Oligosaccharide-debranching enzyme mutant with improved thermal stability and use thereof

Abstract

The application discloses an oligosaccharide debranching enzyme mutant with improved thermal stability and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. Paenibacillus The oligosaccharide debranching enzyme mutant is obtained by mutating the asparagine at the 360th position into lysine or mutating the glutamine at the 364th position into glutamic acid based on the starting mutant V219A / K311M / E405A of the oligosaccharide debranching enzyme derived from sp.STB16. Compared with the starting enzyme, the crude enzyme activity of the V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E mutants is increased by 20.04% and 29.08% respectively, the half-life at 50 DEG C is increased by 82.4% and 55.3% respectively, and the specific enzyme activity is also slightly increased, which indicates that the mutants have higher thermal stability while maintaining the enzyme activity.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a thermostable oligosaccharide debranching enzyme mutant and its applications. Background Technology

[0002] Oligosaccharide debranching enzymes are a class of glycoside hydrolases that can act on the branched structure of oligosaccharides, and they have application value in the directed hydrolysis of glycosidic bonds and related biocatalytic processes. Enzymatic catalysis has the advantages of high specificity and mild conditions, but during scale-up or long-term treatment, enzyme molecules usually need to maintain high structural stability and catalytic activity.

[0003] Insufficient thermal stability is one of the important factors limiting the further application of oligosaccharide debranching enzymes. During heat treatment, enzyme proteins are prone to local conformational loosening, structural disturbance near the active site, or irreversible inactivation, leading to decreased catalytic efficiency, shortened lifespan, and increased enzyme dosage.

[0004] Protein engineering modification of oligosaccharide debranching enzymes is an important means to improve their performance. Previous work has yielded the oligosaccharide debranching enzyme mutant V219A / K311M / E405A, which can serve as the basis for further thermostability modification.

[0005] However, when new amino acid substitutions are introduced based on the starting mutant V219A / K311M / E405A, the effects of the mutation site on enzyme activity and thermostability are not entirely predictable. Some mutations may lead to a decrease in crude enzyme activity or even a near-complete loss of activity. Summary of the Invention

[0006] The technical problem to be solved by this invention is: addressing the issue that there is still room for improvement in the thermal stability of the oligosaccharide debranching enzyme mutant V219A / K311M / E405A, and providing an oligosaccharide debranching enzyme mutant that improves thermal stability while maintaining enzyme activity.

[0007] In screening a group of candidate mutation sites, this invention found that mutations such as S397K, A545P, and N461K did not show an increase in enzyme activity, while N360K and Q364E significantly improved thermal stability while maintaining or increasing enzyme activity. Therefore, obtaining oligosaccharide debranching enzyme mutants with both high enzyme activity and good thermal stability is of practical significance.

[0008] The present invention also provides a nucleic acid molecule encoding the oligosaccharide debranching enzyme mutant, as well as a recombinant plasmid, gene expression cassette and host cell carrying the nucleic acid molecule.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides an oligosaccharide debranching enzyme mutant, wherein the oligosaccharide debranching enzyme mutant is derived from amino acids with the amino acid sequence shown in SEQ ID NO.1. Paenibacillus This was obtained by single-point mutation based on the oligosaccharide debranching mutant V219A / K311M / E405A of sp. STB16.

[0010] The amino acid sequence of the oligosaccharide debranching enzyme has at least 80% identity with the amino acid shown in SEQ ID NO.1 and has oligosaccharide debranching activity.

[0011] The amino acid sequence of V219A / K311M / E405A is shown below (SEQ ID NO.1): MLLFPFESRSRSIPTGGWQMKRAWWKESVVYQIYPRSFQDSNGDGIGDIPGIVSRLDYLQELGVDVVWLCPVYDSPNDDNGYDIRDYRRIMDEFGTLEDWERLLEDLHARGMKLIMDLVVNHSSDEHAWFSESRKSRDGEHRDY YIWRDGKGGAEPNNWSSFFSGSAWKYDGETDQYYLHLFSSKQPDLNWENGKVRREVYNMMAWWLDKGIDGFRMDAINLISKVPGLPDAPGEGRYRSGADYFMNGPRVHEYLQEMNREVLSRYDIMTVGETPGVTPEQAALYVGE DRGELNMVFQFEHMDIDSGPGGMWDVQPWRLTDFKRVMGKWQRELQDRGWNSLYLNNHDQPRMVSRFGDDKNFRKQSAKMLGTLLHTLQGTPYIYQGEELGMTNVRFGSIEDYRDIATLNMYKEATGAGRPAEAVMASVYSKGR DNARTPMQWDGSAHGGFTTGTPWIASNPNYTEINAEDARRDPDSIFHYYRRLIALRKQHDVIVYGRYEALLEEDERIYAYTRMLDGERLLVVLNFFGEEADCSLPEKIRFESAEPLIGNYGNGADRDWRSLKLRPYEALVLRLQG

[0012] The nucleotide sequence encoding V219A / K311M / E405A is shown below (SEQ ID NO.4): In one embodiment of the present invention, the single-point mutation is N360K, that is: based on the oligosaccharide debranching enzyme mutant V219A / K311M / E405A with an amino acid sequence as shown in SEQ ID NO.1, the asparagine at position 360 is mutated to lysine to obtain the V219A / K311M / E405A / N360K mutant.

