High-temperature glucanase mutant having improved alkali resistance and use thereof

Abstract

To provide a high-temperature glucanase mutant having improved alkali resistance, and to provide an application thereof.SOLUTION: The high-temperature glucanase mutant having improved alkali resistance has a specific amino acid sequence. The high-temperature glucanase mutant provided by the present disclosure is obtained by point mutation of a wild-type amino acid of glucanase, and the mutant has a relatively high enzyme activity at neutral and alkaline pH, can withstand high-temperature treatment at 50 °C or higher, and has a high application potential in industries such as hemicellulose degradation, feed, bioenergy, and food.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a high-temperature glucanase mutant with improved alkali tolerance and its application. [Background technology]

[0002] β-Glucan is a non-structured starch polysaccharide widely found in the seeds of higher grasses. Its presence can cause problems in the beer industry, where barley and other grains are used as raw materials, including difficulty in filtration of wort and early whitening of beer, and in the grass-based feed industry, where feed conversion rates are reduced and animal digestibility is poor. β-Glucanase (EC.3.2.1.73) is a general term for enzymes that can degrade glucose polymers linked by β-glycosidic bonds. Depending on their mode of action, they can be divided into two types: endo- and exo-. Among them, endo-β-1,3-1,4-glucanase (EC3.2.1.73) acts specifically on β-1,4 glycosidic bonds linked to β-1,3 bonds, decomposing them into low molecular weight fragments, eliminating hydrophilicity and viscosity, reducing the viscosity of the intestinal contents of monogastric animals, improving the activity of endogenous digestive enzymes, improving the intestinal microbial environment, and improving growth performance and feed conversion rate (Mathlouthi N et al. 2002. Amin Res 51, 395-406.), and is widely used in food brewing.

[0003] Currently, β-glucanases used in the food industry are mainly derived from microorganisms, but they generally suffer from poor thermostability or low enzymatic activity. In biomass decomposition processes, β-glucanases must maintain high catalytic activity under alkaline conditions. In beer processing, β-glucanases must maintain temperature stability at 40-70°C. In bread fermentation, β-glucanases must maintain high activity at moderate temperatures of 28-37°C for at least 2 hours. However, currently selected β-glucanases are often concentrated in acidic (pH 3.5-4.5) and moderate temperatures (50°C) and have poor catalytic performance (thermal stability and pH stability). Therefore, β-glucanases need to be designed and improved to meet the requirements of different process characteristics.

[0004] Because different industries have different needs for the properties of glucanases, research into improving glucanases with potential applications remains of great significance. Screening for the production of β-glucanases with excellent performance and improving the enzyme performance by using genetic engineering or protein engineering to modify the enzyme has important theoretical and practical value for the food industry. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a high-temperature glucanase mutant with improved alkali resistance and its applications, which solves the above-mentioned problems in the prior art. The high-temperature glucanase mutant of the present invention is obtained by point mutation of wild-type amino acids in glucanase. The mutant has relatively high enzymatic activity at both neutral and alkaline pH and can withstand high-temperature treatment above 50°C, and has great potential for application in industrial fields such as hemicellulose degradation, feed, bioenergy, and food. [Means for solving the problem]

[0006] In order to achieve the above object, the present invention provides the following solutions.

[0007] The present invention provides a high-temperature glucanase mutant with improved alkaline tolerance, whose amino acid sequence is shown in SEQ ID NO. 12.

[0008] The present invention further provides a gene encoding the above-mentioned high-temperature glucanase mutant, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0009] The present invention further provides a recombinant vector containing the above gene.

[0010] The present invention further provides a recombinant microorganism containing the above-mentioned recombinant vector.

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

[0012] The present invention further provides an application of the gene, the recombinant vector, or the recombinant microorganism in producing a high-temperature glucanase mutant with improved alkali tolerance.

