A mutant of thermophilic dextranase with improved alkali resistance and its application

Abstract

The present invention discloses an alkali-resistant improved thermophilic glucanase mutant and its application, belonging to the technical field of enzyme engineering. The amino acid sequence of the alkali-resistant improved thermophilic glucanase mutant of the present invention is shown in SEQ ID NO.12. The thermophilic glucanase mutant of the present invention is obtained by point mutation of the amino acids of the wild-type glucanase. This mutant has high enzyme activity at neutral and alkaline pH values, can withstand high-temperature treatment above 50 °C, and has good application potential in industrial fields such as hemicellulose degradation, feed, bioenergy, and food.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and particularly relates to a mutant of thermophilic glucanase with improved alkali resistance and its application. Background Art

[0002] β-glucan is a non-structural starch polysaccharide widely present in the seeds of higher gramineous plants. Its existence brings difficulties in malt juice filtration and early haze turbidity in the beer industry using barley as raw material; it also brings problems such as reduced feed conversion rate and indigestion in animals in the feed industry based on gramineous plants. β-glucanase (EC.3.2.1.73) is a general term for a class of enzymes that can decompose glucose polymers formed by β-glycosidic bond chains. According to different action modes, it can be divided into two categories: endo-type and exo-type. Among them, endo-β-1,3-1,4-glucanase (E.C.3.2.1.73) can specifically act on the β-1,4 glycosidic bond connected to the β-1,3 bond, degrade it into low molecular weight fragments, lose hydrophilicity and viscosity, reduce the viscosity of the intestinal contents of monogastric animals, improve the activity of endogenous digestive enzymes, improve the intestinal microbial environment, and improve growth performance and feed conversion rate (Mathlouthi N et al. 2002. Amin Res 51, 395 - 406.), and it is widely used in food brewing.

[0003] Currently, the β-glucanase applied in the food industry mainly comes from microorganisms, but there are generally problems of low thermal stability or low enzyme activity. In the process of degrading biomass, β-glucanase needs to maintain high catalytic activity under alkaline conditions; in the beer process, β-glucanase needs to maintain temperature stability at 40 - 70°C; in bread fermentation, β-glucanase needs to maintain high activity at 28 - 37°C for more than 2 hours. However, the currently screened β-glucanases mostly focus on acidic (pH 3.5 - 4.5), medium temperature (50°C) and poor catalytic performance (thermal stability and pH stability). Therefore, it is necessary to design and improve β-glucanase to meet the requirements of different process properties.

[0004] Since different industries have different requirements for the properties of glucanase, the research on improving glucanase with application potential is still of great significance. Screening β-glucanase with excellent performance and using genetic engineering or protein engineering to modify the enzyme to improve its performance has important theoretical value and application value for the food industry. Summary of the Invention

[0005] The object of the present invention is to provide a thermophilic glucanase mutant with improved alkali resistance and its application to solve the problems existing in the above-mentioned prior art. The thermophilic glucanase mutant of the present invention is obtained by point mutation of the amino acids of the wild-type glucanase. This mutant has high enzyme activity at neutral and alkaline pH values, can withstand high-temperature treatment above 50 °C, and has good application potential in industrial fields such as hemicellulose degradation, feed, bioenergy, and food.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a thermophilic glucanase mutant with improved alkali resistance, and the amino acid sequence of the thermophilic glucanase mutant is as shown in SEQ ID NO.12.

[0008] The present invention also provides a gene encoding the above thermophilic glucanase mutant, and the nucleotide sequence of the gene is as shown in SEQ ID NO.3.

[0009] The present invention also provides a recombinant vector, and the above gene is contained on the recombinant vector.

[0010] The present invention also provides a recombinant microorganism, and the above recombinant vector is contained in the recombinant microorganism.

[0011] The present invention also provides a method for constructing the above thermophilic glucanase mutant, which includes the step of performing site-directed mutagenesis on the amino acids at positions 73, 209, and 228 based on the wild-type glucanase shown in SEQ ID NO.2.

[0012] The present invention also provides the application of the above gene, or the above recombinant vector, or the above recombinant microorganism in the production of a thermophilic glucanase mutant with improved alkali resistance.