[0013] The amino acid sequence of V219A / K311M / E405A / N360K (SEQ ID NO.2): MLLFPFESRSRSIPTGGWQMKRAWWKESVVYQIYPRSFQDSNGDGIGDIPGIVSRLDYLQELGVDVVWLCPVYDSPNDDNGYDIRDYRRIMDEFGTLEDWERLLEDLHARGMKLIMDLVVNHSSDEHAWFSESRKSR DGEHRDYYIWRDGKGGAEPNNWSSFFSGSAWKYDGETDQYYLHLFSSKQPDLNWENGKVRREVYNMMAWWLDKGIDGFRMDAINLISKVPGLPDAPGEGRYRSGADYFMNGPRVHEYLQEMNREVLSRYDIMTVGETP GVTPEQAALYVGEDRGELNMVFQFEHMDIDSGPGGMWDVQPWRLTDFKRVMGKWQRELQDRGWNSLYLNNHDQPRMVSRFGDDKKFRKQSAKMLGTLLHTLQGTPYIYQGEELGMTNVRFGSIEDYRDIATLNMYKE ATGAGRPAEAVMASVYSKGRDNARTPMQWDGSAHGGFTTGTPWIASNPNYTEINAEDARRDPDSIFHYYRRLIALRKQHDVIVYGRYEALLEEDERIYAYTRMLDGERLLVVLNFFGEEADCSLPEKIRFESAEPLIG NYGNGADRDWRSLKLRPYEALVLRLQG

[0014] In one embodiment of the present invention, the single-point mutation is Q364E, that is: based on the oligosaccharide debranching enzyme mutant V219A / K311M / E405A with an amino acid sequence as shown in SEQ ID NO.1, the glutamine at position 364 is mutated to glutamic acid to obtain the V219A / K311M / E405A / Q364E mutant.

[0015] The amino acid sequence of V219A / K311M / E405A / Q364E (SEQ ID NO.3): MLLFPFESRSRSIPTGGWQMKRAWWKESVVYQIYPRSFQDSNGDGIGDIPGIVSRLDYLQELGVDVVWLCPVYDSPNDDNGYDIRDYRRIMDEFGTLEDWERLLEDLHARGMKLIMDLVVNHSSDEHAWFSESRKSR DGEHRDYYIWRDGKGGAEPNNWSSFFSGSAWKYDGETDQYYLHLFSSKQPDLNWENGKVRREVYNMMAWWLDKGIDGFRMDAINLISKVPGLPDAPGEGRYRSGADYFMNGPRVHEYLQEMNREVLSRYDIMTVGETP GVTPEQAALYVGEDRGELNMVFQFEHMDIDSGPGGMWDVQPWRLTDFKRVMGKWQRELQDRGWNSLYLNNHDQPRMVSRFGDDKNFRKESAKMLGTLLHTLQGTPYIYQGEELGMTNVRFGSIEDYRDIATLNMYKE ATGAGRPAEAVMASVYSKGRDNARTPMQWDGSAHGGFTTGTPWIASNPNYTEINAEDARRDPDSIFHYYRRLIALRKQHDVIVYGRYEALLEEDERIYAYTRMLDGERLLVVLNFFGEEADCSLPEKIRFESAEPLIG NYGNGADRDWRSLKLRPYEALVLRLQG

[0016] In one embodiment of the present invention, the present invention provides a nucleic acid molecule encoding the above-mentioned oligosaccharide debranching enzyme mutant.

[0017] The nucleic acid molecule may be a codon-optimized or uncodon-optimized nucleic acid molecule that can encode the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.3.

[0018] In one embodiment of the present invention, the present invention provides a recombinant plasmid or gene expression cassette carrying the above-mentioned nucleic acid molecules.

[0019] In one embodiment of the present invention, the recombinant plasmid uses pP43NMK as the expression vector backbone.

[0020] In one embodiment of the present invention, the recombinant expression vector is a pET series vector, Duet series vector, pGEX series vector, or pTrc series vector.

[0021] In one embodiment of the present invention, the pET series carrier is pET-24a, pET-28a, pET-29a or pET-30a.

[0022] In one embodiment of the present invention, the Duet series carrier is pRSFDuet-1 or pCDFDuet-1.

[0023] In one embodiment of the present invention, the pTrc series carrier is pTrc99a.

[0024] The present invention provides recombinant microbial cells expressing the above-mentioned oligosaccharide debranching enzyme mutant, or host cells containing the above-mentioned recombinant plasmid or gene expression cassette or recombinant plasmid.

[0025] In one embodiment of the present invention, the host cell is Bacillus subtilis WB600.

[0026] In this invention, the amino acid site numbering is based on the amino acid sequence corresponding to the starting oligosaccharide debranching enzyme mutant V219A / K311M / E405A; the N360K and Q364E mutations may improve the conformational stability of the enzyme protein under heat treatment conditions by introducing charge changes and enhancing local polarity or electrostatic interaction networks.

