A thermostability-enhanced mutant of β-mannanase from Thermococcus thermophilus and its application

Abstract

The present invention relates to a thermophilic tennis beta-mannanase mutant with improved thermal stability and its application, a mutant with improved thermal stability is obtained by random mutagenesis and expressed in Bacillus subtilis. The results show that the optimum temperature of the β-mannanase mutant is 80 ° C, the optimum pH is 5.0, the shake flask fermentation is 4 days, and the konjac gum is used as the substrate, which is 4.1% higher than the wild-type enzyme activity; its half-life at 80 ° C is 5 h, which is 2 h more than the wild-type, and the enzyme is completely inactivated for more than 48 h. In addition, the ability of the β-mannanase mutant to hydrolyze locust bean gum and guar gum is 10.2% and 2.0% higher than the wild-type, respectively. The enzyme has shown good application prospects in the fields of oil extraction and animal breeding.

Description

Technical Field

[0001] The invention relates to a thermophilic Tenchnerella beta-mannanase mutant with improved thermal stability and application thereof. Background Art

[0002] β-Mannanase (EC 3.2.1.78) can randomly hydrolyze the β-1,4-mannosidic bonds of mannan to produce low molecular weight manno-oligosaccharides, which have wide application value in feed, oil extraction, medicine, papermaking, textile and other industries.

[0003] my country has a large proven shallow oil reserve, and low-grade reserves such as low permeability and ultra-low permeability account for a high proportion, reaching about 70%. Fracturing technology is mainly used for low permeability layers to increase oil well production. During drilling operations, fracturing fluid containing guar gum needs to be injected into the oil well to form one or several high-permeability fracture channels in the formation near the bottom of the well; when fracturing is completed, it is necessary to quickly break the gel to help the fracturing fluid flow back to avoid affecting the formation. Conventional oxidative breakers such as potassium persulfate and ammonium persulfate have problems such as incomplete gel breaking and flowback and greater damage to the formation. The application of biological breakers-β-mannanase can not only degrade guar gum in fracturing fluid to achieve the purpose of breaking gel, but also has no harm to the environment, and has gradually become the mainstream product of breakers. Due to the presence of geothermal gradients in deep wells, the development of high-temperature resistant β-mannanase is of great value for its application in oil production.

[0004] In animal husbandry, β-mannanase not only has anti-nutritional effects, but its hydrolyzate is also beneficial to the intestinal health of animals. It is a feed additive permitted by the Ministry of Agriculture and Rural Affairs of my country. β-mannanase needs to be treated at 85℃ for about 3 minutes during feed pelleting. Since the optimum temperature of most β-mannanases is 40~75℃, the enzyme activity is greatly reduced or even completely inactivated at high temperatures. Therefore, screening β-mannanase with high thermal stability is an effective measure to solve the problem of enzyme activity being destroyed during enzyme pelleting. Summary of the invention

[0005] The purpose of the present invention is to provide a thermophilic Tenchnerella beta-mannanase mutant with good thermal stability and high enzyme activity and application thereof.

[0006] The present invention adopts the following technical solution:

[0007] A thermophilic Tenchnerella β-mannanase mutant, the amino acid sequence of which is shown in SEQ ID No.3.

[0008] Furthermore, the half-life of the enzyme activity of the thermophilic Tenchnerella β-mannanase mutant at 80° C. is not less than 5 h.

[0009] Furthermore, the time for the thermophilic Tenchnerella β-mannanase mutant to be completely inactivated at 80° C. is not less than 48 hours.

[0010] Furthermore, after the thermophilic Tenchella β-mannanase mutant was incubated at 80°C for 60 min in the pH range of 5.0-6.5, the residual enzyme activity was higher than 84.8%.

[0011] Furthermore, the thermophilic Tenchnerella β-mannanase mutant had an enzyme activity of 5234.3±87.3 U / mL after fermentation for 72 hours.

[0012] A gene encoding the above-mentioned thermophilic Tenchnerium β-mannanase mutant, whose nucleotide sequence is shown in SEQ ID No.2.

[0013] An expression vector for the above encoding gene.

[0014] A Bacillus subtilis cell encoding the above gene.

[0015] The above-mentioned Bacillus subtilis cells were used for fermentation, and the enzyme activity of the thermophilic Tenchnerella β-mannanase mutant obtained after 72 hours was 5234.3±87.3 U / mL.

[0016] An application of the thermophilic Tenchella β-mannanase mutant in oil production and aquaculture.

[0017] The beneficial effects of the present invention are as follows: the present invention utilizes a random mutation strategy to carry out directed evolution of the thermophilic Tenchnerella β-mannanase gene, screens out β-mannanase mutants with improved thermal stability, and utilizes Bacillus subtilis to achieve its efficient expression and secretion, laying a foundation for the application of the enzyme in the fields of oil extraction, aquaculture, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the PCR results for the β-mannanase gene manA and the vector backbone.

