CN104178472A - Cellulose degradation enzyme, construction and application thereof - Google Patents

Abstract

The invention relates to cellulase, in particular to a cellulose degradation enzyme, construction and application thereof. The cellulose degradation enzyme is shown as the amino acid in sequence table SEQ ID NO:1 or sequence table SEQ ID NO:2. Through rapid expression, biotransformation enzyme derived from cellulose for pyrolysis of cellulose bacteria can be obtained, and the cellulose degradation enzyme provided by the invention has high thermal stability, high specific enzyme activity and long half-life.

Description

Enzyme for Degrading Cellulose and Its Construction and Application

technical field

The present invention relates to cellulase, specifically an enzyme for degrading cellulose and its construction and application.

Background technique

Cellulase has a wide range of applications in industrial fields such as papermaking, food, feed, textile and energy. Cellulase can degrade cellulose into oligosaccharides or monosaccharides. Cellulase can be classified into endo-cellulase, exo-cellulase, and β-glucosidase. Endo-cellulase, also known as endo-1,4-β-glucanase (EC3.2.1.4), specifically cuts the β-1.4-glycosidic linkage in the glucan chain of cellulose, producing low Polymerized dextran or cellooligosaccharides. Lichenanase, also known as 1,3-1,4-β-glucanase (EC3.2.1.73), has strict substrate specificity, and it specifically cuts off 1,3-1,4 - β-1,4-glycosidic linkages to 3-O-glucopyranose groups in β-glucans.

1,3-1,4-β-glucan is a component of the cell wall of gramineous plants, especially in the endosperm cell walls of cereal plants (such as wheat, barley, corn, rice, etc.). Thermophilic lichenases have great advantages in industrial processes, such as in the brewing and animal feed industries. During beer fermentation, the endogenous lichenase of gramineous plants is heat-inactivated; therefore, the addition of exogenous lichenase can enzymatically hydrolyze the high molecular weight β-glucan with mixed linkages, thereby reducing the fermentation rate. The viscosity of the liquid can be improved to increase the filtration speed and extraction yield, and at the same time, it can also avoid precipitation during the beer maturation process. In the animal feed industry, especially in the production of broiler chicken and pig feed industry, adding exogenous lichenase can promote animal digestion and reduce sanitation.

Lignocellulose is mainly composed of cellulose and hemicellulose and is the most abundant source of cellulose on earth. In the biotransformation and processing of lignocellulose, high temperature conditions are often required, and high temperature resistant cellulase can be directly used for biomass processing and conversion under high temperature conditions. Moreover, because these high-temperature-resistant enzymes have the advantages of good thermal stability and long half-life, the use efficiency of enzymes can be improved, the amount of enzymes used in production can be reduced, and production costs can be reduced. Therefore, high temperature resistant cellulase has received more and more attention.

The extreme thermophilic anaerobic bacterium Thermocellulolyticus saccharolyticus belongs to lignocellulose degrading bacteria, has no cellulosome structure, and has free-acting lignocellulosic enzymes, which can be used as an enzyme-producing strain of heat-resistant cellulase. At the same time, microorganisms that produce high-temperature-resistant cellulase have the advantages of not being easily contaminated by other microorganisms during the cultivation process, high-temperature conditions can reduce the viscosity of the fermentation broth and are easy to stir, substrates and products have high solubility, and can reduce cooling costs.

Contents of the invention

The purpose of the present invention is to provide an enzyme for degrading cellulose and its construction and application.

To achieve the above object, the technical solution adopted in the present invention is:

An enzyme for degrading cellulose, the enzyme for degrading cellulose is shown in the amino acid in sequence table SEQ ID NO: 1 or sequence table SEQ ID NO: 2.

The enzyme that degrades cellulose is endocellulase and lichenase; wherein, the amino acid sequence of endocellulase and lichenase is, sequence listing SEQ ID NO: 1 or sequence listing SEQ ID NO: 2 shown in .

The endocellulase and lichenase are fragments having more than 80% homology with the amino acids shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing.

A method for constructing an enzyme for degrading cellulose, introducing recombinant expression vectors containing endocellulase or lichenase genes into host cells, respectively, and expressing to obtain sequence table SEQ ID NO: 1 or sequence table SEQ ID NO: 2 shown in Amino acid sequences of endocellulase and lichenase.

The starting vector as the recombinant expression vector is pEASY-E1, pET-22b, pET28, pET32, pQE-30, pGEX-4T-2, pBR322 or pUC18.

