of water-bath digestions handle 2h, and two target fragments are separately recovered after electrophoresis, are dissolved in 20 ul ddH₂O.Connected with T4 DNA It connects enzyme to be attached, linked system is as follows:

PCR fragment	2ul
		pSU	2ul
10*Buffer	1ul
		T4 DNA ligase	1ul
ddH₂O	4ul
		Total volume	10ul

22 DEG C of connection 1h convert escherichia coli DH5a competence, are coated with LB+AMP plate, grow single bacterium after 37 DEG C of overnight incubations It falls, bacterium colony PCR verifying, which connects correct transformant, extracts plasmid and send sequencing, to get to containing arabinofuranosidase after sequencing correctly The recombinant vector pSU-9969 of glycosidase genes.

The recombinant expression of 3 arabinofuranosidase of embodiment

1, prepared by protoplast:

Inoculated aspergillus niger host strain Su12 boils filtering and removing slag after 20-30min in PDA+U(potato 200g/L；Glucose 2%； Uridine 1%；Agar powder 1.5%) plate, 30 DEG C of culture 5-7d；The fungus block of 2cm × 2cm size is extracted, 100ml liquid is inoculated with PDA+U(potato 200g/L, boils filtering and removing slag after 20-30min；Glucose 2%；Uridine 1%) in culture medium, 30 DEG C of trainings 16h growth mycelium is supported for converting；After the mycelium grown filtering, it is resuspended with 20ml 1.2M Adlerika；It is added 0.2g lysozyme, 30 DEG C, 100rpm culture 2-3h；2 layers of lens wiping paper of cracked mycelia are filtered, 3000rpm is centrifuged 10min Obtain protoplast；Cracked mycelia is filtered with lens wiping paper, centrifugation obtains protoplast；Again with suitable sorbitol solution It is resuspended.

, conversion:

The aspergillus niger Su12 protoplast of above-mentioned acquisition is cleaned 2 times with 1.2M sorbitol solution, then molten with suitable sorbierite Liquid is resuspended, and protoplast concentration is made to reach 10⁸A/ml；The ready recombination of 10ul is separately added into every 200ul protoplast Carrier pSU-9969, is added the PEG6000 of 50ul 25%, and ice bath 20min adds the PEG6000 of 2ml 25%, is placed at room temperature for 5min；4ml sorbitol solution is added to be mixed by inversion, after pouring into 50ml conversion upper layer culture medium, it is flat to pour into 4 conversion lower layers In plate, after the solidification of upper layer culture medium, culture 5d is inverted in 30 DEG C of incubators.

, transformant screening:

After cultivating 5d, the bacterium colony that picking is grown, dibbling carries out secondary screening, 30 DEG C of culture 3d to conversion lower layer's plate.By normal growth Transformant be inoculated into fresh PDA plate, 30 DEG C of culture 5-7d respectively.Each transformant extracts the bacterium of 2cm × 2cm size Block is inoculated with 50ml liquid submerged culture base (maltose 12% respectively；Corn pulp 1.5%；Ammonium sulfate 0.5%；Magnesium sulfate 0.3%；Sulfuric acid Potassium 0.37%；Calcium chloride 0. 1125%；Microelement 0.1%) in fermentation, 30 DEG C of culture 5d.After cultivating 5d, centrifugation thallus is obtained Clear liquid is crude enzyme liquid, carries out the detection of SDS-PAGE protein electrophoresis and the detection of arabinofuranosidase enzyme activity.

The highest positive transformant of arabinofuranosidase enzyme activity is named as aspergillus niger Su12-9969A by applicant (Aspergillus nigerSu12-9969A), arabinofuranosidase vigor is 142U/ in the strain fermentation supernatant ml。

(1) definition of arabinofuranosidase enzyme-activity unit

Under conditions of 50 DEG C, pH value are 4.8, the 4- nitrophenyl alpha-L- Arab furan for being per minute 5 mmol/L from concentration Enzyme amount required for 1 micromole's paranitrophenol of degradation release in the solution of glucosides of muttering is an enzyme activity unit U.

