CN117286125A - Recombinant alpha-L-arabinofuranosidase, coding gene and application thereof - Google Patents
Recombinant alpha-L-arabinofuranosidase, coding gene and application thereof Download PDFInfo
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- CN117286125A CN117286125A CN202311236998.0A CN202311236998A CN117286125A CN 117286125 A CN117286125 A CN 117286125A CN 202311236998 A CN202311236998 A CN 202311236998A CN 117286125 A CN117286125 A CN 117286125A
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- arabinofuranosidase
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a recombinant alpha-L-arabinofuranosidase, a coding gene and application thereof, wherein the amino acid sequence of the recombinant alpha-L-arabinofuranosidase is shown as SEQ ID NO.7, and the coding gene sequence of the recombinant alpha-L-arabinofuranosidase is shown as SEQ ID NO. 6. The optimal pH of the recombinant alpha-L-arabinofuranosidase CcABF is 8.0, and the recombinant alpha-L-arabinofuranosidase CcABF has better tolerance to xylose with higher concentration (1000 mmol/L) and arabinose with lower concentration (400 mmol/L); in addition, the final concentration of the recombinant alpha-L-arabinofuranosidase is 1mmol/L Na + The activity is hardly affected in the ion environment, the residual activity reaches 99 percent, and the activity is simultaneously matched with other gene sourcesHas higher activity than the alpha-L-arabinofuranosidase, and the specific activity is 52.13U/mg.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to recombinant alpha-L-arabinofuranosidase, a coding gene and application thereof.
Background
Hemicellulose is one of the most abundant and inexpensive renewable resources in nature, and its range of existence is very wide, including crop resources and forestry, etc. For the current development goal of 'double carbon', the improvement of the added value of the agricultural waste components represented by hemicellulose is beneficial to the comprehensive utilization of the whole components of agricultural and forestry biomass resources. However, hemicellulose has the characteristics of large molecular weight, multiple components, complex structure and the like, so that the hemicellulose is very limited in application and is difficult to realize large-scale degradation and utilization. The Arabinoxylan (AX) is an important component of hemicellulose in crop resources such as wheat straw, and can be completely degraded under the joint participation of various hydrolytic enzymes such as beta-xylanase, beta-xylosidase, alpha-L-arabinofuranosidase and the like. Compared with beta-xylanase and beta-xylosidase, alpha-L-AFase has relatively few researches and relatively complex structure, and thus becomes one of key enzymes for researching complete degradation of AX.
However, the existing natural alpha-L-arabinofuranosidase has the characteristics of low activity, poor tolerance to special environments such as temperature, pH value and the like, so that the application range of the alpha-L-arabinofuranosidase is greatly limited. Due to the extremely complex structure of substrate AX, some enzymatic reactions are subject to feedback inhibition by the product, namely: as the reaction proceeds, the concentration of the product arabinose or xylose gradually increases, and the increase of the product concentration can lead to the decrease of the reaction rate of the enzyme, so that the degradation efficiency of the substrate and the conversion rate of the substrate are reduced. Therefore, the intensive research of a variety of properties and functions of alpha-L-AFase has guiding significance for exploring and realizing efficient bioconversion of hemicellulose. Chinese patent CN116179516a discloses a vector transformed into escherichia coli BL21 (DE 3) using pEASY-E2, alpha-L-arabinofuranosidase with high ethanol and high salt tolerance, pH6.0, but the highest synergy with commercial xylanase is only 1.11, na at 1mmol/L + The residual enzyme activity is only 23.74 plus or minus 1.48 percent under the optimal conditions. Chinese patent CN111117986B discloses a calcium-dependent thermostable α -L-arabinofuranosidase derived from eupenicillium minutissimum, which has an optimum temperature of 65 ℃ and an optimum pH of 4.5, and which can only convert wheat arabinoxylans to glycans and monosaccharides under acidic conditions in cooperation with xylanase. Chinese patent CN115287291a discloses that alpha-L-arabinofuranosidase is only illustrative of the ability to degrade DDGS, corn husks and wheat branThe substrate does not have the effect of degrading the substrate in cooperation with other enzymes.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a recombinant alpha-L-arabinofuranosidase.
The invention also provides a carrier, engineering bacteria and application of the recombinant alpha-L-arabinofuranosidase.
The technical scheme is as follows: in order to achieve the aim, the invention provides a recombinant alpha-L-arabinofuranosidase, and the amino acid sequence of the recombinant alpha-L-arabinofuranosidase is shown as SEQ ID NO. 7.
Further, the coding gene sequence of the recombinant alpha-L-arabinofuranosidase is shown as SEQ ID NO. 6.
The invention is used for amplifying the coding gene of the recombinant alpha-L-arabinofuranosidase as claimed in claim 2, and the sequence of the used primer pair is shown as SEQ ID NO. 1-2.
Wherein the tolerance pH of the recombinant alpha-L-arabinofuranosidase is 3.0-10.0.
Preferably, the recombinant alpha-L-arabinofuranosidase has an optimal pH of 8.0.
The recombinant vector comprises the recombinant alpha-L-arabinofuranosidase.
The biological material of the recombinant alpha-L-arabinofuranosidase is an expression cassette, a vector and engineering bacteria.
The recombinant alpha-L-arabinofuranosidase or the recombinant vector or the biological material is applied to hemicellulose hydrolysis.
Wherein, the plant fiber is degraded by adopting recombinant alpha-L-arabinofuranosidase.
Further, the plant fiber is wheat bran.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) The optimal pH of the recombinant alpha-L-arabinofuranosidase CcABF is 8.0, and the residual enzyme is remained after the recombinant alpha-L-arabinofuranosidase CcABF is treated for 1h within the pH range of 6.0-8.0The activity reaches more than 82%, the enzyme activity is best in a slightly alkaline environment, the optimal pH environmental condition of the enzyme is enriched, and the degradation of the substrate of the arabinofuranosidase in a neutral or alkaline environment is facilitated; the optimum temperature is 40 ℃ and the stability is better at 20-35 ℃; kinetic parameter K of the enzyme at pH8.0 and temperature 40 DEG C m And v max 3.18mmol/L and 5.43. Mu. Mol/(min.mg), respectively.
