Modified xylanase, DNA molecule, recombinant plasmid and transfected cell

Abstract

The present application relates to the field of enzyme engineering, and more particularly to a modified xylanase, DNA molecule, recombinant plasmid, and transfected cells. The modified xylanase protected by the present application is formed by replacing a deleted segment of a receptor enzyme with a replacement segment. The receptor enzyme is a wild-type xylanase, the sequence of which is SEQ ID NO: 1. The deleted segment includes the carbohydrate-binding module of the receptor enzyme, and the replacement segment is the carbohydrate-binding module of a donor enzyme. The donor enzyme is a carbohydrate-active enzyme, and the sequence of the donor enzyme is one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. By replacing the carbohydrate-binding module of a carbohydrate-active enzyme with a higher activity into the receptor enzyme, the receptor enzyme's ability to bind carbohydrates is improved, thereby increasing the activity of the receptor enzyme.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering, in particular to a modified xylanase, a DNA molecule, a recombinant plasmid and a transfected cell. Background Art

[0002] With the increasing global demand for renewable resources, the potential of plant-based biomass as a clean energy and sustainable resource has attracted considerable attention. Hemicellulose is the second-largest polysaccharide in plant cell walls, second only to cellulose. Xylan, a major component of hemicellulose, plays a crucial role in plant cell walls. Xylanases are enzymes that degrade xylans, which are abundant in nature, by hydrolyzing them into oligosaccharides such as small oligosaccharides and xylobiose, as well as small amounts of xylose and arabinose.

[0003] In the application of xylanase, due to the differences in usage scenarios, there are often requirements that are difficult to achieve with natural xylanase. Therefore, it is imperative to modify natural wild-type xylanase to improve its activity and other properties. Summary of the Invention

[0004] The object of the present invention is to provide a modified xylanase with higher activity, as well as corresponding DNA molecules, recombinant plasmids and transfected cells.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A modified xylanase is formed by replacing a deleted segment of a receptor enzyme with a replacement segment, wherein the receptor enzyme is a wild-type xylanase with a sequence of SEQ ID NO: 1, the deleted segment includes a carbohydrate binding module of the receptor enzyme, the replacement segment includes a carbohydrate binding module of a donor enzyme, the donor enzyme is a carbohydrate-active enzyme, and the sequence of the donor enzyme is one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0007] Optionally, the sequence of the replacement segment is one of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

[0008] Optionally, the sequence of the deleted segment of the receptor enzyme is positions 374 to 413 in the sequence of the receptor enzyme.

[0009] Optionally, the xylose yield from hydrolyzing wood cellulose by the modified xylanase is greater than 4.5%.

[0010] In a second aspect, the present invention further provides a DNA molecule encoding the modified xylanase.

[0011] In a third aspect, the present invention further provides a recombinant plasmid comprising the above-mentioned DNA molecule.

[0012] In a fourth aspect, the present invention also provides a transfected cell, which is prepared by transfecting the above-mentioned DNA molecule into a donor cell.

[0013] Optionally, the donor cell is Pichia pastoris.

[0014] The present invention has the beneficial effects of replacing the carbohydrate-binding module of a highly active carbohydrate-active enzyme with a receptor enzyme, thereby improving the receptor enzyme's ability to bind carbohydrates and enhance its ability to bind carbohydrates and perform enzymatic hydrolysis under harsh conditions, thereby increasing the activity of the receptor enzyme. Furthermore, by limiting the type of donor enzyme, it helps ensure good compatibility between its carbohydrate-binding module and the segment retained by the receptor enzyme, thereby ensuring that the modified xylanase has good activity.

[0015] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram showing the structural analysis results of the receptor enzyme shown in Example 1 of the present invention;

[0017] Figure 2 This is a statistical diagram of the enzyme activity of the acceptor enzyme and various modified xylanases shown in Example 1 of the present invention. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0019] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0020] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0021] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0022] Xylanases typically consist of two components: a catalytic module (CM) and a carbohydrate-binding module (CBM). CBMs are non-catalytic functional modules that bind to xylan substrates, localizing the enzyme's catalytic module to the substrate surface, thereby significantly improving the enzyme's catalytic efficiency. Studies have shown that CBMs play a crucial role in enzyme-substrate interactions, particularly during the degradation of complex lignocellulosic substrates. The presence of CBMs can enhance enzyme affinity and improve the treatment of crystalline substrates.

