Sucrose phosphorylase mutants, nucleic acids encoding same, methods of making, vectors and cells containing the nucleic acids, enzyme preparations containing same, and methods of making glucose-1-phosphate

Abstract

The application relates to the technical field of bioengineering, in particular to a sucrose phosphorylase mutant, a coding nucleic acid and a preparation method thereof, a carrier and a cell containing the coding nucleic acid, an enzyme preparation containing the sucrose phosphorylase mutant, and a preparation method of glucose-1-phosphate. The sucrose phosphorylase mutant has an F156G amino acid substitution relative to a wild-type sucrose phosphorylase. The mutant has a significantly improved enzyme activity relative to the wild-type enzyme, and can be used for preparing glucose-1-phosphate.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to sucrose phosphorylase mutants and their encoding nucleic acids and preparation methods, vectors and cells containing the encoded nucleic acids, enzyme preparations containing them, and methods for preparing glucose-1-phosphate. Background Technology

[0002] Sucrose phosphorylase (SPase, EC 2.4.1.7) belongs to the GH13 family of glycoside hydrolases. Sucrose phosphorylase can catalyze the following three reactions: (1) phosphorylation of sucrose and phosphate as substrates, converting them into fructose and glucose-1-phosphate; (2) hydrolysis of sucrose as substrates, generating glucose and fructose; (3) transglycosylation of sucrose and alcohols as substrates, generating the corresponding glycosides and one molecule of fructose.

[0003] The catalytic mechanism of sucrose phosphorylase (SP) is achieved through a two-site reaction mechanism. The carboxyl residue attacks the anolyte carbon atom of sucrose, generating a covalent β-glucosylase intermediate that releases fructose. Subsequently, the acceptor compound reacts with the intermediate to generate an α-D-glucosyl compound.

[0004] However, the natural enzymatic properties of wild-type SP have low catalytic activity, making it difficult to meet the needs of industrial applications. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide a sucrose phosphorylase mutant and its encoding nucleic acid, a preparation method thereof, a vector and cells containing the encoding nucleic acid, an enzyme preparation containing the same, and a method for preparing glucose-1-phosphate. The technical solutions include the following:

[0006] One or more embodiments of this application provide a sucrose phosphorylase mutant having an F156G amino acid substitution relative to wild-type sucrose phosphorylase.

[0007] In some embodiments of this application, the wild-type sucrose phosphorylase is derived from Bifidobacterium adolescentis;

[0008] Optionally, the amino acid sequence of the wild-type sucrose phosphorylase is shown in SEQ ID NO.1;

[0009] Optionally, the amino acid sequence of the sucrose phosphorylase mutant is shown in SEQ ID NO.3.

[0010] In some embodiments of this application, the N-terminus of the sucrose phosphorylase mutant is attached with a purification tag;

[0011] Optionally, the purification tag includes a His tag.

[0012] One or more embodiments of this application provide a nucleic acid molecule that encodes the sucrose phosphorylase mutant;

[0013] Optionally, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

[0014] One or more embodiments of this application provide a vector comprising the nucleic acid molecule described above;

[0015] Optionally, the vector includes a plasmid.

[0016] One or more embodiments of this application provide cells that express the sucrose phosphorylase mutant and contain the nucleic acid molecule or the vector.

[0017] Optionally, the cells may include microorganisms.

[0018] One or more embodiments of this application provide a method for preparing the sucrose phosphorylase mutant, the method comprising the following steps:

[0019] The cells were fermented, and an enzyme solution containing the sucrose phosphorylase mutant was isolated from the fermentation product.

[0020] In some embodiments of this application, fermentation includes the steps of expanding the cells and inducing the expression of the sucrose phosphorylase mutant;

[0021] Optionally, the fermentation satisfies one or more of the conditions shown in (A1) to (A4) below, or one or more of the conditions shown in (B1) to (B4) below:

[0022] (A1) The amplification culture temperature is 35℃~39℃;

[0023] (A2) Under the condition that the OD600 of the culture system is 0.6~0.8, an inducer is added to induce expression. The final concentration of the inducer in the initial system is 0.1 mmol / L~1 mmol / L.