(2) The alpha-L-arabinofuranosidase has better tolerance to xylose with higher concentration (1000 mmol/L) and arabinose with lower concentration (400 mmol/L), which shows that the improvement of the concentration of two monosaccharides does not influence the activity of the enzyme basically, and is very important for the application of the arabinofuranosidase in degrading substrates; at the same time, the final concentration is 1mmol/L Na + The activity is hardly affected in the ionic environment, the residual activity reaches 99%, and K + 、Ca 2+ 、Mg 2+ 、Cu 2+ 、Mn 2+ 、Al 3+ 、Fe 3+ 、Zn 3+ 、Co 2+ 、Ni 2+ 、Ba 2+ Urea, SDS, EDTA, ethanol, acetic acid, glycerol, and DTT have inhibitory effects on the activity of recombinant enzymes.
(3) Compared with alpha-L-arabinofuranosidase derived from other genes, the recombinase has higher activity, and the specific activity is 52.13U/mg. The highest synergistic degradation rate of the recombinant enzyme for degrading lignocellulose by cooperating with xylanase reaches 3.47, and the enzyme can synergistically degrade arabinoxylan with xylanase and xylosidase, so that the degradation efficiency of the arabinoxylan is improved, and the recombinant enzyme has potential application value in fields of hemicellulose utilization and the like.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis of recombinant α -L-arabinofuranosidase CcABF. (M: standard protein marker;1: purified α -L-arabinofuranosidase CcABF);
FIG. 2 is the optimum temperature for recombinant α -L-arabinofuranosidase CcABF;
FIG. 3 is the temperature stability of recombinant α -L-arabinofuranosidase CcABF;
FIG. 4 is an optimal pH for recombinant α -L-arabinofuranosidase CcABF;
FIG. 5 is a pH stability of recombinant α -L-arabinofuranosidase CcABF;
FIG. 6 is an effect of chemical agents on recombinant α -L-arabinofuranosidase CcABF;
FIG. 7 is the effect of D-xylose and L-arabinose on recombinant α -L-arabinofuranosidase CcABF;
FIG. 8 is a thin layer chromatography of a recombinant α -L-arabinofuranosidase CcABF for enzymatic hydrolysis of Arabinoxylan (AX) (1 is an xylooligosaccharide standard, 2 is a commercial xylanase and a recombinant enzyme of the invention CcABF for enzymatic hydrolysis of AX in concert, 3 is a commercial xylanase and a recombinant enzyme of the invention CcABF for enzymatic hydrolysis of AX in cooperation, 4 is a commercial xylanase and a recombinant enzyme of the invention CcABF for enzymatic hydrolysis of the invention, 5 is a commercial xylanase and a recombinant enzyme of the invention CcABF for enzymatic hydrolysis of the invention, 6 is a commercial xylanase and a recombinant enzyme of the invention CcABF for enzymatic hydrolysis of the invention, 7 is a commercial xylanase and a recombinant enzyme of the invention CcABF for enzymatic hydrolysis of the invention, 8 is a commercial xylanase and a xylosidase for enzymatic hydrolysis of AX simultaneously, 9 is a commercial xylanase only for enzymatic hydrolysis of AX, 10 is a commercial xylanase only for enzymatic hydrolysis of AX of the invention, 11 is a commercial xylanase only for enzymatic hydrolysis of the invention CcABF.
Detailed Description
The invention is further described below with reference to examples and figures.
Materials, reagents and the like used in the following examples were obtained commercially unless otherwise specified.
The gene of the enzyme is derived from NCBI database, and the expression vector and the genetically engineered bacterium are constructed by the inventor. Competent cells of E.coli DH 5. Alpha. And BL21 (DE 3) were purchased from Nanjinouzan Biotechnology Co., ltd, and the gene operating plasmid pET28a-SUMO was purchased from Wuhan vast Ling Biotechnology Co., ltd. SUMO protease was purchased from shanghai bi yunshii biotechnology limited.
Biochemical reagent and consumable: DNA high fidelity polymerase, T4 DNA ligase, plasmid miniprep kit were all purchased from Nanjinouzan Biotechnology Co., ltd; bradford assay kit was purchased from bi yunsan biotechnology limited; aniline, 85% phosphorusAcid, agar powder, wheat bran-derived arabinoxylans, pNPA (p-nitrophenyl-alpha-L-arabinofuranoside), pNPX (p-nitrophenyl-beta-D-xylopyranoside), pNPM (p-nitrophenyl-alpha-D-mannoside), pNPG (p-nitrophenyl-beta-D-glucopyranoside), D-xylose, L-arabinose, kanamycin sulfate, arabinoxylans and xylooligosaccharides are purchased from Shanghai Michelin Biotechnology Co., ltd., beech xylan is purchased from Shanghai Michel Biotechnology Co., ltd., maize cob xylan is purchased from Shanghai Yi Chi laboratory Equipment Co., ltd., bagasse xylan is purchased from Shanghai source leaf Biotechnology Co., diphenylamine, na 2 CO 3 And n-butanol were purchased from national pharmaceutical systems chemical company, ltd. Restriction enzymes, peptones, yeast extract were purchased from the company Sieimer Feichi technology and sodium chloride was purchased from Shanghai Biotechnology Co. Nickel ion affinity chromatography columns were purchased from GE healthcare, USA.
LB medium: 10g of peptone, 5g of yeast extract, 10g of NaCl, 1000mL of distilled water and natural pH (about 6.5-7.0), and 1.5% (w/v) of agar powder were added to the LB solid medium. The culture medium is sterilized at 121deg.C for 20min with high pressure steam.
The molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.
Example 1
Construction of recombinant alpha-L-arabinofuranosidase CcABF expression vector
(1) The gene sequence was designed optimally based on the alpha-L-arabinofuranosidase CcABF gene sequence (NCBI accession number: NC-021487.1: c 1062232-1060745). Extracting recombinant plasmid CcABF-pET30a with target gene alpha-L-arabinofuranosidase CcABF gene by using a plasmid miniprep kit, wherein the CcABF-pET30a is synthesized by Suzhou Ansheng reaching biotechnology Co., ltd; the recombinant plasmid CcABF-pET30a is used as a template, and under the action of 2X Phanta Master Mix high-fidelity enzyme, the PCR amplification is carried out by using Cc-BamHI-F, cc-SaLI-R as a primer, wherein the sequences of Cc-BamHI-F, cc-SaLI-R are respectively shown as SEQ ID NO.1-2, and the target gene fragment Cc-abf is obtained, and the sequence of which is shown as SEQ ID NO. 3.