The application of the enzyme for degrading cellulose, the enzyme shown in the sequence table SEQ ID NO: 1 or the amino acid in the sequence table SEQ ID NO: 2 can be used for the biotransformation of cellulose. The endo-cellulase may act as an exo-enzyme.

The present invention has the advantages that the present invention can quickly express the enzyme for the biotransformation of cellulose derived from pyrolytic cellulolytic bacteria, and has high thermal stability, high specific enzyme activity and long half-life.

Description of drawings

Fig. 1 is the electrophoretic pattern of the molecular weight size of the purified protein detected by SDS-PAGE provided in the embodiment of the present invention.

Detailed ways

Embodiments are carried out on the premise of the technical solutions of the present invention, and detailed implementation methods and specific operation processes are provided, but the protection scope of the present invention is not limited to the following embodiments.

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1, the acquisition of endocellulase and lichenase genes and the construction of their prokaryotic expression vectors

The cloning process of the gene and the construction process of its prokaryotic expression vector comprise the following steps:

1. Cloning of endocellulase and lichenase genes

After whole genome sequencing, endocellulase designed the following primers:

C-F: ATGAAGAAAGAATGTCTTAGAGTTTTTGC

C-R:TTACATCTTTCCTGTAAGTTCTAAAATTTTG

According to the gene of lichenase, the following primers were designed:

E-F:TCCAGTAACAGAGCCAAAATTCC

E-R: TGTTGCTACACACCTCCCTT

Saccharolytic cellulolytic bacteria extracted, such as saccharolytic cellulolytic bacteria DSM8903 (German Collection of Microorganisms and Cell Cultures, DSMZ), saccharolytic cellulolytic bacteria CGMCC1.5183 ( China General Microbiological Culture Collection Management Center, China General Microbiological Culture Collection, CGMCC, address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), total genomic DNA is used as template, and C-F and C-R; E-F and E-R are used as primers respectively Yes, the target gene fragment is amplified by PCR.

The PCR reaction system for amplifying endocellulase is 50 μl, including: bacterial genomic DNA: 1 μl; 10×PCR Buffer: 5 μl; dNTP Mixture (2.5 mM each): 4 μl; primer CF (20 μM), 1 μl; primer CR ( 20μM), 1μl; LA Taq polymerase (5U/μl): 0.5μl; add ddH ₂ O to make the total system 50μl. PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 54°C for 45 sec, extension at 72°C for 150 sec, 30 cycles, and extension at 72°C for 10 min.

The PCR reaction system for amplifying lichenase is 50 μl, including: bacterial genomic DNA: 1 μl; 10×PCR Buffer: 5 μl; dNTP Mixture (2.5 mM each): 4 μl; primer EF (20 μM), 1 μl; primer ER (20 μM ), 1 μl; LA Taq polymerase (5U/μl): 0.5 μl; add ddH ₂ O to make the total system 50 μl. PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 56°C for 45 sec, extension at 72°C for 90 sec, 30 cycles, and extension at 72°C for 10 min.

The endocellulase derived from Thermocellulolyticus saccharolyticus has multiple structural domains, and its catalytic domain belongs to the glycoside hydrolase 5 family. In addition, it also has a carbohydrate binding domain and three S-layer homology structures area.

A lichenase derived from Pyrocellulolyticus saccharolyticus, belonging to the glycoside hydrolase 5 family.

2. Construction of endocellulase and lichenase gene expression vectors

The gene fragments obtained by PCR in step 1 were respectively ligated into the expression vector pEASY-E1, and the ligation products containing endocellulase and lichenase genes were respectively transformed into Escherichia coli E.coli BL21 (DE3), and PCR and sequencing After identification, the positive cloned plasmids containing endocellulase and lichenase genes were named pEASY-E1-JX030399 and pEASY-E1-KC958563 respectively, which can be used to express endocellulase and lichenase.

Embodiment 2, expression and purification of endocellulase and lichenase

The prokaryotic expression vectors pEASY-E1-JX030399 and pEASY-E1-KC958563 obtained in Example 1 that contained endocellulase and lichenase genes were transformed into Escherichia coli E.coliBL21 (DE3) respectively, and positive single clones were selected at 37 Shake the bacteria at °C until the OD ₆₀₀ is 0.5, add 1mMIPTG to induce, and collect the bacteria by centrifugation after 10 hours. For the bacteria containing the target protein, add 4ml of phosphate buffer (50mM NaH ₂ PO ₄ , 300mM NaCl, pH8.0), protease inhibitors, lysozyme (10mg/ml) were added at the same time, and the cells were fully suspended and disrupted by ultrasonication. The obtained cell disruption solution was centrifuged at 4°C and 10000×g for 20 minutes, and the obtained supernatant was filtered through a 0.22 μm filter membrane to obtain the crude enzyme solution.

