CN114410611A

CN114410611A - Kunmu polysaccharide degrading enzyme OUC-BsLam26 and application thereof

Info

Publication number: CN114410611A
Application number: CN202111568019.2A
Authority: CN
Inventors: 毛相朝; 姜宏; 董悦阳; 邱艳君; 全永奕
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2022-04-29
Anticipated expiration: 2041-12-21
Also published as: CN114410611B

Abstract

The invention discloses a Kunmu polysaccharide degrading enzyme OUC-BsLam26, the amino acid sequence is shown in SEQ ID NO. 1. The nucleotide sequence of the gene for coding the Kunmu polysaccharide degrading enzyme OUC-BsLam26 is shown in SEQ ID NO. 2. The laminarin degrading enzyme OUC-BsLam26 can be used for degrading laminarin and preparing laminarin oligosaccharide. The invention also discloses an enzyme preparation containing the Kunmu polysaccharide degrading enzyme OUC-BsLam 26. The invention also discloses a recombinant expression vector and a recombinant engineering bacterium carrying the gene encoding the laminarin degrading enzyme OUC-BsLam 26. The laminarin degrading enzyme OUC-BsLam26 can act as laminarin substrate, and the polymerization degree of the final product laminarin oligosaccharide is 1-4. The laminarin degrading enzyme has excellent enzymology property and specificity, and has important industrial application value and economic value in preparing laminarin oligosaccharide by an enzyme method.

Description

Kunmu polysaccharide degrading enzyme OUC-BsLam26 and application thereof

Technical Field

The invention relates to recombinant expression, preparation and application of Kunmu polysaccharide degrading enzyme OUC-BsLam26, belonging to the technical field of functional gene cloning expression.

Background

Laminarin (Laminarin) is a marine polysaccharide derived from brown algae, and its structure is mainly composed of D-glucopyranose residue connected with beta-1, 3-glycosidic bond and branched chain connected with part of beta-1, 6-glycosidic bond. The laminarin oligosaccharide is a product with the polymerization Degree (DP) between 2 and 10 obtained by degrading laminarin, and has lower molecular weight and higher bioavailability. In recent years, researches prove that the laminarin oligosaccharide has better biological activity than laminarin, and has a plurality of physiological functions of oxidation resistance, anticoagulation, apoptosis resistance, regulation of intestinal flora, bone regeneration and repair and the like. Therefore, how to efficiently prepare laminarin oligosaccharide with a specific polymerization degree is widely concerned.

The method for preparing laminarin oligosaccharide from laminarin mainly comprises a physical method, a chemical method and an enzymatic hydrolysis method. The physical method mainly degrades laminarin by ultrasonic-assisted extraction, gamma ray and other methods; the chemical method mainly degrades laminarin by acid solution and alkali solution. The physical method has high energy consumption and inhomogeneous product, while the chemical method has more severe reaction conditions and uses too many chemical reagents, thus being not in accordance with the concepts of environmental protection and the like. The enzymolysis method has the advantages of mild reaction conditions, high specificity and uniform hydrolysis products. Therefore, the development of a novel laminarin enzyme and the preparation of laminarin oligosaccharide using the same have great research value and development potential.

The related research of human intestinal microorganisms has become a current hot issue. Bacteroides sp is a bacteroidetes member from intestinal microorganisms, which is negatively related to human inflammation, diabetes and other diseases, has the capability of degrading polysaccharides and carbohydrates, and is an ideal excavation host.

Disclosure of Invention

Aiming at the prior art, the invention provides a novel degrading enzyme capable of degrading laminarin to generate laminarin oligosaccharide, namely the laminarin degrading enzyme OUC-BsLam26, and makes up the defects of the existing enzyme gene library.

The invention is realized by the following technical scheme:

the amino acid sequence of the laminarin degrading enzyme OUC-BsLam26 is shown in SEQ ID NO. 1.