[0024] (A3) The induction temperature is 12℃~18℃, and the induction time is 16h~20h;

[0025] (A4) The culture medium used for fermentation contains 8 g / L to 12 g / L tryptone, 8 g / L to 12 g / L sodium chloride and 4 g / L to 6 g / L yeast extract;

[0026] (B1) The amplification culture temperature is 28℃~30℃, and the amplification culture time is 16h~18h;

[0027] (B2) The final concentration of the inducer in the initial system for induced expression is 0.3%–3%;

[0028] (B3) The induction temperature is 28℃~30℃, and the induction time is 4d~7d;

[0029] (B4) The culture medium used for fermentation contains 18 g / L to 22 g / L tryptone, 8 g / L to 12 g / L yeast extract, 18 mL / L to 22 mL / L glycerol, 5 g / L to 8 g / L YNB, 0.3 mg / L to 0.5 mg / L biotin and 0.05 M to 0.15 M potassium phosphate buffer.

[0030] One or more embodiments of this application provide an enzyme preparation comprising the sucrose phosphorylase mutant or the defined enzyme solution.

[0031] One or more embodiments of this application provide a method for preparing glucose-1-phosphate, which uses the sucrose phosphorylase mutant or the defined enzyme solution to catalyze sucrose and phosphate to prepare glucose-1-phosphate.

[0032] Compared with traditional technologies, this application has the following advantages:

[0033] This application provides a sucrose phosphorylase mutant, its encoding nucleic acid, a preparation method thereof, a vector and cells containing the encoding nucleic acid, an enzyme preparation containing the same, and a method for preparing glucose-1-phosphate. Compared to wild-type sucrose phosphorylase, the sucrose phosphorylase mutant has an F156G amino acid substitution. This mutant exhibits significantly enhanced enzyme activity compared to the wild-type enzyme and can be used to prepare glucose-1-phosphate. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0035] Figure 1 Electrophoresis diagram of the target gene for sucrose phosphorylase (SP).

[0036] Figure 2 Electrophoresis diagram of the target gene for sucrose phosphorylase (SP) and pET-28a plasmid after double digestion.

[0037] Figure 3 Electrophoresis image of pET28a-sp recombinant plasmid after double enzyme digestion.

[0038] Figure 4 The specific activity of sucrose phosphorylase (SP) mutant. Detailed Implementation

[0039] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0041] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0042] In this application, the selection scope of "and / or", "or / and", and "and / or" includes any one of two or more related listed items, as well as any and all combinations of the related listed items. "Any and all combinations" includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, "A and / or B" includes three parallel options: A, B, and "a combination of A and B".

[0043] In this application, unless otherwise specified, "multiple", "various", "multiple times", "several", "several", etc., refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0044] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0045] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0046] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0047] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0048] In this application, amino acid residues are named according to the recognized IUPAC nomenclature, using single-letter or three-letter codes. DNA nucleic acid sequences are named according to the recognized IUPAC nomenclature.

[0049] In this application, the mutated amino acid in the SP mutant is represented by "original amino acid + position + substituted amino acid". F156G indicates that the amino acid at position 156 is replaced by Gly from the wild-type SP with Phe. The position number corresponds to the amino acid sequence number of the wild-type SP in SEQ ID NO.1.

[0050] In this application, "nucleic acid" refers to any linearly or sequentially arranged nucleotides and nucleosides, such as cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides, and their derivatives. Nucleic acids may include bacterial plasmid vectors, including expression, cloning, granulation, and transformation vectors, and nucleic acids may include modified or derived nucleotides and nucleosides.

[0051] In this application, "nucleotide sequence" refers to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, and refers to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, and denotes the sense or antisense strand. "Polynucleotide," "oligonucleotide," "nucleotide sequence," and "nucleic acid" are used interchangeably herein and include, but are not limited to, coding sequences. That is, one or more polynucleotide or nucleic acid sequences that, when placed under the control of appropriate regulatory or control sequences, are transcribed and translated into polypeptides in vitro or in vivo; control sequences, such as translation start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, and DNA sequences to which one or more transcription factors bind and positively (inducing) or negatively (inhibiting) alter the promoter activity of a gene, etc.