For protein purification, the specific method is: after the ethanol in the Ni-NTA-Sefinose column flows out, add sterile water with a total volume of 10 ml, 2 ml each time. A total volume of 10 ml of phosphate buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, pH 8.0) was added, 2 ml each time. Add crude enzyme solution and penetrate 3 times. Phosphate buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, pH 8.0) was added until there was no protein in the effluent. Then add 5 mL each of elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, imidazole concentrations of 25 mM, 50 mM, 100 mM, 250 mM, pH 8.0) with gradually increasing imidazole concentration, and collect protein fractions, each time Collect 1 mL. The purified endocellulase and lichenase were verified by SDS-PAGE electrophoresis. The purification results are shown in Figure 1. The obtained endocellulase JX030399 has a molecular weight of 85kDa, and the lichenase KC958563 has a molecular weight of 85kDa. It is 43.7kDa.

SEQ ID NO: 1

MKKECLRVFAVFMVFTFLLSLFPFVTFAQNTAYEKDKYPHLIGNSLVKKPSVAGRLQIIKQNGRRILADQNGEPIQLRGMSTHGLQWFPQIINNNAFAALANDWGCNVIRLAMYIGEGGYATNPQVKDKVIEGIKLAIQNDMYVIVDWHVLNPGDPNAEIYKGAKDFFKEIAQKFPNDFHIIYELCNEPNPTDPGVTNDEAGWKKVKAYAEPIIKMLRQMGNENIIIVGSPNWSQRPDFAIKDPIADDKVMYSVHFYTGTHKVDGYVFENMKMAIEAGVPVFVTEWGTSEASGDGGPYLDEADKWLEYLNANNISWVNWSLTNKNETSGAFVPYISGVSQATDLDPGSDQKWDISELSISGEYVRSRIKGIPYQPIERTLKISQDQVACAPIGQPILPSDFEDGTRQGWDWDGPSGVKGALTIEEANGSNALSWEVEYPEKKLQDGWASAPRLILRNINTTRGDCKYLCFDFYLKPKQATKGELAIFLAFAPPSLNYWAQAEDSFNIDLSNLSTLKKTPDDLYSFKISFDLDKIKEGKIIGPDTHLRDIIIVVADVNSDFKGRMYLDNVRFTNMLFEDVTPQTTGYEAISKLYSKKIVNGISTNLFGPEKAVTRAEVAAMAVRLLDLQEESYNGEFTDVSKNSWYANEVSTAYKAGIILGDGKYIKPEKAVTREEMAVFAMRIYRILTDEKAEATEEIAISDKNSISSWARQDVNAAISLGLMDVFTDGSFGPKAKVARAEATQIIYKILELTGKM

(a) Sequential features:

●Length: 756

●Type: amino acid sequence

●Chain type: single chain

●Topological structure: linear

(b) Molecule Type: Protein

(c) Assumption: No

(d) Antisense: No

(e) Original source: Thermocellulolyticus saccharolyticus

Structural features: 69-325 are glycoside hydrolase 5 family domains, 352-572 are carbohydrate binding domains, 576-618, 636-677, 701-742 are three S-layer homology domains.

SEQ ID NO: 2

SSNRAKIPEIKIASRKIPNNAALKFVKDMKIGWNLGNTFDAAFENPSFDDELLYETAWCGVKTTKQMIDTVKKAGFNTIRIPVSWHNHVTGSNFTISKRWLDRVQQVVDYAMKNKMYVIINIHHDIMPGYYYPNSQHLQTSIKYVKSIWTQVATRFKNYNDHLIFEAVNEPRLTGSRFEWWLDMNNPECRDAVEAINKLNQVFVDTVRSTGGNNVSRYLMVPGYAAAPEYVLIDEFKIPKDSSKYKNRIIISVHAYRPYNFALQAPNESGSVSEWSVNSEESRRDIDYFMDKLYDKFVSKGIPVVIGEFGARDKNGNLQSRVEFAAYYVRAARARGITCCWWDNNAFYGNGENFGLLDRKTLKWVYPEIVSAMMKYAR

(a) Sequential features:

●Length: 378

●Type: amino acid sequence

●Chain type: single chain

●Topological structure: linear

(b) Molecule Type: Protein

(c) Assumption: No

(d) Antisense: No

(e) Original source: Thermocellulolyticus saccharolyticus

Structural features: 54-347 is the glycoside hydrolase 5 family domain

Embodiment 3, enzymatic characteristic detection

Purify according to the method of Example 2 to obtain endocellulase and lichenase respectively, and then use 10kDa ultrafiltration tubes to replace the buffer (200mM acetic acid-acetic acid sodium salt buffer, pH5.6) to remove the enzyme solution The imidazole in the endocellulase and lichenase enzyme solutions were obtained respectively.

