Method for improving activity of arabinofuranosidase based on mutation modification and application
By modifying the amino acid residues of arabinofuranylase to improve its heat resistance, the problem of enzyme inactivation under high temperature conditions was solved, enabling its efficient application in the field of feed additives.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BLUESTAR ADISSEO NANJING CO LTD
- Filing Date
- 2025-12-29
- Publication Date
- 2026-05-01
AI Technical Summary
Arabinofuranosides, whether naturally derived or conventionally produced, are easily inactivated under high temperatures, leading to a decrease in enzyme activity and limiting their effectiveness and economic viability in industries such as feed and food.
By modifying wild-type arabinofuranosylase with amino acid residue mutations, mutant enzymes with high-temperature stability were prepared, including mutations such as N301E, S302P, G303L, G304T, S305C and Y306* or N301C, S302R, G303L, G304C, S305T, Y306H and N307C, which improved the enzyme's heat resistance.
The mutant enzyme maintains high residual enzyme activity at 70℃ and 80℃, achieving stable catalytic ability at high temperatures, which significantly enhances its application potential in the field of feed additives.
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Figure CN121950877A_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of biotechnology, specifically to a method for improving the activity of arabinofuranylase, a protein having arabinofuranylase activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant containing the gene, a method for preparing arabinofuranylase, and the application of arabinofuranylase in the field of feed additives. Background Technology
[0002] Arabinofuranosidase is a key enzyme preparation capable of specifically hydrolyzing the arabinofuranoside bonds in hemicellulose such as xylan and arabinogalactan. It has broad application prospects in various industrial fields, including feed, food, papermaking, and bioenergy. For example, in the feed industry, the addition of arabinofuranosidase can efficiently degrade arabinoxylan in cereal raw materials, disrupting plant cell wall structure, releasing intracellular nutrients, thereby eliminating anti-nutritional factors and improving feed digestibility and absorption. It often works synergistically with xylanase and other enzymes to enhance plant cell wall degradation efficiency, making it valuable in feed and food industrial processes.
[0003] However, arabinofuranosylases from natural sources or conventionally produced arabinofuranosylases face severe challenges in practical industrial applications, especially in processes such as feed pelleting, food cooking, and high-temperature saccharification of biomass. These processes typically require operation at high temperatures (usually above 70°C).
[0004] The optimal operating temperature for most natural arabinofuranosylases is between 40-60°C. When exposed to temperatures above 70°C, the enzyme protein molecules rapidly undergo irreversible denaturation and inactivation, leading to a sharp decline in enzyme activity. This means that enzyme preparations cannot function effectively at critical process temperatures, greatly limiting their application efficacy and economic viability.
[0005] Therefore, there is an urgent need to provide a method for optimizing arabinofuranosidase to improve its thermostability. Summary of the Invention
[0006] The purpose of this disclosure is to provide a method for improving the activity of arabinofuranosaccharide, a protein having arabinofuranosaccharide activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant into which the gene is transformed, a method for preparing arabinofuranosaccharide, and the application of arabinofuranosaccharide in the field of feed additives.
[0007] To achieve the above objectives, a first aspect of this disclosure provides a method for improving the activity of arabinofuranyl glycosidase, the method comprising mutating one or more of the amino acid residues of wild-type arabinofuranyl glycosidase to be mutated, said mutation comprising (1) or (2): (1) N301E, S302P, G303L, G304T, S305C and Y306*; (2) N301C, S302R, G303L, G304C, S305T, Y306H and N307C; The amino acid sequence of the wild-type arabinofuranosidase is shown in SEQ ID NO: 1.
[0008] A second aspect of this disclosure provides a protein having arabinofuranyl glycosidase activity, said protein being a protein derived from the amino acid sequence of wild-type arabinofuranyl glycosidase as shown in SEQ ID NO: 1 by substitution of (1) or (2): (1) N301E, S302P, G303L, G304T, S305C and Y306*; (2) N301C, S302R, G303L, G304C, S305T, Y306H and N307C.
[0009] Optionally, the amino acid sequence of the protein is as shown in SEQ ID NO: 2 or 3.
[0010] This disclosure provides a third aspect of a gene encoding the protein shown in the second aspect, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO: 4 or 5.