[0013] The present invention also provides the application of the above thermophilic glucanase mutant or the thermophilic glucanase mutant constructed by the above construction method in the degradation of glucan, and the temperature during the degradation is 60-80 °C, and the pH is 6.0-9.0.

[0014] The present invention discloses the following technical effects:

[0015] In the present invention, the dextranase PcGlu16B (the amino acid sequence is shown in SEQ ID NO.2) was modified by adopting the enzyme molecule improvement technology of point mutation. The N at the 73rd position was mutated to Y, the S at the 209th position was mutated to K, and the G at the 228th position was mutated to Q, obtaining a dextranase mutant capable of maintaining stable enzyme activity in an alkaline pH environment and within the medium and high temperature range. Compared with the wild type, the pH action range of the dextranase mutant of the present invention shifts 3 pH units towards the alkaline environment, and shows higher enzyme activity in the alkaline environment; the optimal temperature of the dextranase mutant is increased by 10 °C compared with the wild type, and the half-life (t 1 / 2 ) is extended by 2 times compared with the wild type at 50 °C; meanwhile, when barley dextran and lichenan are used as substrates, the catalytic efficiency of the mutant is increased by 110% and 48% respectively compared with the wild type. The mutation method of the present invention belongs to the enzyme molecule improvement technology. Compared with means such as blindly screening bacteria or artificial (natural) mutagenesis, it shortens the time for modifying the enzymatic properties and provides new technical guidance for the development of high-performance dextranase. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 Determination results of the optimal pH of the high-temperature dextranase mutant N73Y / S209K / G228Q with improved alkali resistance and the wild type PcGlu16B;

[0018] Figure 2 Determination results of the pH stability of the high-temperature dextranase mutant N73Y / S209K / G228Q with improved alkali resistance and the wild type PcGlu16B;

[0019] Figure 3 Determination results of the optimal temperature of the high-temperature dextranase mutant N73Y / S209K / G228Q with improved alkali resistance and the wild type PcGlu16B;

[0020] Figure 4 Determination results of the thermal stability of the high-temperature dextranase mutant N73Y / S209K / G228Q with improved alkali resistance and the wild type PcGlu16B at 50 °C. DETAILED DESCRIPTION OF THE INVENTION

[0021] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0025] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0026] The sources of materials that may be used in the embodiments of the present invention are as follows:

[0027] 1. Strains and vectors: The expression host Pichia pastoris GS115 and the expression plasmid vector pPIC9r were purchased from Invitrogen Corporation;

[0028] 2. Enzymes and other biochemical reagents: Restriction enzymes were purchased from Fermentas, ligase was purchased from Promega, and barley glucan was purchased from Sigma; the others are domestic analytical pure reagents (all purchased from Sinopharm Group);

[0029] 3. Media:

[0030] LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

[0031] YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

[0032] MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;

[0033] MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol;

[0034] BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V / V), 1.34% YNB, 0.00004% Biotin;

[0035] BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V / V).

[0036] Example 1: Cloning of the Encoding Gene of a Thermotolerant Glucanase Mutant with Improved Alkaline Resistance

[0037] Using the GH16 family PcGlu16B gene (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2) as the parent, mutations were carried out to obtain the encoding gene of the thermotolerant glucanase mutant with improved alkaline resistance (SEQ ID NO.3). The mutation method and cloning method refer to the literature (Improvement of XYL10C_ΔN catalytic performance through loop engineering for lignocellulosic biomass utilization in feed and fuel industries. Biotechnol Biofuels, 2021, 14:195.). The primer information is shown in Table 1:

[0038] Table 1 Primer Information

[0039] Primer Name Sequence (5'→3') Size (bp) EcoRi-f gtagaattccaatatacccttcagcagg(SEQ ID NO.4) 28 NotI-R attcgcggccgctcaagctgctgaagcagc(SEQ ID NO.5) 30 N73Y-F gttagattgacttcttacaaggcttacgattctggtttg(SEQ ID NO.6) 39 N73Y-R gtaagaagtcaatctaacagaagttctaccgtttggagc(SEQ ID NO.7) 39 S209K-F cctggagatattaagtctggttctcctaatccatc(SEQ ID NO.8) 35 S209K-R cttaatatctccaggaatagaacctcttggaaagaaaaa(SEQ ID NO.9) 39 G228Q-F tgcttactttcaaggtcaaggttgtgattttggttctca(SEQ ID NO.10) 39 G228Q-R ttgaccttgaaagtaagcaactggctgacccc(SEQ ID NO.11) 32