The amplification system and the amplification procedure are shown in tables 1 and 2, respectively.
TABLE 1 PCR reaction System
TABLE 2 PCR amplification procedure
SEQ ID NO.1：5′-TTTGGATCCATGGAAGACGTGCGTATC-3′；
SEQ ID NO.2：5′-TTTGTCGACTCATCTTCGTTTTAACCGGAAC-3′；
SEQ ID NO.3：
ATGGAAGACGTGCGTATCACGATCAAGCCCGATGTCCGCATTGGAACGATATCTCCACGTCTGTACGGACATTTTGCGGAACACCTAGGGCGCTGCTGTTATGATGGACTGTTTTTGAAGCCTACGAGCGCGCCGGTTCCTAACCAAAAAGGTTTTCGCACCGATGTGCTCGAGGCGCTGAAAAACATGCCTATGCCATTGCTGCGTTGGCCTGGGGGGTGTTATGCGGATCACTACCATTGGCGCGATGGTATCGGACCTAAGGATCAGCGCCCTAGAAGGCTCGGGATGTCCTGTGGAGAGGTTGTCGAGGACGACAACAGCTTAGGCACCCATGAGTTTCTTTGGTTTTGTGAGCAGACGGGGGCCGAACCCTATTTGGCTGCAAATGTAGGAACGGGATCGCCTCAAGAGATGTGCGATTGGGTTGAGTACTGCAACAGCGCTCTAGATACAACCCTTACCCGGCTACGGCGGGCAAATGGAGCGGAAAAACCGTTTGGGGTGAAGCTGTGGGGGGTTGGTAACGAAAACTGGGGGTGTGGAGGCAACTTCGATGCGCGCGATTATGCGCTGGAGTATCGGCGCTATGCAACCATGCTTCGGCATGTAGACCCCCACATTGAGCTTGTTGCTTGCGGCTTCGATGAGCCGTGGAATCGGACTCTCCTGCAAACCTTGCGCCACCATCTAGATACCATTGACCATCTTTCAGTGCACCGCTACTGGCACGGAGAAGGGGGAGAGCTCGATCCCTCTGAGAAGGTCTCTTATCGCCTTTTTACCGAGGCATCGCTGACGGAAGCCTTTGTGCAACAGACCGCCGACATCATTCGTGTGGAGCTCGGCGAACGACGCCGTGTGGGGGTTGCTCTGGATGAGTGGGGTATTTGGCATCCGGAGGCGAGACTTCAGTTTGATCCTGATAAACGCGGTGGGCCCTACGAGCAGGTTTCGACCCTACGCGATGCCCTAGCGGCCGCCATTGTTCTGGAAGGCTTTCATCGGCAGTGCCGCGTTTTGAGTTTGGCCAACCTCGCGCAAGTCGTAAATGTGCTTCATGCGCCCATTCAGACGCGAGGGGCTGCTATGTGGCGTACACCCACCTATCATGTCTTTCAGCTTCATGCCCCACATATAGGGGCCACCGCTCTGGAGGTAGAGGTGCATGCAAGTCTTTTGCCCGTTAGTGAGGAGCTGCGCTGCGTTACGGCTACCGCTTCGCTCGATAAGGAGGACAATCTTACACTCACCGTTCTCAATTTGCATTTGGATCAGTCGGCCTCGGTGCATTTCGGCGGCACAGAGGGCCGCGTGCTGTTCGATGCACAGCTGTTGACAGCCGATAACCCCCTCGCTCATAACTCGGCGGAGAAGCCGGACGCGGTGGTACCGCAGCCCATTAAGGCCACTGAGAGCACGCAGGGCGGCTACCTTGTGAATCTGCCGAAACACTCCATGGCGACGTTCCGGTTAAAACGAAGATGA
Double digestion is carried out on the target gene fragment cc-abf and the plasmid pET28a-SUMO by enzyme digestion sites BamHI and SacI, agarose gel electrophoresis is carried out on double digestion products, tapping recovery is carried out, and the recovered digestion products are connected with the target gene cc-abf into the vector pET28a-SUMO through T4 DNA ligase to form connection products. The ligation products were then transformed into DH 5. Alpha. Competent cells as follows:
1) mu.L DH 5. Alpha. Competent cells were placed in 1.5mL EP tube, 10. Mu.L of ligation product was added, mixed well on ice and left for 30min.
2) The EP tube was placed in a 42℃water bath for 45s of heat shock, and then the tube was placed on ice for incubation for 2min.
3) 900mL of LB liquid medium without any antibiotics was then added and incubated at a slow speed in a shaker at 37℃for 1h.
4) The culture was centrifuged to remove most of the supernatant, and the precipitated cells were resuspended and plated on LB solid plates containing kanamycin sulfate at a final concentration of 50. Mu.g/mL, and incubated in an incubator at 37℃for 12-16 hours until single colonies were grown.
