CN118006589A - A combination of acidophilic cellulase and xylanase, its encoding gene and application - Google Patents

Abstract

The invention discloses an acidophilic cellulase and xylanase combination and a coding gene and application thereof; the enzyme combination consists of eosinophilic endoglucanases EGL1, EGL2 and eosinophilic endoxylanase XYN 1; the amino acid sequence of the EGL1 is shown as SEQ ID NO.2, the EGL2 is shown as SEQ ID NO.4, and the amino acid sequence of the XYN1 is shown as SEQ ID NO. 6; the multienzyme combination of the acidophilic endoglucanases EGL1, EGL2 and acidophilic endoxylanase XYN1 has extremely high acid stability (pH is 0.5-4.0), and can stably maintain high-efficiency hydrolase activity in a pH range of 1.5-4.0, so that the special requirements of acid application environments such as juice clarification, animal feed, microbial fertilizer trichoderma spore production and the like on high-efficiency cellulose hydrolysis under low pH value can be met.

Description

A combination of acidophilic cellulase and xylanase, its encoding gene and application

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to a multi-enzyme combination consisting of two acidophilic endoglucanases and one acidophilic endoxylanase, as well as a coding gene and application thereof.

technical background

Plant residues are extremely abundant renewable resources in nature, and their main components include cellulose and xylan. Cellulose is formed by D-glucose residues connected in series through β-1,4-glycosidic bonds to form long-chain molecules, and exists in large quantities in the form of crystalline structures; xylan is formed by D-xylose residues connected in series through β-1,4-glycosidic bonds, and is the most abundant polysaccharide substance after cellulose. In nature, microorganisms can secrete enzymes including endoglucanases and endoxylanases to efficiently hydrolyze the complex cellulose and xylan components in plant residues, thereby playing the role of long-chain breaking and releasing oligosaccharides. Therefore, polysaccharide (cellulose and xylan) hydrolases such as endoglucanases and endoxylanases are widely used in various fields of industry, including papermaking, textiles, bioenergy, feed, food, etc.

With the continuous development of industry, there is an urgent need for cellulase and xylanase products that can still efficiently perform enzymatic hydrolysis in extreme environments. For example, in the field of bioenergy, the acidic environment of plant residue pretreatment, the acidic environment of juice clarification, and the gastric acid environment of animal feed urgently need acid-resistant or acidophilic cellulase and xylanase to efficiently hydrolyze cellulose and xylan in a low pH environment (pH 2-4) to promote saccharification, clarification, and digestion and other key processes. In addition, in the field of agricultural microbial fertilizer manufacturing, acid-treated plant residues are usually fermented under low pH conditions (about 3.0) to prepare Trichoderma functional fungus spore products, but most polysaccharide hydrolases have low activity in this environment. Therefore, the discovery of endoglucanases and endoxylanases that can efficiently perform hydrolysis in an acidic environment (pH 2-4) and have excellent acid stability characteristics can meet the industrial application needs in different fields.

Summary of the invention

The purpose of the present invention is to provide a multi-enzyme combination including two acidophilic endocellulase genes and one acidophilic endoxylanase gene in response to the urgent need for endoglucanases and endoxylanases that can efficiently and stably perform enzymolysis under acidic conditions (pH <4) in different fields, while conventional enzymes are structurally unstable and easily inactivated under such conditions.

Another object of the present invention is to provide a genetically engineered bacterium expressing the three genes.

Another object of the present invention is to provide the application of the three genes. The multi-enzyme preparation produced by the three genes can be used in the fields of juice clarification, animal feed, Trichoderma spore preparation of microbial organic fertilizer, etc., meeting the needs of stable and efficient hydrolysis under low pH conditions, and bringing considerable economic benefits.

The purpose of the present invention can be achieved through the following technical solutions:

The invention discloses an acidophilic endoglucanase gene egl1, whose nucleotide sequence is SEQ ID NO. 1. The full length of the gene (including the start codon and the stop codon) is 663 bp, the G+C content is 49%, and the gene encodes an acidophilic endoglucanase EGL1 composed of 219 amino acids, whose amino acid sequence is SEQ ID NO. 2.

