Patent Application
20240384252
This application claims the priority of Chinese Patent Application No. 202111097561.4, filed with the China National Intellectual Property Administration on Sep. 18, 2021 titled with “High Temperature Resistant Mannanase Mutant”, and the disclosure of which is hereby incorporated by reference in its entirety.
The present invention relates to the field of genetic engineering and protein modification technology, and particularly relates to a mannanase mutant with high-temperature resistance.
Mannan is a natural polymer with high molecular weight, non-toxic, odorless and high viscosity, which is widely used in the fields of pharmaceuticals, foods and cosmetics, etc. However, mannan existing in cell wall or seed of plants is easy to swell when exposing to water, leading to the great increase in viscosity, which will have an adverse impact on the development and utilization of mannan. For example, in the process of animal feeding, mannan in feed can combine with a large amount of water to form a gelatinous substance with high viscosity, which affects the hydrolysis of feed by digestive enzymes, thus reducing the digestion and utilization efficiency of feed by animals. Therefore, mannan has become a common anti-nutritional factor in the field of feeds.
β-mannanase (EC3.2.1.78) is a glycoside hydrolase that can degrade β-1,4-mannosidic bond, and its substrates are linear mannan, glucomannan, galactomannan, galactoglucomannan and the like. Various mannans could be hydrolyzed by β-mannanase and generated mannose and mannan oligosaccharides (MOS, usually consisting of 2-10 monosaccharide residues).
The research, application and development of β-mannanase might be traced back to 1950s. In 1958, Courtios et al. first discovered a β-mannanase from Alfalfa. Williams and Doetsch reported a β-mannanase obtained from anaerobic rumen Streptococci in 1960. Subsequently, Reese and Shibata et al. first systematically proposed the concept of β-mannanase in 1965.
β-mannanases are widely found in plants, animals and microorganisms. For example, β-mannanase is found in intestinal secretions of some lower class animals and germinant seeds of some plants. At present, most of the reported β-mannanases are derived from microorganisms, mainly including bacteria, fungi and actinomycete. For the β-mannanases produced by bacteria, the optimal reaction pH is 5.5-7.0, isoelectric point is 4.0-5.0, and the optimal reaction temperature is 50-70° C. For the β-mannanase from fungi, the isoelectric point is usually 4.0-5.0, the optimum reaction temperature is 55-75° C., and the optimum reaction pH is 4.5-5.5, which belongs to β-mannanase.
The production of β-mannanase by microorganisms has the advantages of not being limited by seasons, high expression level, relatively low production cost and to be easily produced by industrial fermentation, so it has become the main way of industrial production of β-mannanase. However, at present, there is a short-time high temperature stage of 80-90° C. during the production of pellet feed, and the enzyme activity will be greatly lost if β-mannanase, which is not resistant to high temperature, is directly added to animal feed for granulation. Therefore, improving the thermal stability of β-mannanase is of great practical significance in the field of feeds.
The present invention aims to provide a mannanase mutant. Compared with the wild type mannanase, the heat resistance of the mutants is significantly improved, which is beneficial to its wide application in the field of feeds.
In order to achieve the above-mentioned purpose, the present invention provides the following technical solutions.
The present invention provides a mannanase mutant, which comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 1, and compared with SEQ ID NO: 1, the mannanase mutant comprises at least one amino acid substitution at a position selected from the group consisting of: 49, 58, 90, 99, 120, 124, 130, 132, 136, 140, 148, 154, 206, 212, 257, 263, 268, 273, 296, 297, 302, 306, 315, 339, 353, 360 and 362 of SEQ ID NO: 1.
In some embodiments of the present invention, the mutant comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO: 1.
In some more particular embodiments, the mutant comprises an amino acid sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity with SEQ ID NO: 1.