(2) enzyme activity determination method

5 mmol/L 4- nitrophenyl alpha-L- arabinofuranosidase glucosides solution: it is Arabic to accurately weigh 4- nitrophenyl alpha-L- Furanoside 0.0678g, is accurate to 0.0001g, is slowly added into corresponding buffer close to 50ml, magnetic agitation about 10min, uses The citric acid or the corresponding pH value of sodium hydroxide tune of 2mol/L, last constant volume to 50ml, matching while using.

Enzyme solution: being diluted to suitable multiple with the sodium citrate buffer solution of pH4.8, controls light absorption value in 0.2-0.4 range.

The drafting of standard curve: by 10 times of exact dilution of 5mmol/L p-nitrophenyl phenol solution, then respectively dilution 2,4,6,8, 10,12,16 times.

The above-mentioned p-nitrophenol dilution (blank control takes buffer) of 0.5ml is taken, sodium carbonate liquor 2ml is added, is added Substrate solution 0.5ml is uniformly mixed, is returned to zero with blank control, to measure light absorption value in 410nm.

Using the content of p-nitrophenol in system as abscissa (X), light absorption value be ordinate (y), draw standard curve y= kA+b。

Extension rate	Blank control	16	12	10	8	6	4	2
									Content of p-nitrophenol (μm ol) in system	0	0.0156	0.0208	0.025	0.0313	0.0417	0.0625	0.125

Measurement: appropriate amount of substrate, 50 DEG C of preheating 5min are taken；

Four 15*150 test tubes (blank tube, three sample cells) are taken, it is accurate to be added with having diluted respectively into four branch pipes Good enzyme solution 0.5ml；

Four test tubes are placed in 50 ± 0.1 DEG C of water-baths simultaneously, preheat 2min；

0.5ml substrate solution, accurate timing 15min are accurately added into sample tube；

Sodium carbonate liquor 2.0ml is quickly and accurately added into each pipe, substrate solution 0.50ml is accurately added in blank tube, It shakes up.

It is returned to zero with blank tube, at spectrophotometer wavelength 410nm, with 10mm cuvette, is measured respectively.

The absorbance of sample liquid, is averaged in three sample cells.

By looking into standard curve or finding out the content of p-nitrophenol with equation of linear regression.

Enzyme activity calculation formula: A=X × 1/0.5 × n/15

In formula:

A-arabinofuranosidase enzyme activity, U/g (or U/ml)；

X-absorbance checks in the content of p-nitrophenol of (or calculating) on standard curve, μm ol；

1/0.5-enzyme solution the volume being added；

N-enzyme sample extension rate；

15-time scale factors.

4 mutagenesis screening of embodiment

Mutation randomness caused by ultraviolet mutagenesis is very strong, and it is also random for being mutated the effect of generation, it is difficult to be predicted.Therefore, in order to Effective direct mutation is obtained, technical staff usually requires to carry out more wheel ultraviolet mutagenesis, the larger workload of screening, and there are nothings Method obtains a possibility that effective direct mutation.But it because equipment needed for ultraviolet mutagenesis is simple, expense is few, and can obtain in a short time Mass mutation body is obtained, therefore, it is still a kind of common mutagenic breeding method now.

Applicant carries out science of heredity to it by ultraviolet mutagenesis method and changes using aspergillus niger Su12-9969A as starting strain It makes, further increases the yield of its arabinofuranosidase.