[0027] The present invention also provides recombinant microbial cells that express the above-mentioned oligosaccharide debranching enzyme mutant, or carry the above-mentioned gene, or carry the above-mentioned recombinant expression vector.

[0028] In one embodiment of the present invention, the recombinant microbial cells use bacteria or fungi as host cells.

[0029] In one embodiment of the present invention, the recombinant microbial cells use Escherichia coli or Bacillus subtilis as host cells.

[0030] In one embodiment of the present invention, the Escherichia coli is Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli BL21, Escherichia coli S17-1 λpir, Escherichia coli MG1655, Escherichia coli W3110 or Escherichia coli MC1061.

[0031] The present invention also provides a microbial inoculant containing the above-mentioned recombinant microbial cells.

[0032] The present invention also provides a method for improving the enzyme activity or thermal stability of oligosaccharide debranching enzymes, wherein the method comprises mutating asparagine at position 360 to lysine, or glutamine at position 364 to glutamic acid, based on the oligosaccharide debranching enzyme mutant V219A / K311M / E405A with an amino acid sequence as shown in SEQ ID NO.1.

[0033] This invention also provides a method for preparing glucose, the method utilizing glucose mother liquor to prepare glucose, the method comprising: Prepare a glucose stock solution to a mass fraction of 30% (w / w, on a dry basis), equilibrate at 55–60°C for 15–20 min, adjust the pH of the system to 5.5–6.0, and simultaneously add oligosaccharide debranching enzyme mutant V219A / K311M / E405A / N360K or V219A / K311M / E405A / Q364E and amylase. The amount of oligosaccharide debranching enzyme mutant V219A / K311M / E405A / N360K or V219A / K311M / E405A / Q364E added is 20–30 U / g substrate, and the amount of amylase added is 100–120 U / g. The substrate was subjected to a two-enzyme synergistic reaction at 55-60℃ and 200-220r / min for 24-30 h.

[0034] In one embodiment of the present invention, a glucose mother liquor is prepared to a mass fraction of 30% (w / w, on a dry basis), equilibrated at 55°C for 15 min, and the pH of the system is adjusted to 5.5. Simultaneously, oligosaccharide debranching enzyme mutant V219A / K311M / E405A / N360K or oligosaccharide debranching enzyme mutant V219A / K311M / E405A / Q364E and amylase are added. The amount of oligosaccharide debranching enzyme mutant V219A / K311M / E405A / N360K or oligosaccharide debranching enzyme mutant V219A / K311M / E405A / Q364E added is 20 U / g substrate, and the amount of amylase added is 100 U / g substrate. The two enzymes are subjected to a synergistic reaction at 55°C and 200 r / min for 24 h.

[0035] The present invention also provides a recombinant Bacillus subtilis, wherein the recombinant Bacillus subtilis expresses the oligosaccharide debranching enzyme mutant.

[0036] In one embodiment of the present invention, the recombinant Bacillus subtilis is... Bacillus subtilis WB600 is the expression host.

[0037] In one embodiment of the present invention, the recombinant Bacillus subtilis uses pP43NMK as an expression vector.

[0038] The present invention also provides the use of the above-mentioned oligosaccharide debranching enzyme mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cell, or the above-mentioned microbial agent in the preparation of glucose or products containing glucose.

[0039] Beneficial effects Compared with the oligosaccharide debranching enzyme mutant V219A / K311M / E405A, the preferred mutant of the present invention shows improved crude enzyme activity, specific enzyme activity and half-life at 50°C.

[0040] (1) Increased crude enzyme activity: The crude enzyme activity of the starting enzyme V219A / K311M / E405A was 63.20 U / mL; the crude enzyme activity of V219A / K311M / E405A / N360K was 75.87 U / mL, which was 20.04% higher than that of the starting enzyme; the crude enzyme activity of V219A / K311M / E405A / Q364E was 81.58 U / mL, which was 29.08% higher than that of the starting enzyme.

[0041] (2) Increased specific enzyme activity: After purification, the specific enzyme activity of the starting enzyme was 239.36 U / mg; the specific enzyme activity of the N360K mutant was 248.43 U / mg, which was 3.79% higher than that of the starting enzyme; the specific enzyme activity of the Q364E mutant was 249.33 U / mg, which was 4.17% higher than that of the starting enzyme.

[0042] (3) Improved thermal stability: At 50℃, the half-life of the starting enzyme was 10.68 min; the half-life of the N360K mutant was 19.47 min, which was 82.4% higher than that of the starting enzyme; and the half-life of the Q364E mutant was 16.58 min, which was 55.3% higher than that of the starting enzyme.

[0043] (4) The screening results are not obvious: Among the same group of mutations, the crude enzyme activity of mutations such as S136K, S397K, N461K, and A545P decreased or no obvious enzyme activity was detected, indicating that not all amino acid substitutions can improve enzyme activity or stability. The N360K and Q364E mutants obtained by screening in this invention have clear technical effects.