In some embodiments of the present invention, the mannanase mutant comprises at least one amino acid substitution selected from the group consisting of: D49C/I/N, G58D/E/S, H90L, T99Y, K120Y, N124P, T130D/N/P/Q/R/S, Y132F, S136M, S140L/K, T148K, D154P, F206Y, A212N/S/P, L257F, S263R, D268A/E/G/P, G273P, V296I, T297Y, S302M/E, S306M/Q, T315P, N339F, T353L/F, D360E/N/K and A362R
In some embodiments of the present invention, the mannanase mutant comprises an amino acid substitution or a combination thereof selected from the group consisting of: D49C, D491, D49N, G58D, G58E, G58S, H90L, T99Y, K120Y, N124P, T130D, T130N, T130P, T130Q, T130R, T130S, Y132F, S136M, S140L, S140K, T148K, D154P, F206Y, A212N, A212S, A212P, L257F, S263R, D268A, D268E, D268G, D268P, G273P, V296I, T297Y, S302M, S302E, S306M, S306Q, T315P, N339F, T353L, T353F, D360E, D360N, D360K, A362R,
The present invention further provides a DNA molecule encoding the mannanase mutant.
The present invention further provides a recombinant expression plasmid comprising the above DNA molecule.
The present invention further provides a host cell comprising the above recombinant expression plasmid.
The heat resistance of the recombinant mannanase mutant is significantly enhanced, wherein the mannanase mutant is produced by transforming the above plasmid into host cells.
In some embodiments of the present invention, the host cell is Pichia pastoris.
In some embodiments of the present invention, the host cell is Trichoderma reesei.
The present invention further provides the use of the mannanase mutant in the field of feeds.
On the basis of wild-type mannanase M20, the present invention provides a mannanase mutant comprising at least one mutation site selected from the group consisting of: D49C/I/N, G58D/E/S, H90L, T99Y, K120Y, N124P, T130D/N/P/Q/R/S, Y132F, S136M, S140L/K, T148K, D154P, F206Y, A212N/S/P, L257F, S263R, D268A/E/G/P, G273P, S302M/E, S306M/Q, T315P, T353L/F, D360E/N/K and A362R. Compared with wild-type mannanase, the residual enzyme activity of the mutant provided by the present invention generally improved by 69.9%- 429.6%, and the heat resistance is significantly improved, after being treated at 80° C. for 3 minutes. Among them, the mutant comprising a single mutation site of N124P and the mutant comprising a single mutation site of D360K show the best heat resistance, and the residual enzyme activity is as high as 51.81% and 60.69%, respectively, achieving unexpected technical effects.
In summary, the heat resistance of the mannanase mutants provided by the present invention is significantly improved, which is beneficial to its wide application in the field of feeds.
The present invention discloses a mannanase mutant, a preparation method and a use thereof, a DNA molecule encoding the mannanase mutant, a vector, and a host cell. Those skilled in the art can learn from the content of the present invention and make appropriate improvement on the process parameters to achieve the present invention. The method and the use of the present invention have been described through the preferred embodiments, and it is obvious that the method and use described herein may be changed or appropriately modified and combined to realize and use the technology of the present invention by those skilled in the art without departing from the content, spirit and scope of the present invention.
The present invention employs conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as the methods described in MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can adopt other conventional methods, experimental schemes, and reagents in the field on the basis of the technical solutions described in the present invention, and are not limited to the limitations of the particular embodiments of the present invention. For example, the following experimental materials and reagents might be used.
Strains and vectors: Escherichia. coli DH5α, Pichia pastoris GS115, vector pPIC9k, Amp and G418 were purchased from Invitrogen.
Enzymes and kits: PCR enzymes and ligases were purchased from Takara, restriction enzymes were purchased from Fermentas, lyase was purchased from Sigma, plasmid extraction kits and gel purification recovery kits were purchased from Omega, and GeneMorph II random mutagenesis kits were purchased from Beijing Biomars Biological Technology Co., Ltd.
The formula of medium is as follows:
The present invention will be further illustrated below in conjunction with examples.
The genome of Aspergillus niger was used as a template for PCR amplification, and PCR primers M20-F1 and M20-R1 were as follows:
PCR products were recovered from gel, ligated with pEASY-T vector, transformed into E.coli DH5α, and the correct transformants were selected for sequencing. Sequencing results showed that the nucleotide sequence of the gene fragment obtained by amplification was SEQ ID NO: 2 and the encoded amino acid sequence was SEQ ID NO: 1, which indicated that the expression plasmid was successfully constructed and named pPIC9K-M20. Through NCBI BLAST alignment, it was found that the SEQ ID NO: 1 had 100% sequence identity with the sequence of the acidic mannanase from Aspergillus Niger. Thus, it was determined that the gene obtained by PCR was a mannanase gene, named M20.
