阅读说明：本技术 一种β-1,3-1,4-葡聚糖葡萄糖水解酶及其应用 (Beta-1, 3-1, 4-glucan glucohydrolase and application thereof ) 是由 段承杰 冯家勋 姜男 马晓丹 于 2019-09-25 设计创作，主要内容包括：本发明公开了一种β-1,3-1,4-葡聚糖葡萄糖水解酶及其应用。本发明提供的β-1,3-1,4-葡聚糖葡萄糖水解酶,为如下A1)、A2)或A3)：A1)氨基酸序列是序列2的第55-656位的蛋白质；A2)将序列表中序列2的第55-656位的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。本发明的β-1,3-1,4-葡聚糖葡萄糖水解酶可用于水解含有β-1,3糖苷键和/或β-1,4糖苷键的化合物,也可以用来降解纤维素,制备葡萄糖,具有广泛的应用前景。(The invention discloses beta-1, 3-1, 4-glucan glucohydrolase and application thereof. The beta-1, 3-1, 4-glucan glucohydrolase provided by the invention is A1), A2) or A3) as follows: A1) the amino acid sequence is the protein at the 55 th-656 th position of the sequence 2; A2) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence from the 55 th site to the 656 th site of the sequence 2 in the sequence table; A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). The beta-1, 3-1, 4-glucan glucohydrolase of the invention can be used for hydrolyzing compounds containing beta-1, 3 glycosidic bonds and/or beta-1, 4 glycosidic bonds, can also be used for degrading cellulose and preparing glucose, and has wide application prospect.)

1. Use of a protein for the hydrolysis of a compound comprising beta-1, 3 glycosidic and/or beta-1, 4 glycosidic linkages; the protein is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein at the 55 th-656 th position of the sequence 2;

A2) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence from the 55 th site to the 656 th site of the sequence 2 in the sequence table;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

2. Use according to claim 1, characterized in that: A3) the fusion protein is a protein shown in a sequence 2 in a sequence table.

3. Use of a biological material related to a protein according to claim 1 or 2 for the hydrolysis of a compound comprising beta-1, 3 glycosidic and/or beta-1, 4 glycosidic linkages; the biomaterial is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding the protein of claim 1 or 2;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a cell line comprising B1) the nucleic acid molecule or a cell line comprising B2) the expression cassette.

4. Use according to claim 3, characterized in that: B1) the nucleic acid molecule is b11) or b12) or b13) or b14) as follows:

b11) a DNA molecule shown in the 163-1968 site of the sequence 1 in the sequence table;

b12) DNA molecule shown in sequence 1 in the sequence table;

b13) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding the protein of claim 1 or 2;

b14) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined under b11) or b12) or b13) and encodes a protein as claimed in claim 1 or 2.

5. Use according to any one of claims 1 to 4, characterized in that: the compound is polysaccharide or oligosaccharide or pNPC or pNPG.

6. Use according to claim 5, characterized in that: the polysaccharide is cellulose or beta glucan, and the oligosaccharide is cellooligosaccharide or laminaribiose.

7. A method for producing glucose, comprising: carrying out a catalytic reaction on a protein according to claim 1 or 2 using a compound containing a beta-1, 3-glycosidic bond and/or a beta-1, 4-glycosidic bond as a substrate to obtain glucose; the compounds have glucose bound to other moieties via beta-1, 3 glycosidic and/or beta-1, 4 glycosidic linkages.

8. Use of the protein of claim 1 or 2 as a glucan glucohydrolase.

9. Use of the protein of claim 1 or 2 as a β -1,3/1, 4-glucan glucohydrolase.

10. A protein according to claim 1 or 2 or a biomaterial according to claim 3 or 4.

Technical Field

The invention relates to beta-1, 3-1, 4-glucan glucohydrolase and application thereof, belonging to the technical field of biology.

Background

Cellulose is the most abundant biomass on earth, consists of glucose chains connected by beta-1, 4-glycosidic bonds, and forms a highly ordered crystal structure due to a large number of hydrogen bonds among cellulose chains, so that the cellulose is difficult to degrade by cellulase. At least three classes of cellulases are required for effective cellulose degradation, including: endoglucanases (EC 3.2.1.4) which hydrolyze cellulose single strands in amorphous regions randomly, creating more ends; cellobiohydrolases or exoglucanases (EC 3.2.1.91 and 3.2.1.176), which can hydrolyze cellulose in crystalline regions, releasing cellobiose mainly from the reducing and non-reducing ends of the cellulose chains; beta-glucosidase (EC 3.2.1.21), which hydrolyzes cellobiose or cellodextrin to produce glucose.