[0044] The above data fully demonstrate that the mutant obtained in this invention maintains good catalytic activity while also exhibiting significantly enhanced thermal stability. This invention possesses the following outstanding advantages: it achieves a synergistic enhancement of enzyme activity and thermal stability through a site-directed mutagenesis strategy; the technical solution is clear and reproducible, possessing a solid foundation for industrial application; the mutation sites are clearly defined and the effects are significant, providing a new and beneficial combination of sites for the molecular improvement of oligosaccharide debranching enzymes, exhibiting strong technical barriers and originality; the dual enhancement of thermal stability and enzyme activity can significantly reduce the production and use costs of industrial enzyme preparations, expanding their large-scale application prospects in food, feed, and biotransformation fields. Therefore, this invention not only possesses outstanding practicality, advanced technology, and industrialization potential. Attached Figure Description

[0045] Figure 1 The image shows the screening results of crude enzyme activity for single-point mutants of different oligosaccharide debranching enzymes.

[0046] Figure 2 The graph shows the residual enzyme activity changes of oligosaccharide debranching enzyme mutants and preferred mutants at 50°C.

[0047] Figure 3 Electrophoresis diagram of crude enzyme solutions of different oligosaccharide debranching enzyme mutants. Detailed Implementation

[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0049] Technical terms Expression: The term “expression” includes any step involving the production of oligosaccharide debranching enzymes, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0050] Expression vector: The term "expression vector" refers to a straight or circular DNA molecule containing a polynucleotide encoding the oligosaccharide debranching enzyme of the present invention and operatively linked to a control sequence provided for its expression.

[0051] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. Host cells can be any cell type useful in the production of oligosaccharide debranching enzymes, such as prokaryotic or eukaryotic cells. Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus spp. Bacillus Clostridium ( Clostridium ), Enterococcus spp. Enterococcus ), Bacillus spp. Geobacillus Lactobacillus () LactobacillusLactococcus spp. Lactococcus ), Bacillus spp. ( Oceanobacillus Staphylococcus spp. Staphylococcus Streptococcus spp. Streptococcus ) and Streptomyces ( Streptomyces Gram-negative bacteria include, but are not limited to, Campylobacter spp. Campylobacter ), Escherichia coli ( E. coli Flavobacterium ( Flavobacterium ), Fusobacterium spp. Fusobacterium ), Helicobacter spp. Helicobacter ), Colistinia spp. ( Mycobacterium ), Neisseria spp. Neisseria ), Pseudomonas spp. Pseudomonas Salmonella ( Salmonella ), and Ureaplasma genus ( Ureaplasma The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0052] Mutant: As used herein, when referring to variations of the invention, the terms "mutant," "peptide variant," "peptide," or "oligosaccharide debranching enzyme mutant" mean a polypeptide having oligosaccharide debranching enzyme activity and containing alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "parental" oligosaccharide debranching enzyme. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. In the description of variations of the invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used. Substitution: For amino acid substitution, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of arginine at position 88 with glycine is represented as "R88G." Multiple mutations are separated by a symbol (" / "), for example, "R88G / K62P" means that arginine (R) and lysine (K) at positions 88 and 62 are replaced by glycine (G) and proline (P), respectively.

[0053] Amino acid abbreviations: Alanine (Ala); Cysteine ​​(Cys); Aspartic acid (D Asp); Glutamic acid (Glu); Phenylalanine (Phe); Glycine (Gly); Histidine (His); Isoleucine (Ile); Lysine (Lys); Leucine (L) Methionine (M, Met); Asparagine (N, Asn); Proline (P, Pro); Glutamine (Q, Gln); Arginine (R, Arg); Serine (S, Ser); Threonine (T, Thr); Valine (V, Val); Tryptophan (W, Trp); Tyrosine (Y, Tyr). Wild-type enzyme: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). When the parent enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parent enzyme" are used interchangeably.

[0054] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0055] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) is used to determine sequence identity between two amino acid sequences. This algorithm is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277) (preferably version 5.0.0 or later). The parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle-labeled "longest identity" output (obtained using the -nobrief option) is used as the identity percentage and calculated as follows: (identical residues x 100) / (alignment length - total number of vacancies in the alignment) Alternatively, the parameters used can be a vacancy open penalty of 10, a vacancy extension penalty of 0.5, and EDNAFULL (the EMBOSS version of NCBI NUC4.4) to replace the matrix. The output of "Longest Identity" marked with Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows: (identical deoxyribonucleotides × 100) / (alignment length – total number of gaps in the alignment).

[0056] It should be noted that the abbreviations of amino acid residues in this article follow the general definition in this field, but in some instances, the full name is still indicated in parentheses after the abbreviation. In this article, "amino acid residue" and "amino acid" can be used interchangeably. The amino acid sequence in this article starts from the N-terminus.

[0057] The test methods involved in the following embodiments: Methods for determining enzyme activity in fermentation broth: The expression effects and enzyme activities of different oligosaccharide debranching enzyme mutants were compared using the fermentation broth enzyme activity of the oligosaccharide debranching enzyme mutant V219A / K311M / E405A as a control.