[0019] Figure 1 In the figure, lane M is Trans2K plus DNA Marker; lane 1 is β-mannanase manA gene fragment A; and lane 2 is the backbone fragment V of the expression vector.

[0020] Figure 2 The results of SDS-PAGE electrophoresis of the fermentation broth of β-mannanase and its mutant engineered strains DBManA and DBManA-M1.

[0021] Figure 2In the figure, lane 1 is DBManA; lane 2 is DBManA-M1; lane 3 is the protein molecular weight standard.

[0022] Figure 3 Comparison of the optimal temperature results for β-mannanase and its mutants.

[0023] Figure 4 Comparison of the thermal stability of β-mannanase and its mutants at 80 ℃.

[0024] Figure 5 Comparison of the optimal pH results of β-mannanase and its mutants.

[0025] Figure 6 The pH stability results of β-mannanase mutant ManA(Q351Y).

[0026] Figure 7 The effect of metal ions on the activity of β-mannanase ManA(Q351Y).

[0027] Figure 7 In the figure, the same letters in a, b, c, and d indicate that there is no significant difference (P<0.01%) in the effects of different metal ions on the activity of ManA(Q351Y); different letters indicate that there is a significant difference (P<0.01%) in the effects of different metal ions on the activity of ManA(Q351Y).

[0028] Figure 8 Comparison of β-mannanase production and its mutants. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0030] 1. Materials and reagents involved in the present invention

[0031] The plasmids, strains and primers used in the present invention are shown in Table 1, Table 2 and Table 3 respectively. Fastpfu, Hifi Taq, easyTaq, dNTP and the like are products of Beijing Quanshijin Biotechnology Co., Ltd., konjac flour is a product of Hubei Huipu Biochemical Technology Co., Ltd., locust bean gum is a product of Zhejiang Fuxuan Biotechnology Co., Ltd., guar gum is a product of Anhui Weimao Biotechnology Co., Ltd., and other materials are domestic reagents. Biometra Tone PCR instrument is a product of Jena, Germany, and SpectraMaxm i3x multifunctional microplate reader.

[0032] Table 1 Plasmids used in the present invention

[0033] .

[0034] Table 2 Strains used in the present invention

[0035] .

[0036] Table 3 Primers used in the present invention

[0037] .

[0038] 2. Cloning of the β-mannanase gene of Thermococcus thermophilus and construction of expression vector

[0039] According to the literature (Gibbs MD, Reeves RA, Sunna A, Bergquist PL. Sequencing andexpression of a beta-mannanase gene from the extremethermophile Dictyoglomusthermophilum Rt46B.1, and characteristics of the recombinant enzyme. CurrMicrobiol. 1999, 39(6):351-0357.), the β-mannanase gene manA was synthesized by GenScript Biotech Co., Ltd. through codon optimization, and the cloning vector pUC57-manA was constructed.

[0040] Using pUC57-manA plasmid as template, Sgene-1 and Sgene-2 ​​as primers, Fastpfu enzyme was used to amplify manA gene, and the PCR conditions were 95℃ for 2 min; 94℃ for 20 s, 57℃ for 20 s, 72℃ for 20 s, 35 cycles; 72℃ for 5 min. The amplified PCR product was recorded as A.

[0041] Using pWB-manBl (I91N / L211I) plasmid as template and VamyT-F and Vp43-R as primers, the vector backbone was amplified. The PCR conditions were 95 ℃ for 2 min; 94 ℃ for 20 s, 57 ℃ for 20 s, 72 ℃ for 1 min, 35 cycles; 72 ℃ for 5 min. The amplified PCR product was recorded as V.

[0042] The pWB-manA vector was constructed using the POE-PCR method, that is, the purified A and V were mixed in an equimolar ratio, the V fragment was used in an amount of 100 ng, and the PCR conditions were 95 ℃ for 2 min; 94 ℃ for 20 s, 57 ℃ for 30 s, 72 ℃ for 3 min, 35 cycles; 72 ℃ for 5 min.

[0043] The cloned manA and corresponding vector backbone fragments are shown in Figure 1 As shown, lane 1 is β-mannanase manA Lane 2 is the gene fragment A, and lane 3 is the backbone fragment V of the expression vector. The constructed pWB-manA vector was sequenced, and the results showed that D. thermophilum The β-mannanase gene of 1197 bp, as shown in SEQ ID No.1, is an artificially synthesized sequence, and the encoded peptide chain contains 398 amino acids, which is similar to D. thermophilum The β-mannanase sequences of the strains (AAB82454.1) were 100% identical.