[0052] In this application, "overlap PCR" also means "overlap extension PCR", which refers to an in vitro recombination technology that uses primers to design two or more DNA fragments with complementary sequences at their ends, and then uses annealing during the PCR process to "bridge" and pair these fragments, thereby extending them into complete long DNA fragments.

[0053] In this application, "vector" refers to a nucleic acid molecule capable of transporting or transferring foreign nucleic acid molecules, encompassing both expression vectors and transcription vectors. An "expression vector" is a vector capable of expressing an insert in target cells and typically contains control sequences (such as enhancer, promoter, and terminator sequences) driving the expression of the insert. A "transcription vector" is a vector capable of being transcribed but not translated. Transcription vectors are used to amplify their inserts. The foreign nucleic acid molecule is referred to as the "insert" or "transgenic molecule." A vector typically consists of an insert and a larger sequence serving as the vector's backbone. Based on their structure or origin, major types of vectors include plasmid vectors, granular vectors, phage vectors (such as λ phage), viral vectors (such as adenovirus vectors), and artificial chromosomes.

[0054] In this application, "cell" also refers to "host cell," meaning a cell into which an expression vector has been introduced. Host cells may include prokaryotic cells (such as bacteria) and eukaryotic cells (such as fungal, plant, or animal cells). Easily transformable bacteria include members of the Enterobacteriaceae family, such as *Escherichia coli* (e.g., DH5α, BL21(DE3), Rosetta-gami(DE3), JM109, TOP10, etc.) or strains of *Salmonella*; members of the Bacillaceae family, such as *Bacillus subtilis*; *Pneumococcus*; *Streptococcus*; and *Haemophilus influenzae*. Suitable fungal host cells include *Saccharomyces cerevisiae* and *Pichia pastoris*. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0055] This application does not impose any particular limitation on the synthetic method of the mutant. It can be prepared by bioengineering methods or by chemical synthesis (e.g., solid-phase synthesis). It is understood that during the synthesis of the mutant using bioengineering methods, some functional modifications can be made to the mutant for considerations such as stability, purification, and yield.

[0056] Irrational protein design involves inducing numerous mutations in a gene under certain conditions without knowing the protein's three-dimensional structure and mechanism of action, followed by targeted selection of mutants with desired characteristics through multiple rounds of high-throughput screening. Rational molecular design, on the other hand, involves targeted mutations of a gene sequence most likely to affect a protein's function and properties, based on known three-dimensional structure and function. This aims to intentionally alter one or two amino acid residues or modules of the protein, thereby constructing new protein molecules. Compared to irrational design, rational design has the advantages of requiring less work and more readily obtaining effective mutants.

[0057] A first aspect of this application provides a sucrose phosphorylase mutant, which, relative to wild-type sucrose phosphorylase, has an F156G amino acid substitution.

[0058] In some embodiments of this application, the wild-type sucrose phosphorylase is derived from Bifidobacterium adolescentis;

[0059] Optionally, the amino acid sequence of the wild-type sucrose phosphorylase is shown in SEQ ID NO.1;

[0060] Optionally, the amino acid sequence of the sucrose phosphorylase mutant is shown in SEQ ID NO.3.

[0061] In some embodiments of this application, the N-terminus of the sucrose phosphorylase mutant is attached with a purification tag;

[0062] Optionally, the purification tag includes a His tag.

[0063] A second aspect of this application provides a nucleic acid molecule that encodes the sucrose phosphorylase mutant.

[0064] Optionally, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

[0065] A third aspect of this application provides a vector comprising the nucleic acid molecule described above;

[0066] Optionally, the vector includes a plasmid.

[0067] A fourth aspect of this application provides a cell expressing the sucrose phosphorylase mutant, comprising the nucleic acid molecule or the vector.

[0068] Optionally, the cells may include microorganisms.

[0069] A fifth aspect of this application provides a method for preparing the sucrose phosphorylase mutant, the method comprising the following steps:

[0070] The cells were fermented, and an enzyme solution containing the sucrose phosphorylase mutant was isolated from the fermentation product.