[0058] The enzyme activity of oligosaccharide debranching enzyme mutants was determined using the pNPG (p-nitrobenzene-α-D-glucoside) method. Specifically, 10 mM pNPG was prepared as the substrate, and 500 mM PBS solution at pH 6.0 was prepared as the buffer. 800 μL of pNPG and 150 μL of PBS were added to a 5 mL centrifuge tube, and the mixture was preheated at 50 °C for 15 min. Then, 50 μL of serially diluted enzyme solution was added, and the reaction was timed precisely for 5 min (reaction temperature: 50 °C). Immediately afterward, 1 mL of stop solution (1 M Na₂CO₃ solution) was added, and the absorbance was measured at 410 nm. The released p-nitrophenyl (pNP) was determined spectrophotometrically. The enzyme solution inactivated at high temperature served as a blank control.

[0059] Enzyme activity definition: Under the above conditions, the amount of enzyme required to hydrolyze and generate 1 μmol of p-nitrophenol (pNP) per minute is defined as 1 unit of enzyme activity (U). For specific enzyme activity assays, the purified enzyme solution is tested for enzyme activity according to the above method, and the protein concentration is determined using the BCA method. Specific enzyme activity is calculated as the ratio of enzyme activity to protein mass, expressed in U / mg.

[0060] Methods for determining thermal stability and half-life at 50℃: The purified enzyme solution was incubated at 50℃, and samples were taken at different time points and rapidly cooled. The residual enzyme activity was then determined using the method described above. Using the activity of the untreated enzyme solution as 100%, the residual enzyme activity at each time point was calculated, and the half-life t1 / 2 (50℃) was calculated using first-order inactivation kinetics fitting. Specific sampling time points can be supplemented based on the original experimental records.

[0061] Crude enzyme protein electrophoresis detection method: The supernatant from the fermentation of each recombinant Bacillus subtilis was used as the crude enzyme solution sample. This sample was mixed with SDS-PAGE loading buffer, heat-treated, and then loaded onto the plate. SDS-PAGE was used to analyze the protein composition of the crude enzyme solution. After electrophoresis, the gel was stained and destained to observe the expression of target protein bands. The electrophoresis results were then used as the basis for analysis. Figure 3 Specific gel concentration, loading volume, and protein molecular weight standards can be supplemented based on actual electrophoresis records.

[0062] Analysis of saccharification reaction products Oligosaccharide debranching enzyme mutants were applied to the glucose enzymatic production process, and the content of each component in the product was analyzed by high-performance liquid chromatography (HPLC). The analytical conditions were: a ChromCore normal-phase / HILIC column, and 70% (v / v) acetonitrile. The mobile phase was aqueous solution, the flow rate was set to 1.0 mL / min, the column temperature was 30 °C, and the injection volume was 10 μL.

[0063] Raw materials used in the examples: Materials and reagents: The plasmid vector used in this embodiment is pP43NMK, which is used to construct the Bacillus subtilis expression plasmid; the cloning host strain is... Escherichia coli JM109, the expression host strain is Bacillus subtilis WB600. The recombinant plasmid pP43NMK-V219A / K311M / E405A containing the parental enzyme gene was constructed and preserved in our laboratory. The parental enzyme is derived from... Paenibacillus OGA, an oligosaccharide debranching enzyme of sp. STB16. The expression vector was pP43NMK or a functionally equivalent vector; the substrate for assay was p-nitrobenzene-α-D-glucoside (pNPG).

[0064] The plasmid mini-extraction kit, DNA gel recovery kit, agarose gel purification kit, DNA standards, TAE buffer, SDS-PAGE gel electrophoresis preparation kit, Tris-HCl buffer, and antibiotics such as kanamycin used were all commercially available products. The high-fidelity DNA polymerase, dNTP mixture, PCR buffer, and DpnI enzyme used for PCR amplification were all commercially available molecular biology reagents. The primers used were synthesized by a biotechnology company. The raw materials and quantities involved in the examples should be confirmed to comply with relevant laws, regulations, and laboratory management standards.

[0065] The amylase involved is a commercially available product. Aladdin or Merck products can be used. As long as the amount added is guaranteed, the technical effect of this invention can be achieved.

[0066] The culture media involved in the following examples are as follows: All culture media were prepared using ddH2O and sterilized at 115℃ for 15-20 min after preparation.

[0067] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L.

[0068] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar powder 15 g / L.

[0069] TB fermentation medium: yeast extract 24 g / L, tryptone 12 g / L, glycerol 5 g / L, K2HPO4·3H2O 16.43 g / L, KH2PO4 2.32 g / L.

[0070] Kanamycin was added to the culture medium according to the expression vector resistance, with a final concentration of 20 μg / mL.

[0071] The fermentation production method of the strains involved in the following examples is as follows: Recombinant Bacillus subtilis containing different oligosaccharide debranching enzyme mutant genes were inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 12 h to obtain seed culture. Subsequently, the seed culture was inoculated into TB fermentation medium at a 4% (v / v) inoculation rate and fermented at 30℃ and 200 rpm for 96 h. After fermentation, the fermentation broth was centrifuged at 10000 rpm for 10 min, and the supernatant was collected as crude enzyme solution for enzyme activity assay.