An acidophilic endoglucanase gene egl2, whose nucleotide sequence is SEQ ID NO. 3. The full length of the gene (including the start codon and the stop codon) is 1200bp, the G+C content is 49%, and it encodes an acidophilic endoglucanase EGL2 composed of 399 amino acids, whose amino acid sequence is SEQ ID NO. 4.

An acidophilic endoxylanase gene xyn1, whose nucleotide sequence is SEQ ID NO. 5. The full length of the gene (including the start codon and the stop codon) is 750 bp, the G+C content is 54%, and it encodes an acidophilic endoglucanase XYN1 composed of 249 amino acids, whose amino acid sequence is SEQ ID NO.6.

In the first aspect, the present invention protects an enzyme combination, which consists of an acidophilic endoglucanase EGL1, an acidophilic endoglucanase EGL2 and an acidophilic endoxylanase XYN1; the amino acid sequence of the acidophilic endoglucanase EGL1 is shown in SEQ ID NO.2, the acidophilic endoglucanase EGL2 is shown in SEQ ID NO.4, and the acidophilic endoxylanase XYN1 is shown in SEQ ID NO.6.

In the second aspect, the present invention protects a gene combination related to the enzyme combination described above, which comprises gene egl1 encoding acidophilic endoglucanase EGL1, gene egl2 encoding acidophilic endoglucanase EGL2, and gene xyn1 encoding acidophilic endoxylanase XYN1; the nucleotide sequence of gene egl1 is shown in SEQ ID NO.1; the nucleotide sequence of gene egl2 is shown in SEQ ID NO.3; the nucleotide sequence of gene xyn1 is shown in SEQ ID NO.5.

In a third aspect, the present invention protects a recombinant vector combination related to the genes described above, wherein the recombinant vector combination includes a recombinant vector containing the acidophilic endoglucanase gene egl1, a recombinant vector containing the acidophilic endoglucanase gene egl2, and a recombinant vector containing the acidophilic endoxylanase gene xyn1.

In a specific embodiment, the recombinant vector is a recombinant plasmid.

In a more specific embodiment, the present invention discloses three recombinant plasmids, namely pPICZαA-egl1 containing the above-mentioned acidophilic endoglucanase gene egl1, pPICZαA-egl2 containing the acidophilic endoglucanase gene egl2, and pPICZαA-xyn1 containing the acidophilic endoxylanase gene xyn1. All of them are obtained by inserting the cDNA sequences of egl1, egl2 and xyn1 genes into the multiple cloning site of the pPICZαA expression plasmid by double restriction digestion with EcoRI and XbaI, and integrating them into the correct expression frame of the plasmid, and screening the correct mutants by zeocin and sequencing to verify the obtained results.

In a fourth aspect, the present invention also protects genetically engineered bacteria containing the gene combination or the recombinant vector described above.

In a specific embodiment, the genetically engineered bacteria are Pichia pastoris X33-egl1, P. pastoris X33-egl2 and P. pastoris X33-xyn1, which contain the acidophilic endoglucanase genes egl1 and egl2 and the acidophilic endoxylanase gene xyn1, respectively.

In a specific embodiment, the genetically engineered bacteria are constructed by the following method: the recombinant plasmids pPICZαA-egl1, pPICZαA-egl2 and pPICZαA-xyn1 containing the acidophilic endoglucanase genes egl1, egl2 and the acidophilic endoxylanase gene xyn1 are respectively introduced into the host bacteria P. pastoris X33 competent state by electrotransformation, and then mutants are screened on YPD (2% (w/v) peptone, 1% (w/v) yeast powder, 2% (w/v) glucose, 1M sorbitol, pH 6.0) plates containing 25 mg·L ^-1 bleomycin, and transformants are picked after culturing at 30°C for 48 hours. After sequencing verification and pre-fermentation enzyme activity determination, the correct genetically engineered bacteria P. pastoris X33-egl1, P. pastoris X33-egl2 and P. pastoris X33-xyn1 are obtained and stored in glycerol at -80°C.

