CN113151224B

CN113151224B - Exo-glucanase gene CEL1 and application thereof in preparation of kelp hydrolysate

Info

Publication number: CN113151224B
Application number: CN202010074668.6A
Authority: CN
Inventors: 王本新; 姜周
Original assignee: Shandong Hongye Marine Technology Co ltd
Current assignee: Shandong Hongye Marine Technology Co ltd
Priority date: 2020-01-22
Filing date: 2020-01-22
Publication date: 2022-08-30
Anticipated expiration: 2040-01-22
Also published as: CN113151224A

Abstract

The invention discloses an exoglucanase gene CEL1 and application thereof in preparation of kelp hydrolysate. The novel exoglucanase provided by the invention is SEQ ID No.1 or a protein which is substituted and/or deleted and/or added by one or more amino acid residues or has more than 80% of identity with the exoglucanase and the same function, or a fusion protein obtained by connecting a protein tag at the N end and/or the C end of the exoglucanase. The kelp hydrolysate prepared by the exoglucanase and the matching method can obtain 1.2g/L reducing sugar in 8 hours. Therefore, the method provided by the invention has important application value.

Description

Exo-glucanase gene CEL1 and application thereof in preparation of kelp hydrolysate

Technical Field

The invention relates to the fields of kelp processing, industrial enzymes and food industry, in particular to an exoglucanase gene CEL1 and application thereof in preparation of kelp hydrolysate.

Background

The kelp is a large marine brown algae plant growing in low-temperature seawater, and belongs to a seaweed plant. The kelp can be eaten and has wide application value in the fields of medical treatment, health care and the like because the kelp is rich in mineral substances and functional components. In China, the mode of artificial kelp culture is established in the 50 th 20 th century, and with the rapid development of the aquaculture industry in China and the progress of kelp technology, the kelp industry enters a continuous, rapid and healthy development stage. In recent years, the kelp culture scale in China is rapidly developed. The annual output of the Chinese kelp is improved from 398.26 ten thousand tons in 1999 to 539.64 ten thousand tons in 2008, the annual output of the Chinese kelp accounts for about 81-88% of the annual output of the world kelp, and the culture output and the scale of the Chinese kelp are the first in the world seaweed culture.

Kelp contains dozens of nutrient components, mainly including mannitol, algin, etc. Wherein the main functional substances of the kelp are polysaccharide substances. Laminarin has various biological activities and medicinal functions, including enhancing immunity and resisting tumor. The kelp is processed into hydrolysate rich in kelp sugar, and the absorption of functional ingredients of kelp can be promoted. The kelp hydrolysate can be used as a food additive component and a nutrient solution and applied to various fields of kelp sauce, functional beverages and the like. The traditional kelp hydrolysate is subjected to high-temperature cooking, acid-base treatment and other methods, so that the energy consumption is high, a large amount of sewage is discharged, and the requirements of green and environment-friendly processes cannot be met. Based on cellulase (such as exoglucanase), the technology for processing kelp hydrolysate by using an efficient enzyme method is established by combining protease, pectinase and the like, so that the use amount of chemical reagents such as acid, alkali and the like can be greatly reduced. However, how to improve the catalytic efficiency of the enzyme is the key to reduce the cost. The environmental microorganism metagenome is an important source of excellent enzyme genes, and cellulase resources such as novel exoglucanase and the like can be excavated from the environmental microorganism metagenome and used for preparing kelp hydrolysate.

Disclosure of Invention

The invention aims to provide a novel exoglucanase gene and application thereof in preparing kelp hydrolysate.

In a first aspect, the invention claims a protein.

The protein claimed by the invention can be any one of the following:

(A1) protein with amino acid sequence shown as SEQ ID No. 1;

(A2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in (A1) and having the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In a second aspect, the invention claims nucleic acid molecules encoding the proteins described hereinbefore.

The nucleic acid molecule claimed by the invention can be any one of the following DNA molecules:

(B1) DNA molecule shown in SEQ ID No. 2;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the fusion protein;

(B3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined in any one of (B1) to (B2) and encoding the fusion protein.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a third aspect, the invention claims recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria comprising the nucleic acid molecules described above.

In a fourth aspect, the invention claims the use of the protein as described hereinbefore as exoglucanase in the preparation of kelp hydrolysate.

In a fifth aspect, the invention claims a complete set of enzyme preparations for preparing kelp hydrolysate.

The complete enzyme preparation for preparing kelp hydrolysate consists of the exoglucanase and the protease. The exoglucanase is a protein as described above.

In a sixth aspect, the invention claims a kit for preparing kelp hydrolysate.