[0072] Example 1: Design of mutation sites for oligosaccharide debranching enzyme mutants and construction of expression vectors containing the mutants. This embodiment is derived from... Paenibacillus The parent enzyme is the starting mutant V219A / K311M / E405A of the oligosaccharide debranching enzyme OGA of sp. STB16.

[0073] The amino acid sequence of V219A / K311M / E405A is shown below (SEQ ID NO.1): MLLFPFESRSRSIPTGGWQMKRAWWKESVVYQIYPRSFQDSNGDGIGDIPGIVSRLDYLQELGVDVVWLCPVYDSPNDDNGYDIRDYRRIMDEFGTLEDWERLLEDLHARGMKLIMDLVVNHSSDEHAWFSESRKSRDGEHRDY YIWRDGKGGAEPNNWSSFFSGSAWKYDGETDQYYLHLFSSKQPDLNWENGKVRREVYNMMAWWLDKGIDGFRMDAINLISKVPGLPDAPGEGRYRSGADYFMNGPRVHEYLQEMNREVLSRYDIMTVGETPGVTPEQAALYVGE DRGELNMVFQFEHMDIDSGPGGMWDVQPWRLTDFKRVMGKWQRELQDRGWNSLYLNNHDQPRMVSRFGDDKNFRKQSAKMLGTLLHTLQGTPYIYQGEELGMTNVRFGSIEDYRDIATLNMYKEATGAGRPAEAVMASVYSKGR DNARTPMQWDGSAHGGFTTGTPWIASNPNYTEINAEDARRDPDSIFHYYRRLIALRKQHDVIVYGRYEALLEEDERIYAYTRMLDGERLLVVLNFFGEEADCSLPEKIRFESAEPLIGNYGNGADRDWRSLKLRPYEALVLRLQG

[0074] Based on the analysis of the parent enzyme's amino acid sequence, local structural features, and the properties of candidate site amino acids, serine at position 136, asparagine at position 360, glutamine at position 364, serine at position 397, asparagine at position 461, serine at position 535, alanine at position 545, and serine at position 562 were selected as candidate mutation sites. The design of these candidate sites primarily focused on the local structural stability of the enzyme molecule. Mutation strategies included introducing charged amino acids to enhance potential salt bridges or electrostatic interactions, introducing lysine to improve local polar interactions, and introducing proline to restrict local flexible conformations. Since there may be mutual constraints between the thermostability, expression level, and catalytic activity of oligosaccharide debranching enzymes, the above candidate mutants need further screening through expression and enzyme activity tests to determine the preferred mutants.

[0075] 1. The specific construction method is as follows: (1) Construction of recombinant vector expressing V219A / K311M / E405A Using the recombinant plasmid pET-28a-V219A / K311M / E405A (described in CN120424914A) containing the target gene as a template, amplification primers with homologous arms were designed based on the target gene sequence and the linker region of the pP43NMK expression vector. The target gene fragment was obtained by reverse PCR amplification. Simultaneously, using the pP43NMK plasmid as a template, linearization primers were designed, and the linearized pP43NMK vector fragment was obtained by reverse PCR amplification. After agarose gel electrophoresis, the PCR amplification products were detected, and the target gene fragment and the linearized vector fragment were recovered and purified. Subsequently, using the homologous sequences between the ends of the target gene fragment and the ends of the linearized pP43NMK vector, the target gene was ligated into the pP43NMK expression vector via homologous recombination to obtain the recombinant plasmid pP43NMK-V219A / K311M / E405A.

[0076] (2) Using the recombinant plasmid pP43NMK-V219A / K311M / E405A containing the starting oligosaccharide debranching enzyme mutant gene as a template, full plasmid PCR amplification was performed using the site-directed mutagenesis primers containing the mutation sites shown in Table 1. The mutant bases were located in the middle region of the primers, and the two ends of the primers were complementary to the template sequences to ensure that the amplification products could cover the complete expression plasmid and introduce the target mutation.

[0077] The serine at position 136 was mutated to lysine to obtain the S136K mutant (V219A / K311M / E405A / S136K). The asparagine at position 360 was mutated to lysine to obtain the N360K mutant (V219A / K311M / E405A / N360K). The glutamine at position 364 was mutated to glutamic acid to obtain the Q364E mutant (V219A / K311M / E405A / Q364E). The serine at position 397 was mutated to lysine to obtain the S397K mutant (V219A / K311M / E405A / S397K). The asparagine at position 461 was mutated to lysine to obtain the N461K mutant (V219A / K311M / E405A / N461K). The serine at position 535 was mutated to lysine to obtain the S535K mutant (V219A / K311M / E405A / S535K). The alanine at position 545 was mutated to proline to obtain the A545P mutant (V219A / K311M / E405A / A545P). The serine at position 562 was mutated to aspartic acid to obtain the S562D mutant (V219A / K311M / E405A / S562D).

[0078] The site-directed mutagenesis primers for each mutation site are shown in Table 1.

[0079] Table 1: Site-directed mutagenesis primer sequences

[0080] The PCR reaction system includes template plasmid, forward primer, reverse primer, high-fidelity DNA polymerase, dNTP primer, PCR buffer, and sterile water.

