CN114774392A

CN114774392A - Mannase and application thereof

Info

Publication number: CN114774392A
Application number: CN202210378803.5A
Authority: CN
Inventors: 蒋明星; 文孟良; 徐艳; 任禛; 殷根深; 董明华; 易梦镯; 李翠
Original assignee: Kunming University
Current assignee: Kunming University
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2022-07-22

Abstract

The invention discloses mannase ABL-03475, an amino acid sequence of which is shown in SEQ ID NO. 1, and the mannase ABL-03475 is applied to preparation of dendrobe oligosaccharide by utilizing dendrobe polysaccharide, wherein dendrobe polysaccharide with rich sources is taken as a raw material, specific mannase ABL-03475 screened from Aspergillus niger is used, and mannan oligosaccharide with wider activity can be prepared by a biological enzyme conversion method, so that a technical method is provided for further utilizing dendrobe, and a new preparation method is provided for preparing a functional mannan oligosaccharide raw material in a functional food industry.

Description

Mannase and application thereof

Technical Field

The invention relates to mannase ABL-03475 and application thereof in preparation of dendrobe oligosaccharide by utilizing dendrobe polysaccharide.

Background

Mannooligosaccharides (MOS), commonly known as manna-oligosaccharides, are used as oligosaccharides. The small molecular saccharide is formed by connecting several mannose or mannose and glucose through beta-1, 4, beta-1, 6, alpha-1, 2, alpha-1, 6 and other glycosidic bonds. MOS from different sources have different structures, for example, the studied konjac mannan oligosaccharide consists of glucose and mannose with a molecular ratio of 1:1.5, a main chain is connected through beta-1, 4 glycosidic bonds, and a side chain is connected through beta-1, 3 glycosidic bonds. The main chain of the glucomannan oligosaccharide derived from the yeast cell wall is formed by connecting pyranose residues through alpha-1, 6 glycosidic bonds, and the glycosidic bonds of the side chains are alpha-1, 2 and alpha-1, 3.

Besides the functions of oxidation resistance, lipid metabolism regulation and the like, the main physiological function of MOS is to regulate the generation and community structure of intestinal probiotics, so as to inhibit intestinal pathogenic bacteria, improve the immunity of the organism and maintain the health of the organism. Bifidobacteria are the most important dominant flora for maintaining intestinal health, and a large number of experiments show that the intake of MOS can promote the proliferation and the function of the bifidobacteria so as to inhibit the growth and the propagation of harmful bacteria such as clostridium perfringens (clostridium perfringens). MOS can also improve the diversity of intestinal microorganisms, promote the proliferation of intestinal probiotics and further maintain the health of the intestinal tract. The harmful bacteria invasion test shows that MOS can reduce the gene expression of intestinal mucosa cell factors TLR4 and IL-1 beta, and increase the number of immune cells such as intestinal epithelial intercellular lymphocytes and goblet cells, thereby improving the local immune response of the organism and further maintaining the intestinal health.

At present, the main methods for obtaining MOS in laboratories and production are as follows: extraction from natural products, acid hydrolysis or enzymatic hydrolysis of mannans. Mannan is mainly present in cell walls of various microorganisms and plants such as konjac glucomannan and locust bean gum in nature. Due to no charge, complex components and the like, the process of separating and purifying MOS from a natural product is complex, the operation is difficult, and the loss of the product is large; when the MOS is prepared by the acid hydrolysis method, the side reactions are more, so that the product components are complex, the pure product is difficult to obtain, and the yield is low; because the catalytic enzyme has the characteristics of specificity, high efficiency and the like, the MOS prepared by the enzyme method is the most common means with relative safety, high efficiency and simple operation.

Disclosure of Invention

The invention provides mannanase ABL-03475, which is derived from Aspergillus niger (Aspergillus niger), and the nucleic acid sequence of the mannanase is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

The invention also aims to apply the mannase ABL-03475 in preparation of dendrobium oligosaccharide.

According to the invention, a mannase gene ABL-03475 is successfully cloned from Aspergillus niger, the mannase gene ABL-03475 is cloned on a pColdTF vector, and is introduced into BL21 for heterologous expression, the mannase ABL-03475 is successfully obtained in vitro, and the purified mannase ABL-03475 reacts with dendrobe polysaccharide to convert dendrobe polysaccharide into dendrobe oligosaccharide.

