CN118126988A

CN118126988A - Wide Wen Naqie beta-1, 4-glucanase and application thereof

Info

Publication number: CN118126988A
Application number: CN202410397308.8A
Authority: CN
Inventors: 周梅先; 邵宗泽; 王万鹏; 高超琛; 李慧敏; 李增鹏
Original assignee: Third Institute of Oceanography MNR
Current assignee: Third Institute of Oceanography MNR
Priority date: 2024-04-03
Filing date: 2024-04-03
Publication date: 2024-06-04

Abstract

The invention provides a wide Wen Naqie beta-1, 4-glucanase and application thereof, and an amino acid sequence of the wide Wen Naqie beta-1, 4-glucanase is SEQ ID NO. 1. The endo beta-1, 4-glucanase has wide action temperature range, has activity in the range of 0-100 ℃, maintains the enzyme activity to be more than 80% in the range of 15-65 ℃ and maintains the enzyme activity to be more than 90% after heat preservation for 1h in the range of 0-60 ℃; has advantages in the aspects of biological polishing, food processing, extraction of plant bioactive substances and the like under the low-temperature or medium-temperature condition.

Description

Wide Wen Naqie beta-1, 4-glucanase and application thereof

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to a wide Wen Naqie beta-1, 4-glucanase and application thereof.

Background

Cellulose is a main component of plant cell walls, is a high-molecular linear polysaccharide formed by connecting glucose units through beta-1, 4 glycosidic bonds, has the highest content in nature, and can be depolymerized into glucose by cellulase to be applied to fermentation, chemistry and food industry. The natural cellulose raw material contains lignin, hemicellulose and other components, and is usually pretreated by a physical method or a chemical method in the depolymerization process to release cellulose molecules, and then the cellulose molecules are degraded by a biological enzyme method, wherein the enzymolysis process is a speed limiting step of cellulose degradation. Microorganisms have been found to be the major degradation of cellulose in nature, including bacteria and fungi, and the presence of cellulases has also been found in some animal species. The degradation of cellulose to glucose requires the synergistic action of endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase and beta-glucosidase, the endo-beta-1, 4-glucanase produces cellooligosaccharides and lower molecular weight cellulose molecules by randomly cleaving internal glycosidic bonds of amorphous cellulose, while exo-beta-1, 4-glucanase acts on the ends of cellulose molecules to hydrolyze to cellobiose, and the beta-glucosidase then hydrolyzes cellobiose and cellooligosaccharides to glucose. Endo-beta-1, 4-glucanase acts on the initial stages of cellulose degradation and is therefore considered a key enzyme for cellulose degradation.

The enzyme derived from the microorganism has the advantages of high yield, good stability and the like, and is more advantageous from the industrial point of view. The cellulase which is commercially available at present has higher thermal stability, is widely applied to industries such as feed, textile, fruit and vegetable processing, beer brewing, washing and the like, but has low activity under mild conditions or no activity under low temperature conditions, and has limited application range. Although studies have found some low temperature-adaptive endo-beta-1, 4-glucanases, these enzymes are less stable or less active under mild conditions. The wide Wen Naqie beta-1, 4-glucanase with cold adaptability has high activity and high stability under mild conditions and low temperature conditions, and has great application potential in industrial processes such as biological polishing, food processing, plant bioactive substance extraction and the like. The deep sea has the environmental characteristics of low temperature, high salt, high pressure, darkness and the like, contains abundant extreme microorganism resources, and is a good source for obtaining novel endo beta-1, 4-glucanase with excellent characteristics.

Disclosure of Invention

The invention aims to provide a wide Wen Naqie beta-1, 4-glucanase and application thereof, and the wide Wen Naqie beta-1, 4-glucanase (named LsCel) has wider temperature adaptability.

The invention firstly provides an endo beta-1, 4-glucanase, which comprises the following components:

1) Endo beta-1, 4-glucanase with amino acid sequence of SEQ ID NO. 1

CNAQTSENDIAQTGEKPEIEEITLNSAVKEHGQLHVEGSKILDINDNAVQLRGMSLFWSQWIGKYYNPKAIKWLKDDWQCTIVRAAMAVDFDGYLENPDAEKAKVEAVVDAAIEEGIYVIIDWHDHEAQNHVEAAKKFFGEMAQKYGDYPNVIYETYNEPLDVSWTEVLKPYHETVIAEIRKYDPDNIIVCGTRNWSQNVDDVIDNKIDDPNVAYTLHYYAATHKQWLRDIASKALENEVPLFVTEFGTTQASGDEEIDETESKLWWQFLDDNNISWCNWSIADKEELAAALKPGAAAEGGWPETEITTSGKLVRDELKLKNKKY;