Cellulose-degrading microorganisms are the main cause of natural degradation of cellulose. Microorganisms (including bacteria and fungi) use different strategies for utilizing cellulose. Cellulose degrading microorganisms currently use two major mechanisms to degrade cellulose. Most aerobic microorganisms employ a non-complex cellulase system, which secretes a group of soluble extracellular cellulases, including the complete enzyme system of cellulases, which cooperate extracellularly to degrade cellulose; most anaerobic microorganisms use a complex cellulase system, and most of the cellulases produced by the complex cellulase system are combined with the cohesin domain of scaffold protein on the surface of a host cell through a dockerin domain of the cellulase system, so that a huge multienzyme complex (also called cellulase) is formed.

Disclosure of Invention

The present invention provides a protein having beta-1, 3-1, 4-glucan glucohydrolase activity, which is named GGH.

In order to solve the technical problems, the invention firstly provides the application of GGH in hydrolyzing compounds containing beta-1, 3 glycosidic bonds and/or beta-1, 4 glycosidic bonds; GGH is a1), a2) or A3) as follows:

A1) the amino acid sequence is the protein at the 55 th-656 th position of the sequence 2;

A2) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence from the 55 th site to the 656 th site of the sequence 2 in the sequence table;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein of A1), the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence from position 55 to 656 of sequence No. 2 in the sequence listing is labeled as shown in the following table.

Table: sequence of tags

The GGH protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein represented by SEQ ID NO. 55 to 656 of SEQ ID NO. 2 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The GGH protein of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the GGH protein in A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in position 163-1968 of SEQ ID NO. 1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching to the 5 'end and/or 3' end a coding sequence for the tag shown in the above table. Wherein the DNA molecule shown at positions 163-1968 of the sequence 1 encodes the GGH protein shown at positions 55-656 of the sequence 2.

In the application, the fusion protein A3) can be a protein shown as a sequence 2 in a sequence table.

The invention also provides the use of biological material associated with GGH in the hydrolysis of compounds containing beta-1, 3 glycosidic and/or beta-1, 4 glycosidic linkages; the biomaterial is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding GGH;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a cell line comprising B1) the nucleic acid molecule or a cell line comprising B2) the expression cassette.

In the above application, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11) a DNA molecule shown in the 163-1968 site of the sequence 1 in the sequence table;

b12) DNA molecule shown in sequence 1 in the sequence table;

b13) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding GGH;

b14) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined by b11) or b12) or b13) under stringent conditions and codes for GGH.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding a GGH protein of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the isolated GGH protein of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the GGH protein and have the function of the GGH protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence from position 55 to 656 of the coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in 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; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding a GGH protein (GGH gene expression cassette) according to B2) means a DNA capable of expressing a GGH protein in a host cell, which DNA may include not only a promoter which initiates transcription of the GGH gene but also a terminator which terminates transcription of the GGH gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the GGH gene expression cassette can be constructed by using an existing expression vector.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may be pET-30a (+).

B3) The recombinant vector can be pET-GGH. The pET-GGH is a recombinant vector obtained by inserting the 163-.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Among them, the bacterium may be Escherichia coli, such as E.coli Transetta (DE 3).

In the above application, the cell line does not comprise propagation material.

In the above application, the compound may be polysaccharide or oligosaccharide or pNPC or pNPG.

In the above application, the polysaccharide may be cellulose or beta-glucan, and the oligosaccharide may be cellooligosaccharide or laminaribiose.

The cellooligosaccharide may be cellobiose, cellotriose, cellotetraose, cellopentaose, or cellohexaose.

The beta glucan may be barley glucan.

The invention also provides a preparation method of the glucose, which comprises the following steps: taking a compound containing beta-1, 3 glycosidic bonds and/or beta-1, 4 glycosidic bonds as a substrate, and carrying out a catalytic reaction by using GGH to obtain glucose; the compounds have glucose bound to other moieties via beta-1, 3 glycosidic and/or beta-1, 4 glycosidic linkages.

In the above method, the compound contains a glucose residue.

In the above method, the catalytic reaction may be carried out in an environment having a pH of 5.5 to 7.5. Further, the method is simple. The catalytic reaction may be carried out in an environment having a pH of 6 to 7, such as 6.5.