[0013] The present invention further provides application of the above-mentioned high-temperature glucanase mutant or the high-temperature glucanase mutant constructed by the above-mentioned construction method in the degradation of glucan, wherein the degradation temperature is 60 to 80°C and the pH is 6.0 to 9.0. [Effects of the Invention]

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

[0015] The present invention has improved glucanase PcGlu16B (the amino acid sequence of which is shown in SEQ ID NO. 2) using point mutation enzyme molecule improvement technology, mutating N at position 73 to Y, S at position 209 to K, and G at position 228 to Q, thereby obtaining a glucanase mutant strain that can maintain stable enzymatic activity in alkaline pH environments and in the medium-to-high temperature range. Compared with the wild-type, the glucanase mutant of the present invention has a pH action range that is shifted to alkaline environments by 3 pH units and exhibits relatively high enzymatic activity in alkaline environments. The optimum temperature of the glucanase mutant is 10°C higher than that of the wild-type, and the half-life at 50°C (t 1 / 2 ) is two-fold longer than the wild-type, and at the same time, when barley glucan and lichen polysaccharide are used as substrates, the catalytic efficiency of the mutant is improved by 110% and 48%, respectively, compared to the wild-type. The mutation method of the present invention belongs to the technology of enzyme molecule improvement, and shortens the time required to improve enzymatic properties compared to methods such as blind screening of strains or artificial (natural) mutagenesis, providing new technical guidance for the development of high-performance glucanases.

[0016] In order to more clearly describe the embodiments of the present invention or the technical solutions of the prior art, the drawings necessary for describing the embodiments are briefly described below. Of course, the drawings described below are only examples of the embodiments of the present invention, and those skilled in the art can further obtain other drawings based on these drawings without any creative efforts. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the optimum pH values ​​of the high-temperature glucanase mutant N73Y / S209K / G228Q, which has improved alkaline tolerance, and the wild-type PcGlu16B. [Figure 2] FIG. 2 shows the pH stability of the high-temperature glucanase mutant N73Y / S209K / G228Q, which has improved alkaline tolerance, and the wild-type PcGlu16B. [Figure 3]FIG. 3 shows the optimum temperatures of the high-temperature glucanase mutant N73Y / S209K / G228Q, which has improved alkaline tolerance, and the wild-type PcGlu16B. [Figure 4] FIG. 4 shows the results of measuring the thermostability at 50° C. of the high-temperature glucanase mutant N73Y / S209K / G228Q, which has improved alkali resistance, and the wild-type PcGlu16B. DETAILED DESCRIPTION OF THE INVENTION

[0018] Several exemplary embodiments of the present invention are described in detail below, and the detailed descriptions should not be construed as limiting the present invention, but should be understood as further illustrating certain aspects, features and embodiments of the present invention.

[0019] It should be understood that the terms used in the present invention are merely for describing particular embodiments and are not intended to limit the present invention. Numerical ranges in the present invention should also be understood to more specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within that range is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range.

[0020] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The present invention merely describes preferred methods and materials, but any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents mentioned herein are incorporated by reference for the purpose of disclosing and describing the methods and / or materials related to said documents. In the event of a conflict with any incorporated document, the contents of this specification shall control.

[0021] It will be apparent to those skilled in the art that various modifications and variations can be made to the specific embodiments of the specification of the invention without departing from the scope or spirit of the invention. Other embodiments of the invention taken from the specification of the invention will be apparent to those skilled in the art. The specification and examples of the invention are illustrative only.

[0022] As used herein, the terms "including," "including," "having," "containing," and the like are all open-ended terms, i.e., meaning "including, but not limited to."

[0023] The origin of materials that may be used in the examples of the present invention is as follows: First, strains and vectors The expression host Pichia pastoris GS115 and the expression plasmid vector pPIC9r were purchased from Invitrogen. Second, enzymes and other biochemical reagents The endoenzyme was purchased from Fermentas, the ligase was purchased from Promaga, the barley glucan was purchased from Sigma, and the rest were analytical pure reagents made in China (they were purchased from Sinopharm). Third, the medium LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0; YPD medium is 1% yeast extract, 2% peptone, and 2% glucose. MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% biotin; MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% biotin, 0.5% methanol; BMGY medium contains 1% yeast extract, 2% peptone, 1% glycerin (V / V), 1.34% YNB, and 0.00004% biotin. For BMMY medium, 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, and 0.5% methanol (V / V).

[0024] Example 1: Cloning of genes encoding high-temperature glucanase mutants with improved alkaline tolerance The GH16 family PcGlu16B gene (nucleotide sequence shown in SEQ ID NO. 1, amino acid sequence shown in SEQ ID NO. 2) was used as a base for mutation to obtain a gene (SEQ ID NO. 3) encoding a high-temperature glucanase variant with improved alkaline tolerance. The mutation and cloning methods were described in 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 used is shown in Table 1.