Using the endocellulase and lichenase obtained above, carry out the following reactions respectively to identify the enzymatic characteristics:

Using filter paper, sodium carboxymethylcellulose, xylan, microcrystalline cellulose, lichenin and barley dextran as substrates to determine protease activity, use 3.5-dinitrosalicylic acid (DNS) method to determine the content of reducing sugar Generation volume. When detecting the enzyme activity of the filter paper, add 5 mg of Whatman NO.1 filter paper as a substrate to the 150 μl reaction system, and the total addition amount of other substrates is 1% (w/v).

Take 50 μl each of the endocellulase solution with a concentration of 3.2 μg/ml and the lichenase solution with a concentration of 2 μg/ml, and add it to acetic acid-sodium acetate buffer (200 mM acetic acid-sodium acetate buffer, pH 5.6) After reacting at 75°C for 30 minutes, add 200 μl of DNS solution, boil for 5 minutes, add 650 μl of water after cooling and mix well, take 200 μl and add it to the microtiter plate, and measure its absorption value at 540 nm. With glucose or xylose at 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1mg/ml as standard samples, draw a standard curve with the absorbance value measured by the DNS method and the sugar concentration, according to the standard The curve calculates the amount of reducing sugar in the obtained sample.

Glucose standard curve: y=9.783x+0.0009 R ² =0.9978

Xylose standard curve: y=11.406x-0.0049 R ² =0.9987

Among them, x is the concentration of glucose or xylose, mg/ml; y is the absorption value at 540 nm under the corresponding sugar concentration.

One unit of enzyme activity is defined as the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of reducing sugar per minute. The specific enzyme activity is defined as the ratio of the enzyme activity to the corresponding protein amount. The enzyme activity reaction system is shown in Table 1.

Table 1 enzyme activity reaction system

components blank Experimental control group sample group Sample control group buffer 150μl 50μl 100μl substrate 100μl 100μl Enzyme solution 50μl 50μl

Then with 1mM p-nitrophenyl-β-D-galactopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC) and p-nitrophenyl-β-D-pyranoside Xyloside (pNPX) solution was used as the substrate to determine the enzyme activity.

Take the appropriately diluted enzyme solution and add it to the acetic acid-sodium acetate buffer, react at 75°C for 30 minutes, add 450 μl of saturated sodium borate solution and mix well, take 200 μl and add it to the microplate, and measure its absorbance at 405 nm. Dilute the 1mg/ml p-nitrophenol standard sample with Tris-HCl (50mM, pH9.0) solution to make the final concentration 0-20μg/μl, take 200μl and add it to the microtiter plate, and measure its absorption at 405nm Value to draw a standard curve, according to the standard curve to calculate the amount of p-nitrophenol enzymatic hydrolysis reaction.

p-nitrophenol standard curve: y=0.086x+0.0803 R ² =0.999

Wherein, x is the concentration ug/ml of p-nitrophenol; y is the absorption value at 405 nm corresponding to the concentration of p-nitrophenol.

One enzyme activity unit is defined as the amount of enzyme needed to hydrolyze the substrate to produce 1 μmol of p-nitrophenol per minute. The specific enzyme activity is defined as the ratio of the enzyme activity to the corresponding protein amount. The enzyme activity reaction system is shown in Table 1.

Experiment 1. Determination of the optimum reaction pH value, optimum reaction temperature and half-life of endocellulase and lichenase under optimum reaction conditions

Determination of protein at 75°C in different pH ranges (200mM acetic acid-sodium acetate buffer for pH 4.6, 5, 5.6; PC buffer (50mM phosphoric acid, 12mM citric acid) for pH 6, 6.6, 7, 7.6, and 8) to determine its optimum reaction pH value; measure its enzyme activity in different temperature ranges (55, 60, 65, 70, 75, 80°C) under its optimum reaction pH value to determine its optimum temperature reflex. The method for determining the half-life under the optimum reaction conditions is: place the enzyme under its optimum reaction conditions, measure the residual enzyme activity at intervals, and the sampling time when the residual enzyme activity is 50% is the half-life under this condition.