The production methods of the acidophilic endoglucanase proteins EGL1 and EGL2 and the acidophilic endoxylanase protein XYN1 are as follows:

1) A small amount of genetically engineered bacteria P. pastoris X33-egl1, P. pastoris X33-egl2 and P. pastoris X33- ^xyn1 were inoculated into BMGY liquid culture medium (1% (w/v) yeast powder, 2% (w/v) peptone, 4×10 ^-5 % (w/v) biotin, 1.34% (w/v) YNB, 1% (v/v) glycerol) containing 100 mg·L -1 bleomycin, cultured in a shaking incubator at 28° C. and 250 rpm for 24 h in the dark, centrifuged at 8000 rpm for 5 min, and the supernatant was removed to harvest the bacteria.

2) Each engineered bacterial cell was re-transferred into BMMY liquid culture medium containing 1% methanol (1% (w/v) yeast powder, 2% (w/v) peptone, 4× ^10-5 % (w/v) biotin, 1.34% (w/v) YNB, 1% (v/v) methanol), and cultured in a shaking incubator at 28°C and 250 rpm in the dark. Methanol (1% (v/v) content) was added once every 24 h. After 72 h, the cells were centrifuged at 8000 rpm for 5 min to remove the bacterial precipitate, and EGL1, EGL2 and XYN1 protein fermentation broths were obtained, respectively.

3) A small amount of fermentation broth was verified by SDS-PAGE gel electrophoresis and DNS enzyme activity assay. After confirmation, it was purified in large quantities using molecular sieve technology. After purification, each protein was dissolved in 100 mM potassium phosphate solution (pH 6.6). The protein content was determined by BCA method and was between 1.0-2.0 g·L ^-1 . The protein was stored at -80°C for use.

In a fifth aspect, the present invention protects the use of the combination or individual of the acidophilic endoglucanases EGL1, EGL2 and the acidophilic endoxylanase XYN1 encoded by the genes shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 in the production of acidic environments such as juice clarification, animal feed or biological organic fertilizer Trichoderma spores.

In a sixth aspect, the present invention also protects the use of the aforementioned gene combination, recombinant vector combination or genetically engineered bacteria combination in the production of acidic environments such as juice clarification, animal feed or biological organic fertilizer Trichoderma spores.

The beneficial effects of the present invention are as follows:

The invention provides an acidophilic multi-enzyme combination, a gene combination, a vector combination and an engineered bacterium thereof, comprising acidophilic endoglucanase genes egl1 and egl2 and acidophilic endoxylanase gene xyn1, wherein the expressed proteins EGL1 and EGL2 can hydrolyze cellulose that is difficult to degrade by microorganisms into soluble cellooligosaccharides, and XYN1 can hydrolyze xylan into soluble xylo-oligosaccharides. The optimum reaction temperature of EGL1, EGL2 and XYN1 is 50°C, and the optimum reaction pH value is in the range of 3.0-3.5. EGL1, EGL2 and XYN1 can maintain more than 80% of enzyme activity in the range of pH 3.0-4.0, pH 2.5-4.0 and pH2.0-4.0, respectively. EGL1 can maintain stable structure and enzyme activity in an extremely acidic environment (pH 0.5-4.0), while EGL2 and XYN1 can maintain stable structure and enzyme activity under the condition of pH≥2.0. The multi-enzyme combination formed by the three can achieve stable and efficient hydrolysis of cellulose, xylan and other components under pH 2.0-4.0 conditions, with significant synergistic effects. This feature is highly suitable for acidic environments such as juice clarification, animal feed, and microbial organic fertilizer Trichoderma spore production, thereby enhancing juice clarity, promoting animal feed absorption, and improving Trichoderma fermentation efficiency and spore production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an SDS-PAGE image of acidophilic endoglucanases EGL1, EGL2 and acidophilic endoxylanase XYN1.

A: Acidophilic endoglucanase EGL1; B: Acidophilic endoglucanase EGL2; C: Acidophilic endoxylanase XYN1.

Fig. 2 Optimal temperature and temperature stability determination of acidophilic endoglucanases EGL1, EGL2 and acidophilic endoxylanase XYN1.

A: Optimum temperature of EGL1. An appropriate amount of EGL1 was mixed with citric acid buffer (pH 3.5) containing 0.5% sodium carboxymethyl cellulose (CMC-Na), and reacted at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C in a water bath for 30 min. The enzyme activity was determined by the DNS method.