And (3) picking a single colony to an LB liquid culture medium containing kanamycin sulfate with a final concentration of 50 mug/mL for culture, and extracting plasmids in the bacterial cells by using a plasmid small extraction kit when the bacterial cells grow to a platform stage to obtain a recombinant expression plasmid pET28a-SUMO-CcABF, wherein sequencing verification of the recombinant plasmid is completed by Suzhou Anshengzha biotechnology limited company. Finally, the recombinant plasmid pET28a-SUMO-CcABF contains recombinant alpha-L-arabinofuranosidase CcABF and a full-length fragment of the gene of the pro-expression tag SUMO, and the N end of the SUMO contains 6 histidine tags for subsequent purification. The nucleotide sequence of the SUMO gene is shown as SEQ ID NO.4, and the nucleotide sequence of the CcABF-SUMO fusion gene is shown as SEQ ID NO.5 (5 '. Fwdarw.3'):
SEQ ID NO.4：
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGA
SEQ ID NO.5：
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGATCCATGGAAGACGTGCGTATCACGATCAAGCCCGATGTCCGCATTGGAACGATATCTCCACGTCTGTACGGACATTTTGCGGAACACCTAGGGCGCTGCTGTTATGATGGACTGTTTTTGAAGCCTACGAGCGCGCCGGTTCCTAACCAAAAAGGTTTTCGCACCGATGTGCTCGAGGCGCTGAAAAACATGCCTATGCCATTGCTGCGTTGGCCTGGGGGGTGTTATGCGGATCACTACCATTGGCGCGATGGTATCGGACCTAAGGATCAGCGCCCTAGAAGGCTCGGGATGTCCTGTGGAGAGGTTGTCGAGGACGACAACAGCTTAGGCACCCATGAGTTTCTTTGGTTTTGTGAGCAGACGGGGGCCGAACCCTATTTGGCTGCAAATGTAGGAACGGGATCGCCTCAAGAGATGTGCGATTGGGTTGAGTACTGCAACAGCGCTCTAGATACAACCCTTACCCGGCTACGGCGGGCAAATGGAGCGGAAAAACCGTTTGGGGTGAAGCTGTGGGGGGTTGGTAACGAAAACTGGGGGTGTGGAGGCAACTTCGATGCGCGCGATTATGCGCTGGAGTATCGGCGCTATGCAACCATGCTTCGGCATGTAGACCCCCACATTGAGCTTGTTGCTTGCGGCTTCGATGAGCCGTGGAATCGGACTCTCCTGCAAACCTTGCGCCACCATCTAGATACCATTGACCATCTTTCAGTGCACCGCTACTGGCACGGAGAAGGGGGAGAGCTCGATCCCTCTGAGAAGGTCTCTTATCGCCTTTTTACCGAGGCATCGCTGACGGAAGCCTTTGTGCAACAGACCGCCGACATCATTCGTGTGGAGCTCGGCGAACGACGCCGTGTGGGGGTTGCTCTGGATGAGTGGGGTATTTGGCATCCGGAGGCGAGACTTCAGTTTGATCCTGATAAACGCGGTGGGCCCTACGAGCAGGTTTCGACCCTACGCGATGCCCTAGCGGCCGCCATTGTTCTGGAAGGCTTTCATCGGCAGTGCCGCGTTTTGAGTTTGGCCAACCTCGCGCAAGTCGTAAATGTGCTTCATGCGCCCATTCAGACGCGAGGGGCTGCTATGTGGCGTACACCCACCTATCATGTCTTTCAGCTTCATGCCCCACATATAGGGGCCACCGCTCTGGAGGTAGAGGTGCATGCAAGTCTTTTGCCCGTTAGTGAGGAGCTGCGCTGCGTTACGGCTACCGCTTCGCTCGATAAGGAGGACAATCTTACACTCACCGTTCTCAATTTGCATTTGGATCAGTCGGCCTCGGTGCATTTCGGCGGCACAGAGGGCCGCGTGCTGTTCGATGCACAGCTGTTGACAGCCGATAACCCCCTCGCTCATAACTCGGCGGAGAAGCCGGACGCGGTGGTACCGCAGCCCATTAAGGCCACTGAGAGCACGCAGGGCGGCTACCTTGTGAATCTGCCGAAACACTCCATGGCGACGTTCCGGTTAAAACGAAGA
(2) Analysis of results: according to sequencing results and sequence analysis, the gene of expressing recombinant alpha-L-arabinofuranosidase CcABF-SUMO fusion protein has the total length of 1851bp, the theoretical molecular weight of 69.33kDa and the theoretical isoelectric point (pI) of 6.01.
The fusion protein gene SUMO-CcABF constructed according to the method consists of a recombinant alpha-L-arabinofuranosidase CcABF gene and a SUMO gene. The fusion protein gene is introduced into snapgene software for analysis, and the gene length of recombinant alpha-L-arabinofuranosidase CcABF=the gene length of SUMO-CcABF fusion protein-SUMO gene length is utilized to obtain the gene full length 1491bp of the recombinant alpha-L-arabinofuranosidase CcABF, the theoretical molecular weight of mature CcABF is 55.63kDa, the theoretical isoelectric point (pI) is 6.02, and the nucleotide sequence is shown as SEQ ID NO. 6.
SEQ ID NO.6：
TCCATGGAAGACGTGCGTATCACGATCAAGCCCGATGTCCGCATTGGAACGATATCTCCACGTCTGTACGGACATTTTGCGGAACACCTAGGGCGCTGCTGTTATGATGGACTGTTTTTGAAGCCTACGAGCGCGCCGGTTCCTAACCAAAAAGGTTTTCGCACCGATGTGCTCGAGGCGCTGAAAAACATGCCTATGCCATTGCTGCGTTGGCCTGGGGGGTGTTATGCGGATCACTACCATTGGCGCGATGGTATCGGACCTAAGGATCAGCGCCCTAGAAGGCTCGGGATGTCCTGTGGAGAGGTTGTCGAGGACGACAACAGCTTAGGCACCCATGAGTTTCTTTGGTTTTGTGAGCAGACGGGGGCCGAACCCTATTTGGCTGCAAATGTAGGAACGGGATCGCCTCAAGAGATGTGCGATTGGGTTGAGTACTGCAACAGCGCTCTAGATACAACCCTTACCCGGCTACGGCGGGCAAATGGAGCGGAAAAACCGTTTGGGGTGAAGCTGTGGGGGGTTGGTAACGAAAACTGGGGGTGTGGAGGCAACTTCGATGCGCGCGATTATGCGCTGGAGTATCGGCGCTATGCAACCATGCTTCGGCATGTAGACCCCCACATTGAGCTTGTTGCTTGCGGCTTCGATGAGCCGTGGAATCGGACTCTCCTGCAAACCTTGCGCCACCATCTAGATACCATTGACCATCTTTCAGTGCACCGCTACTGGCACGGAGAAGGGGGAGAGCTCGATCCCTCTGAGAAGGTCTCTTATCGCCTTTTTACCGAGGCATCGCTGACGGAAGCCTTTGTGCAACAGACCGCCGACATCATTCGTGTGGAGCTCGGCGAACGACGCCGTGTGGGGGTTGCTCTGGATGAGTGGGGTATTTGGCATCCGGAGGCGAGACTTCAGTTTGATCCTGATAAACGCGGTGGGCCCTACGAGCAGGTTTCGACCCTACGCGATGCCCTAGCGGCCGCCATTGTTCTGGAAGGCTTTCATCGGCAGTGCCGCGTTTTGAGTTTGGCCAACCTCGCGCAAGTCGTAAATGTGCTTCATGCGCCCATTCAGACGCGAGGGGCTGCTATGTGGCGTACACCCACCTATCATGTCTTTCAGCTTCATGCCCCACATATAGGGGCCACCGCTCTGGAGGTAGAGGTGCATGCAAGTCTTTTGCCCGTTAGTGAGGAGCTGCGCTGCGTTACGGCTACCGCTTCGCTCGATAAGGAGGACAATCTTACACTCACCGTTCTCAATTTGCATTTGGATCAGTCGGCCTCGGTGCATTTCGGCGGCACAGAGGGCCGCGTGCTGTTCGATGCACAGCTGTTGACAGCCGATAACCCCCTCGCTCATAACTCGGCGGAGAAGCCGGACGCGGTGGTACCGCAGCCCATTAAGGCCACTGAGAGCACGCAGGGCGGCTACCTTGTGAATCTGCCGAAACACTCCATGGCGACGTTCCGGTTAAAACGAAGATGA
Example 2
Construction of recombinant alpha-L-arabinofuranosidase CcABF genetically engineered bacterium
The recombinant plasmid pET28a-SUMO-CcABF prepared in the embodiment 1 with correct sequencing is introduced into escherichia coli BL21 (DE 3) for transformation, and the specific transformation process is as follows:
(1) mu.L of recombinant plasmid pET28a-SUMO-CcABF was taken and added to an EP tube containing 50. Mu.L of E.coli competent cells (BL 21) DE3, and after mixing well, placed on ice for 30min.