In order to improve the heat resistance of acidic mannanase M20, a large number of mutants of the enzyme were screened by directed evolution technique.
M20 gene was served as the template, and the primers M20-F1 and M20-R1 were used to perform PCR amplification by GeneMorph II Random Mutagenesis Kit (Beijing Biomars), followed by recovering the PCR product from gel. After being digested with EcoRI and NotI, the PCR product was ligated with pET21a vector digested with the same enzymes. The ligation product was transformed into E. coli BL21 (DE3) and then the cells were spread on LB+Amp plate for culturing upside down at 37° C. After the transformation, single colonies were picked one by one with toothpicks and transferred to a 96-well plate. Each well on the plate was added with 150 μl of LB+Amp medium containing 0.1 mM IPTG, followed by cultured at 37° C. for about 6 hours with shaking at 220 rpm. Afterwards, the culture was centrifuged and the supernatant was discarded. The bacteria cells were resuspended in buffer, and frozen and thawed repeatedly to obtain E. coli cell lysate containing mannanase.
30 μL of lysate was taken from each well and transferred to two new 96-well plates, respectively. Then, one of the plates was treated at 75° C. for 5 min. Afterwards, 30 μL of substrate was added to each well in both 96-well plates, and reaction was carried out at 37° C. for 30 min. The reducing sugar was determined by DNS method, and the enzyme activity levels of different mutants were calculated respectively.
The experimental results showed that different mutants exhibited different activities after high temperature treatment. Some mutations had no effect on the heat resistance of mannanase M20, while some made it even worse. In addition, there were some mutations, which improved the heat resistance of mannanase, but changed its enzymatic properties significantly. These mutants did not meet the desired requirements. Finally, mutation sites that not only significantly improved the heat resistance of mannanase but also did not affect its original enzymatic properties were selected, which were as follows: D49C, D491, D49N, G58D, G58E, G58S, H90L, T99Y, K120Y, N124P, T130D, T130N, T130P, T130Q, T130R, T130S, Y132F, S136M, S140L, S140K, T148K, D154P, F206Y, A212N, A212S, A212P, L257F, S263R, D268A, D268E, D268G, D268P, G273P, S302M, S302E, S306M, S306Q, T315P, T353L, T353F, D360E, D360N, D360K and A362R.
Based on the wild-type mannanase M20, the present invention provides mannanase mutants which comprises one mutation selected from the group consisting of D49C, D49I, D49N, G58D, G58E, G58S, H90L, T99Y, K120Y, N124P, T130D, T130N, T130P, T130Q, T130R, T130S, Y132F, S136M, S140L, S140K, T148K, D154P, F206Y, A212N, A212S, A212P, L257F, S263R, D268A, D268E, D268G, D268P, G273P, S302M, S302E, S306M, S306Q, T315P, T353L, T353F, D360E, D360N, D360K and A362R.
The present invention further provided a mannanase mutant comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 mutations selected from the group consisting of D49C/I/N, G58D/E/S, H90L, T99Y, K120Y, N124P, T130D/N/P/Q/R/S, Y132F, S136M, S140L/K, T148K, D154P, F206Y, A212N/S/P, L257F, S263R, D268A/E/G/P, G273P, V296I, T297Y, S302M/E, S306M/Q, T315P, T353L/F, D360E/N/K and A362R.
Referring to the amino acid sequence of the mutants, the nucleotide sequences encoding the mannanase mutants were obtained.
According to the codon bias of Pichia pastoris, the sequences of wild-type mannanase M20 and the mutants were optimized. The genes were synthesized by Shanghai Generay Biotechnology Co., Ltd., and two restriction sites of enzymes EcoRI and NotI were added to the 5′ and 3′ ends of the synthetic sequence.