B: Optimum temperature of EGL2, the method is the same as EGL1;

C: Optimum temperature of XYN1. An appropriate amount of XYN1 was mixed with citric acid buffer (pH 3.5) containing 1% oat xylan, and reacted at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C in a water bath for 10 min. The enzyme activity was determined by the DNS method.

D: Temperature stability of EGL1, the EGL1 protein solution was incubated at different temperatures (30°C, 40°C, 50°C, 60°C, 70°C, 80°C) for different time periods (15 min, 30 min, 45 min, 60 min), and the residual enzyme activity was determined by the DNS method;

E: EGL2 temperature stability, the method is the same as EGL1;

F: XYN1 stabilization, the method is the same as EGL1.

Figure 3 Optimal pH and pH stability determination of acidophilic endoglucanases EGL1, EGL2 and acidophilic endoxylanase XYN1.

A: The optimal pH of EGL1, different pH values (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 10.6) of buffer containing 0.5% CMC-Na were prepared, and the appropriate amount of EGL1 was added to react at 50℃ for 30min, and the enzyme activity was determined by DNS method;

B: Optimal pH for EGL2, the method is the same as that for EGL1;

C: The optimal pH of XYN1, different pH values (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 10.6) of buffer containing 1% oat xylan were prepared, and after adding appropriate amount of XYN1, the reaction was carried out at 50℃ for 10min, and the enzyme activity was determined by DNS method;

D: pH stability of EGL1. EGL1 was incubated in buffers with different pH values at 4 °C for 1 h, followed by hydrolysis with 0.5% CMC-Na at 50 °C and pH 3.5 for 30 min. The residual enzyme activity was determined by the DNS method.

E: pH stability of EGL2, the method is the same as that of EGL1;

F: pH stability of XYN1. XYN1 was incubated in buffers with different pH values at 4°C for 1 h, and then hydrolyzed 1% oat xylan at 50°C and pH 3.0 for 10 min. The residual enzyme activity was determined by the DNS method.

Figure 4 Acidophilic endoglucanases EGL1, EGL2 and acidophilic endoxylanase XYN1 efficiently and synergistically decompose corn cobs at low pH.

A: At pH 2.5, 2 times the fixed volume of enzyme solution (2×EGL1, 2×EGL2 or 2×XYN1), 1 times the fixed volume of enzyme solution in combination of two (EGL1+EGL2, EGL1+XYN1 or EGL2+XYN1), 3 times the fixed volume of enzyme solution (3×EGL1, 3×EGL2 or 3×XYN1) and 1 times the fixed volume of enzyme solution mixed together (EGL1+EGL2+XYN1) were used to hydrolyze 3 mL of 2% (w/v) corn cob particles at 40°C for 10 h. The results were compared with the differences in the amount of reducing sugar released.

B: At pH 3.0, the treatment is the same as above.

Biomaterial Deposit Information

Trichoderma harzianum NJAU4742 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.12166. As it has been disclosed in many documents, this application does not provide a deposit certificate.

Penicillium singorens ZL10 is deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.40202 and the deposit date is May 23, 2022.

Detailed ways

The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

Example 1 Preparation of Acidophilic Endoglucanase EGL1, EGL2 and Acidophilic Endoxylanase XYN1 1. Cloning of Acidophilic Endoglucanase Genes egl1, egl2 and Acidophilic Endoxylanase Gene xyn1

After Trichoderma NJAU4742 or Penicillium ZL10 were cultured in an inorganic salt medium with 2% (w/v) glucose as the sole carbon source at 150 rpm for 48 h, the mycelium was transferred to an inorganic salt medium containing 1% (w/v) crystalline fiber and induced at 150 rpm for 12 h. Then, the cells were ground with liquid nitrogen and total RNA of Trichoderma NJAU4742 or Penicillium ZL10 induced by crystalline fibers was extracted using RNeasy Plant Mini Kit (QIAGEN), and cDNA was synthesized by reverse sequencing using PrimeScript RT-PCR Kit (TAKARA), and the primer pairs F-egl1 (5′-CCGgaattcCAAACCAGCTGCGAACAGTATG-3′, SEQ ID NO.7) and R-egl1 (5′-CTAGtctagaTTAGTTGATAGATGCGGTCCAGG-3′, SEQ ID NO.8), F-egl2 (5′-CCGgaattcCAGCAAACTGTTTGGGGGC-3′, SEQ ID NO.9) and R-egl2 (5′-CTAGtctagaCTATTTCCGGGAAAGGCATGAG-3′, SEQ ID NO.10). NO.10) was PCR amplified from the cDNA sample of Trichoderma NJAU4742 to obtain the cDNA sequences of egl1 and egl2 genes respectively; the cDNA sequence of the xyn1 gene was PCR amplified from the cDNA sample of Penicillium ZL10 by primer pair F-xyn1 (5'-CCGgaattcATGAACACCCCCAACTCAC-3', SEQ ID NO.11) and R-xyn1 (5'-CCGgaattcCTAGCGTATTTCGGAGTACGTTTTG-3', SEQ ID NO.12). After gel excision and recovery, it was dissolved in double distilled water and stored at -20°C.