The kit for preparing kelp hydrolysate claimed by the invention can contain any one of the following components:

(C1) the enzyme preparation kit and kelp powder described hereinbefore;

(C2) the nucleic acid molecule or expression cassette or recombinant vector or recombinant bacterium or recombinant cell, protease and kelp powder.

In a seventh aspect, the invention claims the use of the enzyme kit as described above or the kit for the preparation of kelp hydrolysate.

In an eighth aspect, the present invention claims a method for preparing kelp hydrolysate.

The method for preparing kelp hydrolysate as claimed in the invention can comprise the following steps: adding exoglucanase and protease into the kelp powder for reaction; the exoglucanase is a protein as described above.

The reaction is carried out using a reaction buffer having a pH of 4.5 to 5.5 (e.g., pH5.0) and a reaction temperature of 42 to 47 deg.C (e.g., 45 deg.C).

In a specific embodiment of the present invention, the reaction buffer is specifically 0.1M acetic acid-sodium acetate buffer at pH 5.0.

In the process, the reaction time period may be 8 to 10 hours (e.g., 8 hours); the rotational speed during the reaction may be 200 rpm.

Correspondingly, the proportion of the exoglucanase, the protease and the kelp powder can be 10000IU (calculated by cellulase activity) and 10000IU:50 g.

In a specific embodiment of the invention, the final concentration of the exoglucanase in the reaction system is about 10000IU/L (on cellulase activity basis); the final concentration of the protease in the reaction system is about 10000 IU/L; the final concentration of the kelp powder in the reaction system is 50 g/L.

In a particular embodiment of the invention, the exoglucanase is 95-99% (e.g., 96%) pure; the protease is a commodity with the product number of P0029 of Beijing Yinuoka science and technology Limited.

In each of the above aspects, the exoglucanase may be prepared according to a method comprising the steps of: introducing a nucleic acid molecule as described in the second aspect above (a nucleic acid molecule encoding a protein as described above) into an E.coli recipient cell to obtain a recombinant E.coli; and culturing the recombinant escherichia coli to obtain the exoglucanase.

Wherein the nucleic acid molecule can be introduced into the E.coli recipient cell in the form of a recombinant vector.

In a specific embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing a small fragment between the cleavage sites NotI and BamHI of the pET28a vector with the nucleic acid molecule (SEQ ID No. 2).

Further, the recombinant E.coli was cultured under conditions of 2 hours at 30 ℃ followed by inoculation of IPTG to a final concentration of 0.1mM, followed by culture for 16 hours. And collecting the thallus after culture, and extracting protein by a nickel affinity chromatography after ultrasonic crushing to obtain the exoglucanase protein solution.

In the invention, the particle size of the kelp powder is less than 40 meshes.

Further, the kelp powder can be obtained by drying fresh kelp at 60 ℃, crushing and sieving with a 40-mesh sieve.

Experiments prove that 1.2g/L reducing sugar can be obtained in 8 hours by using the exoglucanase and a matching method to prepare kelp hydrolysate. Therefore, the method provided by the invention has important application value.

Drawings

FIG. 1 shows the results of producing kelp hydrolysate using exoglucanase CEL 1. T1 represents experimental group; c represents a control group. The ordinate g/L refers to the reducing sugar content per liter of fermentation broth.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Example 1 preparation of exoglucanase cel1 Gene

1. The whole gene was synthesized to obtain SEQ ID No.2 (Nanjing Jinzhi Biotech Co., Ltd.) and cel1 gene.

The gene sequence is from the metagenome library of the hot spring microorganism of the river of Jilin pine. Wherein, SEQ ID No.2 codes the amino acid sequence shown in SEQ ID No. 1.

2. The cel1 gene was constructed on pET28a plasmid by the Gibson method

(1) PCR amplification of the cel1 gene. A plasmid containing the cel1 gene (SEQ ID No.1) provided by Nanjing Jinwei Zhi Biotechnology Limited is used as a template, C1-F and C1-R are used as primers, and a high fidelity TransStart Fastpfu DNA polymerase (Beijing Quanji Biotechnology Limited, Cat. No. AP221) is used for PCR amplification to obtain a gene fragment cel 1.