Following the method described in Example 1, the DNA of the synthesized mutant was digested with EcoRI and NotI, and then ligated with the pPIC-9K vector (digested with the same enzymes) at 16° C. overnight. The ligation product was transformed into E. coli DH5a and then spread on LB+Amp plate for culturing upside down at 37° C. After the transformants appeared, the positive clones were verified by colony PCR, and the correct expression plasmid of recombinant mutant was obtained after sequencing verification.
Pichia pastoris GS115 strain was activated on an YPD plate. After culturing at 30° C. for 48 hours, single clone of the activated GS115 was inoculated into 6 mL of YPD liquid medium and cultured at 220 rpm at 30° C. for about 12 hours. After that, the cells were transferred into an Erlenmeyer flask containing 30 mL of YPD liquid medium and cultured at 220 rpm at 30° C. for about 5 hours. The cell density was detected by an ultraviolet spectrophotometer. After the OD600 value was in the range of 1.1-1.3, 4 mL of cells was collected into a sterilized EP tube by centrifugation at 4° C., 9,000 rpm for 2 min. The supernatant was carefully discarded, and the remaining supernatant was absorbed with sterile filter paper. Subsequently, the cells were resuspended in 1 mL of pre-cooled sterilized water, followed by centrifugation at 4° C., 9,000 rpm for 2 min. The supernatant was carefully discarded, and the cells were washed with 1 mL sterile water, followed by centrifugation at 4° C., 9,000 rpm for 2 min. The supernatant was carefully discarded, and the cells were resuspended in 1 mL of pre-cooled sorbitol (1 mol/L), followed by centrifugation at 4° C., 9,000 rpm for 2 min. The supernatant was carefully discarded, and the cells were gently resuspended in 100-150 μl of the pre-cooled sorbitol (1 mol/L).
The expression plasmids constructed in Example 1 and Example 3.1 were linearized with SacI. After the linearized fragments were purified and recovered, they were transformed into Pichia pastoris GS115 by electroporation. The recombinant strains of Pichia pastoris were screened on MD plates, and then transformants with multiple copies were screened on an YPD plate containing different concentrations of geneticin (0.5 mg/mL-8 mg/mL).
The obtained transformants were transferred to BMGY medium and cultured with shaking at 30° C. and 250 rpm for 1 d; then transferred to BMMY medium and cultured with shaking at 250 rpm, 30° C. 0.5% methanol was added every day to induce expression for 4 days. The cells were removed by centrifugation at 9000 rpm for 10 min to obtain supernatants from fermentation containing wild-type mannanase M20 and mutants respectively.
At 37° C. and pH 5.5, the amount of enzyme required to degrade and release 1 μmol reducing sugar per minute from a 3 mg/mL mannan solution is defined as one enzyme-activity unit U.
4.0 mL of acetic acid-sodium acetate buffer solution was taken and added to 5.0 mL of DNS reagent. The obtained mixture was heated in boiling water bath for 5 min then cooled to room temperature by tap water. The volume was adjusted to 25.0 mL with water to make a standard blank sample.
1.00 ml, 2.00 ml, 3.00 ml, 4.00 ml, 5.00 ml, 6.00 ml and 7.00 mL of mannose solution (pH 5.5) were taken respectively, and the total volume was adjusted to 100 mL with acetic acid-sodium acetate buffer solution respectively, to prepare D-mannose standard solutions with concentrations of 0.10-0.70 mg/ml.
2.00 mL of mannose standard solutions of the above concentration series (in two replicates) were taken and added into calibration test tubes, respectively. And then 2 mL of acetic acid-sodium acetate buffer solution and 5 ml of DNS reagent were added to each tube. The test tubes were subjected to electromagnetic oscillation for 3 s, heated in boiling water bath for 5 min and cooled to room temperature by tap water. The volume of each tube was adjusted to 25 mL with water. The standard blank sample was used as control for zero adjustment, and the OD value was measured at 540 nm.
The standard curve was plotted with mannose concentration as y axis and OD value as the x axis. The standard curve should be re-plotted every time when a new batch of DNS reagent was prepared.
10.0 mL of mannan solution was taken and equilibrated at 37° C. for 10 min.
10.0 mL of properly diluted enzyme solution was taken and equilibrated at 37° C. for 10 min.