The first step of the reverse transcription reaction system was (dNTP Mixture (10mM each) 1μL; Oligo dTPrimer (2.5μM) 1μL; Template RNA 1μg; RNase Free dH ₂ O up to 10μL) with reaction conditions of 65℃ for 5min and 4℃ for 10min. The second step was (reaction solution of the first step 10μl; 5× PrimeScript ^TM Buffer 4μL; RNase Inhibitor (40U·μL ^-1 ) 0.5μl; PrimeScript ^TM RTase (for 2Step) 0.5μL; RNase Free dH ₂ O 5μL) with reaction conditions of 42℃ for 30min, 95℃ for 5min and 4℃ for 10min. The amplification system of cDNA sequence of each gene was (Primer star enzyme (5U/μL) 0.5μL; 5×PCR Buffer II (Mg ²⁺ Plus) 10μl; dNTP Mixture (2.5mM each) 5μl; template cDNA 1μL; upstream primer (20μM) 1μL; downstream primer (20μM) 1μL; sterile double distilled water to 50μL; PCR amplification program: denaturation at 98℃ for 10sec, annealing at 60℃ for 10sec, extension at 72℃ for 1.5min, for 30 cycles; extension at 72℃ for 10min; cooling at 4℃ for 10min.

2. Construction of expression vectors pPICZαA-egl1, pPICZαA-egl2 and pPICZαA-xyn1

The c DNA sequences of the acidophilic endoglucanase genes egl1, egl2 and the acidophilic endoxylanase gene xyn1 and the pPICZαA plasmid sequence were digested with EcoRI and XbaI, respectively. The digestion reaction system was (EcoRI 1μL; XbaI 1μL; 10×M Buffer 5μL; gene fragment or expression plasmid 20μL; sterilized distilled water was added to 50μL), and the reaction was carried out in a 37°C water bath for about 3h. The digestion products were recovered by agarose gel electrophoresis. The recovered egl1, egl2 or xyn1 gene fragments and pPICZαA linearized plasmid fragments were mixed in a ratio of 10:1, and reacted in a 16°C water bath overnight under the action of T4 ligase. The enzyme ligation system is (0.5 μL of pPICZαA linearized plasmid after double enzyme digestion; 4.5 μL of egl1, egl2 or xyn1 gene fragment after double enzyme digestion; 5 μL of Ligate Solution I; add sterile double distilled water to 10 μL).

3. Enzyme-linked product transformation and screening of positive clones

10 μL of the above enzyme-linked product was mixed with 200 μL of E. coli DH5α competent cells and placed in an ice bath for 30 min; then heat-shocked in a 42°C water bath for 90 s, quickly transferred to an ice bath and cooled for 1-2 min; then 800 μL of liquid LLB medium was added and incubated at 37°C at 100 rpm for 1 h. Then, centrifuged at 4000 rpm for 3 min, most of the supernatant was removed, and the remaining approximately 200 μL of competent cells was spread on an LLB agar plate containing 25 mg·L ^-1 bleomycin, and incubated at 37°C for 16 h. About 10 single colonies were selected and numbered, and then inoculated into a 3 mL LLB test tube containing 25 mg·L ^-1 bleomycin. After the colony suspension grew, the plasmid was sequenced to verify its accuracy. The correct plasmids obtained were named pPICZαA-egl1, pPICZαA-egl2, and pPICZαA-xyn1, respectively.