The mannase ABL-03475 has the optimum temperature of 35 ℃, the optimum pH of 7.5 and the highest enzyme activity of 68U/mL; the mannase ABL-03475 can hydrolyze dendrobium polysaccharide into mannan oligosaccharide.

The invention has the advantages and technical effects that:

the mannase ABL-03475 provided by the invention has high catalytic efficiency and strong specificity. The dendrobe polysaccharide can be almost completely converted into dendrobe oligosaccharide after reaction for 30min under the condition of neutral pH at 30-40 ℃, and the whole reaction operation process is simple, short in reaction time and low in pollution, so that the method is a mild, environment-friendly and efficient dendrobe oligosaccharide production technology and is suitable for industrial production.

Drawings

FIG. 1 is an agarose electrophoresis picture of Aspergillus niger total RNA, wherein M: BM5000 DNA Marker; lanes 1, 2: aspergillus niger RNA;

FIG. 2 is an agarose electrophoresis picture of the target gene abl-03475, wherein M: DL2000 DNA Marker; lane 1: gene abl-03475;

FIG. 3 is a clone validation agarose electrophoresis of the target gene abl-03475, wherein M: DL2000 DNA Marker; lanes 1-3: verifying the gene abl-03475 by PCR;

FIG. 4 shows the recovery of the target gene gel and the extraction of pCold TF plasmid, wherein (a) M: DL2000 DNA Marker; lane 1: recovering abl-03475 gene PCR amplification glue; (b) m: DL5000DNA Marker; lanes 1-2: extracting pCold TF (5769bp) plasmid;

FIG. 5 shows the recovery of the target gene and pCold TF plasmid after double enzyme gel cutting, wherein (a) M: DL5000DNA Marker; lane 1: recovering abl-034751(1,008bp) gene double-enzyme gel cutting; (b) m: DL5000DNA Marker; lane 1: recovering pColdTF vector + Kpn I and Xba I by double-enzyme gel cutting;

fig. 6 is the result of PCR verification of recombinant plasmid, wherein M: DL2000 DNA Marker; lanes 1-4: PCR verification of the pColdTF-abl-03475 recombinant plasmid;

FIG. 7 shows the SDS-PAGE analysis of the recombinant protein, wherein M: protein Marker; lane 1: pColdTF-ABL-03475 recombinant protein;

FIG. 8 shows the SDS-PAGE analysis of the purified recombinant protein, M: a protein Marker; lanes 1-3: pColdTF-ABL-03475 crude protein; lane 4: pCold TF-ABL-03475 is loaded on a column and then collected by using 10mM imidazole washing liquid; lane 5: purified ABL-03475 protein;

FIG. 9 shows the relative enzyme activities of heterologous expression recombinase ABL-03475 at different pH values;

FIG. 10 shows the relative enzyme activities of heterologous expression recombinase ABL-03475 at different temperatures;

FIG. 11 shows the TLC method for detecting the product of the hydrolysis of dendrobii polysaccharide by recombinant protease during purification: 1: a dendrobe polysaccharide reference substance; 2: reacting the crude enzyme solution with a substrate before purification; 3: a dendrobe polysaccharide reference substance; 4: the pure enzyme solution reacts with the substrate.

Detailed Description

The present invention is further described in detail with reference to the following drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples, unless otherwise specified, are conventional reagents and conventional methods;

in the examples pCold TF: expression vectors, available from baozhi bioengineering (Dalian); coli Trans 1-T1: cloning hosts, purchased from Beijing Quanji Biotech, Inc.; coli BL21(DE 3): expression hosts, purchased from Beijing Quanjin Biotechnology Ltd;

LB medium: adding yeast extract of 5g/L, peptone of 10g/L and NaCl of 10g/L into deionized water of 800mL, stirring thoroughly to dissolve, diluting to 1000mL, sterilizing at 121 ℃ for 20min, and storing at room temperature for later use; the concentration of kanamycin added to the LB kanamycin-resistant medium is 50 mug/mL; if the solid LB culture medium is prepared, 20g/L agar is added for screening positive clones.

Antibiotics: kanamycin (Kanamycin) stock (50 mg/mL).