2) Endo-beta-1, 4-glucanase amplified from Latifer and having a homology of not less than 80% with an enzyme having an amino acid sequence of SEQ ID NO. 1;

preferably, the homology is not less than 90%, most preferably not less than 99%;

the invention also provides a gene for encoding the endo beta-1, 4-glucanase, and a specific nucleotide sequence of the gene is SEQ ID NO. 2:

tgtaatgcgcagacctcagaaaatgatattgcacaaaccggggagaaaccagaaatcgaagaaatcacgctaaattctgcagttaaagaacatggccagctacacgtggaaggttccaaaattcttgacataaacgataacgctgtgcaactgcgcggcatgtctcttttctggagccaatggattgggaaatattacaaccctaaagcgataaaatggctaaaagatgactggcagtgtacgattgtgcgtgccgcaatggcagtagattttgatggatatcttgaaaacccagatgccgaaaaagctaaagtagaagccgttgtagatgctgctattgaagaaggtatttatgtaattattgactggcacgatcatgaggcacaaaatcatgtggaagcagctaaaaagttctttggtgagatggcacagaagtatggtgattatcccaatgtgatctatgagacttataatgagccacttgatgtatcgtggacagaagttctaaaaccatatcacgaaactgtaattgcagaaatacgtaaatatgatcctgataatataattgtatgtggcacgcgcaattggtcacaaaatgtagatgatgtgatcgacaataaaattgatgatcccaatgtcgcttataccctgcattattatgccgccacacataaacaatggcttagagacatagcgagcaaagcgcttgagaatgaagttccactttttgtaacagaatttggtaccacacaggcttctggagatgaagagatagatgaaaccgaatctaaactctggtggcagtttctcgatgataacaacatatcttggtgcaactggtctattgcagacaaagaagaactggctgcagctttaaaaccgggtgctgcagcagaaggcggctggccagaaactgaaattacaacttcaggaaaattagtgcgtgacgaattaaaattaaagaacaaaaaatattga(SEQ ID NO:2);

furthermore, the above genes are optimized for the expression host, and the optimized sequences are as follows:

tgcaatgcccagaccagtgaaaatgatattgcccagaccggcgaaaaaccggaaattgaagaaattaccctgaat

agtgcagtgaaagaacatggtcagctgcatgttgaaggtagcaaaattctggatattaatgataacgccgttcagct

gcgcggcatgagtctgttttggagccagtggattggcaaatattataatccgaaagcaatcaaatggctgaaagatg

attggcagtgtaccattgttcgcgccgcaatggccgttgattttgatggctatctggaaaatccggatgccgaaaaa

gccaaagttgaagccgtggtggatgccgccattgaagaaggcatttatgttattattgactggcatgatcacgaagc

acagaatcatgtggaagccgcaaaaaaattttttggtgaaatggcacagaagtatggtgattatccgaatgttattt

acgaaacctataacgaaccgctggatgtgagctggaccgaagttctgaaaccgtatcatgaaaccgtgattgcagaa

attcgcaaatatgatccggataatattatcgtgtgcggcacccgcaattggagccagaatgttgatgatgttattga

taataagatcgacgatccgaatgttgcctataccctgcattattatgcagcaacccataaacagtggctgcgtgata

ttgcaagcaaagcactggaaaatgaagttccgctgtttgtgaccgaatttggcaccacccaggcaagcggtgatgaa

gaaattgatgaaaccgaaagcaaactgtggtggcagtttctggatgataataatattagttggtgcaattggagtat

cgccgataaagaagaactggccgcagcactgaaaccgggtgcagccgccgaaggtggctggcctgaaaccgaaattaccaccagcggtaaactggtgcgtgatgaactgaaactgaaaaataaaaagtac(SEQ ID NO:3).

In a further aspect, the present invention provides a recombinant expression vector for recombinant expression of the endo- β -1, 4-glucanase described above;

the invention also provides an engineering strain transformed with the recombinant expression vector, and the engineering strain is an escherichia coli engineering strain as one specific record of the embodiment.

The invention also provides application of the endo beta-1, 4-glucanase in the aspects of biological polishing, food processing, plant bioactive substance extraction and the like.