[0081] The PCR system consisted of: 25 μL high-fidelity DNA polymerase, 2 μL forward primer, 2 μL reverse primer, 1 μL template plasmid, and 20 μL sterile water.

[0082] 2: Construction of expression plasmid for oligosaccharide debranching enzyme mutant After PCR amplification, the amplification products were digested with DpnI enzyme to remove methylated parental template plasmids; subsequently, the digestion products were transformed. Escherichia coli JM109 competent cells were plated on LB agar containing kanamycin and incubated upside down at 37°C. Single clones were picked for colony PCR identification, and positive clones were sent for sequencing verification. After sequencing confirmed the presence of the target mutation and the absence of additional mutations, recombinant plasmids were extracted to obtain recombinant expression plasmids for each mutant.

[0083] Example 2: Expression and screening of crude enzyme activity of oligosaccharide debranching enzyme mutants The recombinant expression plasmids of the correctly sequenced mutants obtained in Example 1 were transformed respectively. Bacillus subtilis WB600 competent cells were used to screen and obtain recombinant Bacillus subtilis expressing different oligosaccharide debranching enzyme mutants.

[0084] Recombinant Bacillus subtilis containing different oligosaccharide debranching enzyme mutant genes were inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 12 h to obtain seed culture. Subsequently, the seed culture was inoculated into TB fermentation medium at a 4% (v / v) inoculation rate and fermented at 30℃ and 200 rpm for 96 h. After fermentation, the resulting fermentation broth was centrifuged at 10000 rpm for 10 min, and the supernatant was collected as crude enzyme solution for enzyme activity assay.

[0085] The fermentation supernatant was prepared as crude enzyme solution according to the above fermentation production method, and the crude enzyme activity was determined by the pNPG method.

[0086] The results of crude enzyme activity screening are shown in Table 2. Figure 1 , Figure 3 ).

[0087] Table 2: Screening results of crude enzyme activity of different oligosaccharide debranching enzyme mutants

[0088] The results show: Using the starting enzyme V219A / K311M / E405A as a control, the crude enzyme activities of the V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E mutants increased to 75.87 U / mL and 81.58 U / mL, respectively, which were 1.20 times and 1.29 times that of the starting enzyme; most other mutants did not show significant improvement.

[0089] Table 2 lists the results of the candidate mutants whose crude enzyme activity has been determined in this round. Based on these screening results, V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E, with crude enzyme activity higher than the starting enzyme and a significant increase, were selected as preferred mutants for subsequent purification, enzyme activity comparison, and thermostability evaluation.

[0090] Example 3: Purification, enzyme activity and thermal stability determination of the preferred mutant Based on the crude enzyme activity screening results of Example 2, two mutants, V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E, were selected for further purification.

[0091] Following the method in Example 2, crude enzyme solutions of V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E were prepared respectively. Purification of crude enzyme solution: Oligosaccharide debranching enzymes were purified in two steps using an ion-exchange column and a hydrophobic column. The first step involved ion-exchange column purification: the equilibration buffer (Solution A, pH=7.0) was a 20 mM Tris-HCl buffer, and the eluent (Solution B, pH=7.0) was a 20 mM Tris-HCl buffer containing 1 M NaCl. The crude enzyme solution was adjusted to pH=7.0. The ion-exchange column was equilibrated with buffer A, and then the target protein was loaded onto the column. After loading, elution was performed with buffer B, and the corresponding eluent was collected based on the elution peaks. The second step involved hydrophobic column purification: the equilibration buffer (Solution A) was a pH 7.0, 20 mM Tris-HCl buffer containing 20% ​​saturated (NH4)2SO4, and the eluent (Solution B) was pure water. The enzyme solution obtained in the previous step was then added to a final concentration of 20% saturated (NH4)2SO4. First, equilibrate the hydrophobic column with buffer A and load the sample; then elute with buffer B (pure water), and collect the corresponding eluent based on the elution peak to obtain the purified oligosaccharide debranching enzyme.

[0092] Pure enzyme solutions of V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E were obtained, and protein concentration was determined by the BCA method. Specific enzyme activity and half-life at 50℃ were then calculated. The results are shown in Table 3. Figure 2 ).

[0093] Table 3: Specific enzyme activity and half-life at 50°C of the starting enzyme and the preferred mutant

[0094] Compared with the starting enzyme, the V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E mutants showed slightly increased specific enzyme activity and significantly prolonged half-life at 50℃, indicating that the mutations can improve thermal stability without reducing catalytic activity.

[0095] Example 4: Structural analysis of mutation sites Structural analysis was performed on the mutation sites of oligosaccharide debranching enzyme mutants V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E.

[0096] The V219A / K311M / E405A / N360K mutation replaces neutral asparagine at position 360 with a positively charged lysine residue, which may enhance its local interaction with neighboring acidic or polar residues, thereby improving local structural stability.

[0097] The V219A / K311M / E405A / Q364E mutation replaces glutamine at position 364 with glutamate, potentially enhancing the polar or electrostatic interaction network in this region. The above structural analysis is used to interpret the experimental results; specific interaction distances and conformational changes can be further supplemented based on structural models.