[0023] Although CBMs have a significant impact on the catalytic activity of xylanases, naturally derived CBMs may not fully meet specific industrial needs in some cases. For example, some naturally derived CBMs have low substrate binding affinity, limiting the enzyme's performance on difficult-to-degrade substrates. Some CBMs also exhibit poor stability at high temperatures or extreme pH conditions, impacting the enzyme's overall applicability. CBM modules from different sources exhibit structural diversity, and different CBMs can significantly influence the enzyme's binding capacity, thermal stability, and catalytic efficiency. For example, some CBMs have stronger binding to crystalline xylan, while others exhibit greater environmental tolerance. Therefore, by introducing CBM modules from diverse sources, xylanases can be endowed with new functional properties or enhance their existing performance, making them more suitable for specific industrial applications. However, CBM modules are often incompatible with other components of wild-type xylanases. Even if a CBM module from a donor enzyme with better properties is replaced with a wild-type xylanase, it is difficult to guarantee optimal performance of the wild-type xylanase.

[0024] The present invention applies to protect a modified xylanase, which is formed by replacing the deleted segment of the acceptor enzyme with the replacement segment, the acceptor enzyme is a wild-type xylanase, and its sequence is SEQ ID NO: 1, the deleted segment includes the carbohydrate binding module of the acceptor enzyme, the replacement segment includes the carbohydrate binding module of the donor enzyme, the donor enzyme is a carbohydrate-active enzyme, and the sequence of the donor enzyme is one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0025] By replacing the carbohydrate-binding module of a highly active carbohydrate-active enzyme with the acceptor enzyme, the acceptor enzyme's ability to bind carbohydrates is improved, and its ability to bind carbohydrates and perform enzymatic hydrolysis under harsh conditions is enhanced, thereby increasing the acceptor enzyme's activity. By limiting the type of donor enzyme, it helps ensure good compatibility between its carbohydrate-binding module and the chain segment retained by the acceptor enzyme, thereby ensuring that the modified xylanase has good activity.

[0026] Please refer to the following examples for details.

[0027] Example 1:

[0028] The modified xylanase shown in a preferred embodiment of the present application is formed by replacing the carbohydrate binding module (CBM) of the receptor enzyme and the non-functional short peptides before and after the module with a replacement segment. In this embodiment, the receptor enzyme is the wild-type xylanase PuXyn from Penicillium species, whose amino acid sequence is SEQ ID NO: 1 and NCBI accession number is KAF7714546.1. The sequence of the receptor enzyme is analyzed by protein structure analysis software. The protein structure analysis software in this embodiment is the Conserved Domain Search (CD-Search) online server, which can be accessed from the NBCI official website. The sequence SEQ ID NO: 1 was analyzed by CD-Search, and the analysis results can be seen in Figure 1 , it can be seen that the receptor enzyme contains two domains: a CM corresponding to the glycoside hydrolase 10 family (Glyco_hydro_10) and a CBM belonging to the CBM_1 superfamily. The CBM is located between positions 381 and 409 of the receptor enzyme sequence. The CBM and the non-functional short peptides before and after it at positions 374 to 380 and 410 to 413, respectively, together form a deletion segment at positions 374 to 413.

[0029] The same protein structure analysis software was used to analyze the structures of various carbohydrate-active enzymes with high enzymatic activity and hydrolysis capacity, and enzymes with CBMs in their structures were selected as donor enzymes. In this example, a total of six donor enzymes were provided, and their specific information is shown in Table 1 below.

[0030]

[0031] The CBMs of the six donor enzymes were fully synthesized according to their corresponding amino acid sequences. The synthesized genes were then optimized according to the codon preference of Pichia pastoris to form nucleotide fragments. Each nucleotide fragment was then inserted between the EcoRI and NotI restriction sites of the pPIC9K plasmid to generate plasmid vectors carrying the corresponding CBM genes. The sequences and nomenclature of the donor enzymes and corresponding nucleotide fragments are shown in Table 2 below.

[0032]

[0033] The sequence of SEQ ID NO: 1 was fully synthesized, the synthesized gene was optimized according to the codon preference of Pichia pastoris, and the synthesized nucleotide fragment SEQ ID NO: 20 was inserted between the EcoRI and NotI restriction sites of the pPIC9K plasmid, thereby obtaining the plasmid vector pPIC9K-PuXyn expressing the wild-type xylanase PuXyn.

[0034] Based on the sequence information of SEQ ID NO: 14 to SEQ ID NO: 20, a variety of primers for PCR amplification of various modified xylanases were designed. The names and sequences of the primers are shown in Table 3 below.