[0044] 3. Construction and expression of the expression vector of the thermophilic Tenchella β-mannanase mutant

[0045] Random mutagenesis of the β-mannanase gene of the thermophilic tennis fungus manA (Q351Y) was obtained. The pUC57-manA plasmid was used as a template, Sgene-1 and Sgene-2 ​​were used as primers, and the easyTaq enzyme was used to amplify the manA mutant gene manA (Q351Y). The concentration of each PCR component was based on Huang Liuyu et al., Principles, Methods and Applications of the Latest PCR Technology, Beijing: Chemical Industry Press, 2004, and the conditions were as follows: 94 ℃ 30 s, 57 ℃ 30 s, 72 ℃ 2 min. The vector fragment was amplified using VamyT-F and Vp43-R as primers, pWB-manA vector as a template, and Fastpfu was used for PCR amplification. The construction of the manA mutant plasmid was also carried out by POE-PCR. The nucleotide sequence and amino acid sequence of the β-mannanase mutant manA (Q351Y) gene are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

[0046] The expression vectors of manA gene and mutant gene constructed by POE-PCR technology were transformed into B. subtilis The obtained transformants were fermented in DB104 competent cells, and the expression of β-mannanase and its mutants was studied by SDS-PAGE electrophoresis.

[0047] Fermentation culture method: stored at -70℃ B. subtilis DB104 was streaked on LB solid medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, 15 g / L agar powder), incubated upside down at 37°C for 16 h, and a single clone was picked and cultured in LB liquid medium. Escherichia coli The culture of ManA strain is basically the same as B. subtilisDB104, but add ampicillin to the LB medium at a final concentration of 100 mg / L.

[0048] B. subtilis Each engineered strain DBManA and DBManA-M1 was streaked in LB solid medium containing 10 mg / L kanamycin at 37°C, and a single clone was picked and cultured overnight in LB liquid medium at 200 rpm for 12 h. The clone was then transferred to fermentation medium (2.0% yeast extract, 2.5% egg white, 0.3% K+) at a ratio of 10%. 2 HPO 4 and 3.0% glucose), 200 rpm, 37°C for 4 d.

[0049] The expression results of each gene are as follows Figure 2 The results showed that in the fermentation broth of DBManA (lane 1) and DBManA-M1 (lane 2), a specific band with a high concentration between 30 and 40 kDa appeared, which was consistent with the theoretical molecular weight of β-mannanase reported in the literature, 37 kDa, preliminarily indicating that β-mannanase and its mutants B. subtilis DB104 successfully achieved expression and secretion. The gene manA and mutant manA(Q351Y) were expressed in Bacillus subtilis for the first time.

[0050] 4. Study on the enzymatic properties of β-mannanase mutants

[0051] (1) Optimal substrate for the enzyme

[0052] The enzyme activity was determined at pH 5.0 and 80°C using 0.05% (w / v) konjac gum, locust bean gum, guar gum, xanthan gum and gum arabic as substrates.

[0053] The enzyme activity determination method (DNS method) is as follows: refined konjac gum is dissolved in pH 5.0 sodium phosphate buffer to prepare a 5 g / L solution as a substrate, 0.03 mL of appropriately diluted crude enzyme solution (supernatant after fermentation culture) is added to 0.27 mL of substrate, incubated in a water bath at 80°C for 10 min, 0.6 mL of DNS reagent is added, mixed and incubated in a boiling water bath for 5 min for color development, immediately placed in ice water to cool, and the absorbance at 540 nm is measured using a SpectraMaxm i3x multi-function microplate reader.

[0054] Definition of enzyme activity: At a certain temperature and pH, the amount of enzyme required to catalyze the hydrolysis of substrate to produce 1 μmol D-mannose per minute is defined as one enzyme activity unit (U).

[0055] Relative enzyme activity: The relative enzyme activity was calculated by taking 0.05% (w / v) konjac gum as substrate, the enzyme activity measured at pH 5.0 and 80 ℃ as 100%.

[0056] Table 4 Relative enzyme activities of ManA and optimal ManA (Q351Y) against different substrates

[0057] .

[0058] The results are shown in Table 4. The optimal substrate for ManA and ManA(Q351Y) is konjac gum. ManA(Q351Y) has a better effect on hydrolyzing locust bean gum and guar gum than ManA.

[0059] (2) Optimal temperature and thermal stability of enzymes

[0060] Under pH 5.0, the enzyme activity was measured at different temperatures of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, and 95 ℃ to determine the optimal reaction temperature of the enzyme. The enzyme activity measured at 80 ℃ and pH 5.0 was taken as 100%, and the relative enzyme activity under different pH conditions was calculated.