4. The acidophilic endoglucanase genes egl1, egl2 and the acidophilic endoxylanase gene xyn1 were highly expressed in P. pastoris X33

The pPICZαA-egl1, pPICZαA-egl2 and pPICZαA-xyn1 recombinant plasmids containing the acidophilic endoglucanase genes egl1, egl2 and the acidophilic endoxylanase gene xyn1 were linearized with the restriction endonuclease PmeI. 10 μL of high-concentration linearized plasmid (>400 ng·μL ^-1 ) was thoroughly mixed with 200 μL of P.pastoris X33 competent cells and electroporated. Immediately after electroporation, 1 mL of 1 M sorbitol solution was added, and the mixture was allowed to stand at 30°C for 1 hour. After centrifugation at 4000 rpm, most of the supernatant was removed and about 100 μL of bacterial solution was retained. The mixture was spread on YPD medium containing 25 mg·L ^-1 bleomycin. After culturing at 30°C for 2-3 days, positive clones were picked and transferred to fresh YPD medium containing 25 mg·L ^-1 bleomycin. After culturing at 30°C for 3 days, mutants were verified by colony PCR.

The above positive clones were cultured in BMGY medium at 30°C and 200rpm for 20h, centrifuged at 4000rpm for 5min to obtain the bacteria, transferred to an equal volume of fresh BMMY liquid medium, continued to be cultured at 30°C and 200rpm, and methanol solution was added again at a volume ratio of 1% every 24h. After the minimum induction for 48h, the fermentation broth was centrifuged to remove the precipitate, which was the crude enzyme solution of acidophilic endoglucanase EGL1, EGL2 or acidophilic endoxylanase XYN1. 10μL of EGL1 or EGL2 purified protein was added to 1mL of citric acid buffer (pH 3.5) containing 0.5% sodium carboxymethyl cellulose, reacted at 50°C for 30min, and then 1mL of DNS reagent was added to mix well and boiled in water bath for 10min. The enzymatic efficiency was determined by the absorbance value at a wavelength of 520nm, and the standard curve was made using glucose solution. The enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute. The specific activities of EGL1 and EGL2 were 23.85 U·mg ^-1 and 31.61 U·mg ^-1 , respectively. 2 μL of XYN1 purified protein was added to 1 mL of citric acid buffer (pH 3.0) containing 1% oat xylan. After reacting at 50°C for 10 minutes, the enzyme activity was determined by the DNS method, and the standard curve was made with different concentrations of xylose solution. The specific activity of XYN1 was measured to be 1631.28 U·mg ^-1 .

Example 2 Detection of the Optimal Reaction Temperature and Temperature Stability of Acidophilic Endoglucanases EGL1, EGL2 and Acidophilic Endoxylanase XYN1

2 μL of purified acidophilic endoglucanase EGL1 or EGL2 was added to 1 mL of citric acid buffer (pH 3.5) containing 0.5% CMC-Na, and reacted for 30 min at a water bath temperature of 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C, with 3 replicates at each temperature. 2 μL of purified acidophilic endoxylanase XYN1 was added to 1 mL of citric acid buffer (pH 3.0) containing 1% oat xylan, and reacted for 10 min at a water bath temperature of 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C, with 3 replicates at each temperature. After the reaction is completed, 1 mL of DNS reagent is added immediately and mixed thoroughly. After boiling in a water bath for 10 minutes, 200 μL of the solution is drawn and the absorbance at a wavelength of 520 nm is detected by a spectrophotometer. The optimal reaction temperature of the acidophilic endoglucanase EGL1 or EGL2 with CMC-Na as a substrate and the acidophilic endoxylanase XYN1 with oat xylan as a substrate is determined by the absorbance value. Take an appropriate amount of EGL1, EGL2 or XYN1 enzyme solution and incubate in a water bath at different temperatures (30-80°C, with a gradient of 10°C). The enzyme solution is taken out at the time points of 15 minutes, 30 minutes, 45 minutes, and 60 minutes of incubation and placed on ice for cooling. Then, under the conditions of the optimal temperature (50°C) and the optimal pH (3.5 or 3.0), the release of enzymatic reducing sugars is detected by the DNS method to calculate the residual enzyme activity. The results showed that the optimal reaction temperature of acidophilic endoglucanases EGL1, EGL2 and acidophilic endoxylanase XYN1 was 50°C. In the temperature range of 30°C-50°C, EGL1 and EGL2 could maintain more than 80% activity. In terms of temperature stability, EGL1, EGL2 and XYN1 could maintain stable enzyme activity at 50°C and below.