[0035]

[0036] The plasmid vector corresponding to each donor enzyme was used as the template DNA, and KOD Fx Neo high-fidelity DNA polymerase (Toyobo Shanghai Biotechnology Co., Ltd., KFX-201) was used for amplification with primers 1 and 2. After gel recovery and purification, the nucleotide fragment encoding the CBM of each donor enzyme was formed. The PCR system is shown in Table 4 below.

[0037]

[0038] In the above PCR experiment, the corresponding relationship between the template DNA, primer 1, primer 2, and the donor enzyme corresponding to the CBM encoded by the amplified nucleotide fragment is shown in Table 5 below.

[0039]

[0040] The plasmid vector pPIC9K-PuXyn was then used as a DNA template, with primer Puc-1 as primer 1 and primer 373_ga2 as primer 2, for amplification using the same PCR system. After gel recovery and purification, the nucleotide fragment PuXyn-C corresponding to the receptor enzyme after the original CBM was deleted was generated. The plasmid vector pPIC9K-PuXyn was digested with restriction endonucleases EcoRI and NotI, and the plasmid vector gene fragment pPIC9K-B was obtained after gel recovery and purification.

[0041] Using a homologous recombination kit (Beijing Quanshijin Biotechnology Co., Ltd., CU101-01), the nucleotide fragments PuXyn-B and PuXyn-C were ligated with the nucleotide fragments encoding the CBM of each donor enzyme. The ligation products were transformed into competent Escherichia coli Top10 cells (Beijing Zhuangmeng International Biogene Technology Co., Ltd., ZC104) and plated on LBK kanamycin-resistant plates for screening. Correct transformants, identified by colony PCR, were inoculated into LB medium, and plasmids were extracted. After sequencing verification at Shanghai Sangon Biotechnology Co., Ltd., various recombinant plasmids containing modified xylanases with CBM replacements were obtained.

[0042] Buffered Glycerol-complex Medium (BMGY) and Buffered Methanol-complex Medium (BMMY) are pre-prepared. BMGY medium is primarily used for the first stage of Pichia pastoris culture, namely the cell proliferation stage. In this example, the BMGY medium consists of 1% yeast extract, 2% tryptone, 1.34% amino-free yeast nitrogen source, 10% 1 mol / L potassium phosphate buffer (pH 6.0), and 1% glycerol. After preparation, it is sterilized at high temperature and high pressure before use. All concentrations are expressed in mass-volume percentages. In BMGY medium, glycerol as a carbon source, tryptone as a nitrogen source, and various trace elements provide a rich supply of nutrients for Pichia pastoris growth. In BMMY medium, 1% (v / v) glycerol is replaced with 1% (v / v) methanol in the BMGY medium to induce Pichia pastoris to express exogenous proteins. Methanol is an inducer that can initiate exogenous gene expression. Methanol was added to BMMY medium after autoclaving.

[0043] The recombinant plasmid pPIC9K-PuXyn and various modified xylanase recombinant plasmids with CBM replaced were linearized with restriction endonuclease SalI, and various linearized vectors were purified and recovered and electroporated into Pichia pastoris GS115 competent cells purchased from Life Technologies Corporation, USA. Positive transformants were screened on histidine nutrient-deficient plates, and the obtained positive transformants were inoculated into BMGY medium. After shaking and culturing in a shaker set at 30°C and 250 rpm for 20 hours, the cells were centrifuged at 4°C and 6000 rpm for 5 minutes, the bacterial pellets were collected and resuspended in BMMY medium, and the concentration was adjusted so that the OD of the suspension was 0. 600The value of was about 0.5, and the culture was placed in the shaker again for culture and fermentation, and methanol was added to a final concentration of 1% every day. After five days of fermentation, the mixture was centrifuged again at 4°C and 6000 rpm for 5 minutes, and the supernatant was recovered to obtain an enzyme solution containing the acceptor enzyme or modified xylanase. The acceptor enzyme was named PuXyn-WT, the modified xylanase with a donor enzyme sequence of SEQ ID NO: 2 was named PuXyn-XCBM, the modified xylanase with a donor enzyme sequence of SEQ ID NO: 3 was named PuXyn-XCBM6, the modified xylanase with a donor enzyme sequence of SEQ ID NO: 4 was named PuXyn-CBM22-2, the modified xylanase with a donor enzyme sequence of SEQ ID NO: 5 was named PuXyn-CBM6, the modified xylanase with a donor enzyme sequence of SEQ ID NO: 6 was named PuXyn-CBM9-2, and the modified xylanase with a donor enzyme sequence of SEQ ID NO: 7 was named PuXyn-CrCBM2. By combining the sequences of the acceptor enzyme, the deleted segment and the CBM of each donor enzyme, the sequences of each modified xylanase were obtained, as shown in Table 6 below.