[0061] The results showed that β-mannanase ManA and its mutant ManA(Q351Y) had enzyme activity at 30℃~95℃, and the optimum temperature for both was 80℃. The relative enzyme activities of both enzymes exceeded 70% in the range of 60℃~85℃. The residual enzyme activity of β-mannanase mutant ManA(Q351Y) was slightly higher than that of wild-type ManA at different temperatures (see Figure 3 ).

[0062] The crude enzyme solution was placed at 80 °C and the residual enzyme activity after heat incubation was measured at different times, such as 0 h, 1 h, 2 h, 3 h, and 4 h, to determine the thermal stability of the enzyme. The enzyme activity measured at 80 °C and pH 5.0 was taken as 100%, and the relative enzyme activity under different pH conditions was calculated.

[0063] The results of the thermal stability test of the two enzymes at 80°C are shown in Figure 4 The half-life of wild-type ManA at 80℃ is 3 h; the mutant ManA(Q351Y) has a higher thermal stability at 80℃ than the wild-type ManA, with a half-life of about 5 h. After incubation at 80℃ for 24 h, the residual enzyme activity still reaches 22.9%, and the enzyme is completely inactivated for more than 48 h. The thermal stability of ManA(Q351Y) is higher than that of most β-mannanases, and it has important value for oil extraction and other applications in high temperature environments.

[0064] (3) Optimal pH and pH stability of enzymes

[0065] At 80°C, the enzyme activity was measured at pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5 and pH 7.0 to determine the optimal pH of the enzyme. The enzyme activity measured at 80°C and pH 5.0 was taken as 100%, and the relative enzyme activity under different pH conditions was calculated.

[0066] The relative enzyme activities of β-mannanase ManA and its mutant ManA(Q351Y) between pH 4.0 and 8.0 are shown in Figure 5 The optimum pH for both is 5.0. Between pH 4.5 and pH 6.5, the relative enzyme activities are greater than 58%. At pH 4.0 and pH 8.0, the relative enzyme activities of the two enzymes are less than 10%.

[0067] The β-mannanase mutant ManA(Q351Y) was mixed with pH 4.5, pH 5.0, pH 6.0, and pH 6.5 buffer solutions at a volume ratio of 1:1, incubated at 80°C for 60 min, and the residual enzyme activity was determined. The enzyme activity measured at 80°C and pH 5.0 was taken as 100%, and the relative enzyme activity under different pH conditions was calculated to determine the pH stability of the mutant enzyme.

[0068] Figure 6 The results showed that the residual relative activity of the β-mannanase mutants exceeded 84.8% after incubation at 80 ℃ for 60 min under different pH conditions. Among them, the relative enzyme activity after incubation at pH 5.0 for 1 h was increased by 31.2% compared with that without incubation.

[0069] (4) Effects of metal ions on the activity of β-mannanase mutants

[0070] 100 mM Mg 2+ , K + , Ca 2+ , Mn 2+ , Fe 3+ 、Co 2+ , Cu 2+ 、Zn 2+ The metal ions were added to the ManA(Q351Y) enzyme solution to a final concentration of 5 mM. After standing at room temperature for 1 h, the activity of the β-mannanase mutants was determined. The relative enzyme activity of different metal ions was calculated, with the enzyme activity without metal ions added as 100%.

[0071] 5 mM Mg 2+ , K + , Cu 2+ and Zn 2+There was no significant effect on β-mannanase ManA (Q351Y), Ca 2+ and Mn 2+ The activity of ManA(Q351Y) was reduced by 13.5% and 14.7%, respectively, while Fe 3+ and Co 2+ The activity of ManA(Q351Y) was increased by 9.3% and 9.7% respectively (see Figure 7 ), and reached a very significant level.

[0072] (5) Changes in the activity of β-mannanase and its mutants with fermentation time

[0073] Within 72 h of fermentation, the enzyme activity of β-mannanase produced by the engineered strains DBManA and DBManA-M1 and their mutants increased with the fermentation time (see Figure 8 ), at 72 h of fermentation, the enzyme activities of the engineered strains DBManA and DBManA-M1 were 5026.1±263.7 U / mL and 5234.3±87.3 U / mL, respectively, and the enzyme production of DBManA-M1 was 4.1% higher than that of DBManA.

Claims

1. A thermophilic β-mannanase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID No.

3.

2. A gene encoding the thermophilic β-mannanase mutant of claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

2.

3. An expression vector comprising the coding gene according to claim 2.

4. A Bacillus subtilis cell comprising the encoding gene of claim 2.

5. Use of the thermophilic Tenchnerella β-mannanase mutant according to claim 1 in oil production and aquaculture.

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