1, lethality is determined:

Inoculated aspergillus niger Su12-9969A is in PDA plate, 30 DEG C of culture 5-7d.When bacterium colony surface generates a large amount of spores, draw The sterile water elution of 5ml obtains spore liquid, is resuspended after centrifugation with sterile water, is counted with blood counting chamber.Take a 90mm culture Ware, the spore suspension that 5ml has diluted is added, and (concentration is 1 × 10⁷/ ml), rotor, which is added, and stirs on magnetic stirring apparatus makes spore Sub- liquid is in uniform state.In aseptic superclean bench, shone with the ultraviolet lamp that power is 9w in the top of vertical range 20cm It penetrates, irradiates 30s, 45s, 60s, 75s, 90s, 105s, 120s respectively, the spore liquid after taking irradiation dilutes 10,100,1000 times, takes 100ul is coated with PDA plate, counts after 30 DEG C of culture 2-3d, is control with non-irradiated spore liquid, calculates lethality.Wherein according to When penetrating 105s, lethality 96% chooses the irradiation time and carries out subsequent Mutagenesis experiments.

2, first round mutagenesis screening

A 90mm culture dish is taken, the spore suspension that 5ml has diluted is added, and (concentration is 1 × 10⁷/ ml), rotor is added and in magnetic Stirring makes spore liquid be in uniform state on power blender.In aseptic superclean bench, the ultraviolet lamp for being 9w with power is in vertical The directly top irradiation of distance 20cm, dilutes 1000 times after irradiating 105s, 100ul is taken to be coated with PDA plate, 30 DEG C of culture 2-3d.

The first round screens and is coated with 180 pieces of PDA plates altogether, and after 30 DEG C of culture 2-3d, each plate grows 20-40 bacterium colony, Colonial morphology is first passed through, the mutant of short branch is screened.Applicant's picking colony form is smaller, mycelia is fine and close, periphery of bacterial colonies suede Hair is mutant bacteria totally 85 shorter, is inoculated into PDA plate, 30 DEG C of culture 5-7d respectively.It is big that each mutant bacteria extracts 2cm × 2cm Small fungus block is inoculated in 50ml liquid submerged culture base ferments respectively, 30 DEG C of culture 5d.After cultivating 5d, centrifugation thallus is obtained Clear liquid is crude enzyme liquid, carries out protein electrophoresis detection and the detection of arabinofuranosidase enzyme activity respectively.Simultaneously to go out bacterium germination Aspergillus niger Su12-9969A is as a control group.

The results show that in the 85 plant mutant bacterium that first round Uv-induced screening obtains, without a plant mutant bacterium fermentation supernatant Arabinofuranosidase enzyme activity is higher than bacterium germination in liquid；Wherein, the enzyme activity and bacterium germination out of 78 plant mutant bacterium are substantially suitable, remaining The enzyme activity of mutant bacteria even reduces 6-11% than going out bacterium germination.

Applicant has continued 8 wheel mutagenesis screenings according to the method described above, finally obtains one plant of arabinofuranosidase Yield is significantly higher than the mutant strain of bacterium germination out, is named as aspergillus niger Su12-9969B(Aspergillus niger Su12- 9969B).Arabinofuranosidase enzyme activity reaches 239U/ml in the mutant strain shake flask fermentation supernatant, mentions than going out bacterium germination It is high by 68.3%.

Applicant will further go out bacterium germination aspergillus niger Su12-9969A and mutant bacteria aspergillus niger Su12-9969B and carry out respectively The fermentation of 20L tank, fermentation diagram is as shown in Fig. 2, the SDS-PAGE electrophoresis detection result of fermented supernatant fluid is as shown in Figure 3.Fermentation After 160h, arabinofuranosidase enzyme activity reaches 751U/ml in bacterium germination fermented supernatant fluid out, and mutant bacteria aspergillus niger Su12- Arabinofuranosidase enzyme activity is up to 1281U/ml in 9969B fermented supernatant fluid, improves 70.6% than going out bacterium germination, achieves Unexpected technical effect.

Applicant is on June 5th, 2019 by mutant strain aspergillus niger Su12-9969B(Aspergillus niger Su12-9969B it) is preserved in the China typical culture collection administrative center of Wuhan, China Wuhan University, deposit number is CCTCC NO:M2019433。

Sequence table

<110>Qingdao Weilan Biology Group Co., Ltd.