[0044]

[0045] The activity of the acceptor enzyme and each modified xylanase was tested using enzyme solution. Under pH 5.0 and 50°C water bath conditions, 100 μL of 1% beechwood xylan substrate was mixed with 100 μL of enzyme solution of the same concentration. After reacting for 30 minutes, 600 μL of DNS solution was added to terminate the reaction and the protein component in the liquid was removed by boiling in a water bath for 5 minutes. After cooling, the absorbance value A540 at a wavelength of 540 nm was measured, and the content of released reducing sugars was read against the standard curve. 1 unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions. For test results, please refer to Figure 2 It can be seen that compared with the acceptor enzyme, the specific enzyme activity of 4 mutants among the 6 modified xylanases with replaced CBM was improved, among which the PuXyn-XCBM mutant had the largest increase, reaching 1.41 times that of the acceptor enzyme, indicating that replacing CBM is an effective method to improve the catalytic activity of xylanase, and not all replacements of the CBM of carbohydrate-active enzymes with higher enzyme activity can improve the enzymatic activity of the acceptor enzyme.

[0046] The ability of the acceptor enzyme and each modified xylanase to hydrolyze wood cellulose was tested by enzyme solution.

[0047] Weigh 2.616 g of wheat straw for delignification pretreatment to yield 1 g of dry matter. Add water and mix thoroughly. Add 2.5 mL of 1 mol / L acetic acid-sodium acetate buffer and 500 U of the acceptor enzyme or any modified xylanase. Add water to 50 mL to achieve a dry matter content of 2%. The hydrolysis system was placed in a 50°C shaker water bath for 5 h. After enzymatic hydrolysis, the system was removed and separated using a 300-mesh mesh bag to obtain a first hydrolyzate. An equal volume of 8% (m / v) sulfuric acid was added to the first hydrolyzate, and the system was acid-hydrolyzed in an autoclave at 121°C for 60 min. Sodium hydroxide was added to the acid hydrolyzate to adjust the pH to a range of 5-7. The sugar content of the resulting complete hydrolyzate was determined according to the method in GB / T 35545-2017, and the sugar yield was calculated using the following formula:

[0048] Xylose yield = xylose content / dry matter content × 100%.

[0049] It should be noted that due to solvent peak interference at the elution position of xylooligosaccharides in the HPLC analysis of the first hydrolyzate, the acid hydrolysis method described above was used to hydrolyze xylooligosaccharides into xylose to characterize the degree of hemicellulose degradation. Therefore, the xylose yield after the first hydrolysis is the sum of the xylooligosaccharide yield and the xylose yield. The sugar yields obtained by hydrolysis using different xylanases are shown in Table 7 below.

[0050]

[0051] As can be seen from Table 7, the hydrolysis ability of the modified xylanase PuXyn-CBM6 was significantly improved compared with the wild-type xylanase, and the xylose yield was increased by about 1.15 times. Figure 2 The specific enzymatic activity of the modified xylanase was approximately 1.32-fold higher than that of the wild-type xylanase. These results demonstrate that by replacing the carbohydrate-binding module, mutants with enhanced catalytic activity and hydrolysis capacity can be obtained. Furthermore, this replacement requires that the portion of the acceptor enzyme after deleting the carbohydrate-binding module be highly compatible with the carbohydrate-binding module of the donor enzyme.

[0052] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0053] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A modified xylanase, characterized in that The deletion segment of the receptor enzyme is replaced by the replacement segment, the receptor enzyme is a wild-type xylanase with a sequence of SEQ ID NO: 1, the sequence of the deletion segment is positions 374 to 413 in the sequence of the receptor enzyme, and the sequence of the replacement segment is one of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:

11.

2. The modified xylanase according to claim 1, wherein The xylose yield of the modified xylanase hydrolyzing wood cellulose is greater than 4.5%.

3. A DNA molecule, characterized in that The DNA molecule encodes the modified xylanase according to claim 1 or 2.

4. A recombinant plasmid, characterized in that The recombinant plasmid comprises the DNA molecule according to claim 3.

5. A transfected cell, characterized in that Prepared by transfecting the DNA molecule as claimed in claim 3 into donor cells.

6. The transfected cell according to claim 5, characterized in that The donor cell is Pichia pastoris.

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