2.00 mL of properly diluted enzyme solution (after equilibration at 37° C.) was taken and added to the calibration test tube, and 5 ml of DNS reagent was added. The test tube was subjected to electromagnetic oscillation for 3 s. 2.0 mL of mannan solution was added to the tube, kept at 37° C. for 30 min, heated in boiling water bath for 5 min and cooled to room temperature by tap water. The volume was adjusted to 25 mL, and then subjected to electromagnetic oscillation for 3 s. The standard blank sample was used as the blank control, the absorbance AB was determined at 540 nm.
2.00 mL of properly diluted enzyme solution (after equilibration at 37° C.) was taken and added to the calibration test tube, and 2.0 ml of mannan solution (after equilibration at 37° C.) was also added. The test tube was subjected to electromagnetic oscillation for 3 s and kept at 37° C. for 30 min. 5.0 mL DNS reagent was added. The test tube was subjected to electromagnetic oscillation for 3 s and enzymatic hydrolysis was carried out. Then, the tube was heated in boiling water bath for 5 min and cooled to room temperature by tap water. The volume was adjusted to 25 mL and subjected to electromagnetic oscillation for 3 s. The standard blank sample was used as the blank control, the absorbance AE was determined at 540 nm.
The calculation formula of enzyme activity is as follow:
In the formula: XD is the activity of mannanase in diluted enzyme solution, U/ml; AE is the absorbance of the enzyme reaction solution; AB is the absorbance of enzyme blank solution; K is the slope of the standard curve; C0 is the intercept of the standard curve; M is the molar mass of xylose, 180.2 g/mol; t is the reaction time of enzymolysis, min; N is the dilution multiple of enzyme solution; 1000 is the conversion factor, 1 mmol=1000 μmol.
The enzyme activity was measured according to the above method. The results showed that the enzyme activity of the fermentation supernatant from the recombinant Pichia pastoris strain expression mannanase M20 or mutants was 200-500 U/mL.
According to the codon bias of Trichoderma reesei, the sequences of wild-type mannanase M20 and the mutants were optimized. The genes were synthesized by Shanghai Generay Biotechnology Co., Ltd., and two restriction sites of enzymes KpnI and MluI were added to the 5′ and 3′ ends of the synthetic sequence.
The synthesized mannanase gene fragment and pSC1G vector were digested with restriction enzymes KpnI and MluI, respectively. The digested products were purified by gel purification kit and ligated together using T4 DNA ligase. The ligation products were transformed into E.coli DH5α. The clones were selected by ampicillin and verified by sequencing. After sequencing verification, the recombinant plasmid comprising mannanase gene was obtained.
The spore suspension from Trichoderma reesei UE host cells was inoculated on a PDA plate, and cultured at 30° C. for 6 days. After the spores were abundant, a colony of about 1 cm×1 cm was cut and transferred into liquid culture medium containing 120 mL of YEG+U (0.5% yeast powder, 1% glucose, and 0.1% uridine), and cultured with shaking at 30° C. and 220 rpm for 14-16 h.
The mycelia were collected by filtration with sterile gauze, and washed once with sterile water. The mycelia were transferred into an Erlenmeyer flask containing 20 mL of 10 mg/mL lysing enzyme solution (Sigma L1412) and cultured at 30° C., 90 rpm for 1-2 h. The process of protoplast transformation was observed and detected with a microscope.
20 mL of pre-cooled 1.2 M sorbitol (1.2 M sorbitol, 50 mM Tris-Cl, 50 mM CaCl2) was added to the above flask and shaken well gently. The filtrate was collected by using the sterile Miracloth filtration material and centrifuged at 3000 rpm at 4° C. for 10 min. The supernatant was discarded, and 5 mL of pre-cooled sorbitol solution (1.2M) was added to resuspend the cells. The mixture was centrifuged at 3000 rpm at 4° C. for 10 min, the supernatant was discarded, and appropriate amount of the pre-cooled sorbitol (1.2M) was added to resuspend the cells and the cells were aliquoted (200 μL/tube, with a concentration of 108 protoplast/mL).