Other reagents: t is a unit of₄DNA ligase, Fastpfu DNA polymerase, was purchased from Beijing Quanjin Biotechnology Ltd. BM5000, DL2000, high purity plasmid extraction kit and nucleic acid purification kit were purchased from Beijing Baitake biotechnology, Inc., and RNA extraction kit Fungal RNAKit (OMEGA) was purchased from Kunming mercy-Summit technology, Inc. Restriction enzymes: kpn I and Xba I were purchased from Beijing Quanji gold Biotechnology Ltd.

Example 1: cloning, recombinant plasmid construction and transformation of mannanase ABL-03475

1. Aspergillus niger total RNA extraction

Washing spores of Aspergillus niger NG1306 with sterile water to obtain spore suspension, inoculating into 150mL triangular flask containing 50mL liquid culture medium, shake culturing at 30 deg.C and 180r/min for 4d, extracting total RNA according to OMEGA kit instruction (RNA is easily degradable and is not suitable for overnight storage), and obtaining the result shown in FIG. 1; a band consistent with the expected fragment size is visible in the figure, indicating that Aspergillus niger RNA was successfully extracted and can be used as a template for RT-PCR amplification.

2. Amplification of mannanase gene

The extracted Aspergillus niger total RNA is subjected to RT-PCR by adopting a method and steps of a reverse transcription Kit PrimeScriptTM One Step RT-PCR Kit Ver.2; RT-PCR amplification conditions (one-step method): 50 ℃, 30min, pre-denaturation 94 ℃,2 min; denaturation at 94 ℃ for 30 s; annealing at 56 ℃ for 30 s; extension at 72 ℃, 90s, 35 cycles; re-extension at 72 ℃ for 10min, and RT-PCR system as shown in Table 1:

TABLE 1 RT-PCR System

3.PCR product purification

The procedures of the agarose gel DNA recovery kit purchased from Baitach corporation in laboratory are referred to the instruction for operation and electrophoresis detection, the Aspergillus niger total RNA is amplified by RT-PCR, the product gel is recovered, 5 microlitre of gel recovery product is taken for agarose gel electrophoresis detection (Marker is DL5000), and the result is shown in figure 2. As can be seen from the figure, the target gene abl-03475(1008bp) is successfully amplified.

4. Subcloning and sequencing of genes of interest

(1) Connecting: mixing 4 μ L of the target fragment with 1 μ L of pEASY-T1 Simple vector, and connecting at 37 deg.C for 20 min;

(2) and (3) transformation: taking out E.coli Trans1-T1 competent cells stored at-80 ℃, putting the competent cells on ice, adding the ligation product, uniformly mixing, and carrying out ice bath for 20-30 min; heat shock is carried out for 90s at 42 ℃, ice bath is carried out for 3min, and then 1mL of LB liquid culture medium is added; incubating in a shaking table at 37 ℃ for 1h, centrifuging at 5000rpm for 5min, discarding the supernatant, adding 200 μ L sterile water, mixing, uniformly coating on LB culture medium containing kanamycin with final concentration of 100 μ g/mL, and inversely placing in an incubator at 37 ℃ for culturing for 14-16 h;

(3) extracting plasmids according to the steps of the Baitach plasmid extraction kit, and performing PCR verification by taking the plasmids as a template;

(4) sequencing: the PCR is verified to be positive and sent to be sequenced, and the sequencing result is compared with the template sequence;

subcloning a target gene in a pEASY-T1 Simple Cloning vector, transforming Escherichia coli E.coli DH5 alpha, selecting a grown positive clone, culturing at 37 ℃ and 180rpm for 8-12h, extracting a plasmid, and then extracting a specific primer PCR amplification verification result as shown in figure 3, wherein the positive clone respectively selects transformants to Kunming Shuoqing biology Limited for sequencing, and a sequencing primer is an M13 universal primer; as can be seen from the figure, the transformants picked up by abl-03475 all amplified bands consistent with the size of the target fragment, which indicates that all transformants were positive, and the target gene was successfully transferred into the cloning host.

5. Design of target gene primer

Comprehensively analyzing enzyme cutting sites contained in related genes, primer annealing temperature and other factors, designing primers, designing front and rear primers respectively containing Kpn I and Xba I enzyme cutting site base sequences, and entrusting Kunming Optimoku Biotechnology Limited to synthesize the primers;

TABLE 2 abl-03475 primers with cleavage site

Note: underlined in the tables indicate the restriction sites

After obtaining the target gene abl-03475 by PCR, double digestion was performed with Kpn I and Xba I, and the pCold TF vector was double digested with the same enzymes, followed by cloning the target gene into the pCold TF vector using T4 ligase.