[0011] The fourth aspect of this disclosure provides a recombinant vector, which is a recombinant expression vector, wherein the recombinant vector is inserted with the gene described in the third aspect.
[0012] The fifth aspect of this disclosure provides a transformant in which the introduced gene includes the gene described in the third aspect, or the transformant contains the recombinant vector described in the fourth aspect.
[0013] The sixth aspect of this disclosure provides a method for preparing arabinofuranyl glycosidase, wherein the transformant described in the fifth aspect is inoculated into a culture medium and cultured to obtain the cultured material.
[0014] The seventh aspect of this disclosure provides the application of arabinofuranylase in the field of feed additives, said arabinofuranylase containing the protein shown in the second aspect.
[0015] Optionally, the application includes arabinofuranylase hydrolyzing arabinofuranyl bonds on the side chains of non-starch polysaccharides in feed; The non-starch polysaccharides include arabinoxylan and / or arabinogalactan.
[0016] Optionally, the feed may contain non-starch polysaccharide substances including corn, corn husks, wheat, wheat bran, barley, and soybean meal.
[0017] Through the above technical solutions, this disclosure provides a method for improving the activity of arabinofuranosylase, a protein with arabinofuranosylase activity, a gene encoding the protein, a recombinant vector with the inserted gene, a transformant containing the gene, a method for preparing arabinofuranosylase, and the application of arabinofuranosylase in the field of feed additives. The two mutants prepared by the method of this disclosure maintain high residual enzyme activity at 70℃ and 80℃, thus achieving stable catalytic ability at high temperatures, and have good application prospects in the field of feed additive preparation.
[0018] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description
[0019] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings: Figure 1 The specific enzyme activity and heat-treated specific enzyme activity of arabinofuranyl glycosidase Abf6 are characterized. Figure 2 These are the results of the molecular dynamics simulation (RMSF) of arabinofuranyl glycosidase Abf6; Figure 3 The specific enzyme activity and thermostability of the arabinofuranyl glycosidase mutant are characterized. Figure 4 It is the effect of arabinofuranyl glycosidase mutant synergistic complex enzyme preparation on wheat bran degradation. Detailed Implementation
[0020] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.
[0021] The first aspect of this disclosure provides a method for improving the activity of arabinofuranyl glycosidase, the method comprising mutating one or more of the amino acid residues of wild-type arabinofuranyl glycosidase to be mutated, said mutation comprising (1) or (2): (1) N301E, S302P, G303L, G304T, S305C and Y306*; (2) N301C, S302R, G303L, G304C, S305T, Y306H and N307C; The amino acid sequence of the wild-type arabinofuranosidase is shown in SEQ ID NO: 1.
[0022] In this disclosure, the inventors use a combination of regional saturation mutation and high-throughput screening to target substances derived from... Talaromyces pinophilus By modifying and screening arabinofuranyl glycosidase, two mutants were obtained that still maintained high residual enzyme activity at 70℃ and 80℃, thus achieving stable catalytic ability at high temperatures, which has good application prospects in the field of feed additive preparation.
[0023] In this disclosure, the terms "protein with arabinofuranylase activity", "protein" and "mutant" are used interchangeably and refer to arabinofuranylase that has been mutated.
[0024] In this disclosure, mutations refer to amino acid substitutions; for example, "N301E" indicates that asparagine at position 301 is replaced by glutamic acid (which can also be labeled Asn→Glu); "Y306*" indicates that due to the premature presence of a terminator, the tyrosine residue (Tyr) at position 306 and the amino acid sequence after position 306 are deleted; "*" represents a terminator (stop codon). Other amino acid substitutions described in this disclosure follow a similar nomenclature. Specifically: (1) N301E (Asn→Glu), S302P (Ser→Pro), G303L (Gly→Leu), G304T (Gly→Thr), S305C (Ser→Cys) and Y306* (Tyr→stop codon); (2) N301C (Asn→Cys), S302R (Ser→Arg), G303L (Gly→Leu), G304C (Gly→Cys), S305T (Ser→Thr), Y306H (Tyr→His) and N307C (Asn→Cys).