(2) The EP tube was immediately removed, placed in a 42℃water bath for 45s of heat shock, and then placed on ice for 2-3min.
(3) After addition of 900. Mu.L of LB liquid medium (no resistance), incubation was carried out for 1h at 37℃in an environment of 180 rpm.
(4) Centrifuging at 8000rpm for 5min at 4deg.C, discarding 860 μl supernatant, re-suspending the rest liquid, and absorbing 15 μl fungus liquid, coating on LB solid plate (containing kanamycin sulfate with final concentration of 50 μg/mL), and culturing at 37deg.C for 15-16 hr.
After single colony grows out of the flat plate, picking a single colony by using a toothpick, inoculating the single colony into LB liquid culture medium (containing kanamycin sulfate with the final concentration of 50 mug/mL), and culturing to obtain recombinant genetically engineered bacterium CcABF-SUMO-BL21.
Example 3
Preparation of recombinant alpha-L-arabinofuranosidase CcABF
(1) The experimental method comprises the following steps: inoculating the constructed genetic engineering strain (CcABF-SUMO-BL 21) in 50mL LB liquid medium (containing kanamycin sulfate with the final concentration of 50 mug/mL), and culturing at 180rpm and 37 ℃ overnight for 12-14h; the next day was inoculated in 1L of fresh LB medium (containing kanamycin sulfate at a final concentration of 50. Mu.g/mL) at a transfer rate of 5%, and the OD was reached 600 When the concentration is 0.8-1.0, IPTG with the final concentration of 0.2mmol/L is added for induction, and the induction condition is 180rpm and the induction is carried out at 25 ℃ for 16-20 h. Purification of CcABF is split into two steps: firstly, separating and purifying target fusion enzyme by using a nickel column affinity chromatography method, collecting eluent in the eluting process, and finally, storing pure enzyme in a storage buffer (20 mmol/L Tris-HCl,50mmol/L NaCl). And secondly, cutting the CcABF-SUMO fusion protein by using SUMO protease, purifying by using nickel column affinity chromatography again, and collecting a penetrating fluid in the loading process to obtain the single enzyme protein CcABF. The purified protein was detected by SDS-PAGE. Protein quantification was performed using Bradford assay kit.
The reagents and methods used for the nickel ion affinity chromatography column are as follows:
1) Reagent(s)
Protein purification equilibration buffer (buffer a): 20mmol/L Tris-HCl,500mmol/L NaCl,20mmol/L imidazole, pH 8.0;
protein purification elution buffer (buffer B): 20mmol/L Tris-HCl,500mmol/L NaCl,500mmol/L imidazole, pH 8.0.
2) Step (a)
(1) Filtering the crude protein solution, buffer A, buffer B and ultrapure water by using a microporous filter membrane with the diameter of 0.22 mu m;
(2) flushing the AKTA system, the pump head A and the pump head B by ultrapure water;
(3) and (3) column loading: placing the pump head A into ultrapure water, regulating the flow rate to 1mL/min, and loading a nickel ion affinity column on an AKTA protein purifier;
(4) punching a column: and placing the pump head A in the buffer A, and placing the pump head B in the buffer B. The flow rate is set to 3mL/min, 3-5 column volumes are flushed with buffer B, and then the column is flushed with buffer A until the baseline is balanced;
(5) loading: the pump head A is placed in crude protease solution for loading, and the penetrating solution is collected. And after the sample is loaded, the pump head A is replaced in the buffer A until the baseline is balanced.
(6) Eluting: gradient elution was performed on the target protein using buffer B, and the elution time was set to 15min. During this period, when the ultraviolet absorption peak was increased, the target protein was collected in a volume of 2 mL/tube.
(7) The collected target proteins were concentrated using a 10kDa ultrafiltration centrifuge tube and buffer A or buffer B was replaced with enzyme storage buffer.
(2) Analysis of results: as shown in FIG. 1, recombinant alpha-L-arabinofuranosidase CcABF is successfully expressed in escherichia coli BL21 (DE 3), and is purified to form a single band, the amino acid sequence of the single band is shown as SEQ ID NO.7, and the actual molecular weight is close to the theoretical molecular weight (55.63 kDa and including histidine tag). It was calculated that 4.48. Mu.g of pure enzyme could be obtained per ml of fermentation broth.