Table 2 Enzymatic property detection of JX030399 protein and KC958563 protein

Experiment 2. Determination of substrate specificity of endocellulase and lichenase

The substrate specificity of JX030399 protein was determined by DNS method and pNP method under the optimal conditions (75°C, pH5.6), respectively, and the reaction system is shown in Table 1. The experimental results are shown in Table 3. When the JX030399 protein uses sodium carboxymethyl cellulose as the substrate, its enzyme activity is 17.11U/mg, which use microcrystalline cellulose Avicel PH-101 and p-nitrophenyl cellobioside (pNPC) as substrates respectively When the enzyme activity is 10.11U/mg and 10.43U/mg. The results indicated that JX030399 protein was a processive endo-cellulase with high exo-cellulase activity.

The substrate specificity analysis of table 3JX030399 protein

The substrate specificity of KC958563 protein was determined by DNS method and pNP method under the optimal conditions (80°C, pH5.6). The reaction system is shown in Table 1. The experimental results are shown in Table 3. KC958563 protein had higher enzyme activity when barley glucan and lichenin were used as substrates, 1284IU/mg and 627IU/mg respectively, and other enzyme activities were lower.

The substrate specificity analysis of table 4KC958563 protein

substrate Specific enzyme activity (IU/mg) barley glucan 1284.03±114.84 Lichenan 627±8.37 Xylan 6.49±0.26 Phosphoric Acid Swellable Cellulose 1.79±0.01 p-nitrophenyl-β-D-cellobioside 0.36±0.02 Sodium carboxymethyl cellulose 0.19±0.03 Microcrystalline Cellulose PH-101 0.18±0.11

Example 4

Utilize the above-mentioned Example 2 to purify endo-cellulase to degrade microcrystalline cellulose or unpretreated wheat straw:

Specifically, the unpretreated wheat straw or microcrystalline cellulose was used as a substrate, and the endo-cellulase purified by the above-mentioned Example 2 was subjected to enzymatic hydrolysis treatment respectively;

Dissolve 10 μg of JX030399 protein in the acetate buffer system of pH 5.6, add 50 mg of solid substrate, the total reaction system is 1 ml, the enzymolysis temperature is 75°C, and the enzymolysis time is 1, 3, 6, 12, 24, 36 hours. The glucose and cellobiose contents in the supernatant were determined at different enzymatic hydrolysis times.

The amount of glucose obtained by enzymatic hydrolysis of AvicelPH-101 was 3.3 μmol/nmol enzyme, and the amount of cellobiose produced was 9.1 μmol/nmol enzyme. The amount of glucose produced by enzymatic hydrolysis of unpretreated wheat straw was 3.5 μmol/nmol enzyme, and the amount of cellobiose produced was 0.93 μmol/nmol enzyme.

Claims (7)

1. An enzyme for degrading cellulose, characterized in that: the enzyme for degrading cellulose is shown in amino acid in sequence table SEQ ID NO: 1 or sequence table SEQ ID NO: 2. 2. The enzyme for degrading cellulose according to claim 1, characterized in that: the enzyme for degrading cellulose is endocellulase and lichenase; wherein, endocellulase and lichenase The amino acid sequence is shown in the sequence listing SEQ ID NO: 1 or the sequence listing SEQ ID NO: 2. 3. The enzyme for degrading cellulose according to claim 1 or 2, characterized in that: the endocellulase and lichenase are the same as the sequence table SEQ ID NO: 1 or the sequence table SEQ ID NO: 2 Fragments with more than 80% homology of amino acids shown in . 4. A method for constructing an enzyme for degrading cellulose according to claim 1, characterized in that: the recombinant expression vector containing endocellulase or lichenase gene is introduced into host cells respectively, and the expression obtains the sequence table SEQ ID The amino acid sequence of endocellulase and lichenase shown in NO: 1 or SEQ ID NO: 2 in the sequence table. 5. The construction method of the enzyme degrading cellulose according to claim 4, characterized in that: the starting vector as the recombinant expression vector is pEASY-E1, pET-22b, pET28, pET32, pQE-30, pGEX- 4T-2, pBR322 or pUC18. 6. An application of the enzyme degrading cellulose according to claim 1, characterized in that: the enzyme shown in the amino acid in the sequence table SEQ ID NO: 1 or sequence table SEQ ID NO: 2 can be used as an enzyme for fiber Biotransformation of hormones. 7. The application of the enzyme for degrading cellulose according to claim 6, characterized in that: the endo-cellulase can be used as an exo-enzyme.