Amino acid sequence of Kunmu polysaccharide degrading enzyme OUC-BsLam26 (SEQ ID NO. 1):

MKKIFYSLILSTLMFGACSASDDGVPQTPEDPDGTQEPQEEPVTYTGIQKRSVKRGVSYSFQLPKEDTQMLGSAIS WAYNWGTEISSDLSTEFSKQQIDYCPMSWNANPDIAPKIRRYVNAHPECKYLLAYNEPNLTDQARMTPQEAAAQWPALRA LASELNLKLISPAMNYGTLPDYSDPIKWLDEFFSNVPLSDVDGIAIHCYMESPSSLINYVSMFKKYGKPIWLTEFCAWPS SDKISISSQMNYMSETLHYLESDPDVFRYAWFIPRGIGPTDPSTGTTSNSLLPGKPNALTDLGTVFVNMSTLDKTAYYNK NQVIPAEHYSSINTVENLTSVSAHLRPTTDISGILDVYDLKQEQWLKYQLDAPKAGTYQLDIRYTSFRDNKVEITIDKGT AATLDLPNVDSKWTTVTTNIQLKAGKQTLRIKVTTGNIALNWLRFKD。

a gene for coding the above Kunmu polysaccharide degrading enzyme OUC-BsLam26, the nucleotide sequence of which is shown in SEQ ID NO. 2.

Nucleotide sequence of the gene encoding Kunmu polysaccharide degrading enzyme OUC-BsLam26 (SEQ ID NO. 2):

5’-ATGAAAAAGATATTCTATTCACTCATACTATCCACACTGATGTTCGGAGCCTGTTCCGCTTCGGATGACGGA GTTCCTCAAACACCTGAAGACCCCGACGGGACACAGGAACCGCAGGAAGAGCCGGTCACCTACACCGGTATACAGAAACG TAGTGTAAAACGTGGAGTCAGCTATAGTTTCCAGCTCCCCAAAGAAGACACGCAAATGTTGGGAAGTGCCATATCCTGGG CTTACAACTGGGGGACAGAGATTTCTTCCGATTTAAGTACTGAGTTCAGCAAGCAGCAAATCGACTATTGCCCAATGTCA TGGAATGCCAATCCAGACATCGCTCCCAAGATTCGCAGGTACGTCAACGCACACCCGGAATGTAAATATCTACTTGCATA CAATGAGCCTAATCTGACAGACCAGGCAAGAATGACACCCCAGGAAGCTGCCGCACAATGGCCTGCACTGCGTGCATTAG CTTCTGAACTAAATCTGAAGCTCATATCTCCGGCTATGAACTACGGAACATTGCCGGATTATAGTGACCCGATCAAATGG TTGGACGAGTTTTTCAGCAACGTTCCCCTCAGTGATGTAGACGGTATAGCTATCCATTGTTACATGGAAAGCCCTTCTTC TCTTATCAACTATGTAAGCATGTTCAAAAAATATGGAAAACCTATTTGGTTAACAGAATTCTGTGCATGGCCCAGTAGTG ATAAGATTAGCATATCAAGCCAGATGAACTATATGAGCGAGACTCTTCATTATCTTGAGTCCGATCCTGACGTATTCCGC TATGCCTGGTTCATCCCAAGAGGTATTGGACCGACCGATCCAAGTACCGGTACAACCAGCAACAGCTTATTACCCGGTAA GCCCAATGCATTGACTGACCTCGGAACAGTGTTTGTGAATATGTCCACTCTGGATAAAACTGCTTATTACAATAAGAATC AAGTTATTCCCGCCGAACACTACAGTTCAATAAACACAGTTGAGAATTTAACATCAGTCAGCGCACACCTGCGCCCTACA ACCGATATTTCGGGCATATTGGATGTTTATGATTTGAAACAGGAACAATGGCTGAAATATCAACTGGATGCTCCTAAAGC CGGAACTTACCAGTTGGATATCCGCTATACCAGTTTCAGAGATAACAAGGTTGAGATAACTATAGACAAAGGTACGGCAG CAACACTAGACCTGCCTAATGTAGACAGCAAATGGACTACTGTCACTACCAACATCCAACTAAAAGCAGGTAAGCAAACG CTACGTATAAAAGTCACTACAGGCAATATTGCATTAAATTGGCTGCGTTTCAAAGACTAA-3’。

the laminarin degrading enzyme OUC-BsLam26 can be used for degrading laminarin and preparing laminarin oligosaccharide.