SEQ ID NO.7：
SMEDVRITIKPDVRIGTISPRLYGHFAEHLGRCCYDGLFLKPTSAPVPNQKGFRTDVLEALKNMPMPLLRWPGGCYADHYHWRDGIGPKDQRPRRLGMSCGEVVEDDNSLGTHEFLWFCEQTGAEPYLAANVGTGSPQEMCDWVEYCNSALDTTLTRLRRANGAEKPFGVKLWGVGNENWGCGGNFDARDYALEYRRYATMLRHVDPHIELVACGFDEPWNRTLLQTLRHHLDTIDHLSVHRYWHGEGGELDPSEKVSYRLFTEASLTEAFVQQTADIIRVELGERRRVGVALDEWGIWHPEARLQFDPDKRGGPYEQVSTLRDALAAAIVLEGFHRQCRVLSLANLAQVVNVLHAPIQTRGAAMWRTPTYHVFQLHAPHIGATALEVEVHASLLPVSEELRCVTATASLDKEDNLTLTVLNLHLDQSASVHFGGTEGRVLFDAQLLTADNPLAHNSAEKPDAVVPQPIKATESTQGGYLVNLPKHSMATFRLKRR
Example 4
Enzymatic Properties of recombinant alpha-L-arabinofuranosidase CcABF
1. Activity assay of recombinant alpha-L-arabinofuranosidase CcABF
(1) And (3) manufacturing a standard curve: the pNP standard solution 0, 1, 2, 3, 4, 5uL to 1.5mL EP tube was accurately aspirated using pure water and an amount of pNP (p-nitrophenol) powder to a final concentration of 0.5mg/mL, and filled to 100uL with phosphate buffer pH=7.7. Respectively at different concentrations100. Mu.L of 1mol/L Na was added to the pNP diluent of (C) 2 CO 3 Mixing well, sucking 200 μl sample, and measuring absorbance at 405nm, pNP concentration and A concentration 405nm In a linear relationship. On the abscissa, the pNP solution concentration and OD 405 Reading is vertical coordinate, and drawing a standard curve.
(2) Activity measurement System: methods for measuring enzyme activities are described in Peng Cheng et al (Peng Cheng, et al 2021), pNPA and Na 2 CO 3 The concentration, amount of buffer used and reaction time were slightly modified. The activity of alpha-L-arabinofuranosidase was determined using pNPA (p-nitrophenyl-alpha-L-arabinofuranosid) as substrate in phosphate buffer pH 8.0. The method comprises the following steps: 150. Mu.L of phosphate buffer (20 mmol/L, pH 8.0) was added to 25. Mu.L of pNPA at a final concentration of 0.5mg/mL, 25. Mu.L of the recombinant alpha-L-arabinofuranosidase prepared in example 3 was further added, the mixture was reacted at 40℃for 10min, and 100. Mu.L of 1mol/L Na was immediately added 2 CO 3 The reaction was terminated.
Control group: the recombinant alpha-L-arabinofuranosidase prepared in example 3 was treated at 100deg.C for 5min for inactivation using the inactivated recombinase liquid as a control. 150. Mu.L of phosphate (20 mmol/L, pH 8.0) buffer was added with 25. Mu.L of pNPA at a final concentration of 0.5mg/mL, 25. Mu.L of the prepared inactivated recombinase was added, the mixture was reacted at 40℃for 10min, and 100. Mu.L of 1mol/L Na was immediately added 2 CO 3 The reaction was terminated.
The amount of enzyme required to hydrolyze to 1. Mu. MoL of p-nitrophenol (pNP) per minute was defined as 1 unit of enzyme activity (U). The standard curve was determined as: y=5.0274x+0.1328, where y is OD 405 The reading, x, is pNP concentration (. Mu.mol/L). The specific activity of arabinofuranosidase in the samples was calculated to be 52.13U/mg according to the standard curve.
2. Determination of optimum temperature and temperature stability of recombinant alpha-L-arabinofuranosidase CcABF
The activity of the recombinase at different temperatures was measured and the optimum catalytic temperature was determined. 150. Mu.L of phosphate buffer (20 mmol/L, pH 8.0) was added to 25. Mu.L of pNPA at a final concentration of 0.5mg/mL, followed by 25. Mu.L of example 3The prepared recombinant alpha-L-arabinofuranosidase liquid is reacted with the mixture at different temperatures (20-85 ℃) for 10min respectively, and 100 mu L of 1mol/L Na is added immediately 2 CO 3 The reaction was terminated. The control and the part 1 activity assay system of this example were identical to those of the control using inactivated recombinase liquid, except that the mixture was reacted at (20-85 ℃) for 10min, respectively. As shown in FIG. 2, the optimal temperature of the recombinant α -L-arabinofuranosidase CcABF is 40 ℃.
The temperature stability of the recombinase at different temperatures was determined. Taking 25 mu L of recombinant alpha-L-arabinofuranosidase purified in example 3, placing in 20mmol/L phosphate buffer solution with pH of 8.0, respectively incubating for 1h at different temperatures (20-85 ℃), taking 150 mu L of phosphate buffer solution after incubation at different temperatures, respectively adding 25 mu L of pNPA with final concentration of 0.5mg/mL, reacting the mixture at 40 ℃ for 10min, immediately adding 100 mu L of 1mol/L Na 2 CO 3 The reaction was terminated, and the residual enzyme activity was measured, and the control group and the part 1 activity measuring system of this example were the same as those of the inactivated recombinase liquid as a control group, and the temperature stability of the enzyme was analyzed, assuming that the activity of the original enzyme liquid which had not been treated for 1h as described above was 100%. As shown in FIG. 3, the activity of the recombinant enzyme is still maintained at more than 85.00% after incubation for 1h at 20-35 ℃.
3. Determination of optimum pH and pH stability of recombinant alpha-L-arabinofuranosidase CcABF
Phosphate buffers with different pH values are respectively: 20mmol/L glycine-hydrochloric acid buffer (pH 2.0-5.0); 20mmol/L phosphate buffer (pH 3.0-7.0); 20mmol/L glycine-sodium hydroxide buffer (pH 8.0-10.5).
Measuring the activity of the enzyme at different pH (2.0-10.5) to determine the optimum pH; respectively taking 150 mu L of phosphate buffer solutions with different pH values, respectively adding 25 mu L of pNPA with final concentration of 0.5mg/mL, further adding 25 mu L of recombinant alpha-L-arabinofuranosidase solution prepared in example 3, reacting the mixture at 40 ℃ for 10min, immediately adding 100 mu L of 1mol/L Na 2 CO 3 The reaction was terminated. The control group and the part 1 activity assay system of this example were identical to those of the control group using the inactivated recombinase liquid, except that phosphate buffer solutions of different pH were used. As a result, the recombinant α -L-arabinofuranosidase has an optimal pH of 8.0 as shown in FIG. 4.
The pH stability of the recombinant enzyme was determined at different pH phosphate buffers. Firstly, placing recombinant enzyme alpha-L-arabinofuranosidase in different pH buffers to treat for 24 hours at 4 ℃, respectively taking 150 mu L of different pH buffers, respectively adding 25 mu L of pNPA with the final concentration of 0.5mg/mL, reacting the mixture at 40 ℃ for 10 minutes, and immediately adding 100 mu L of 1mol/L Na 2 CO 3 The reaction was terminated. The activity of the enzyme was measured in the same manner as in the case of the inactivated recombinant enzyme solution as a control group by using the activity measurement system of the part 1 of this example, and the pH stability of the enzyme was analyzed by taking the activity of the original enzyme solution, which had not been subjected to the above-mentioned treatment for 24 hours, as 100%. As a result, as shown in FIG. 5, the relative enzyme activity was maintained at 90.00% or more after 24 hours of treatment with the buffer solution at pH 8.0.