Example 3 Optimal pH and pH stability test of acidophilic endoglucanases EGL1, EGL2 and acidophilic endoxylanase XYN1

Different pH buffers (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 10.6) containing 0.5% CMC-Na or 1% oat xylan were prepared, and 2 μL of purified acidophilic endoglucanase EGL1, EGL2 or acidophilic endoxylanase XYN1 were added and mixed respectively. The mixture was reacted in a water bath at 50°C for 30 min. The amount of reducing sugar released was determined by DNS method and the enzyme activity was calculated, so as to compare the ability of acidophilic endoglucanase EGL1, EGL2 or acidophilic endoxylanase XYN1 to hydrolyze substrate CMC-Na or oat xylan under different pH conditions. In addition, EGL1, EGL2 or XYN1 were incubated in a buffer solution with a pH value of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 10.6 at 4°C for 1 hour, and then the residual enzyme activity was determined by the DNS method under the optimum temperature (50°C) and the optimum pH (3.5 or 3.0). It was finally concluded that the optimum reaction pH values of acidophilic endoglucanases EGL1 and EGL2 were both 3.5, and the optimum reaction pH value of acidophilic endoxylanase XYN1 was 3.0. EGL1, EGL2 and XYN1 were able to maintain more than 80% of the enzyme activity in the pH 3.0-4.0, pH 2.5-4.0 and pH 2.0-4.0 ranges, respectively. EGL1 can maintain stable structure and enzyme activity in extremely acidic environment (pH 0.5-4.0), while EGL2 and XYN1 can maintain stable structure and enzyme activity under pH ≥ 2.0.

Example 4 Multi-enzyme synergistic detection of acidophilic endoglucanases EGL1, EGL2 and acidophilic endoxylanase XYN1

pH 2.5 and pH 3.0 were selected as the pH values for synergistic effect detection, and a 2% (w/v) corncob particle suspension was prepared at this pH value, and 3 mL corncob particle buffer was used as a standard reaction volume. First, an appropriate amount of EGL1, EGL2 or XYN1 was added to 3 mL corncob buffer, and the reducing sugar release was determined by the DNS method after reacting at 40°C for 10 hours. At the same time, the enzyme amount of EGL1, EGL2 or XYN1 was continuously increased, and the above experiment was repeated to draw a dynamic curve of the reducing sugar release of 3 mL 2% (w/v) corncob particles with the increase of each enzyme amount. The enzyme amount corresponding to the inflection point of each curve was selected as the standard fixed volume of EGL1, EGL2 or XYN1, that is, the increase in the enzyme amount would not cause a significant increase in the amount of reducing sugar.

Then, different combinations were set up at pH 2.5 or pH 3.0 to enzymolyze 3 mL of 2% (w/v) corn cob particles at 40°C for 10 h, and the multi-enzyme synergy of EGL1, EGL2 and XYN1 was detected by comparing the amount of reducing sugar released. The combinations included: 2 times the fixed volume of enzyme solution (2×EGL1, 2×EGL2 or 2×XYN1), 1 times the fixed volume of enzyme solution in pairs (EGL1+EGL2, EGL1+XYN1 or EGL2+XYN1), 3 times the fixed volume of enzyme solution (3×EGL1, 3×EGL2 or 3×XYN1) and 1 times the fixed volume of enzyme solution mixed with all three (EGL1+EGL2+XYN1). The test results showed that there was a significant synergistic effect between the acidophilic cellulases EGL1, EGL2 and the acidophilic xylan XYN1. The hydrolysis effect of the three enzymes in combination of two or three on the substrate corncob particles under low pH conditions (2.5-3.0) was significantly higher than that of any single enzyme.

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims (10)

1. An enzyme combination, characterized in that the enzyme combination consists of an eosinophilic endoglucanase EGL1, an eosinophilic endoglucanase EGL2 and an eosinophilic endoxylanase XYN 1; the amino acid sequence of the eosinophilic endoglucanase EGL1 is shown as SEQ ID NO.2, the eosinophilic endoglucanase EGL2 is shown as SEQ ID NO.4, and the eosinophilic endoxylanase XYN1 is shown as SEQ ID NO. 6.