A method for degrading laminarin/preparing laminarin oligosaccharide comprises: degrading laminarin with the laminarin degrading enzyme OUC-BsLam26 to obtain laminarin oligosaccharide product containing glucose, laminarin trisaccharide and laminarin tetrasaccharide.

Further, the degradation conditions are as follows: the laminarin solution has a concentration of 0.2-0.3% (m/v, unit g/ml), a temperature of 30-50 deg.C, a pH of 3.0-8.0, and a time of 10 minutes or more, preferably 0.5 hours or more. Preferably, the degradation conditions are: the temperature is 45 ℃, the pH value is 6.0, and the time is more than 0.5 hour. Preferred degradation conditions may also be: carrying out enzymolysis for more than 48 hours at the temperature of 35-40 ℃ and under the condition of pH value of 3.0-10.0.

Further, the laminarin oligosaccharide is a single laminarin tetrasaccharide. After enzymolysis, separating by preparative silica gel column chromatography, wherein the mobile phase is as follows: n-butanol: acetic acid: water 2: 1: 1(v/v/v), obtaining eluent, and obtaining single laminariptan in the eluent after detection, thereby realizing the separation and preparation of the laminariptan.

The gene for coding the laminarin degrading enzyme OUC-BsLam26 is applied to the preparation of enzyme preparations for degrading laminarin/preparing laminarin oligosaccharide.

An enzyme preparation comprises the above Kunbuterol polysaccharide-degrading enzyme OUC-BsLam 26.

The enzyme preparation can be used for degrading laminarin and preparing laminarin oligosaccharide.

A recombinant expression vector carrying the above gene encoding Kunmu polysaccharide-degrading enzyme OUC-BsLam 26.

A recombinant engineered bacterium having the above gene encoding Kunmu sugar-degrading enzyme OUC-BsLam26 inserted into its genome and capable of expressing Kunmu sugar-degrading enzyme OUC-BsLam 26.

The recombinant engineering bacteria can be applied to the preparation of laminarinase OUC-Bs-26.

The Kunmu polysaccharide degrading enzyme OUC-BsLam26 is derived from human intestinal microorganisms Bacteroides sp, and is identified to belong to a glycoside hydrolase GH128 family. The invention constructs the recombinant vector containing the laminarin enzyme gene, realizes the heterologous expression in the large intestine bacillus and provides good foundation for the industrial production and application of the enzyme. The enzyme has high catalytic activity under the conditions of 45 ℃ and pH 6.0, and the specific enzyme activity after nickel column purification can reach 3.42U/mg. The enzyme can efficiently hydrolyze laminarin, and the hydrolysis products mainly comprise laminariptan, laminaripriose, laminaribiose and glucose. The laminarin enzyme preparation has high activity and stability (the enzyme activity can still be kept for more than 90 percent when the laminarin enzyme preparation is placed at 40 ℃ for 48 hours, the enzyme activity can still be kept for more than 50 percent when the laminarin enzyme preparation is placed in a buffer solution with the pH value of 3.0-10.0 for 48 hours), and the laminarin enzyme preparation has high efficiency, high purity and high yield, and the prepared oligosaccharide can be used for adjusting intestinal flora, treating obesity and the like and has the potential of realizing industrial utilization.

In addition, the prior literature can only detect the hydrolysate of laminarin, but not prepare oligosaccharides with specific polymerization degrees, compared with the prior art, the laminarin enzyme of the invention can not only detect the hydrolysate, but also separate and prepare oligosaccharides with specific polymerization degrees, thereby having important significance for subsequent industrial application and research on the activity of oligosaccharides with specific polymerization degrees.