The endo beta-1, 4-glucanase has wide action temperature range, has activity in the range of 0-100 ℃, maintains the enzyme activity to be more than 80% in the range of 15-65 ℃ and maintains the enzyme activity to be more than 90% after heat preservation for 1h in the range of 0-60 ℃; has advantages in the aspects of biological polishing, food processing, extraction of plant bioactive substances and the like under the low-temperature or medium-temperature condition.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of the purified enzyme protein;

FIG. 2 is a graph showing the effect of different temperatures on enzyme activity;

FIG. 3 is a graph of stability assays for enzymes under different temperature conditions;

FIG. 4 is a graph showing the effect of different pH on enzyme activity;

FIG. 5 is a graph showing the effect of enzymes on hydrolysis of various substrates and analysis of the hydrolysis products of the enzymes.

Detailed Description

The information of the experimental materials used in the examples of the present invention is as follows:

Coli E.coli TOP10 and E.coli BL21 (DE 3) (from ThermoFisher Co.); expression vector pET-28a (+) (available from Novagen); restriction enzymes BamHI and XhoI (available from full formula gold Co.); t4 ligase (available from Takara Co.); LB medium (10 g/liter with peptone, 5g yeast extract, 10g NaCl); binding buffer (1 XPBS buffer: 10mM phosphate, pH 7.4); rinsing buffer (500 mM NaCl, 10-50 mM imidazole, 20mM phosphate, pH 7.4); elution buffer (500 mM NaCl,500mM imidazole, 20mM phosphate, pH 7.4); 3, 5-dinitrosalicylic acid (DNS) and kanamycin (available from Shanghai, inc.); ni Sepharose ^TM Fast Flow kit (purchased from Taijing Co.); dialysis bag MD25 (available from beijing solebao corporation); carboxymethyl cellulose (Carboxymethylcellulose, CMC) and Laminarin (LAMINARIN) (from Sigma), barley beta-glucan (Barley beta-glucan) and lichenin (Lichenan) (from Megazyme). The LB medium containing kanamycin used in the present invention had a kanamycin concentration of 50. Mu.g/mL.

The present invention will be described in detail with reference to the following examples and the accompanying drawings.

Example 1: screening of endo-beta-1, 4-glucanase and recombinant expression

1. Obtaining endo beta-1, 4-glucanase gene

The inventor separates Latifolia (Leeuwenhoekiella sp.BCO5) from western Pacific deep sea sediment, based on Latifolia BCO5 genome sequencing data, analyzes and obtains mature protein gene LsCel of endo beta-1, 4-glucanase, and the coded amino acid sequence is SEQ ID NO 1; the nucleotide sequence is SEQ ID NO. 2. The mature protein sequence of the endo-beta-1, 4-glucanase disclosed by the invention has the highest homology of not more than 80 percent (the amino acid sequence identity with the protein from Lutibacter sp.B1 of glycoside hydrolase family 5 is 77.49 percent) with the homologous protein by using NCBI on-line BlastP tool analysis, and is a novel endo-beta-1, 4-glucanase.

The sequence of the mature protein gene LsCel of the endo beta-1, 4-glucanase is optimized, so that the endo beta-1, 4-glucanase is suitable for being expressed in escherichia coli, and the optimized endo beta-1, 4-glucanase gene sequence is obtained through an artificial synthesis method, and the nucleotide sequence of the optimized endo beta-1, 4-glucanase gene is shown as SEQ ID NO. 3.

2. Construction of recombinant plasmids

Respectively introducing two enzyme cutting sites of BamHI and XhoI at the 5 'and 3' ends of the artificially synthesized optimized endo beta-1, 4-glucanase gene sequence, carrying out glue recovery after the DNA fragments of the artificially synthesized endo beta-1, 4-glucanase gene with the BamHI and XhoI enzyme cutting sites are subjected to restriction enzyme BamHI and XhoI enzyme cutting, carrying out enzyme cutting and glue recovery on a pET-28a (+) vector by using restriction enzyme BamHI and XhoI, and connecting the DNA fragments of the enzyme-cut beta-1, 4-glucanase gene with the pET-28a (+) vector by using T4 ligase; mixing the connection product with recipient bacterium E.coli Top10 in a competent mode, placing on ice for 30min, carrying out heat shock for 90s at 42 ℃, adding 400 mu L of LB liquid medium, resuscitating for 45min at 37 ℃ and 160rpm, centrifuging, coating on LB solid medium containing kanamycin, culturing overnight at 37 ℃, screening recombinant plasmids, and carrying out double enzyme digestion and sequencing verification on the recombinant plasmids to obtain recombinant plasmids containing target genes.