<120>a kind of Aspergillus niger strain of high yield arabinofuranosidase

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Met Pro Ser Arg Arg Thr Leu Leu Gly Leu Ala Ala Thr Gly Thr Leu

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Val Ser Ala Gly Pro Cys Asp Ile Tyr Ala Ser Gly Gly Ala Pro Cys

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Ile Ala Ala His Ser Thr Thr Arg Ala Leu Tyr Ser Ala Tyr Ser Gly

35 40 45

Pro Leu Tyr Gln Val Ile Arg Gly Ser Asp Ser Ala Thr Thr Asp Ile

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Lys Pro Leu Thr Ala Gly Gly Val Ala Asn Ala Ala Ala Gln Asp Thr

65 70 75 80

Phe Cys Ser Gly Thr Thr Cys Leu Ile Ser Ile Ile Tyr Asp Gln Ser

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Gly Ser Gly Asn Asp Leu Thr Gln Ala Pro Pro Gly Gly Phe Asp Gly

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Pro Glu Ala Asn Gly Tyr Asp Asn Leu Ala Ser Ala Ile Gly Ala Pro

115 120 125

Val Thr Leu Asn Gly Gln Lys Ala Tyr Gly Val Phe Val Ser Pro Gly

130 135 140

Thr Gly Tyr Arg Asn Asn Asp Ala Asn Gly Thr Ala Thr Gly Asp Glu

145 150 155 160

Pro Glu Gly Met Tyr Ala Val Leu Asp Gly Thr His Tyr Asn Asp Ala

165 170 175

Cys Cys Phe Asp Tyr Gly Asn Ala Glu Val Ser Ser Thr Asp Thr Gly

180 185 190

Asn Gly His Met Glu Ala Ile Tyr Tyr Gly Thr Ser Lys Thr Trp Gly

195 200 205

Tyr Gly Ser Gly Ser Gly Pro Trp Val Met Ala Asp Leu Glu Asn Asn

210 215 220

Leu Phe Ser Gly Gln Ala Glu Gly Leu Asn Ser Ala Asp Pro Ser Ile

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Ser Tyr Arg Phe Val Thr Ala Ala Val Lys Gly Glu Pro Asn Gln Trp

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Ala Ile Arg Gly Gly Asn Ala Ala Ser Gly Ser Leu Thr Ser Tyr Tyr

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Ser Gly Ala Arg Pro Thr Val Ser Gly Tyr Asn Pro Met Ser Lys Glu

275 280 285

Gly Ala Ile Ile Leu Gly Ile Gly Gly Asp Asn Ser Asn Gly Ala Gln

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Gly Thr Phe Tyr Glu Gly Val Met Thr Ser Gly Tyr Pro Ser Asp Ala

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Thr Glu Asn Ser Val Gln Ala Asn Ile Val Ala Ala Lys Tyr Ala Thr

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Thr Ser Leu Thr Ser Gly Ser Ala Leu Thr Ala Gly Ser Ser Ile Ser

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Leu Arg Val Thr Thr Thr Gly Tyr Thr Thr Arg Tyr Leu Ala His Asn

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Thr Thr Asn Val Ile Thr Ser Val Val Ser Ser Ser Ser Ser Ser Thr

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Leu Gln Lys Gln Ala Ser Trp Thr Val Val Ala Gly Leu Ala Asn Ser

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Gly Cys Phe Ser Phe Glu Ser Val Asp Thr Pro Gly Ser Tyr Ile Arg

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His Tyr Asn Phe Glu Leu Leu Leu Ala Ala Ser Asp Gly Thr Lys Gln

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Phe Lys Glu Asp Ala Thr Phe Cys Pro Glu Ser Gly Leu Ser Gly Ser

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Gly Thr Ser Ile Arg Ser Trp Asn Tyr Pro Thr Arg Trp Phe Arg His

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Tyr Ser Asn Val Gly Tyr Ile Ala Ser Asn Gly Gly Val His Thr Phe