The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.

Drawings

FIG. 1: the purified pure enzyme SDS-PAGE electrophoresis picture of the laminarin degrading enzyme is shown in the invention, wherein M is a standard protein Marker; 1 is crude enzyme protein; 2 is purified laminarin protein.

FIG. 2: schematic diagram of the effect of temperature and pH changes on relative enzyme activity, wherein a: the effect of temperature on relative enzyme activity; b: the influence on relative enzyme activity is caused by placing the enzyme at different temperatures for 48 hours; c: the influence of pH on the relative enzyme activity; d: effect on relative enzyme activity by standing at different pH for 48 h.

FIG. 3: schematic diagram of the effect of metal ions and chemical reagents and substrate specificity on relative enzyme activity, wherein, a; the effect of metal ions and chemical agents on relative enzyme activity; b: results of the substrate specificity study are shown schematically.

FIG. 4: TLC pattern of the inventive laminarin degradation enzymatic product.

FIG. 5: liquid phase diagram of enzymatic hydrolysate of laminarin degrading enzyme of the present invention.

FIG. 6: the invention relates to a mass spectrogram of a laminarin degradation enzymolysis product.

FIG. 7: TLC pattern of the purified product of the present invention isolated by a laminarin degrading enzyme.

FIG. 8: TLC pattern of isolated and purified product of Kunbutero polysaccharide-degrading enzyme of the invention (example 13).

Detailed Description

The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

Example 1 cloning of laminarin degrading enzyme Gene OUC-BsLam26

The laminarin degrading enzyme gene OUC-BsLam26 of the present invention is obtained by whole-gene synthesis and is obtained by mining from NCBI library (laminarin is mainly derived from microorganisms, and also from animals, higher plants, viruses, etc., but laminarin derived from plants has a small content under normal growth environment and has low laminarin activity compared with laminarin derived from microorganisms. related experiments prove that Bacillus, Flavobacterium, Cellulomonas, Paenibacillus, etc. derived from bacteria have laminarin activity, and furthermore, experiments prove that Akkermanisa mucophila bacteria derived from intestinal microorganisms have a gene encoding laminarin and can realize heterologous laminarin expression in Escherichia coli, so that the present invention excavates a strain from intestinal microorganisms to verify whether the strain has a protein capable of expressing laminarin activity), it is derived from intestinal microorganisms Bacteroides sp.CBA7301 with the sequence number of QIU 93826.1. The fragment contains 1332 base sequences shown as SEQ ID NO.2 and encodes 443 amino acid sequences shown as SEQ ID NO. 1. According to sequence alignment and phylogenetic tree analysis, it has the highest homology with endo-beta-1, 3-glucanase from Pseudomonas viridiflava CFBP 1590, and the predicted protein sequence similarity value is 39.70%, the laminarinase OUC-Bs-26 belongs to glycoside hydrolase family 128 (GH 128). The invention expresses and purifies the enzyme for the first time and carries out preparation related research.

Example 2 construction of expression vector containing laminarin enzyme degrading enzyme Gene

The gene fragment and pET-28a cloning vector are connected by adopting a seamless cloning technology, a connection product is transferred into E.coli DH5 alpha competent cells, the competent cells are coated on an LB culture medium solid plate (containing 50 mu g/mL kanamycin), after the culture is carried out for 16 hours in an incubator at 37 ℃, a single clone is picked up to an LB liquid culture medium containing 50 mu g/mL kanamycin, and the culture is carried out for 12 hours in a shaking table at 37 ℃ with the rotating speed of 220 rpm. Sequencing was performed after positive validation of the monoclonal. The successfully verified plasmid was extracted and designated pET28a-OUC-BsLam26 and stored at-20 ℃ for use.