6. Target gene amplification and extraction of expression plasmid pCold TF

The subclone-positive strain and the strain containing pCold TF vector were inoculated into LB medium containing kanamycin and ampicillin at final concentrations of 50. mu.g/mL and 50. mu.g/mL, respectively, cultured at 37 ℃ and 180rpm for 12 hours, and then plasmids were extracted using the kit. The high fidelity Fastpfu DNA polymerase is adopted to amplify the target gene, and the amplification system is shown in Table 3.PCR amplification conditions: pre-denaturation at 95 deg.C for 2 min; denaturation at 95 ℃ for 20 s; annealing at 56 deg.C for 20 s; extension 72 ℃, 90s, 35 cycles, and finally extension for 10min at 72 ℃. Purifying the PCR product by using a nucleic acid purification kit (Beijing Baitaike biotechnology, Inc.); the PCR conditions are shown in Table 3:

TABLE 3 PCR reaction conditions

PCR amplification (a primer with a restriction enzyme site) and pColdTF empty plasmid extraction products are carried out by taking a plasmid which is successfully subcloned as a template, and agarose gel electrophoresis detection (DL 5000 is Marker) is carried out on a 5 mu L gel recovery product, and the result is shown in figure 4.

7. Digestion and ligation of plasmid

The enzyme digestion reaction system is shown in Table 4, and the ligation reaction system is shown in Table 5; respectively carrying out overnight enzyme digestion gel recovery on the target gene and the plasmid by using corresponding restriction enzymes; detecting the recovered product of the identification gel by using 1.2% agarose gel electrophoresis, connecting for about 16h as shown in figure 5, and then carrying out transformation;

TABLE 4 double digestion reaction system for abl-03475 and pCold TF vectors

TABLE 5 DNA fragment ligation System

8. Preparation of competent cells of Escherichia coli BL21

(1) Taking out the preserved strain from a refrigerator at minus 80 ℃, diluting and coating or marking an LB solid flat plate, and inversely placing the flat plate in an incubator at 37 ℃ for culturing for 16-24 h;

(2) selecting a single colony, inoculating the single colony in 15mL LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12-16 h;

(3) inoculating the culture solution at 1% ratio into 100mL LB liquid culture medium, 37 deg.CCulturing at 200rpm for 2-3h to OD₆₀₀0.4-0.6, ice-bath for 10 min;

(4) the ice-cooled culture solution was dispensed into two 50mL centrifuge tubes, centrifuged at 5000rpm for 5min at 4 deg.C, the supernatant was discarded, and 30mL of pre-cooled 0.1M CaCl was added to each tube₂The solution was gently resuspended, centrifuged at 5000rpm at 4 ℃ for 5min, and the supernatant was discarded.

(5) 30mL of precooled 0.1M CaCl was added to each tube₂After the solution is gently resuspended, the solution is iced for 30min and centrifuged for 5min at 5000rpm and 4 ℃;

(6) the supernatant was aspirated off, and 2mL of pre-cooled 15% glycerol plus 0.1M CaCl were added to each tube₂The solution was gently resuspended and dispensed into sterile 1.5mL centrifuge tubes at 100. mu.L/tube and stored at-80 ℃.

9. Transformation of competent cells of E.coli

(1) Taking out Escherichia coli competence from refrigerator at-80 deg.C, thawing on ice, adding 5 μ L ligation product into 50 μ L competent cell, gently mixing, standing in ice bath for 30min, and performing the process under strict aseptic condition;

(2) floating the centrifuge tube in a water bath at 42 ℃ for 30s to perform heat shock, standing on ice for incubation for 2min, and avoiding shaking in the moving process;

(3) adding 0.5mL of sterilized LB culture medium into a centrifuge tube, and recovering and culturing for 1-2h in a shaking table at the temperature of 37 ℃ and the rpm of 200;

(4) the cells were collected by centrifugation at 4000rpm for 5min, the supernatant was discarded, 100. mu.L of sterile water was added to resuspend the cells, the cells were spread on LB plates containing ampicillin resistance at a final concentration of 50. mu.g/mL by aseptic technique, and the cells were cultured in an inverted incubator at 37 ℃.