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Gly Phe Ala Ser Ala

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atgccttcac gacgaaccct cctcgggctg gcggccacgg gcaccctggt cagcgccgga 60

ccctgcgaca tctatgcctc cggtggggcg ccctgtatcg ctgcgcacag caccactcgt 120

gccttgtaca gtgcctattc cggtcccctt taccaggtca tccgtggctc ggacagtgct 180

accaccgaca tcaagccgct caccgctggt ggggttgcca atgctgccgc ccaggacacc 240

ttctgctctg gcacgacctg cctgatctcg atcatctacg accagtccgg tagtggaaac 300

gacctcaccc aggctcctcc gggcggcttc gatgggcccg aggccaacgg ctacgacaac 360

ctggccagcg cgatcggcgc ccccgtcacg ttgaacggac agaaggcgta cggtgtcttc 420

gtctcgcccg gcaccggcta ccgcaacaac gatgccaacg gcactgccac cggcgatgag 480

cccgagggca tgtacgcggt cctggacggc acgcactaca acgatgcctg ctgcttcgac 540

tacggcaacg ccgaggtcag cagcaccgac acgggcaacg gccacatgga ggccatctac 600

tacggtacca gcaagacctg gggctatggc tcgggcagtg gcccgtgggt gatggccgat 660

ctcgagaaca acctgttctc cgggcaggcg gagggcctga actcggcgga cccgtcgatc 720

tcctaccggt tcgtgacggc ggcggtcaag ggcgagccca accagtgggc gatccgcggt 780

ggcaacgcgg cctcgggctc gctgacgagc tattacagcg gcgcgcggcc cacggtgagc 840

ggctacaacc cgatgagcaa ggagggggcg atcatcctgg ggatcggcgg cgacaacagc 900

aacggcgcgc agggcacctt ctacgagggg gtgatgacct cgggctaccc gtcggacgcg 960

acggagaact cggtgcaggc caacatcgtg gcggccaagt acgcgacgac ctcgctgacc 1020

agcgggtcgg cgctgacggc gggctcctcg atctcgctgc gcgtcacgac cacggggtac 1080

acgacccgct acctcgccca caacaccacc aacgtcatca cctcggtcgt ctcctcgtcc 1140

agctcctcca ccctgcagaa gcaggccagc tggaccgtcg tcgcggggct ggccaactcc 1200

ggctgctttt ccttcgagtc cgtcgacacc cccggcagct acatccgcca ctacaacttc 1260

gagctgctgc tcgccgccag cgacggcacc aagcagttca aagaggacgc caccttctgc 1320

ccggagtccg gcctcagcgg ctccggcacc tccatccgct cctggaacta ccccacccgt 1380

tggttccgcc actacagcaa cgtgggctac atcgccagca acggcggcgt gcacaccttc 1440

gactccaaga cttcgttcaa tgcggatgtg acctggtcga tcagctcggg ctttgcgtcc 1500

gcgtag 1506

Claims

1. a kind of aspergillus niger engineering bacteria strain, which is characterized in that the aspergillus niger engineering bacteria strain carry for recombinantly express Ah Draw the recombinant plasmid of primary furanoside enzyme.

2. aspergillus niger engineering bacteria strain as described in claim 1, which is characterized in that the amino of the arabinofuranosidase Acid sequence is SEQ ID NO:1.

3. aspergillus niger engineering bacteria strain as claimed in claim 1 or 2, which is characterized in that the arabinofuranosidase, Coding nucleotide sequence is SEQ ID NO:2.

4. a kind of aspergillus niger mutant strain, which is characterized in that the aspergillus niger mutant strain is to as claimed in claim 3 black Aspergillus engineered strain screens acquisition after carrying out ultraviolet mutagenesis.

5. aspergillus niger mutant strain as claimed in claim 4, which is characterized in that the preservation of the aspergillus niger mutant strain is compiled Number be CCTCC NO:M2019433.

6. application of the aspergillus niger engineering bacteria strain described in claim 1 in production arabinofuranosidase.

7. application of the aspergillus niger mutant strain described in claim 4 or 5 in production arabinofuranosidase.