Example 3 construction of recombinant plasmid and engineered bacterium containing laminarin degrading enzyme Gene

The plasmid pET28a-OUC-BsLam26 extracted in the example 2 is transformed into a host E.coli BL21 competent cell, and the constructed engineering bacteria grow on a kanamycin sulfate resistant plate to obtain a recombinant expression strain.

Example 4 preparation of recombinant Kunmu polysaccharide degrading enzyme Using engineered Escherichia coli

The recombinant Escherichia coli strain was activated in 5mL of LB liquid medium (containing 50. mu.g/mL kanamycin) for 12 hours, inoculated at 1% of the inoculum size into ZYP-5052 medium containing kanamycin sulfate (50. mu.g/mL), incubated at 20 ℃ and 200rpm for 48 hours, and self-induced to express the Kunmu polysaccharide-degrading enzyme.

After fermentation, the bacterial liquid is centrifuged at 8000g and 4 ℃ for 10 minutes, then thalli are collected, cells are suspended in 50mM Tirs-HCl buffer solution with the pH value of 7.0, then the cells are placed in ice water bath for ultrasonic disruption for 30min (40% power, 3s on and 3s off), then centrifugation is carried out again at 8000g and 4 ℃ for 10min, and the collected supernatant is crude enzyme liquid. The laminarinase contains 6 His purification tags, and the crude enzyme solution is subjected to affinity chromatography using Ni-NTA column, wherein the column is first equilibrated with an equilibration buffer solution (500mM NaCl,50mM Tris-HCl), then the heteroprotein with poor binding capacity is eluted with a 20mM imidazole solution (20mM imidazole, 500mM NaCl,50mM Tris-HCl), the target protein is eluted with a 100mM imidazole solution (100mM imidazole, 500mM NaCl,50mM Tris-HCl), and the buffer elution component of the fraction is collected to obtain a purified recombinant laminarin degrading enzyme solution (protein content of 0.3 mg/mL). SDS-PAGE is used for detecting the purity and molecular weight of the protein, whether the band is single or not and whether the size is accurate or not are verified (figure 1), and the result shows that the size of the recombinant protein is about 63KDa and is consistent with the prediction result, so that the target protein is obtained.

Example 5 measurement of specific enzyme Activity of recombinant laminarinase

The standard assay for Kunmu polysaccharide degrading enzyme OUC-BsLam26 activity was: a250 microliter reaction system containing 50. mu.L of enzyme solution and 200. mu.L of 0.2% (m/v) laminarin substrate prepared from citric acid buffer solution with pH of 6.0 was reacted at 35 ℃ for 10min, and then centrifuged at 12000rpm in a mini centrifuge for 1min after inactivating the enzyme in a boiling water bath for 10 min. Then 180. mu.L of the supernatant solution was taken out and mixed with 270. mu.L of DNS reagent, and boiled in a boiling water bath for 5min for color development. The absorbance was then measured at 540nm after centrifugation at 12000rpm for 1min in a mini centrifuge. Enzyme activity is defined as the amount of enzyme required to produce 1. mu.M reducing sugar per min under standard conditions. Through determination, the activity of the purified Kunmu polysaccharide degrading enzyme can reach 3.42U/mg. Compared with the enzyme activity of laminarin GluB from Lysobacter enzymogenes, the enzyme activity of laminarin Lcf from chlamys farreri can reach 1.67U/mg under the conditions of pH 6.0 and 44 ℃, and the enzyme activity of the laminarin degrading enzyme OUC-BsLam26 is obviously higher. Under the same enzymolysis efficiency, the enzymolysis speed of the invention is faster, and the required time is shorter (the time is not more than 10 minutes).