3. Induction expression of genes and preparation of crude enzyme solution

The obtained recombinant plasmid is transformed into E.coli BL21 (DE 3) to obtain recombinant strain containing endo beta-1, 4-glucanase gene. The recombinant strain was inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL kanamycin for overnight culture, inoculated into 500mL of LB liquid medium containing kanamycin at an inoculum size of 1%, cultured at 37℃at 180rpm until OD ₆₀₀ was about 0.6, induced by adding IPTG to a final concentration of 0.5mmol/L, induced and expressed at 18℃at 180rpm for 18h, and centrifuged at 800rpm for 10min to collect the cells. The thalli is washed for 2 times by using a binding buffer solution, then the thalli is resuspended in 50mL of the binding buffer solution, a proper amount of lysozyme is added for 3 hours at 4 ℃, the thalli is crushed by ultrasonic with the power of 300W for 25 minutes at the working/clearance time of 3s/5s, the thalli is centrifuged at 5000rpm for 10 minutes at the 4 ℃, and the supernatant is collected to obtain crude enzyme solution.

4. Purification of enzymes

The prepared crude enzyme solution is added into a pre-balanced His purification column Ni Sepharose ^TM fast flow, evenly mixed for 2.5h at 4 ℃, and then the waste liquid is discarded. The cells were rinsed 1 time with 20mL of a rinse buffer containing 10mM imidazole, 1 time with 20mL of a rinse buffer containing 20mM imidazole, and 1 time with 10mL of a rinse buffer containing 50mM imidazole. After rinsing, the eluate was eluted 5 times (the first elution volume was 2mL, the second and third elution volumes were 4mL, the fourth elution volume was 6mL, and the fifth elution volume was 4 mL) with an elution buffer containing 500mM imidazole, and the eluate was collected by tube separation and subjected to SDS-PAGE to examine the purity of the eluate (FIG. 1). The eluate was dialyzed overnight with dialysis bag MD25 to remove imidazole and high concentration NaCl to obtain a purified enzyme solution.

Example 2: enzymatic Properties of endo-beta-1, 4-glucanase

1. Method for measuring activity of endo beta-1, 4-glucanase

The reducing sugar content was determined by the DNS method, 25. Mu.L of diluted enzyme solution was mixed with 100. Mu.L of 1% carboxymethyl cellulose, and reacted at 60℃for 30min. After the reaction is completed, 200 mu L of DNS reagent is added, boiled for 5min, immediately placed in ice water for cooling, supernatant is taken after short centrifugation, OD ₅₄₀ value is measured (inactivated enzyme liquid is used as a reference), and the reducing sugar amount and enzyme activity are calculated according to a standard curve. Definition of enzyme activity unit: under the above assay conditions, the amount of enzyme required to hydrolyze carboxymethyl cellulose to produce 1. Mu. Mol of reducing sugar per minute is defined as one enzyme activity unit (U).

2. Enzyme action temperature

The diluted enzyme solution is subjected to enzyme activity measurement at the temperature of 0-100 ℃ by taking 1% of carboxymethyl cellulose prepared by 50mM Sodium acetate (pH5.5) buffer solution as a substrate, and the relative enzyme activity at different temperatures is calculated by taking the maximum enzyme activity as 100%. The results show that the endo beta-1, 4-glucanase has activity in the range of 0-100 ℃ under the condition of pH5.5, the optimal reaction temperature is 60 ℃, the enzyme activity is maintained at more than 80% in the range of 15-65 ℃, the enzyme activity is maintained at more than 70% in the range of 0-70 ℃, and the enzyme activity is maintained at more than 50% in the range of 75-95 ℃, and the results are shown in figure 2.

3. Stability of enzymes under different temperature conditions

The enzyme solution is incubated for 1h at different temperatures (0-90 ℃), and then reacts with 1% carboxymethyl cellulose prepared by 50mM Sodium acetate (pH5.5) buffer solution to perform enzyme activity measurement, and the relative activity of the enzyme treated differently is calculated by taking the activity of untreated enzyme as 100%. The result shows that the enzyme has good temperature stability, the enzyme activity is maintained to be more than 90% after the temperature is kept for 1h within the range of 0-60 ℃, and the residual enzyme activity is maintained to be more than 60% after the temperature is kept for 1h under the condition of 65-80 ℃, and the result is shown in figure 3.