2. A combination of genes related to the combination of enzymes of claim 1, characterized in that the combination of genes comprises a gene EGL1 encoding an eosinophilic endoglucanase EGL1, a gene EGL2 encoding an eosinophilic endoglucanase EGL2 and a gene XYN1 encoding an eosinophilic endoxylanase XYN 1; the nucleotide sequence of the gene egl1 is shown as SEQ ID NO. 1; the gene egl2 is shown as a nucleotide sequence SEQ ID NO. 3; the nucleotide sequence of the gene xyn1 is shown as SEQ ID NO. 5.

3. A recombinant vector combination related to the gene combination of claim 2, characterized in that the recombinant vector combination comprises a recombinant vector comprising an eosinophilic endoglucanase gene egl1, a recombinant vector comprising an eosinophilic endoglucanase gene egl2 and a recombinant vector comprising an eosinophilic endoxylanase gene xyn 1.

4. The recombinant vector combination according to claim 3, wherein the recombinant vector is a recombinant plasmid.

5. The recombinant vector combination according to claim 4, wherein the recombinant plasmid is based on the pPICZ alpha A expression plasmid.

6. A genetically engineered bacterium comprising the gene combination of claim 2 or the recombinant vector of claim 3.

7. The genetically engineered bacterium of claim 6, wherein the genetically engineered bacterium is Pichia pastoris X-egl 1, pichia pastoris X-egl 2, and Pichia pastoris X-xyr 1, respectively.

8. The genetically engineered bacterium of claim 7, wherein the genetically engineered bacterium Pichia pastoris X-egl 1, pichia pastoris X-egl 2, and Pichia pastoris X33-xyr1 are constructed as follows: three recombinant plasmids according to claim 5 are respectively and electrically transduced into an expression host bacterium P.pastoris X33 after being linearized by PmeI restriction endonuclease, and then are screened by YPD flat plates containing 25 mg.L ^-1 bleomycin, are cultured for 48 hours at 30 ℃, and single colonies are picked up and are stored after sequencing to verify that the gene sequence is correct.

9. The method for preparing acidic endoglucanase proteins EGL1 and EGL2 and eosinophilic endoglucanase protein XYN1 by using the genetically engineered bacterium of claim 7 or 8 comprises the following steps:

1) Respectively inoculating genetically engineered bacteria P.pastoris X33-egl1, P.pastoris X33-egl2 and P.pastoris X33-xyn1 into BMGY liquid culture medium containing 100 mg.L ^-1 bleomycin, shake culturing at 28 ℃ for 24 hours in dark condition, centrifuging to remove supernatant, and obtaining the bacterial cells;

2) Transferring each engineering bacteria body into BMMY liquid culture medium containing 1% methanol, shake culturing at 28deg.C in dark, adding methanol every 24 hr, centrifuging after 72 hr to remove bacterial precipitate, and respectively obtaining EGL1, EGL2 and XYN1 protein fermentation liquor;

3) Taking fermentation liquor, performing SDS-PAGE gel electrophoresis and DNS method enzyme activity measurement verification, purifying in a large scale by using a molecular sieve technology after confirming that the fermentation liquor is error-free, dissolving each purified protein in 100mM potassium phosphate solution with pH of 6.6, and preserving at-80 ℃ for standby when the protein content measured by BCA method is between 1.0 and 2.0 g.L ^-1;

Preferably, the BMGY liquid medium comprises the following components: 1% w/v yeast powder, 2% w/v peptone, 4X 10 ^-5% w/v biotin, 1.34% w/vYNB,1% v/v glycerol;

preferably, the BMMY liquid medium comprises the following components: 1% w/v yeast powder, 2% w/v peptone, 4X 10 ^-5% w/v biotin, 1.34% w/vYNB,1% v/v methanol.

The application of the combination of the eosinophilic endoglucanases EGL1, EGL2 and the eosinophilic endoxylanase XYN1 which are encoded by the genes shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 or the independent application in fruit juice clarification, animal feed or production of bio-organic trichoderma spores.