C1-F：5’-GCCGCGCGGCAGCCAT-ATGAGCCTGATTAGCAGCGTGCTGCTGATT-3’；

C1-R：5’-GTCGACGAGCTCGAATTCG-TTATTTTTTCGCAAAGGTGCTGCCAATCGG-3’。

(2) Constructing a recombinant expression vector containing the cel1 gene. Carrying out agarose gel electrophoresis on the PCR amplification fragment obtained in the step (1), and recovering a target fragment; the vector pET28a (Wuhan vast Ling Biotech Co., Ltd., product No. VT0331-01) was digested with NotI and BamHI at the same time, and the vector large fragment ET28a was recovered. The cel1 fragment was ligated to ET28a fragment using the Gibson assembly method (Gibson DG, Young L, ET al. enzymatic assembly of DNA molecules up to segmented cloned nucleic acids. Nat. methods. 2009; 6(5): 343-345). With CaCl ₂ Escherichia coli DH 5. alpha. competent cells (Beijing Quanjin Biotechnology Co., Ltd., cat # CD201) were transformed by the method. This was spread evenly on LB plates containing kanamycin and cultured overnight at 37 ℃. Clones were selected and sequenced, and the resulting positive plasmid was designated pETC 01.

The structure of pETC01 is described as: and (3) replacing a small fragment between the cleavage sites NotI and BamHI of the pET28a vector by the DNA fragment shown in SEQ ID No. 2.

(3) Construction of E.coli strain EC 01: the pETC01 plasmid was transformed into Transetta (DE3) competent cells (Beijing Quanjin Biotechnology Co., Ltd., product No. CD801-01) by calcium chloride transformation, and cultured overnight at 37 ℃ on an LB plate containing kanamycin to select clones, thereby obtaining an E.coli strain EC01 expressing CEL1 protein.

Preparation of CEL1 protein

After activation of the EC01 strain, the strain was inoculated at a ratio of 1:100 into a 5L fermentor (3L of LB medium was cultured at 30 ℃ C., and IPTG concentration was added to a final concentration of 0.1mM at 2 hours after the transfer) and cultured for 16 hours. The cells were centrifuged at 5000rpm and collected. After the cells were sonicated, proteins were extracted by nickel affinity chromatography (Beijing Quanyu gold Biotechnology Co., Ltd., product No. DP101-01) (all methods were as described in the specification). The protein was then analyzed for molecular weight and purity by SDS-PAGE. As a result, it was confirmed that the molecular weight of the purified protein was about 49KD, which substantially coincided with the theoretical molecular weight of the protein represented by SEQ ID No. 1. SDS-PAGE shows that the purified product has no impurity band (the purity reaches 96%). It was demonstrated that purified CEL1 protein has been obtained. Then, the concentration of CEL1 protein was measured using Brandford reagent (product number C600641) according to the product instructions. The determination of the exoglucanase enzyme activity is carried out by adopting a pNPG method according to the literature (bud inclusion, screening, cloning and identification of cellulose degrading enzymes from uncultured microorganisms in rumens of Chinese yaks, doctor's academic thesis of university of Compound denier, 2010), and the CEL1 protein after determination and purification has exoglucanase enzyme activity which can reach 50IU/mg (the enzyme activity unit is defined as the enzyme quantity which catalyzes the pNPG to hydrolyze at the conditions of pH5 and 45 ℃ to generate 1 micromole of p-nitrophenol per minute and is an activity unit).

Example 2 production of sea tangle hydrolysate Using CEL1 protein

Cleaning fresh herba Zosterae Marinae (Weihai, etc.) and oven drying at 60 deg.C overnight, pulverizing in a high-power pulverizer, and sieving with 40 mesh sieve to obtain herba Zosterae Marinae powder.

Reaction in a 5L reaction tank:

the reaction system comprises the following components:

CEL1 protein obtained in example 1, final enzyme concentration: the activity of the exoglucanase (the activity of cellulase) is about 10000IU/L, wherein IU is defined as the enzyme amount which catalyzes the hydrolysis of sodium carboxymethylcellulose to generate 1 micromole of reducing sugar per minute under the optimal condition (pH5 and 45 ℃) and is one activity unit;

protease (Beijing Yinaoka science and technology Co., Ltd., cat # P0029), final enzyme concentration: about 10000IU/L, wherein IU is defined as enzyme which catalyzes hydrolysis of casein to generate 1 micromole of tyrosine per minute at the conditions of pH5 and 45 ℃ as one activity unit;

50g/L of kelp powder.

Reaction conditions of the reaction tank:

reaction buffer: 0.1M sodium acetate/acetic acid buffer (pH 5)

Reaction temperature: 45 ℃;

the reaction time is as follows: 8 h;

reaction speed: 200 rpm.

The test group T1 was set for containing all the components, and the control group C was set for containing no CEL1 component.

The reducing sugar detection method comprises the following steps:

taking out a sample, centrifuging at 13000rpm for 5min, taking the supernatant, boiling water bath for 5min, taking 100 μ l of the supernatant, adding 100 μ l of DNS solution (the formula is that sodium hydroxide 21g and DNS 6.3g are fully dissolved in 500mL of distilled water, adding potassium sodium tartrate 182g, phenol 5g and sodium metabisulfite 5g into the solution, stirring until the solution is completely dissolved, fixing the volume to 1000mL, keeping the solution out of the sun), boiling water bath for 5min, centrifuging, taking 100 μ l of the supernatant, adding the supernatant into a 96-well plate, and measuring the content of reducing sugar in an enzyme labeling instrument.