Example 6 determination of optimal reaction conditions and stability of Kunbuterol polysaccharide-degrading enzyme

The purified laminarin degrading enzyme obtained in example 4 was reacted at different temperatures and pH, and the effect of temperature and pH on enzyme activity was determined. Selecting the temperature of 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, reacting for 10min according to the method for measuring specific enzyme activity of laminarin degrading enzyme in example 5, measuring optimum temperature, and measuring enzyme activity by DNS method. And (3) at 45 ℃, selecting buffer solutions with pH of 3.0-10.0 as different pH determination buffer solutions for enzyme reaction, and determining the optimum pH of the laminarin degrading enzyme according to the enzyme activity of the laminarin degrading enzyme. Incubating the enzyme at 35 deg.C, 40 deg.C, 45 deg.C, and 50 deg.C, and determining the residual enzyme activity under optimum condition of 45 deg.C and pH of 6.0 to obtain its temperature stability. Mixing with buffer solutions with different pH values, incubating at 4 deg.C, and measuring the residual enzyme activity at optimum temperature to obtain pH stability. The highest enzyme activity is taken as 100%, the relative enzyme activities under different conditions are calculated, the result is shown in figure 2, the optimal reaction temperature of the recombinant laminarin degrading enzyme is 45 ℃, the optimal pH is 6.0, and meanwhile, the optimal temperature measurement experiment result shows that the enzyme activity of the recombinant laminarin degrading enzyme is higher at 40-50 ℃. The recombinant thallus laminariae polysaccharide degrading enzyme is placed at 35 deg.C and 40 deg.C for 48h, and the enzyme activity can still be maintained above 90%; the enzyme activity can be kept for more than 50 percent in the buffer solution with the pH value of 3.0-10.0 for 48 hours, which shows that the enzyme activity stability is better.

Example 7 determination of the Effect of Metal ions and chemical reagents on the Activity of recombinant laminarin degrading enzyme

Adding different metal ions and chemical reagents SDS and Na2EDTA into pure enzyme of the laminarin degrading enzyme OUC-BsLam26 respectively to make the concentration of the final system be 1mM and 10mM respectively, standing for 1h at 25 ℃, then adding 0.2% (w/v) laminarin substrate prepared by using citrate with pH 6.0 respectively, standing at 45 ℃, reacting for 30min, and measuring the enzyme activity by using a DNS method. As shown in FIG. 3A, most of the reagents had some effect of promoting the enzyme activity, but the high concentration of Fe³⁺And Cu²⁺All inhibit the activity of the enzyme.

Example 8 substrate selectivity Studies for determination of Kunmu polysaccharide-degrading enzyme OUC-BsLam26

Laminarin, pachyman, yeast beta-glucan, and curdlan were formulated at 0.2% (w/v) with citrate buffer pH 6.0, respectively. Respectively taking 200 mu L of different reaction substrates, then adding 50 mu L of pure enzyme of the Kunbu polysaccharide degrading enzyme OUC-BsLam26, uniformly mixing, reacting under the optimal condition, and measuring the enzyme activity by using a DNS method. As shown in FIG. 3B, laminarin degrading enzyme OUC-BsLam26 has the strongest catalytic activity on laminarin, followed by curdlan, yeast β -glucan, which is inactive on pachyman, sodium carboxymethylcellulose (CMC-Na), and agarose.

Example 9 identification of degradation products of Kunmu polysaccharide-degrading enzyme OUC-BsLam26 by TLC

The laminarin degrading enzyme OUC-BsLam26 obtained in example 4 was reacted with 0.2% laminarin at 45 ℃ for various times, and then the product was checked by Thin Layer Chromatography (TLC). The specific method comprises the following steps: developer (n-butanol: acetic acid: water: 2: 1: 1) and developer of 0.2% (w/v)3, 5-dihydroxytoluene in 10% (v/v) H₂SO₄Developing at 75 deg.C. As shown in FIG. 4, the degradation product of laminarinase OUC-BsLam26 was analyzed by TLC using glucose as a reference, and the enzymatic degradation product consisted mainly of four fractions, i.e., glucose, laminaribiose, laminaripriose and laminariptan.