[0098] Example 5: Application of the mutant in glucose mother liquor Oligosaccharide debranching enzyme mutants V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E were used in synergistic treatment with amyloglucosidase for the enzymatic hydrolysis of glucose mother liquor. The preparation method of the oligosaccharide debranching enzyme mutants V219A / K311M / E405A / N360K and V219A / K311M / E405A / Q364E was the same as in Example 3.

[0099] Specifically, using glucose mother liquor as the substrate, the glucose mother liquor was diluted to a substrate mass fraction of 30% (w / w, on a dry basis), placed in a four-necked flask, and equilibrated in a constant temperature water bath at 55°C for 15 min. The pH of the reaction system was then adjusted to 5.5.

[0100] Based on the dry weight of the substrate, add 20 U / g of oligosaccharide debranching enzyme mutant V219A / K311M / E405A / N360K or V219A / K311M / E405A / Q364E and 100 U / g of amylase (commercially available product) to the above reaction system, and carry out the synergistic reaction of the two enzymes at 55°C and a stirring speed of 200 r / min for 24 h.

[0101] After the reaction was completed, the reaction solution was placed in a boiling water bath for 20 minutes to inactivate the enzyme, and the enzymatically hydrolyzed glucose mother liquor was obtained.

[0102] High-performance liquid chromatography (HPLC) was used to analyze the sugar components in the reaction solution. The chromatographic analysis was performed using a Hypersil APS2 column with a 70% (v / v) acetonitrile aqueous solution as the mobile phase, a column temperature of 30 °C, and a flow rate of 1.0 mL / min.

[0103] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An oligosaccharide debranching enzyme mutant, characterized in that, The oligosaccharide debranching enzyme mutant is obtained by mutating asparagine at position 360 to lysine, or glutamine at position 364 to glutamic acid, based on the oligosaccharide debranching enzyme mutant V219A / K311M / E405A with the amino acid sequence shown in SEQ ID NO.

1.

2. The gene encoding the oligosaccharide debranching enzyme mutant of claim 1.

3. A recombinant expression vector carrying the gene of claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is a pET series vector, Duet series vector, pGEX series vector, or pTrc series vector. Preferably, the pET series carrier is pET-24a, pET-28a, pET-29a or pET-30a; Preferably, the Duet series carrier is pRSFDuet-1 or pCDFDuet-1; Preferably, the pTrc series carrier is pTrc99a.

5. Recombinant microbial cells expressing the oligosaccharide debranching enzyme mutant of claim 1.

6. A recombinant microbial cell carrying the gene of claim 2.

7. Recombinant microbial cells carrying the recombinant expression vector of claim 3 or 4.

8. The recombinant microbial cell according to any one of claims 5 to 7, characterized in that, The recombinant microbial cells use bacteria or fungi as host cells.

9. The recombinant microbial cell according to claim 8, characterized in that, The recombinant microbial cells use Escherichia coli or Bacillus subtilis as host cells.

10. The recombinant microbial cell according to claim 9, characterized in that, The Escherichia coli is Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli BL21, Escherichia coli S17-1 λpir, Escherichia coli MG1655, Escherichia coli W3110 or Escherichia coli MC1061.

11. A microbial inoculant containing any one of the recombinant microbial cells as described in claims 5 to 10.

12. A method for improving the enzymatic activity or thermal stability of oligosaccharide debranching enzymes, characterized in that, The method involves mutating asparagine at position 360 to lysine, or glutamine at position 364 to glutamic acid, based on the oligosaccharide debranching mutant V219A / K311M / E405A with an amino acid sequence as shown in SEQ ID NO.

1.

13. The use of the oligosaccharide debranching enzyme mutant of claim 1, the recombinant microbial cell of any one of claims 5 to 10, or the microbial agent of claim 11 in the hydrolysis of oligosaccharides into glucose.

14. The application according to claim 13, characterized in that, The oligosaccharides include one or more of maltose, maltotriose, isomaltose, and isomalttriose.

15. A method for hydrolyzing oligosaccharides into glucose, characterized in that, The oligosaccharide debranching enzyme mutant of claim 1, the recombinant microbial cells of any one of claims 5 to 10, or the microbial agent of claim 11 are added to the reaction system with oligosaccharides as the substrate.

16. The method according to claim 15, characterized in that, The oligosaccharides include one or more of maltose, maltotriose, isomaltose, and isomalttriose.

17. A recombinant Bacillus subtilis, characterized in that, The recombinant Bacillus subtilis expressed the oligosaccharide debranching enzyme mutant of claim 1.

18. The recombinant Bacillus subtilis according to claim 17, characterized in that, The recombinant Bacillus subtilis is... Bacillus subtilis WB600 is the expression host.

19. The recombinant Bacillus subtilis according to claim 17 or 18, characterized in that, The recombinant Bacillus subtilis was expressed using pP43NMK as the expression vector.

20. The use of the oligosaccharide debranching enzyme mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3 or 4, or the recombinant microbial cell of any one of claims 5 to 10, or the microbial agent of claim 11 in the preparation of glucose or products containing glucose.

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