10. Coli DH5 alpha transformed with recombinant plasmid and verified

Carrying out plasmid enzyme digestion verification according to the operation of the kit, carrying out nucleic acid sequence sequencing verification after the verification is correct, wherein the sequencing primer uses universal primers pCold TF-F (5'-CCACTTTCAACGAGCTGATG-3') and pCold TF-R (5'-GGCAGGGATCTTAGATTCTG-3') of pCold TF; transformants of abl-03475 were picked and cultured overnight at 37 ℃ and the quality-improved pellets were verified by PCR using universal primers, the PCR results are shown in FIG. 6.

11. Transformation of recombinant plasmid into E.coli BL21(DE3)

And transferring the positive recombinants which are verified to be correct into E.coli BL21(DE3) for induced expression, and storing the strain at-80 ℃.

12. Prokaryotic expression and enzyme activity analysis of target protein

And (3) inducing expression of the target protein: the constructed recombinant strain is inoculated in 15mL LB liquid culture medium containing 50 mug/mL of ampicillin with final concentration, and cultured for 12h at 37 ℃ and 180 rpm; then inoculating at a ratio of 1%, culturing at 37 deg.C and 180rpm for 2-3 hr until OD₆₀₀At 0.6, IPTG was added to a final concentration of 0.5mmol/L, and the mixture was induced at 180rpm and 15 ℃ for 24 hours. After the induction, the cells were centrifuged at 5000rpm for 5min at 4 ℃ and then harvested with ddH₂And O, washing the thalli twice, then re-suspending with 10mL PBS, carrying out cell disruption by adopting a nano homogenizer, detecting the protein expression condition by using 10% SDS-PAGE electrophoresis, and detecting the enzyme activity by using dendrobe polysaccharide as a substrate.

13. SDS-PAGE analysis

5% of the concentrated gum and 10% of the separating gum were prepared, the formulation being shown in Table 7:

TABLE 7 formulation of Polyacrylamide gels

Manufacturing a gel plate:

firstly, cleaning and drying a glass rubber plate for assembly, then preparing separation glue according to the upper table system, fully and uniformly mixing the separation glue, quickly filling the separation glue between the glass rubber plates to a scale mark, adding absolute ethyl alcohol, completely removing the absolute ethyl alcohol on the separation glue after gel is completely polymerized, adding concentrated glue prepared according to the upper table system, and inserting a comb between the glass plates;

② sample treatment

Adding SDS-PAGE Loading Buffer into the sample according to the proportion of 10%, fully and uniformly mixing, and boiling for 5 min;

(iii) sampling

Adding 20 mu L of the treated sample into a sample loading groove, and simultaneously taking 5 mu L of protein standard substance as a control;

(iv) electrophoresis

Connecting a power supply to carry out electrophoresis, wherein the gel concentration current is 20mA and is about 30min, the gel separation current is 60mA and is about 90min, and the electrophoresis is carried out until bromophenol blue moves to the lower end of an electrophoresis tank;

dyeing

Taking the glue out of the glass plate, adding a proper amount of Coomassie brilliant blue staining solution, and slightly oscillating and staining for 30min at 37 ℃;

sixth of color removal

Taking out the glue, recovering the dyeing liquid, putting the glue in the decoloring liquid, and decoloring for many times at 50 ℃ until the protein band is clear.

SDS-PAGE analysis shows that the expression protein (the molecular weight of the target protein is 37kDa and the carrier tag protein is 52kDa to 89kDa) of BL21/pCold TF-ABL-03475 recombinant plasmid detects that the target band is 89kDa, and as shown in figure 7, recombinase ABL-03475 is obviously expressed.

14. Recombinant protein purification

His-Tag in this experiment was bound to the C-terminus of the target protein. His-Tag can be combined with Ca²⁺、Mg²⁺、Ni²⁺、Cu²⁺，Fe³⁺And the like, so that the separation and purification of the target protein are realized. The experiment adopts a nickel column of a polyhistidine tag (9His-tag), and the specific steps of protein purification are as follows:

1) column assembling: according to the amount of protein to be purified, a proper amount of resuspended medium is filled into the chromatographic column, and the chromatographic column is kept still for precipitation.

2) Balancing: the column is equilibrated with 5-10 column volumes of equilibration buffer. For His label recombinant protein with strong binding capacity, the balance buffer solution is properly added with 10-20mM low-concentration imidazole, so that the specific binding can be improved.