[0025] [Table 1]

[0026] SEQ ID NO. 1 is SEQ ID NO. 2 is QYTLEDDYLANGFFDQFSFFTSGDPTHGFVQYVDQGTANSNGLISSSGSQAVMRVDSTNTAPNGRTSVRLTSNKAYDSGLVIADIAHMPGGICGVWPAFWMVGPNWPSNGEIDIIEGVNDQSTNDMTLHTSDGCSIGSGGMSGYVVTSNCYINAPGQSSNQGCQIGTGDTSTYGSGFNANGGGVYATEFTSSGVKIFFFPRGSIPGDISSGSPNPSSWGQPVAYFQGGGCDFGSHIKQQQIVFDTTFCGDWAGAVWGNGGCASRAGSCNDFVANNPSAFSDAYWAVNGLKVYQNYGSTSFDLESPPSNSSASSSSASVLKESKKERFRRHLAEHRNSGAELF, SEQ ID NO. 3 is

[0027] Example 2 Preparation of a high-temperature glucanase mutant with improved alkaline tolerance The expression vector pPIC9r was double-digested (EcoR I + Not I), and the gene encoding the high-temperature glucanase mutant with improved alkali tolerance was double-digested (EcoR I + Not I). The cleaved mature gene fragment (with the signal peptide fragment removed) encoding the high-temperature glucanase mutant with improved alkali tolerance was ligated into the cleaved expression vector pPIC9r to obtain a recombinant vector containing the high-temperature glucanase mutant gene with improved alkali tolerance, which was then transformed into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / N73Y / S209K / G228Q.

[0028] The recombinant plasmid-containing strain GS115 / N73Y / S209K / G228Q was inoculated into 200 mL of BMGY medium in a 1 L Erlenmeyer flask and cultured on a rocking platform at 30°C and 220 rpm for 48 h. The 200 mL culture was then centrifuged for 5 min, the supernatant discarded, and the precipitate resuspended in 200 mL of BMMY medium containing 0.5% methanol. The culture was then cultured again at 30°C and 220 rpm for induction. Every 12 h, 0.5 mL of methanol was added to maintain a 0.5% methanol concentration in the culture, and the supernatant was collected for enzyme activity detection. Finally, the supernatant was concentrated to 30 mL and desalted in dibasic sodium phosphate-citric acid buffer (pH 6.0, 10 mM) for enzymatic property measurement and comparison. The protein was purified by anion exchange after desalting in 10 mM disodium hydrogen phosphate-citric acid buffer. After purification, the expressed glucanase N73Y / S209K / G228Q had a protein content of more than 98% of the total protein, and the amino acid sequence of this glucanase is shown in SEQ ID NO. 12.

[0029] SEQ ID NO. 12 is QYTLEDDYLANGFFDQFSFFTSGDPTHGFVQYVDQGTANSNGLISSSGSQAVMRVDSTNTAPNGRTSVRLTSYKAYDSGLVIADIAHMPGGICGVWPAFWMVGPNWPSNGEIDIIEGVNDQSTNDMTLHTSDGCSIGSGGMSGYVVTSNCYINAPGQSSNQGCQIGTGDTSTYGSGFNANGGGVYATEFTSSGVKIFFFPRGSIPGDIKSGSPNPSSWGQPVAYFQGQGCDFGSHIKQQQIVFDTTFCGDWAGAVWGNGGCASRAGSCNDFVANNPSAFSDAYWAVNGLKVYQNYGSTSFDLESPPSNSSASSSSASVLKESKKERFRRHLAEHRNSGAELF.

[0030] Example 3 Activity analysis of high-temperature glucanase mutants with improved alkaline tolerance and wild-type glucanase 1. Measurement of enzyme activity by DNS method The specific method is as follows: 50 μL of diluted enzyme solution (2 μg / mL) was mixed with 450 μL of substrate (5 mg / mL) at pH 4.0 and 50°C for 10 minutes, and the reaction was terminated by adding 0.75 mL of DNS. The mixture was then boiled for 5 minutes. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) was defined as the amount of enzyme required to decompose glucan to produce 1 μmol of reducing sugar per minute under the given conditions.

[0031] 2. Performance measurement of high-temperature glucanase mutants with improved alkali tolerance and wild-type glucanase First, the method for determining the optimum pH is as follows. The purified high-temperature glucanase mutant N73Y / S209K / G228Q with improved alkali tolerance and the wild-type glucanase PcGlu16B were subjected to enzymatic reactions at different pH levels to determine their optimum pH. Barley glucan, the substrate, was dissolved in 0.1 mol / L citric acid-disodium hydrogen phosphate buffers at different pH levels, and glucanase activity was measured at 50°C. The results are shown in Figure 1, revealing that the high-temperature glucanase mutant with improved alkali tolerance had an optimum pH of 7.0, a three-unit shift toward an alkaline environment compared to the optimal reaction pH of the wild-type.