The catalytic reaction can be carried out at 20-35 ℃. Further, the method is simple. The catalytic reaction may be carried out at a temperature of from 25 to 35 deg.C, such as from 30 to 35 deg.C.

The invention also provides the application of GGH as glucan glucohydrolase.

The invention also provides application of GGH as beta-1, 3/1, 4-glucan glucohydrolase.

GGH or the biological material, also belongs to the protection scope of the invention.

The GGH has beta-1, 3/1, 4-glucan glucohydrolase activity, can be used for hydrolyzing compounds containing beta-1, 3 glycosidic bonds and/or beta-1, 4 glycosidic bonds, can also be used for degrading cellulose and preparing glucose, and has wide application prospect.

Drawings

FIG. 1 is a map of pET-GGH by electrophoresis.

FIG. 2 is an SDS-PAGE electrophoresis of purified GGH recombinant protein solutions.

FIG. 3 shows the HPLC detection results of GGH recombinant protein hydrolyzed laminaribiose, gentiobiose and sophorose. a: gentiobiose (. beta. -1, 6); b: hydrolysate of GGH recombinant protein after acting on gentiobiose; c: sophorose (β -1, 2); d: hydrolysate of GGH recombinant protein after action on sophorose disaccharide; e: laminaribiose (. beta. -1, 3); f: hydrolysate of GGH recombinant protein after acting on laminabiose; g: and (5) glucose standard.

FIG. 4 shows the HPLC detection results of the products of GGH recombinant proteolytic sugars. a: the sugar standard products G1-G6 are glucose, cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose respectively; b-g: hydrolysis products of glucose, cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose which are respectively hydrolyzed by GGH recombinant protein; h: the GGH recombinant protein hydrolyzes the hydrolysate of barley glucan.

FIG. 5 shows the HPLC detection results of GGH recombinant protein hydrolysis of fiber pentasaccharide at different time points. G2-G5 are cellobiose, cellotriose, cellotetraose and cellopentaose respectively, and Glucose is Glucose.

FIG. 6 shows the TLC thin layer chromatography detection results of GGH recombinant protein on p-NPC. G1 and G2 are glucose and cellobiose, respectively; m: standard samples, 1-8 are hydrolysates of pNPC with recombinant GGH proteins at 0, 1 minute, 5 minutes, 30 minutes, 1 hour, 4 hours, 12 hours and 24 hours, respectively.

FIG. 7 shows the results of the glucose tolerance assay of the GGH recombinant protein.

FIG. 8 shows the results of the detection of the optimum pH of GGH recombinant protein.

FIG. 9 shows the results of the detection of the optimum temperature of GGH recombinant protein.

FIG. 10 shows the results of detection of pH tolerance of GGH recombinant proteins.

FIG. 11 shows the results of the detection of temperature tolerance of GGH recombinant proteins.

FIG. 12 shows the results of the detection of temperature tolerance of GGH recombinant proteins.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples were carried out in triplicate, and the results were averaged. In the following examples,% is by mass unless otherwise specified. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

p-nitrophenyl-beta-D-glucopyranoside (p-NPG) and p-nitrophenyl cellobioside (p-NPC), both available from SIGMA corporation as N8700 and N5759, respectively.

Carboxymethyl cellulose (CMC), microcrystalline cellulose (Avicel) and Hydroxyethyl cellulose (2-Hydroxyethyl cellulose) are products of SIGMA, and the product numbers are C5678, 11356 and 09368 respectively.

Barley glucan (barley glucan), lichenin (lichenin), beech xylan (beechwood xylan), Mannan (Mannan), Xyloglucan (Xyloglucan) and laminarin (laminarin) are all products of Megazyme company, and the product numbers are I-AZBGL, P-LICHN, I-AZXBE, I-AZGMA, I-AZXYG and L8030 respectively.

The Acid-swellable cellulose (ASC) is prepared by the following method:

(1)10 grams of microcrystalline cellulose suspended in 85% H₃PO₄The mixture was stirred at 1 ℃ for 1 hour in the aqueous solution.

(2) The mixture was poured into 4L of ice-cold (temperature range 2-8 ℃) water and left for 30 min.

(3) Washing the microcrystalline cellulose treated in step (2) with pre-cooled water (temperature range 2-8 deg.C) for several times, and then with 1% NaHCO₃Washing with water solution, and washing with pre-cooled water (at 2-8 deg.C) until acid-base balance is achieved to obtain acid-expanded cellulose.

(4) Acid swollen cellulose was stored in 5mM NaN₃Storing in water solution at 1 deg.C.