3) Sampling: the sample is centrifuged or filtered using a 0.45 μm filter to prevent clogging of the column. And selecting an ultrafiltration tube with proper size for buffer solution replacement, so that the sample buffer solution is as consistent as possible with the balance buffer solution, and the binding efficiency of the target protein His tag is improved.

4) Washing: after the sample loading is finished, washing with 5-10 times of column volume of equilibrium liquid, and collecting effluent liquid.

5) And (3) elution: and (3) carrying out gradient elution on the target protein by using imidazole with different concentrations, and collecting effluent to carry out SDS-PAGE analysis.

Equilibration buffer: 300mM NaCl, 50mM NaH₂PO₄、10mM Mimidazole、10mM Tris base，pH＝8.0。

The purification results are shown in FIG. 8, and recombinase ABL-03475 was successfully purified.

15. Recombinase ABL-03475 property study

15.1 relative enzyme Activity under different pH conditions

Buffers at different pH values: 50mM disodium hydrogenphosphate-citric acid buffer (pH 2.0-6.0), 50mM disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH 7.0-8.0), and 50mM sodium carbonate-sodium bicarbonate buffer (pH 8.5-10).

Adding 5 μ L pure enzyme and 5g/L substrate dissolved in different buffer solutions into 100 μ L reaction system, performing enzymolysis reaction at 37 deg.C for 30min, adding 100 μ L DNS to terminate the reaction, boiling for 7min, and detecting absorbance at 563 nm. The pH optimum activity was defined as 100%. Mixing 5 μ L pure enzyme with buffer solution of pH 4-9, standing at 4 deg.C for 24h, reacting at 30 deg.C for 30min to detect enzyme residual activity, and determining enzyme stability in buffer solution of different pH.

The results are shown in FIG. 9, and the optimum pH is obtained by detecting the enzyme activity of the recombinant protein in a buffer solution with pH 2-10 and reacting at 35 ℃ for 30 min. The results showed that the enzyme had an optimum pH of 7.5 and was relatively stable in a buffer of pH 6-9, confirming that the enzyme is suitable for a more neutral environment.

15.2 relative enzyme Activity at different temperatures

Adding 5 μ L pure enzyme and 5g/L substrate into 100mL reaction system, performing enzymolysis reaction in buffer solution with optimum pH at 10-60 deg.C for 30min, adding 100mL DNS to stop reaction, boiling for 7min, and detecting light absorption value at 540 nm. The activity at the optimum temperature was defined as 100%.

The result is shown in figure 10, and the optimum temperature is obtained by detecting the enzyme activity of the recombinant protein at 10-60 ℃ for 30min under the optimum pH condition by using the dendrobium polysaccharide as a substrate. The results showed that the optimum temperature of the enzyme was 35 ℃.

15.3 analysis of recombinant proteolysis of Dendrobium polysaccharide products

The method comprises the steps of centrifuging the cells subjected to ultrasonic disruption to obtain a crude enzyme solution, reacting the crude enzyme solution with a substrate of 5g/L of dendrobe polysaccharide, wherein the reaction system is 100 mu L of the crude enzyme solution and 400 mu L of the substrate, reacting for 30min at 35 ℃, and detecting a hydrolysis product by TLC (thin layer chromatography), and the result is shown in figure 11, wherein the ABL-03475 is the same as a product obtained by hydrolyzing dendrobe polysaccharide with the crude enzyme solution obtained by solid fermentation of Aspergillus niger, and the dendrobe polysaccharide can be hydrolyzed into dendrobe oligosaccharide in a short time.

