Alkaline beta-mannase, encoding genes thereof and application of encoding genes

[0048] The method for preparing alkaline β-mannanase provided by the invention comprises: cultivating the recombinant strain provided by the invention, inducing the expression of the gene encoding alkaline β-mannanase; separating and purifying the expressed alkaline β-mannanase Glycanase. The culture conditions are conventional culture conditions, such as using LB medium (the solvent is water, the solute and its final concentration are respectively: Tryptone10g/L, yeast extract 5g/L, NaCl10g/L), at 35-37°C Grow to OD 600 is 0.6. Since the recombinant strain provided by the invention contains the gene encoding the alkaline β-mannanase, it can efficiently express the alkaline β-mannanase. After culturing, the high-purity alkaline β-mannanase can be obtained through separation and purification. Methods known to those skilled in the art can be used for separation and purification (for example, adding isopropyl-β-d-thiogalactopyranoside (IPTG) to the culture medium to a final concentration of 0.8mM, and continuing to shake at 37°C for 5 hours Afterwards, the cells were collected, suspended with 20 mM Tris-HCl buffer solution of pH 7.9 and disrupted by ultrasonic, and then purified to obtain alkaline β-mannanase), which will not be repeated here.

[0049] The composition for degrading mannan provided by the present invention contains the alkaline β-mannanase of the present invention as an active ingredient, based on the total weight of the composition, the alkaline β-mannanase The content is 10-90% by weight. The composition may also contain solvents known to those skilled in the art (such as glycerin, carbohydrates, and protease inhibitors and other protein protectants), agonists and the like.

[0050] The present invention also provides the application of the above-mentioned alkaline β-mannanase, gene, recombinant vector, recombinant strain and composition of the present invention in degrading mannan.

[0054] Example 1

[0055] Acquisition of alkaline β-mannanase and its coding gene

[0056] (1) Cloning of the gene encoding alkaline β-mannanase (ManA)

[0057] Take the Bacillus clausii S10 isolated from the alkaline hot spring sample in Inner Mongolia, use the genome extraction kit to extract the total DNA of the Bacillus clausii S10, and measure the purity of the DNA with a UV spectrophotometer. The result is: A260/A280 = 1.88, A260/A230 = 2.13. 10 μl of the total DNA solution (about 50 μg DNA) was taken, partially digested with restriction endonuclease Sau3AI, and a 2-8 kb DNA fragment was recovered by agarose gel electrophoresis. Then carry out ligation reaction, 4°C ligation reaction for 16 hours, the ligation system is as follows (20 μl):

[0058]

[0059]

[0060] Transform competent Escherichia coli DH5α with the product of the ligation reaction, and then apply it to a solid at pH 8.0 containing 60 μg/ml ampicillin (Amp), 20 μg/ml IPTG, 40 μg/ml galactoside (X-gal) and 0.5% konjac powder Incubate on LB medium for 16-18 hours at 37°C, then incubate at 50°C for 1 hour, then carefully pour over high-temperature sterilized 0.1% Congo red solution to develop color, and the colony with a transparent circle around it is a positive clone. Carefully blot dry the Congo red solution, pick out the positive colony with a sterile toothpick, streak a solid LB plate (containing 60 μg/ml of Amp) and incubate overnight at 37°C, pick a single colony of the positive clone that was streaked overnight, and place it on the Amp- After culturing in LB liquid medium at 37°C for 16 hours, the positive single clones with alkaline β-mannanase activity were determined by activity test.

[0061] The recombinant plasmids in the positive clones were sequenced, and the results showed that in the recombinant plasmids, a DNA fragment was inserted into the pUC118 DNA backbone. This DNA fragment contained a 954bp open reading frame (ORF), and its corresponding nucleotide sequence was as follows: As shown in SEQ ID NO: 4, the amino acid sequence encoded by it is the amino acid sequence shown in SEQ ID NO: 3, and the protein with the amino acid sequence shown in SEQ ID NO: 3 is named ManA-S.