EXAMPLE 10 identification of degradation products of Kunmu polysaccharide-degrading enzyme OUC-BsLam26 by liquid phase assay

The laminarin degrading enzyme OUC-BsLam-26 obtained in example 4 and 0.2% laminarin were incubated at 45 ℃ for different periods of time, respectively, and then the enzymatic hydrolysate was detected by high performance liquid chromatography. As shown in FIG. 5, the enzymatic products of laminarinase OUC-BsLam26 are mainly laminaribiose, laminariose, laminaripentaose, and laminaripentaose.

Example 11 definition of the DP composition of recombinant Kunmu polysaccharide-degrading enzyme products

The laminarin degrading enzyme OUC-BsLam26 obtained in example 4 was reacted with 0.2% laminarin at 45 ℃ and the product was then examined by ESI-MS. As shown in FIG. 6, the results showed that the laminarin oligosaccharide contained in the product contained laminarin trisaccharide, laminarin tetrasaccharide and laminarin pentasaccharide.

Example 12 identification of products after complete conversion of recombinant Kunbutero polysaccharide-degrading enzymes

The laminarin degrading enzyme OUC-BsLam26 obtained in example 4 was reacted with 1% laminarin at 45 ℃ for 6h, and then the product was checked by TLC. As shown in FIG. 7, the results indicated that the enzymatic products were glucose, laminaribiose, laminariose and laminariptan.

EXAMPLE 13 preparation of Laminarin oligosaccharide by Kuncuba polysaccharide-degrading enzyme

The laminarin degrading enzyme OUC-BsLam26 obtained in example 4 was reacted with laminarin of 1% concentration at 45 ℃ for 6 hours, separated and prepared by preparative silica gel column chromatography with the mobile phase: n-butanol: acetic acid: water 2: 1: 1(v/v/v), and then the product was checked by TLC. As shown in FIG. 8, the results showed that the enzymatic hydrolysate contained glucose, laminaribiose, laminariose and laminariptan, 4 contained 4 kinds of oligosaccharides, 5 and 6 contained laminariose and laminariptan, and 7 and 8 produced a single laminariptan, realizing the separation production of laminariptan.

Example 14 preparation of enzyme preparation Using recombinant Kunbu polysaccharide-degrading enzyme

Preparation of an enzyme preparation using the recombinant laminarin degrading enzyme prepared in example 4: purifying the fermented and crushed solution, replacing imidazole with a buffer solution, freeze-drying and storing enzyme powder.

Example 15 preparation of Laminaria oligosaccharide Using recombinant Kuncuba polysaccharide degrading enzyme

Laminarin oligosaccharides were prepared using the recombinant laminarin degrading enzyme prepared in example 4: separating and purifying the enzymolyzed oligosaccharide to obtain thallus laminariae oligosaccharide, lyophilizing, and storing thallus laminariae oligosaccharide powder.

The above examples are provided to enable those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art will be within the scope of the appended claims.

Sequence listing

<110> China oceanic university

<120> Kunmu polysaccharide degrading enzyme OUC-BsLam26 and application thereof

<141> 2021-12-16

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Gln Ile Asp Tyr Cys Pro Met Ser Trp Asn Ala Asn Pro Asp Ile Ala

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Pro Lys Ile Arg Arg Tyr Val Asn Ala His Pro Glu Cys Lys Tyr Leu

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Leu Ala Tyr Asn Glu Pro Asn Leu Thr Asp Gln Ala Arg Met Thr Pro

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Gln Glu Ala Ala Ala Gln Trp Pro Ala Leu Arg Ala Leu Ala Ser Glu

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Leu Asn Leu Lys Leu Ile Ser Pro Ala Met Asn Tyr Gly Thr Leu Pro

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Asp Tyr Ser Asp Pro Ile Lys Trp Leu Asp Glu Phe Phe Ser Asn Val

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Pro Leu Ser Asp Val Asp Gly Ile Ala Ile His Cys Tyr Met Glu Ser

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Pro Ser Ser Leu Ile Asn Tyr Val Ser Met Phe Lys Lys Tyr Gly Lys