[0040] SEQ ID NO.1:

[0041] CAATACACCTTGGAAGATGACTACTTGGCTAATGGTTTCTTTGATCAATTTTCTTTCTTC

[0042] ACCTCTGGTGACCCAACTCATGGTTTCGTTCAATACGTTGATCAAGGTACCGCTAATTCT

[0043] AACGGTTTGATTTCTTCTTCCGGTTCTCAAGCTGTTATGAGAGTTGATTCTACTAACACTG

[0044] CTCCAAACGGTAGAACTTCTGTTAGATTGACTTCTAATAAGGCTTACGATTCTGGTTTGG

[0045] TTATTGCTGATATTGCTCATATGCCTGGTGGTATTTGTGGTGTTTGGCCAGCTTTTTGGATG

[0046] GTTGGTCCTAATTGGCCATCCAACGGAGAAATCGATATTATTGAAGGTGTTAATGACCAG

[0047] TCTACTAACGATATGACTTTGCATACTTCTGATGGTTGTTCTATTGGTTCTGGTGGTATGTC

[0048] TGGTTACGTTGTTACTTCTAATTGTTACATTAACGCTCCAGGTCAATCATCTAACCAAGGT

[0049] TGTCAAATTGGAACCGGTGATACTTCTACTTACGGTTCTGGTTTTAATGCTAACGGTGGT

[0050] GGTGTTTACGCTACTGAATTTACTTCTTCTGGTGTTAAGATCTTTTTCTTTCCAAGAGGTT

[0051] CTATTCCTGGAGATATTTCTTCTGGTTCTCCTAATCCATCTTCTTGGGGTCAGCCAGTTGC

[0052] TTACTTTCAAGGTGGTGGTTGTGATTTTGGTTCTCATATTAAGCAGCAACAAATTGTTTTC

[0053] GATACTACTTTTTGTGGTGATTGGGCTGGTGCTGTTTGGGGTAATGGTGGTTGTGCTTCT

[0054] AGAGCTGGTTCTTGTAACGATTTTGTTGCTAATAACCCTTCTGCTTTTTCTGATGCTTATT

[0055] GGGCTGTTAATGGTTTGAAGGTTTATCAAAATTACGGTTCTACTTCTTTCGATTTGGAATC

[0056] TCCACCATCTAACTCTTCTGCTTCTTCTTCTTCTGCTTCCGTTTTGAAGGAATCCAAGAAAGAAAGATTTAGAAGACATTTGGCTGAACATAGAAACTCTGGTGCTGAATTGTTT;

[0057] SEQ ID NO.2:

[0058] QYTLEDDYLANGFFDQFSFFTSGDPTHGFVQYVDQGTANSNGLISSSGSQAVMRVDSTNTAPNGRTSVRLTSNKAYDSGLVIADIAHMPGGICGVWPAFWMVGPNWPSNGEIDIIEGVNDQSTNDMTLHTSDGCSIGSGGMSGYVVTSNCYINAPGQSSNQGCQIGTGDTSTYGSGFNANGGGVYATEFTSSGVKIFFFPRGSIPGDISSGSPNPSSWGQPVAYFQGGGCDFGSHIKQQQIVFDTTFCGDWAGAVWGNGGCASRAGSCNDFVANNPSAFSDAYWAVNGLKVYQNYGSTSFDLESPPSNSSASSSSASVLKESKKERFRRHLAEHRNSGAELF;

[0059] SEQ ID NO.3:

[0060]

[0061] Preparation of a Thermotolerant Glucanase Mutant with Improved Alkaline Resistance, Example 2

[0062] The expression vector pPIC9r was double digested (EcoR I + Not I), and at the same time, the gene encoding the thermotolerant glucanase mutant with improved alkaline resistance was double digested (EcoR I + Not I). The cut gene fragment encoding the mature thermotolerant glucanase mutant with improved alkaline resistance (removing the signal peptide fragment) was ligated to the cut expression vector pPIC9r to obtain a recombinant vector containing the gene of the thermotolerant glucanase mutant with improved alkaline resistance, and then transformed into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / N73Y / S209K / G228Q.