[0025] A second aspect of this disclosure provides a protein having arabinofuranyl glycosidase activity, said protein being a protein derived from the amino acid sequence of wild-type arabinofuranyl glycosidase as shown in SEQ ID NO: 1 by substitution of (1) or (2): (1) N301E, S302P, G303L, G304T, S305C and Y306*; (2) N301C, S302R, G303L, G304C, S305T, Y306H and N307C.
[0026] In one embodiment, the amino acid sequence of the protein is as shown in SEQ ID NO: 2 or 3.
[0027] The third aspect of this disclosure provides a gene encoding the protein shown in the second aspect, the nucleotide sequence of which is shown in SEQ ID NO: 4 or 5.
[0028] The fourth aspect of this disclosure provides a recombinant vector, which is a recombinant expression vector, wherein the recombinant vector is inserted with the gene described in the third aspect.
[0029] The fifth aspect of this disclosure provides a transformant in which the introduced gene includes the gene described in the third aspect, or the transformant contains the recombinant vector described in the fourth aspect.
[0030] The host of the transformant can be any commonly used by those skilled in the art; for example, the host of the transformant can be *Escherichia coli*. E. coli Any one of the following: strains, Pichia pastoris, and Bacillus subtilis.
[0031] The sixth aspect of this disclosure provides a method for preparing arabinofuranyl glycosidase, wherein the transformant described in the fifth aspect is inoculated into a culture medium and cultured to obtain the cultured material.
[0032] The culture conditions include: induction culture in BMMY medium, shaking culture at a temperature of 28-30℃, and periodic methanol supplementation for induced expression for 72-120 hours. Preferably, the temperature is 30℃, the time is 96 hours, and 1% methanol is added to the medium every 24 hours.
[0033] The seventh aspect of this disclosure provides the application of arabinofuranylase in the field of feed additives, said arabinofuranylase containing the protein shown in the second aspect.
[0034] In one embodiment, the application includes the hydrolysis of arabinofuranyl glycosidase on the arabinofuranyl bonds of the side chains of non-starch polysaccharides in feed; The non-starch polysaccharides include arabinoxylan and / or arabinogalactan.
[0035] In one embodiment, the feed contains non-starch polysaccharide substances including corn, corn husks, wheat, wheat bran, barley, and soybean meal.
[0036] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.
[0037] All raw materials used in the embodiments can be obtained through commercial purchase.
[0038] Example 1 This embodiment provides a source Talaromyces pinophilus Cloning and expression methods of GH62 family arabinofuranosylase (Abf6): The amino acid sequence of Abf6 is shown in SEQ ID NO: 1, wherein the leader sequence MISLAPALLLLLPAVSG is predicted to be its natural signal peptide. This is for use in Pichia pastoris (…). Pichia pastoris To achieve efficient secretory expression, the signal peptide portion was removed and codons preferred by Pichia pastoris were optimized. The recombinant plasmid pPIC9K-abf6 was constructed by inserting the restriction enzyme sites EcoRI and NotI after the α-factor signal peptide of the Pichia pastoris expression vector pPICK9K.
[0039] After verification, the plasmid was linearized and transformed into Pichia pastoris strain GS115. Positive clones were screened to obtain the recombinant strain GS115_Abf6.
[0040] The recombinant strain was inoculated into BMGY medium and cultured for 16-24 h. It was then transferred to BMMY medium and cultured at 30℃ in a shaker at 200 rpm. Methanol was added to the medium every 24 h until the final concentration reached 0.5%. After induced fermentation, the supernatant was collected for SDS-PAGE analysis, which showed that Abf6 was successfully expressed in Pichia pastoris.
[0041] Enzyme activity was detected using the α-L-arabinofuranylase activity assay kit (BC4765) provided by Solarbio Science, using p-nitrophenol-α-L-arabinofuranylglycoside as the substrate. Abf6 activity was calculated by measuring absorbance changes at 400 nm. Results are as follows: Figure 1 As shown.
[0042] Depend on Figure 1 It can be seen that the specific enzyme activity of Abf6 is 302.4 U / mg; the residual enzyme activities after treatment at 70℃ for 1 min and 3 min are 273.7 U / mg and 130.8 U / mg, respectively; and the residual enzyme activities after treatment at 75℃ for 1 min and 3 min are 174.1 U / mg and 78.4 U / mg, respectively, indicating that its activity decreases significantly under high temperature conditions.