4. PH range of enzyme action

The enzyme activity was measured at 60℃using 1% carboxymethyl cellulose prepared from different buffers (50 mM Na ₂HPO₄ -CITRIC ACID buffer, pH 3.5-4.5;50mM Sodium acetate buffer, pH 4.0-6.0; 50mM Na ₂HPO₄-NaH₂PO₄ buffer, pH 6.0-8.0; 50mM Tris-HCl buffer, pH 7.0-9.0; 50mM Glycine-NaOH buffer, pH 9.0-10.5) as a substrate, and the relative activities of the enzymes at different pH values were calculated with the maximum enzyme activity being 100%. The results show that the enzyme has activity in the pH range of 4.0-9.5, the optimal pH value is 5.5, the enzyme activity is maintained to be more than 80% in the pH range of 4.5-6.5, the enzyme activity is maintained to be more than 50% in the pH range of 7.0-8.0, and under the same pH condition, the Na ₂HPO₄-NaH₂PO₄ buffer solution is obviously superior to the Tris-HCl buffer solution, and the result is shown in figure 4.

5. Enzyme substrate specificity and hydrolysate analysis

Enzyme activity was measured at 60℃and pH5.5 using 1% of carboxymethyl cellulose, barley beta-glucan, lichenin and laminarin as substrates, respectively, and relative activities of endo-beta-1, 4-glucanase to hydrolyze different substrates were calculated using 100% of carboxymethyl cellulase activity. The results show that the enzyme has degradation activity on carboxymethyl cellulose, barley beta-glucan and lichenin, wherein the effect on barley beta-glucan degradation is the best, but has little activity on laminarin, which indicates that the enzyme is beta-1, 4-glucanase, and the result is shown in figure 5A.

The endo-beta-1, 4-glucanase products were analyzed by Thin Layer Chromatography (TLC). Pure enzyme was added to 1% carboxymethyl cellulose prepared with 50mM Sodium acetate (pH 5.5) buffer solution, and the reaction was completed at 60℃for 12 hours, and the reaction supernatant was taken for thin layer chromatography analysis. The spreading agent is isopropanol: ethyl acetate: water=3:1:1 (V: V), the developer is N- (1-naphthyl) ethylenediamine hydrochloride: methanol: concentrated sulfuric acid=0.3:100:5 (W: V). The results show that the final product of the enzyme hydrolysis of carboxymethyl cellulose is mainly cellotriose and an oligosaccharide mixture above cellotriose, and only contains trace cellobiose, which indicates that the enzyme is an endo beta-1, 4-glucanase, and the result is shown in figure 5B.

The result shows that the optimal reaction temperature of the screened enzyme is 60 ℃, the screened enzyme has activity within the range of 0-100 ℃, the enzyme activity is maintained to be more than 80% within the range of 15-65 ℃, and the enzyme activity is maintained to be more than 90% after the screened enzyme is preserved for 1h within the range of 0-60 ℃; the optimal pH of the enzyme is 5.5, and the enzyme activity is maintained to be more than 80% within the pH range of 4.5-6.5; the enzyme has degradation activity on carboxymethyl cellulose, barley beta-glucan and lichenin, and has good application prospects in the aspects of cellulose resource development and utilization, biological polishing, food processing, plant bioactive substance extraction and the like.

Claims

1. An endo-beta-1, 4-glucanase, characterized in that the endo-beta-1, 4-glucanase comprises:

1) Endo beta-1, 4-glucanase with an amino acid sequence of SEQ ID NO. 1;

2) Endo-beta-1, 4-glucanase having a homology of not less than 80% with an enzyme having an amino acid sequence of SEQ ID NO. 1, amplified from Latifer.

2. The endo- β -1, 4-glucanase of claim 1, wherein the homology is not less than 90%.

3. The endo- β -1, 4-glucanase of claim 1, wherein the homology is not less than 99%.

4. A gene encoding the endo- β -1, 4-glucanase of any of claims 1-3.

5. The gene according to claim 4, wherein the nucleotide sequence of the gene is SEQ ID NO. 2.

6. The gene according to claim 4, wherein the nucleotide sequence of the gene is SEQ ID NO. 3.

7. A recombinant expression vector for recombinant expression of the endo- β -1, 4-glucanase of any of claims 1-3.

8. A recombinant engineering strain, wherein the recombinant engineering strain is transformed with the recombinant expression vector of claim 7.

9. The recombinant engineering strain of claim 8, wherein the recombinant engineering strain is a prokaryotic engineering strain or a eukaryotic engineering strain.

10. Use of an endo-beta-1, 4-glucanase according to any of claims 1-3 for biopolishing, food processing, extraction of plant bioactive substances.