[0063] The GS115 / N73Y / S209K / G228Q strain containing the recombinant plasmid was inoculated into a 1 L Erlenmeyer flask with 200 mL of BMGY medium and cultured at 30 °C and 220 rpm on a shaker for 48 h; then 200 mL of the culture solution was centrifuged for 5 min, the supernatant was discarded, and the precipitate was resuspended with 200 mL of BMMY medium containing 0.5% methanol and then induced to culture again at 30 °C and 220 rpm. 0.5 mL of methanol was added every 12 h to keep the methanol concentration in the bacterial solution at 0.5%, and at the same time, the supernatant was taken for enzyme activity detection. Finally, the supernatant was concentrated to 30 mL, desalted with sodium hydrogen phosphate-citric acid buffer (pH 6.0, 10 mM), and then purified by anion exchange method to obtain the protein for enzymatic property determination and comparison. After purification, the content of the expressed glucanase N73Y / S209K / G228Q reached more than 98% of the total protein. The amino acid sequence of this glucanase is shown in SEQ ID NO.12.

[0064] SEQ ID NO.12:

[0065] QYTLEDDYLANGFFDQFSFFTSGDPTHGFVQYVDQGTANSNGLISSSGSQAVMRVDSTNTAPNGRTSVRLTSYKAYDSGLVIADIAHMPGGICGVWPAFWMVGPNWPSNGEIDIIEGVNDQSTNDMTLHTSDGCSIGSGGMSGYVVTSNCYINAPGQSSNQGCQIGTGDTSTYGSGFNANGGGVYATEFTSSGVKIFFFPRGSIPGDIKSGSPNPSSWGQPVAYFQGQGCDFGSHIKQQQIVFDTTFCGDWAGAVWGNGGCASRAGSCNDFVANNPSAFSDAYWAVNGLKVYQNYGSTSFDLESPPSNSSASSSSASVLKESKKERFRRHLAEHRNSGAELF。

[0066] Example 3 Activity Analysis of the Alkaline-Resistant High-Temperature Glucanase Mutant and the Wild-Type Glucanase

[0067] I. Determination of Enzyme Activity by DNS Method

[0068] The specific method is as follows: At pH 4.0 and 50 °C, 50 μL of the diluted enzyme solution (2 μg / mL) was mixed with 450 μL of the substrate (5 mg / mL), reacted for 10 min, and 0.75 mL of DNS was added to terminate the reaction, and then boiled in water for 5 min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose glucan to generate 1 μmol of reducing sugar per minute under the given conditions.

[0069] II. Performance Determination of the Alkaline-Resistant High-Temperature Glucanase Mutant and the Wild-Type Glucanase

[0070] 1. The method for determining the optimal pH is as follows:

[0071] The alkaline-resistant high-temperature glucanase mutant N73Y / S209K / G228Q purified in Example 2 and the wild-type glucanase PcGlu16B were subjected to enzymatic reactions at different pH values to determine their optimal pH. The substrate barley glucan was dissolved in 0.1 mol / L citric acid-disodium hydrogen phosphate buffer with different pH values, and the glucanase activity was measured at 50 °C. The results are as Figure 1 , indicating that the optimal reaction pH of the alkaline-resistant high-temperature glucanase mutant shifted 3 units towards the alkaline environment compared with the wild-type, and the optimal pH was 7.0.

[0072] 2. The method for determining pH stability is as follows:

[0073] The mutant N73Y / S209K / G228Q of thermophilic glucanase with improved alkali resistance and the enzyme solution of wild-type PcGlu16B glucanase were incubated in buffer solutions with different pH values (1 - 12) at 37 °C for 1 hour, and then the remaining enzyme activity was measured to show the pH stability of the enzyme. The results are as Figure 2 , indicating that the mutant of thermophilic glucanase with improved alkali resistance still maintains a relative enzyme activity of more than 80% in the range of pH 6.0 - pH 9.0, and its stability is significantly better than that of the wild-type.