4. Determination of the influence of different Metal ions and chemical reagents on the Activity of recombinant alpha-L-arabinofuranosidase CcABF
Firstly, metal ions (Na) with final concentration of 1mmol/L and 10mmol/L are respectively added into a phosphate buffer solution with the pH of 20mmol/L and 8.0 + 、K + 、Ca 2+ 、Mg 2+ 、Cu 2+ 、Mn 2+ 、Al 3+ 、Fe 3+ 、Zn 3+ 、Co 2+ 、Ni 2+ 、Ba 2+ ) 150. Mu.L of pH buffer containing different metal ions was taken, 25. Mu.L of pNPA was added thereto at a final concentration of 0.5mg/mL, 25. Mu.L of the recombinant alpha-L-arabinofuranosidase solution prepared in example 3 was further added thereto, the mixture was reacted at 40℃for 10 minutes, and 100. Mu.L of 1mol/L Na was immediately added thereto 2 CO 3 The reaction was terminated. The control and the part 1 activity assay system of this example were identical with the inactivated recombinase solution as the control, except that different metal ions were added to the phosphate buffer at different final concentrations.
In addition, chemical reagents (urea, SDS, EDTA, ethanol, acetic acid, glycerol, DTT) with a final concentration of 1% (V/V) were added to 20mmol/L of phosphate buffer solution at pH8.0, 150. Mu.L of pH buffer solution containing different chemical reagents was respectively added, 25. Mu.L of pNPA with a final concentration of 0.5mg/mL was respectively added, and 25. Mu.L of recombinant alpha-L-arabinofuranosidase solution, the mixture was reacted at 40℃for 10min, and 100. Mu.L of 1mol/L Na was immediately added 2 CO 3 The reaction was terminated. The control group and the part 1 activity assay system of this example were identical with the inactivated recombinase solution as the control group, except that the inactivated recombinase of the reaction system added with the different chemical reagent groups was used as the control group in the phosphate buffer solution, and the other processes were unchanged.
The effect on the recombinant enzyme activity was studied, taking the activity of the enzyme in the reaction system to which no metal ion was added as 100%. The change in recombinase activity was measured at 40℃and pH8.0, and the relative recombinase activity was calculated. As a result of analysis, as shown in FIG. 6, a low concentration (1 mmol/L) of metal ion K + 、Ca 2+ 、Mg 2+ 、Cu 2+ 、Mn 2+ 、Al 3+ 、Fe 3+ 、Zn 3+ 、Co 2+ 、Ni 2+ 、Ba 2+ All have inhibitory effect on the activity of the enzyme, wherein Fe 3+ 、Cu 2+ 、Ba 2+ And Co 2+ The metal ion inhibition effect is most obvious, and the relative enzyme activity only remains 1.15% -9.26%; na (Na) + Has little influence on the enzyme activity, and the residual enzyme activity can still be kept at 99.07 percent, which indicates that the recombinase has no influence on Na + The tolerance is high. Under the action of high-concentration (10 mmol/L) metal ions, only Na + And Ca 2+ Under the action of the recombinant enzyme, the activity of the recombinant enzyme is kept at 50.44% and 58.54%, and the relative activity of the recombinant enzyme is reduced to 0.00% -8.75% after the rest metal ions act. The addition of the chemical reagent has an inhibition effect on the activity of the recombinase, and the activity is reduced to 12.20% -49.50%. The results demonstrate that recombinant alpha-L-arabinofuranosidase CcABF is resistant to Na + And other metal ions and chemical agents can inhibit the activity of the recombinase to different degrees.
5. Enzymatic kinetic parameter determination of the recombinase alpha-L-arabinofuranosidase CcABF
The dynamic parameters are measured at 20mmol/L, phosphate buffer with pH of 8.0 and different concentrations of pNPA as substrates at 40 ℃ and 0.16-2.60 mmol/L, and K is obtained by calculation by using the Lineweaver-Burk method m And v max Values.
The results show that, by measurement, the enzyme K m And v max The values were 0.16mmol/L and 0.10. Mu. Mol/(min. Mg), respectively
Example 5
Application of recombinant alpha-L-arabinofuranosidase CcABF
1. Effect of D-xylose and L-arabinose on recombinant alpha-L-arabinofuranosidase CcABF
150. Mu.L of phosphate buffer having pH8.0 was added respectively to 25. Mu.L of D-xylose and L-arabinose in final concentrations of 50, 100, 200, 400, 800 and 1000mmol/L, 25. Mu.L of pNPA in final concentration of 0.5mg/mL was added respectively, 25. Mu.L of the recombinant alpha-L-arabinofuranosidase solution prepared in example 3 was further added, the mixture was reacted at 40℃for 10min, and 100. Mu.L of Na in 1mol/L was immediately added 2 CO 3 The reaction was terminated. The control and the part 1 activity assay system of this example were identical with the inactivated recombinase liquid as the control, except that D-xylose and L-arabinose were added at different concentrations. The effect of monosaccharides on recombinase activity was analyzed with the activity of the experimental group without any added sugar set at 100%.
As a result, as shown in FIG. 7, at a low concentration (50 mmol/L), the feedback inhibition by the presence of L-arabinose on the alpha-L-arabinofuranosidase CcABF was minimal, and the relative activity was 77.34%. With further increase of the concentration, the presence of L-arabinose has an inhibitory effect on the activity of alpha-L-arabinofuranosidase. D-xylose has small inhibition effect on the enzyme, and the activity of alpha-L-arabinofuranosidase is improved along with the increase of the concentration, and the relative activity is still maintained to be more than 80.00% when the concentration is 800-1000 mmol/L. The result shows that the enzyme has better tolerance to high-concentration D-xylose and has strong potential in hemicellulose hydrolysis application.