[0043] Example 2 Mutant library design and construction: To obtain the Abf6 mutant with higher enzyme activity retention under high temperature conditions, based on its three-dimensional structure prediction model, the root mean square fluctuation value (RMSF) of each amino acid residue was calculated by molecular dynamics simulation to evaluate the flexible region (e.g., Figure 2 (As shown).
[0044] The results showed that the RMSF values of residues at positions 89–93, 115–117, 146–149, 225–229, and 301–307 were high, indicating that these regions have greater structural flexibility. Therefore, these five regions were selected as mutation hotspots, and a regional saturation mutation strategy (NNK codon saturation) was adopted to construct locally saturated mutation libraries for different regions, which were then constructed into the pPIC9K vector to form expression libraries.
[0045] Example 3 Mutant screening: The mutant library obtained in Example 2 was electroporated into Pichia pastoris GS115 competent cells. After transformation, the cells were incubated for 2 hours at 30°C and 200 rpm in a mixture of YPD and sorbitol (1:1). Then, the cells were transferred to 5 mL of MD medium and cultured overnight at 30°C in a shaker.
[0046] The following day, the bacterial suspension was washed and resuspended in BMMY induction medium, and a droplet stabilizer was added to generate microfluidic droplets. The generated droplets were then incubated at 30°C for 3–5 days, and microscopic observation confirmed normal bacterial growth within the droplets.
[0047] The cultured droplets were divided into two batches and heat-treated at 85°C for 3 min and 5 min, respectively. Subsequently, the heat-treated droplets were fused with the substrate 4-methylumbelliferone-α-L-arabinofuranoside to generate new droplets, and the reaction was carried out at 40°C.
[0048] The substrate releases 4-methylumbelliferone (4-MU) under the catalysis of arabinofuranosides, which generates a fluorescent signal under excitation at 365 nm and emission at 450 nm. The fluorescence intensity of the droplets was recorded using a fluorescence detection system at the corresponding wavelengths, and droplets that still exhibited high fluorescence signals (i.e., high residual activity) after heat treatment were screened and printed in 96-well plates.
[0049] The top 0.1% of droplets by fluorescence intensity were selected for PCR verification and sequencing. The results showed two types of mutations: EPLTC*F at positions 301–307, and CRLCTHC at positions 301–307.
[0050] Based on this, two mutants were constructed: Abf6-S1 (N301E, S302P, G303L, G304T, S305C, Y306*), amino acid sequence as shown in SEQ ID NO: 2, nucleotide sequence as shown in SEQ ID NO: 4. Due to the presence of a terminator in the mutation, all sequences after amino acid 306 were deleted. Abf6-S3 (N301C, S302R, G303L, G304C, S305T, Y306H, N307C), amino acid sequence as shown in SEQ ID NO: 3, nucleotide sequence as shown in SEQ ID NO: 5.
[0051] The two nucleotide sequences were constructed into Pichia pastoris expression vectors to obtain recombinant strains GS115-Abf6-S1 and GS115-Abf6-S3.
[0052] Example 4 Enzyme activity and heat resistance tests of mutants: The GS115-Abf6-S1 and GS115-Abf6-S3 strains were induced to express and purified under the same conditions as in Example 1.
[0053] The specific enzyme activity of each mutant was tested using the same substrate as in Example 1. Residual enzyme activity was measured after treatment at 70℃ and 80℃ for 1, 3, 5, and 10 min, respectively, to evaluate the performance after high-temperature treatment. The results are shown in Table 1 and... Figure 3 As shown: Table 1
[0054] From Table 1 and Figure 1 , 3 It was found that, compared to the wild-type Abf6, both mutants Abf6-S1 and Abf6-S3 showed significantly increased specific enzyme activity and residual enzyme activity after high-temperature treatment. Specifically, without heat treatment, the specific enzyme activity of Abf6-S1 increased to 12.61 times that of the wild-type, and the specific enzyme activity of Abf6-S3 increased to 2.54 times that of the wild-type. After heat treatment at 80℃ for 1 min, both mutants maintained higher enzyme activities than the untreated Abf6, demonstrating a significant increase in enzyme activity under high-temperature conditions.