The results are shown in FIG. 1: the experimental group T1 produced about 1.2g/L of reducing sugar, while the control group C produced about 0.1g/L of reducing sugar. The method for preparing the kelp hydrolysate by using the CEL1 protein has obvious advantages.

<110> Shandong Hongye ocean science and technology Co Ltd

<120> exoglucanase gene CEL1 and application thereof in preparation of kelp hydrolysate

<130> GNCLN200365

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<170> PatentIn version 3.5

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Met Ser Leu Ile Ser Ser Val Leu Leu Ile Ser Ala Ser Leu Phe Ala

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Thr Ser Val Val Leu Asp Ala Asn Tyr Arg Trp Leu His Tyr Thr Thr

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Gly Tyr Thr Asn Cys Tyr Thr Gly Asn Leu Trp Asp Ala Thr Leu Cys

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Pro Asp Pro Val Thr Cys Ala Arg Asn Cys Ala Ile Trp Gly Val Pro

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

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Ser Leu Ser Leu Lys Phe Val Thr Lys Ser Gln Ala Gln Thr Asn Ile

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Gly Pro Arg Thr Tyr Leu Leu Ala Ser Asn Gly Asp Leu Thr Thr Tyr

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Arg Met Phe Gly Leu Lys Asn Lys Glu Phe Thr Phe Asp Val Asp Val

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Ser Lys Val Pro Cys Gly Val Asn Ser Ala Leu Tyr Phe Ile Glu Met

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Glu Ala Asp Gly Gly Ala Ser Ser Lys Tyr Pro Ser Ser Thr Asn Lys

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Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ala Gln Cys Pro Ala

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Glu Pro Gly Lys Phe Ile Asn Gly Leu Ala Asn Ser Lys Asp Trp Thr

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Pro Ile Pro Gly Asp Gly Ala Cys Cys Asn Glu Met Asp Ile Trp Glu

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Ala Asn Asn Thr Leu Ile Thr Ala Glu Phe Pro His Pro Cys Asn Ile

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Thr Gly Tyr Gly Gln Cys Asp Pro Tyr Gly Cys Asp Phe Asn Pro Tyr

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agcggcacct atggcattac caccagcaac ggcaacagcc tgagcctgaa atttgtgacc 360

aaaagccagg cgcagaccaa cattggcccg cgcacctatc tgctggcgag caacggcgat 420

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gatcatgcgg tgaacatgct gtggatggat aacgaaccga acgtggcgcg cggcccgtgc 1200

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Claims

1. A protein, which is any one of:

(A1) protein with amino acid sequence shown as SEQ ID No. 1;

(A2) and (C) attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in (A1).

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown in SEQ ID No. 2.

4. An expression cassette comprising the nucleic acid molecule of claim 2 or 3.

5. A recombinant vector comprising the nucleic acid molecule of claim 2 or 3.

6. A transgenic cell line comprising the nucleic acid molecule of claim 2 or 3; the transgenic cell line is a non-animal or plant variety.

7. A recombinant bacterium comprising the nucleic acid molecule of claim 2 or 3.

8. Use of the protein of claim 1 as an exoglucanase in the preparation of a kelp hydrolysate.

9. Use of the nucleic acid molecule of claim 2 or 3 or the expression cassette of claim 4 or the recombinant vector of claim 5 or the transgenic cell line of claim 6 or the recombinant bacterium of claim 7 for the preparation of kelp hydrolysate.

10. A complete set of enzyme preparation for preparing sea tangle hydrolysate comprises exoglucanase and protease; the exoglucanase is the protein of claim 1.

11. A kit for preparing kelp hydrolysate, which comprises any one of the following components:

(C1) the enzyme kit as claimed in claim 10 and kelp powder;

(C2) the nucleic acid molecule of claim 2 or 3 or the expression cassette of claim 4 or the recombinant vector of claim 5 or the transgenic cell line of claim 6 or the recombinant bacterium of claim 7, protease and kelp powder.

12. Use of the enzyme kit according to claim 10 or the kit according to claim 11 for the preparation of a kelp hydrolysate.

13. A method for preparing kelp hydrolysate comprises the following steps: adding exoglucanase and protease into the kelp powder for reaction;

the pH value of the reaction buffer solution adopted for the reaction is 4.5-5.5, and the reaction temperature is 42-47 ℃;

the exoglucanase is the protein of claim 1.