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Pro Ile Trp Leu Thr Glu Phe Cys Ala Trp Pro Ser Ser Asp Lys Ile

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Ser Ile Ser Ser Gln Met Asn Tyr Met Ser Glu Thr Leu His Tyr Leu

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Glu Ser Asp Pro Asp Val Phe Arg Tyr Ala Trp Phe Ile Pro Arg Gly

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Ile Gly Pro Thr Asp Pro Ser Thr Gly Thr Thr Ser Asn Ser Leu Leu

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

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atgaaaaaga tattctattc actcatacta tccacactga tgttcggagc ctgttccgct 60

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tgggggacag agatttcttc cgatttaagt actgagttca gcaagcagca aatcgactat 300

tgcccaatgt catggaatgc caatccagac atcgctccca agattcgcag gtacgtcaac 360

gcacacccgg aatgtaaata tctacttgca tacaatgagc ctaatctgac agaccaggca 420

agaatgacac cccaggaagc tgccgcacaa tggcctgcac tgcgtgcatt agcttctgaa 480

ctaaatctga agctcatatc tccggctatg aactacggaa cattgccgga ttatagtgac 540

ccgatcaaat ggttggacga gtttttcagc aacgttcccc tcagtgatgt agacggtata 600

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gtgtttgtga atatgtccac tctggataaa actgcttatt acaataagaa tcaagttatt 960

cccgccgaac actacagttc aataaacaca gttgagaatt taacatcagt cagcgcacac 1020

ctgcgcccta caaccgatat ttcgggcata ttggatgttt atgatttgaa acaggaacaa 1080

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accagtttca gagataacaa ggttgagata actatagaca aaggtacggc agcaacacta 1200

gacctgccta atgtagacag caaatggact actgtcacta ccaacatcca actaaaagca 1260

ggtaagcaaa cgctacgtat aaaagtcact acaggcaata ttgcattaaa ttggctgcgt 1320

ttcaaagact aa 1332

Claims

1. The amino acid sequence of the laminarin degrading enzyme OUC-BsLam26 is shown in SEQ ID NO. 1.

2. The nucleotide sequence of the gene for coding the Kunmu polysaccharide degrading enzyme OUC-BsLam26 is shown in SEQ ID NO. 2.

3. Use of the laminarin degrading enzyme OUC-BsLam26 of claim 1 for degrading laminarin/preparing laminarin oligosaccharides.

4. A method for degrading laminarin/preparing laminarin oligosaccharide is characterized in that: degrading laminarin with the laminarin degrading enzyme OUC-BsLam26 of claim 1 to obtain laminarin oligosaccharide.

5. The method for degrading laminarin/preparing laminarin oligosaccharide according to claim 4, wherein: the laminarin oligosaccharide is laminarin.

6. The method for degrading laminarin/preparing laminarin oligosaccharide according to claim 4 or 5, wherein: the degradation conditions are as follows: the concentration of the laminarin solution is 0.2-0.3%, the temperature is 30-50 ℃, the pH value is 3.0-8.0, the time is more than 10 minutes, and preferably more than 0.5 hour;

or: the temperature is 45 ℃, the pH value is 6.0, and the time is more than 0.5 hour;

or: carrying out enzymolysis for more than 48 hours at the temperature of 35-40 ℃ and under the condition of pH value of 3.0-10.0.

7. An enzyme preparation comprising the Kunbuterol polysaccharide-degrading enzyme OUC-BsLam26 of claim 1.

8. A recombinant expression vector carrying the gene encoding the laminarin degrading enzyme OUC-BsLam26 of claim 2.

9. A recombinant engineered bacterium having the gene encoding Kunmu sugar-degrading enzyme OUC-BsLam26 of claim 2 inserted into its genome and capable of expressing Kunmu sugar-degrading enzyme OUC-BsLam 26.

10. Use of the recombinant expression vector of claim 8 or the recombinant engineered bacterium of claim 9 in the preparation of Kunmu polysaccharide degrading enzyme OUC-BsLam 26.