[0055] Example 5 In vitro tests for feed degradation: To verify the application potential of mutants in feed systems, wheat bran was used as a model substrate to test the effects of different arabinofuranylases on the dry matter loss rate of feed ingredients.
[0056] The compound enzyme preparation product Romosin (purchased from Adisseo, one of its main components is xylanase) was added to wheat bran at a concentration of 1 mg / g dry matter, and 0.5 mg / g Romosin was mixed with 0.5 mg / g of different arabinofuranylases. A pH 5.5 dipotassium hydrogen phosphate solution was added to the reaction system, and the mixture was incubated overnight at 40℃ and 200 rpm. After the reaction, the supernatant was discarded by centrifugation, and the mixture was washed once with pure water, then centrifuged again and the supernatant was discarded. The empty aluminum foil cup was weighed and recorded; simultaneously, wheat bran was washed with pure water, poured into the aluminum foil cup, placed in an oven until completely dry, weighed, and the weight was recorded. The weight of the dried precipitate was obtained by subtracting the weight of the aluminum foil cup, and the dry matter loss rate was calculated. Dry matter loss rate = (dry weight of raw material - weight of precipitate after drying) / dry weight of raw material × 100%.
[0057] The results are as follows Figure 4 As shown, compared with the blank control, the dry matter loss rate of wheat bran increased from 2.79% to 11.74% when only Luo Meibao product was added. After replacing half of Luo Meibao with Abf6, the dry matter loss rate of the raw material was further increased to 13.29%. After replacing Abf6 with the mutants Abf6-S1 and Abf6-S3 respectively, the dry matter loss rate was further increased to 15.90% and 17.38% respectively.
[0058] The results showed that both the mutants Abf6-S1 and Abf6-S3 significantly improved the dry matter loss rate of feed and exhibited better synergistic effects with xylanase, demonstrating good potential for industrial application in feed.
[0059] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure. It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this disclosure will not further describe the various possible combinations.
[0060] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.
Claims
1. A method for increasing the activity of arabinofuranylase, characterized in that, The method includes mutating one or more of the amino acid residues of the wild-type arabinofuranyl glycosidase to be mutated, said mutation comprising (1) or (2): (1) N301E, S302P, G303L, G304T, S305C and Y306*; (2) N301C, S302R, G303L, G304C, S305T, Y306H and N307C; The amino acid sequence of the wild-type arabinofuranosidase is shown in SEQ ID NO:
1.
2. A protein possessing arabinofuranyl glycosidase activity, wherein, The protein is a protein derived from the amino acid sequence of the wild-type arabinofuranase shown in SEQ ID NO: 1 by substitution of (1) or (2): (1) N301E, S302P, G303L, G304T, S305C and Y306*; (2) N301C, S302R, G303L, G304C, S305T, Y306H and N307C.
3. The protein according to claim 2, wherein, The amino acid sequence of the protein is shown in SEQ ID NO: 2 or 3.
4. A gene encoding the protein of claim 2 or 3, wherein, The nucleotide sequence of the gene is shown in SEQ ID NO: 4 or 5.
5. A recombinant vector, characterized in that, The recombinant vector is a recombinant expression vector, and the recombinant vector is inserted with the gene described in claim 4.
6. A transformant, characterized in that, The gene introduced into the transformant includes the gene of claim 4, or the recombinant vector of claim 5 is introduced into the transformant.
7. A method for preparing arabinofuranosidase, characterized in that, The transformant according to claim 6 is inoculated into a culture medium and cultured to obtain the cultured material.
8. The application of an arabinofuranase in the field of feed additives, wherein, The arabinofuranase contains the protein shown in claim 2 or 3.
9. The application according to claim 8, wherein, The application includes the hydrolysis of arabinofuranyl bonds on the side chains of non-starch polysaccharides in feed by arabinofuranylase; The non-starch polysaccharides include arabinoxylan and / or arabinogalactan.
10. The application according to claim 9, wherein, The feed contains non-starch polysaccharides, including corn, corn husks, wheat, wheat bran, barley, and soybean meal.