Sequence listing

<110> Kunming academy of academic

<120> mannase and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1008

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<400> 1

atgttcgcca aattgtccct tctttcgctt cttttcagct ctgctgcgct gggcgcctcc 60

aaccagactc tgtcctatgg caacattgac aagtctgcca cccccggcgc cagagcgctc 120

ctgaagtaca tccagcttca gtatggatcg cactacatat ctggacagca ggacatcgac 180

agctggaact gggtcgagaa gaacattggt gtggcccctg ccatcctcgg cagcgacttc 240

acctactact cgccatcggc tgttgcccac ggcggcaagt ctcacgcggt cgaggatgta 300

atccagcacg ccggtcgcaa tggaatcaat gccctggttt ggcattggta cgctcccacc 360

tgtctgctcg ataccgataa agagccgtgg tacaagggat tctacaccga ggccacctgc 420

ttcaacgtgt ctgaagccgt caacgaccat ggcaacggca ccaactacaa gctcctgctg 480

cgtgatatcg acgccattgc tgctcagatc aagcgcctgg atcaggccaa cgtgcccatt 540

ctcttccgcc cgctccacga gcccgagggt ggctggttct ggtggggtgc ccagggtcct 600

gctcccttca agaagctgtg ggatattctc tacgaccgca tcactcgcta ccacaacctc 660

cacaacttgg tctgggtttg caacactgct gatccggcct ggtatcccgg aaacgacaaa 720

tgcgacatcg ccaccatcga tcactatccc gccgttggtg accacggagt cgcggccgac 780

cagtacaaga aactccagac cctgaccaag aacgagaggg ttttggctat ggcagaagtt 840

ggccccattc cggaccccga tatgcaggct cgtgagaatg tcaactgggc ttactggatg 900

gtttggtccg gtgagttcat tgaggatggt aagcagaacc ctaaccagtt cttgcacaag 960

gtgtacaacg acacccgggt tgtggctctg aactgggaag gggcttaa 1008

<210> 2

<211> 335

<212> PRT

<213> Aspergillus niger (Aspergillus niger)

<400> 2

Met Phe Ala Lys Leu Ser Leu Leu Ser Leu Leu Phe Ser Ser Ala Ala

1 5 10 15

Leu Gly Ala Ser Asn Gln Thr Leu Ser Tyr Gly Asn Ile Asp Lys Ser

20 25 30

Ala Thr Pro Gly Ala Arg Ala Leu Leu Lys Tyr Ile Gln Leu Gln Tyr

35 40 45

Gly Ser His Tyr Ile Ser Gly Gln Gln Asp Ile Asp Ser Trp Asn Trp

50 55 60

Val Glu Lys Asn Ile Gly Val Ala Pro Ala Ile Leu Gly Ser Asp Phe

65 70 75 80

Thr Tyr Tyr Ser Pro Ser Ala Val Ala His Gly Gly Lys Ser His Ala

85 90 95

Val Glu Asp Val Ile Gln His Ala Gly Arg Asn Gly Ile Asn Ala Leu

100 105 110

Val Trp His Trp Tyr Ala Pro Thr Cys Leu Leu Asp Thr Asp Lys Glu

115 120 125

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

130 135 140

Glu Ala Val Asn Asp His Gly Asn Gly Thr Asn Tyr Lys Leu Leu Leu

145 150 155 160

Arg Asp Ile Asp Ala Ile Ala Ala Gln Ile Lys Arg Leu Asp Gln Ala

165 170 175

Asn Val Pro Ile Leu Phe Arg Pro Leu His Glu Pro Glu Gly Gly Trp

180 185 190

Phe Trp Trp Gly Ala Gln Gly Pro Ala Pro Phe Lys Lys Leu Trp Asp

195 200 205

Ile Leu Tyr Asp Arg Ile Thr Arg Tyr His Asn Leu His Asn Leu Val

210 215 220

Trp Val Cys Asn Thr Ala Asp Pro Ala Trp Tyr Pro Gly Asn Asp Lys

225 230 235 240

Cys Asp Ile Ala Thr Ile Asp His Tyr Pro Ala Val Gly Asp His Gly

245 250 255

Val Ala Ala Asp Gln Tyr Lys Lys Leu Gln Thr Leu Thr Lys Asn Glu

260 265 270

Arg Val Leu Ala Met Ala Glu Val Gly Pro Ile Pro Asp Pro Asp Met

275 280 285

Gln Ala Arg Glu Asn Val Asn Trp Ala Tyr Trp Met Val Trp Ser Gly

290 295 300

Glu Phe Ile Glu Asp Gly Lys Gln Asn Pro Asn Gln Phe Leu His Lys

305 310 315 320

Val Tyr Asn Asp Thr Arg Val Val Ala Leu Asn Trp Glu Gly Ala

325 330 335

<210> 3

<211> 30

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

ggggtaccat gttcgccaaa ttgtcccttc 30

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence (Artificial)

<400> 4

gctctagatt aagccccttc ccagttcag 29

Claims

1. The mannase ABL-03475 has an amino acid sequence shown in SEQ ID NO. 1.

2. The use of the mannanase ABL-03475 of claim 1 in the preparation of dendrobe oligosaccharide from dendrobe polysaccharide.