[0062] Through the analysis of the signal peptide online prediction software SignalP4.1Server, it is concluded that the 1st to 28th positions in SEQIDNO: 3 are the signal peptide sequence, so the mature alkaline β-mannanase has a total of 289 amino acids, named ManA, and the sequence is as shown in SEQIDNO : 1, the nucleotide sequence of its coding gene is shown in SEQ ID NO: 2.

[0063] (2) Construction of expression vector and recombinant strain of ManA

[0064] According to the nucleotide sequence shown in SEQIDNO: 2, the primer pair is designed as follows:

[0065]Forward primer: 5'-CTA GCTAGC CAAAGCGGCTTTCACGTAAAAG-3' (SEQ ID NO: 10), the underlined part is the NheI restriction site; reverse primer: 5'-CCC AAGCTT TTAATCACGTTTGAGCCCATTTTC-3' (SEQ ID NO: 11), the underlined part is the HindIII restriction site.

[0066] Using the total DNA of Bacillus clausii S10 as a template, PCR amplification was performed with the designed primer pair, and the PCR reaction system was as follows (50 μl):

[0067]

[0068] PCR amplification conditions were: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles; and final extension at 72°C for 10 min. The yield and specificity of the PCR product were detected by 1% agarose gel electrophoresis, and purified with a DNA purification kit (ultra-thin spin column type, produced by Tiangen Company). The purified PCR product is sequenced to detect whether it is the gene fragment shown in SEQ ID NO:2. The correctly sequenced PCR product and the plasmid pET28a (purchased from Novogen) were both digested with NheI and HindIII and recovered by agarose electrophoresis, and then the two digested products were ligated to obtain a recombinant plasmid. The ligation condition was 4°C After 16 hours, the ligation reaction system was as follows (10 μl):

[0069]

[0070] The correct recombinant plasmid verified by sequencing was named pET28a-ManA, and it was used to transform Escherichia coli BL21 (DE3) competent cells, spread on LB solid plates containing 50 μg/ml kanamycin, and cultivate overnight at 37°C to obtain pET28a-containing -ManA recombinant engineering bacteria.

[0071] (3) Preparation and purification of ManA

[0072] The recombinant engineered bacterium that obtains is inoculated in the LB medium that contains 50 μ g/ml kanamycin, and 37 ℃ of overnight culture activations obtain seed liquid, then seed liquid is inoculated in the fresh LB medium of 100ml by the amount of 1% ( Containing 50μg/ml kanamycin), cultivated at 37°C for about 3 hours to OD 600 =0.6, IPTG was added to a final concentration of 0.8 mM, and induction culture was continued for 5 hours at 37°C. Centrifuge the culture solution at 6000g for 10min to collect the bacteria, suspend in 10ml of solution A (20mM Tris-HCl, pH7.9, 0.5MNaCl, 5mM imidazole), and ultrasonically break in an ice bath (60w, 15min; ultrasonic for 3s, stop for 3s), Cell debris was then removed by centrifugation at 12000 g for 5 min. Then heat-treated in a water bath at 60°C for 10 minutes, and then centrifuged at 15,000g for 10 minutes to remove heat-unstable impurities. The supernatant was passed through a Ni-IDA His Bind Superflow purification column (Novogen), washed with 5ml of solution A, and then washed with 10ml of solution B (20mM Tris-HCl , pH7.9, 0.5M NaCl, 60mM imidazole) rinse, and then eluted with 2ml solution C (20mM Tris-HCl, pH7.9, 0.5MNaCl, 1M imidazole), and the eluate was collected. The eluate was desalted with a desalting buffer (20 mM Tris-HCl, pH 7.9) on an AKTA FPLC (fast protein liquid chromatography) system to obtain purified ManA. SDS-PAGE electrophoresis showed that the molecular weight of the purified ManA was about 35kDa (see figure 1 , wherein the M channel is Marker, the 1st channel is the supernatant after the expression of the recombinant engineered bacteria and the cells are broken, the 2nd channel is the supernatant after the heat treatment, and the 3rd channel is the purified ManA), which basically conforms to the theoretical value (34kDa, containing His -tag purification tag).