2. Degradation ability of recombinant alpha-L-arabinofuranosidase CcABF on different substrates
150. Mu.L of phosphate (20 mmol/L, pH 8.0) buffer was added, 25. Mu.L of different substrates were added, respectively, at a final concentration of 0.5mg/mL, 25. Mu.L of the recombinant alpha-L-arabinofuranosidase prepared in example 3 was further added, the mixture was reacted at 40℃for 10min, and 100. Mu.L was immediately added 1mol/L Na 2 CO 3 The reaction was terminated. The substrates are respectively as follows: pNPA, p-nitrophenyl B-D-glucopyranoside (pNPG), p-nitrophenyl-beta-D-xylopyranoside (pNPX), p-nitrophenyl-alpha-D-mannoside (pNPM) and 0.2% (V/N) of wheat bran-derived arabinoxylans, beech xylan, corncob xylan, bagasse xylan at a final concentration of 0.5 mg/mL.
The enzyme activity of p-nitrophenyl alpha-L-arabinofuranoside (pNPA) serving as a substrate is set to be 100%, and the degradation capacities of different substrates are measured. The results show that the recombinant alpha-L-arabinofuranosidase only specifically decomposes pNPA and 0.2% (V/N) of wheat bran-derived arabinoxylans, with activities of 52.13U/mg and 9.33U/mg, respectively, and no activity on other substrates.
3. Determination of the product composition of the recombinant alpha-L-arabinofuranosidase CcABF in combination with commercial xylanase and commercial xylosidase degradation arabinoxylans.
The reaction solution cooled after boiling (enzyme solution/0.2% arabinoxylan substrate solution=1/1:v:v) was centrifuged at 8000r/min for 5min, and the supernatant was filtered through a 0.22 μm filter membrane. N-butanol: ethanol: the water is 5:3:2 (V: V) as developing agent, and Thin Layer Chromatography (TLC) analysis was performed using 1g of diphenylamine, 1mL of aniline, 5mL of 85% phosphoric acid and 50mL of acetone as a developing agent.
As shown in FIG. 8, the generation of the products in the arabinoxylan degradation experiment, and thus, the recombinant alpha-L-arabinofuranosidase CcABF and the commercial xylanase as well as the commercial xylosidase (shown as 3 and 2 in FIG. 8) have different degrees of synergistic degradation capability, and the products are xylooligosaccharides. Wherein, no matter which mode of action, the synergistic degradation products of the recombinant alpha-L-arabinofuranosidase CcABF and the commercial xylanase are most obvious, and the synergistic degradation rate of the recombinant alpha-L-arabinofuranosidase CcABF and the commercial xylanase is higher than that of the commercial xylanase to degrade AX.
4. Synergistic degradation efficiency determination of recombinant alpha-L-arabinofuranosidase CcABF in conjunction with commercial xylanase (Xylan)
(1) The experimental method comprises the following steps: degradation of arabinoxylans 11.2U/mL of recombinant alpha-L-arabinofuranosidase CcABF and 11.2U/mL of commercial Xylan or different combinations thereof (5 combinations below) were added to 0.2% arabinoxylans and the synergy rate was calculated.
Combination 1: adding 11.2U/mL Xylan into 0.2% arabinoxylan, and reacting at 40 ℃ and pH of 8.0 for 8 hours;
combination 2: adding only 11.2U/mL recombinant alpha-L-arabinofuranosidase CcABF into 0.2% arabinoxylan, and reacting at 40 ℃ and pH of 8.0 for 8h;
combination 3: adding 11.2U/mL of recombinant alpha-L-arabinofuranosidase CcABF into 0.2% of arabinoxylan, reacting at 40 ℃ and pH5.0 for 4 hours, boiling for 15 minutes to inactivate enzyme liquid, cooling, adding 11.2U/mL of Xylan, reacting for 4 hours, and boiling for 15 minutes to inactivate enzyme liquid;
combination 4: adding 11.2U/mL of Xylan into 0.2% of arabinoxylan, reacting for 4 hours at 40 ℃ and pH5.0, boiling for 15 minutes, inactivating the enzyme solution, cooling, adding 11.2U/mL of recombinant alpha-L-arabinofuranosidase CcABF, reacting for 4 hours, and boiling for 15 minutes;
combination 5: meanwhile, 11.2U/mL of recombinant alpha-L-arabinofuranosidase CcABF and 11.2U/mL of Xylan are added into 0.2% of arabinoxylan to react for 8 hours at 40 ℃ and pH5.0, and the mixture is boiled for 15 minutes to inactivate enzyme liquid;
the reducing sugar content was determined by DNS method using arabinoxylan without any enzyme solution as a control.
(2) Analysis of results: as shown in Table 1, recombinant alpha-L-arabinofuranosidase CcABF is capable of degrading arabinoxylans in cooperation with the commercial xylanase Xylan, wherein the mode of action of simultaneous addition of Xylan and recombinant alpha-L-arabinofuranosidase CcABF is at a maximum of 3.47 and the yield of reducing sugar is 0.26mg/mL. Therefore, the alpha-L-arabinofuranosidase CcABF has better synergistic effect on degrading arabinoxylan with commercial xylanase.
TABLE 1 synergistic effect of alpha-L-arabinofuranosidase CcABF with commercial xylanase (Xylan)
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Claims (9)
1. The recombinant alpha-L-arabinofuranosidase is characterized in that the amino acid sequence of the recombinant alpha-L-arabinofuranosidase is shown as SEQ ID NO. 7.
2. The coding gene of the recombinant alpha-L-arabinofuranosidase according to claim 1, wherein the coding gene sequence of the recombinant alpha-L-arabinofuranosidase is shown in SEQ ID NO. 6.
3. A method for amplifying a gene encoding the recombinant alpha-L-arabinofuranosidase according to claim 2, wherein the primer pair is set forth in SEQ ID NO. 1-2.
4. The recombinant α -L-arabinofuranosidase according to claim 1, wherein the recombinant α -L-arabinofuranosidase has a tolerance pH of 3.0-10.0.
5. A recombinant vector comprising the recombinant α -L-arabinofuranosidase of claim 1.
6. A biological material containing the recombinant alpha-L-arabinofuranosidase according to claim 1, wherein the biological material is an expression cassette, a vector or an engineering bacterium.
7. Use of the recombinant α -L-arabinofuranosidase of claim 1 or the recombinant vector of claim 5 or the biological material of claim 6 in hemicellulose hydrolysis.
8. The use according to claim 7, characterized in that the plant fiber is degraded by recombinant α -L-arabinofuranosidase.
9. The use according to claim 8, wherein the plant fiber is wheat bran.
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