[0074] 3. The method for determining the optimum temperature is as follows:

[0075] The mutant N73Y / S209K / G228Q of thermophilic glucanase with improved alkali resistance and the wild-type PcGlu16B glucanase were subjected to enzymatic reactions in a 0.1 mol / L citric acid - disodium hydrogen phosphate buffer solution (pH 4.0) system at different temperatures. The results are as Figure 3 , indicating that the optimum temperature of the mutant (60 °C) is 10 °C higher than that of the wild-type (50 °C), and the relative enzyme activity of the mutant at high temperatures (65 - 80 °C) (45% - 96%) is significantly higher than that of the wild enzyme (0.7% - 21%).

[0076] 4. The thermal stability at 50 °C was determined as follows:

[0077] The detection method refers to the literature (Functional analysis of a highly active β - glucanase from Bispora sp. MEY - 1 using its C - terminally truncated mutant. J Agric Food Chem. 2018, 66(37):9728 - 9737.), and the results are as Figure 4 , indicating that the thermal stability of the mutant N73Y / S209K / G228Q of thermophilic glucanase with improved alkali resistance is significantly better than that of the wild-type. The half-life at 50 °C (238 min) is 2.0 times longer than that of the wild-type (80 min), and after treatment at 50 °C for 5 hours, the remaining enzyme activity of the mutant is 20 times that of the wild-type.

[0078] 4. The method for determining kinetic parameters is as follows:

[0079] The detection method referred to the literature (Functional analysis of a highly active β-glucanase from Bispora sp. MEY-1 using its C-terminally truncated mutant. J Agric Food Chem. 2018, 66(37):9728-9737.), measured the first-order reaction time of the reaction, and determined K m and V max The reaction time was 5 min. Then, different concentrations of dextran (1.25, 1.0, 0.8, 0.4, 0.2, 0.15, and 0.1%) were used as substrates, and the enzyme activity was measured under standard conditions (50 °C, pH 4.0), the corresponding reaction rates were calculated, and K m and V max .

[0080] When barley dextran was used as the substrate, the K m values of the wild type and the mutant were 3.4 and 1.6 mg / mL, respectively; the catalytic efficiencies (k cat / K m ) were 3100 and 6500 mL / s·mg, respectively; when lichenan was used as the substrate, the K m values of the wild type and the mutant were 2.8 and 1.8 mg / mL, respectively; the catalytic efficiencies (k cat / K m ) were 2300 and 3400 mL / s·mg, respectively (see Table 2).

[0081] Table 2 Specific activities and kinetic parameters of the mutant of the high-temperature glucanase with improved alkali resistance and the wild-type glucanase

[0082]

[0083]

[0084] Note: The kinetic values are shown as mean standard deviations (n = 3).

[0085] The above results indicate that the mutant N73Y / S209K / G228Q of the high-temperature glucanase with improved alkali resistance can withstand high-temperature treatment above 50 °C, has very high enzyme activity under alkaline and neutral pH, and has good application potential in industrial fields such as feed, bioenergy, and food.

[0086] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A high-temperature glucanase mutant with improved alkali resistance, characterized in that: The amino acid sequence of the thermophilic glucanase mutant is shown in SEQ ID NO.

12.

2. A gene encoding the thermoglucanase mutant according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 2.

4. A recombinant microorganism, characterized in that The recombinant microorganism contains the recombinant vector according to claim 3.

5. A method for constructing the thermoglucanase mutant according to claim 1, characterized in that: The method comprises the steps of simultaneously performing site-directed mutagenesis on the amino acids at positions 73, 209 and 228 based on the wild type of glucanase as shown in SEQ ID NO.2; The method of site-directed mutation of the amino acid at position 73 is to mutate from N to Y; The method of site-directed mutation of the amino acid at position 209 is to mutate from S to K; The site-directed mutation of the amino acid at position 228 is performed by mutating G to Q.

6. Use of the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant microorganism according to claim 4 in producing a high-temperature glucanase with improved alkali resistance.

7. Use of the thermoglucanase mutant according to claim 1 or the thermoglucanase mutant constructed by the construction method according to claim 5 in degrading glucan, characterized in that: The temperature during the degradation is 60-80° C. and the pH is 6.0-9.0; The high temperature glucanase mutant is β-glucanase.

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