[0073] (4) Preparation and purification of ManA-S

[0074] According to the nucleotide sequence shown in SEQ ID NO: 4, ManA-S was obtained by referring to the same method as steps (2) and (3).

[0075] Example 2

[0076] Detection of Enzymatic Properties of Alkaline β-Mannanase (ManA)

[0077] (1) Standard enzyme activity assay method

[0078] Take 10 μl of the ManA enzyme solution obtained in Example 1 (diluted to 1.5 μg/ml) and 190 μL of glycine-NaOH buffer solution with a pH value of 9.5 containing 0.4% locust bean gum, mix well, react at 75°C for 10 minutes, and add 200 μL Dinitrosalicylic acid solution (DNS) terminated the reaction (edited by Zhang Longxiang et al., "Biochemical Experimental Methods and Techniques", Higher Education Press, 1996), and then measured the absorbance at 540nm after reacting in a boiling water bath for 5 minutes.

[0079] (2) Determination of the optimum pH value and pH stability of ManA

[0080] At 75°C, the ManA enzyme solution was subjected to enzymatic reactions in buffer solutions with different pH values ​​(pH5.5-11.5) to determine its optimum pH value. The remaining conditions were the same as (1), and the buffer solution used was pH5.5 -7.5Na 2 HPO 4 - Citrate buffer, 50 mM Tris-HCl buffer at pH 7.5-8.5, 50 mM Glycine-NaOH buffer at pH 8.5-10.5 and KCl-NaOH buffer at pH 10.5-11.5. The result is as figure 2 As shown, the optimum pH value of ManA is 9.5.

[0081] Treat the enzyme liquid in buffers with different pH (pH4.5-12.0) at 30°C for 6 hours, then measure the enzyme activity to study the pH stability of the enzyme. The other specific conditions are the same as (1), and the buffer used is pH4 .5-7.5 Na 2 HPO 4 - Citrate buffer, 50 mM Tris-HCl buffer at pH 7.5-8.5, 50 mM Glycine-NaOH buffer at pH 8.5-10.5 and KCl-NaOH buffer at pH 10.5-12.0. The result is as image 3 As shown, ManA is very stable between pH 7.5-12.0, maintaining more than 70% of the enzyme activity.

[0082] (3) Determination of the optimum temperature and thermal stability of ManA

[0083] Enzyme-catalyzed reaction was carried out in 50mM glycine-NaOH buffer system (containing 0.4% locust bean gum) at pH 9.5 and different temperatures (40-90° C.) to determine the optimum temperature of ManA, and the other conditions were the same as (1). Enzyme optimum temperature determination results (see Figure 4 ) shows that the optimum temperature of ManA is 75℃.

[0084] ManA was diluted to a concentration of 1 μg/ml with 50 mM glycine-NaOH buffer solution at pH 9.0, and then incubated at different temperatures (50°C, 60°C, 70°C and 80°C) for different times to determine the residual enzyme activity, The other specific determination conditions are the same as (1), and the thermostability curve of the enzyme is drawn. Results (see Figure 5 ) shows that under the alkaline condition of pH 9.0, ManA still has 90% enzyme activity after being treated at 50°C and 60°C for 180 minutes, and still has 80% enzyme activity after being treated at 70°C for 60 minutes, so ManA has relatively Good thermal alkali stability.

[0085] (4) Enzyme activity of ManA on different mannans

[0086] Under the most suitable reaction conditions (75°C, pH9.5), with locust bean gum, konjac flour, guar gum and Tianqing gum as substrates, and the other conditions are the same as (1), the specific activities of ManA are 1600U/mg , 1800U/mg, 330U/mg and 470U/mg.

[0087] Simultaneously ManA vs. C 3-6 The mannan oligosaccharides also have hydrolytic activity, but have no hydrolytic activity to other polysaccharides such as cellulose, xylan, chitin, soluble starch, pectin, dextran, etc.