[0032] Second, the method for measuring pH stability is as follows. The enzyme solutions of the high-temperature glucanase mutant N73Y / S209K / G228Q with improved alkali tolerance and the wild-type glucanase PcGlu16B were incubated in buffers of different pHs (1-12) at 37°C for 1 hour, and the residual enzyme activity was then measured to demonstrate the pH stability of the enzymes. The results are shown in Figure 2, which demonstrate that the high-temperature glucanase mutant with improved alkali tolerance still maintained over 80% of its relative enzyme activity within the pH range of 6.0 to 9.0, demonstrating significantly greater stability than the wild-type.

[0033] Third, the method for measuring the optimum temperature is as follows. The high-temperature glucanase mutant N73Y / S209K / G228Q, which has improved alkaline tolerance, and the wild-type glucanase PcGlu16B were subjected to enzymatic reactions in a 0.1 mol / L citrate-disodium hydrogen phosphate buffer solution (pH 4.0) at different temperatures. The results, shown in Figure 3, revealed that the optimum temperature of the mutant (60°C) was 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%) was significantly higher than that of the wild-type enzyme (0.7%-21%).

[0034] Fourth, the thermal stability at 50°C was measured as follows. The detection method was based on 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.). The results are shown in Figure 4. The high-temperature glucanase mutant N73Y / S209K / G228Q, which has improved alkali resistance, has significantly better thermal stability than the wild-type. Its half-life at 50°C (238 min) was 2.0 times longer than that of the wild-type (80 min). After treatment at 50°C for 5 hours, the remaining enzyme activity of the mutant was 20 times that of the wild-type.

[0035] Fifth, the method for measuring kinetic parameters is as follows. The detection method was based on 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 primary reaction time was measured to determine the K m and V max The reaction time was determined as 5 min. Then, the enzyme activity was measured under standard conditions (50 °C, pH 4.0) using different concentrations of glucan (1.25, 1.0, 0.8, 0.4, 0.2, 0.15, and 0.1%) as substrates, and the corresponding reaction rates were calculated. The K was calculated using GraFit7 software. m and V max was calculated.

[0036] When barley glucan was used as a substrate, the K m The values ​​were 3.4 and 1.6 mg / mL, respectively, and the catalytic efficiency (k cat / K m ) were 3100 and 6500 mL / s mg, respectively, and when lichen polysaccharide was used as a substrate, the K mThe values ​​were 2.8 and 1.8 mg / mL, respectively, and the catalytic efficiency (k cat / K m ) were 2300 and 3400 mL / s·mg, respectively (see Table 2).

[0037] [Table 2] NOTE: Kinetic values ​​are shown as the mean standard deviation (n = 3).

[0038] The above results revealed that the high-temperature glucanase mutant N73Y / S209K / G228Q, which has improved alkali resistance, can withstand high-temperature treatment above 50°C, has extremely high enzymatic activity at both alkaline and neutral pH, and has great potential for application in industrial fields such as feed, bioenergy, and food.

[0039] The above-described embodiments are merely illustrative of the preferred means of the present invention and do not limit the scope of the present invention. Any modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit and scope of the design of the present invention should be included in the scope of protection 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 is shown in SEQ ID NO.

12.

2. 2. The gene encoding the high-temperature glucanase mutant according to claim 1, characterized in that the nucleotide sequence is shown in SEQ ID NO.

3.

3. A recombinant vector comprising the gene according to claim 2.

4. A recombinant microorganism, characterized by containing the recombinant vector according to claim 3.

5. 2. The method for constructing a high-temperature glucanase mutant according to claim 1, characterized in that it is based on the wild-type glucanase shown in SEQ ID NO. 2 and comprises the steps of site-directed mutagenesis of the amino acids at positions 73, 209 and 228.

6. 10. The use of the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant microorganism according to claim 4 in the production of a high-temperature glucanase with improved alkali tolerance.

7. The application of the high-temperature glucanase mutant described in claim 1 or the high-temperature glucanase mutant constructed by the construction method described in claim 5 in the degradation of glucan, characterized in that the temperature during degradation is 60 to 80°C and the pH is 6.0 to 9.0.

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