The following operations were all performed on ice. 10 μg of the recombinant plasmids constructed above was added to 7 mL sterile centrifuge tube containing 200 μL of protoplast solution, and then 50 μL of 25% PEG (25% PEG, 50 mM Tris-Cl, 50 mM CaCl2) was added. The mixture was mixed well by flicking the bottom of the tube, and placed on ice for 20 minutes. 2 mL of 25% PEG was added, mixed well and allowed to stand for 5 minutes at room temperature. 4 mL of 1.2 M sorbitol was added and mixed gently. The mixture was added to the upper medium that had been melted and kept at 55° C. After being mixed gently, the mixture was spread on the pre-prepared lower medium plate and cultured at 30° C. for 5-7 days until transformants appeared. The transformants were picked and transferred to the lower medium plate for re-screening, and the colonies with smooth edges were the positive transformants.
According to the above method, the recombinant Trichoderma reesei strains expressing wild-type mannanase M20 or mutants were respectively constructed.
The engineered Trichoderma reesei strains constructed above were inoculated on the PDA solid plate respectively, and cultured upside down in a constant temperature incubator at 30° C. for 6-7 days. After the spores were abundant, two colonies with a diameter of 1 cm were taken and inoculated into 250 mL Erlenmeyer flask containing 50 mL fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH4)2SO4, 0.09% MgSO4, 2% KH2PO4, 0.04% CaCl2, 0.018% Tween-80, 0.018% trace element), cultured at 30° C. for 48 hours and then cultured at 25° C. for 48 hours. The fermentation solutions were centrifuged to obtain fermentation supernatants containing wild-type mannanase M20 and mutants respectively.
According to the above method, the enzyme activity was detected, and the results showed that the enzyme activity of the fermentation supernatants from recombinant Trichoderma reesei strains expressing wild-type mannanase M20 or mutant was 230-510 U/mL.
Disodium hydrogen phosphate-citric acid buffers with pH values of 2.0, 2.5, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 8.0 were respectively used to dilute the supernatants after fermentation of recombinant Pichia pastoris strains constructed above, and mannan substrate was also prepared using buffers with corresponding pH values, respectively. The enzyme activity of mannase was measured at 37° C. and the relative enzyme activity was calculated by taking the highest enzyme activity as 100%.
The results showed that the optimal pH value of both wild-type mannanase M20 and mutants was 4.0, indicating that the mutations did not change the optimal pH value of mannanase M20.
The enzyme activity of mannanase in the supernatants after fermentation of the recombinant Pichia pastoris strains constructed above was determined at various temperatures of 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., under pH of 5.5. The relative enzyme activity was calculated by taking the highest enzyme activity as 100%.
The results showed that the optimal reaction temperature of wild-type mannanase M20 was 70° C. The optimal reaction temperature of mannanase mutants was 72-77° C.
The fermentation supernatants of recombinant Pichia pastoris strains were diluted to about 20 U/mL with acetic acid-sodium acetate buffer with pH 5.5, and treated at 80° C. for 3 min. The enzyme activity of mannanase was determined, and the residual enzyme activity was calculated by taking enzyme activity of the untreated sample as 100%. The results are shown in Table 1.
As can be seen from the results shown in Table 1, compared with wild-type mannanase M20, the residual enzyme activities of the mutants with single mutation were increased by 69.9%- 429.6%, after being treated at 80° C. for 3 min, indicating that the heat resistance of these mutants was significantly improved. Among them, N124P mutant and D360K mutant have the best heat resistance. After being treated at 80° C. for 3 minutes, the residual enzyme activities was as high as 51.81% and 60.69% of the untreated enzyme, respectively, showing unexpected technical effect.
The high temperature resistant mannanase provided by the present invention is suitable to be widely used in the field of feeds.
The high temperature-resistant mannanase provided by the present invention has been introduced in detail above. The principle and implementation of the present invention are illustrated by using specific embodiments herein. The above descriptions of the embodiments are only used to facilitate understanding of the method and the core idea of the present invention. It should be noted that, several improvements and modifications may be made by those skilled in the art to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111097561.4 | Sep 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/119328 | 9/16/2022 | WO |