[0071] In some embodiments of this application, fermentation includes the steps of expanding the cells and inducing the expression of the sucrose phosphorylase mutant;

[0072] Optionally, the fermentation satisfies one or more of the conditions shown in (A1) to (A4) below, or one or more of the conditions shown in (B1) to (B4) below:

[0073] (A1) The amplification culture temperature is 35℃~39℃, for example, 35℃, 36℃, 37℃, 38℃, 39℃;

[0074] (A2) Under the condition that the OD600 of the culture system is 0.6~0.8, an inducer is added to induce expression, for example, 0.6, 0.7, 0.8. The final concentration of the inducer in the initial system for induction expression is 0.1 mmol / L~1 mmol / L, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mmol / L;

[0075] (A3) The induction temperature is 12℃~18℃, for example 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, and the induction time is 16h~20h, for example 16.0h, 16.5h, 17.0h, 17.5h, 18.0h, 18.5h, 19.0h, 19.5h, 20.0h;

[0076] (A4) The culture medium used for fermentation contains 8 g / L to 12 g / L tryptone, 8 g / L to 12 g / L sodium chloride and 4 g / L to 6 g / L yeast extract. For example, the culture medium used for fermentation contains 10 g / L tryptone, 10 g / L sodium chloride and 5 g / L yeast extract.

[0077] (B1) The amplification culture temperature is 28℃~30℃, and the amplification culture time is 16h~18h, for example, culture at 37℃ for 12h;

[0078] (B2) The final concentration of the inducer in the initial system for induced expression is 0.3% to 3%, for example, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%;

[0079] (B3) The induction temperature is 28℃~30℃, and the induction time is 4d~7d, for example, induction at 30℃ for 6d;

[0080] (B4) The fermentation medium contains 18 g / L to 22 g / L tryptone, 8 g / L to 12 g / L yeast extract, 18 mL / L to 22 mL / L glycerol, 5 g / L to 8 g / L YNB, 0.3 mg / L to 0.5 mg / L biotin and 0.05 M to 0.15 M potassium phosphate buffer. For example, the fermentation medium contains 20 g and 10 g yeast extract, 20 mL glycerol, 6.7 g YNB, 0.4 mg biotin and 0.1 M potassium phosphate buffer.

[0081] A sixth aspect of the embodiments of this application provides an enzyme preparation comprising the sucrose phosphorylase mutant or the defined enzyme solution.

[0082] A seventh aspect of this application provides a method for preparing glucose-1-phosphate, wherein the sucrose phosphorylase mutant or the defined enzyme solution is used to catalyze sucrose and phosphate to prepare glucose-1-phosphate.

[0083] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0084] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0085] The following are some examples.

[0086] Example 1: Construction, expression and preparation of sucrose phosphorylase in Escherichia coli BL21

[0087] (1) LB (Luria-Bertani) medium: 10 g / L tryptone, 10 g / L sodium chloride (NaCl) and 5 g / L yeast extract, sterilized at 121℃ for 20 min.

[0088] (2) LA medium (Luria-Agar): Add agar powder to LB at a ratio of 2.0% and sterilize at 121℃ for 20 min.

[0089] (3) 0.5 M Tris-HCl solution (pH 7.0): 68.5 g Tris (tris(hydroxymethyl)aminomethane) was dissolved in ddH2O, and hydrochloric acid was added to adjust the pH to 7.0, and the volume was adjusted to 500 mL.

[0090] (4) Lysis Buffer solution: 0.7 g imidazole, 1.21 g Tris, and 14.63 g NaCl are dissolved in ddH2O. Hydrochloric acid is added to adjust the pH to 7.0 and the volume is adjusted to 500 mL.

[0091] (5) Wash Buffer solution: 1.75 g imidazole, 1.21 g Tris, and 14.63 g NaCl are dissolved in ddH2O. Hydrochloric acid is added to adjust the pH to 7.0 and the volume is brought up to 1 L.

[0092] (6) Elution Buffer: 13.6 g imidazole, 2.42 g Tris, and 29.25 g NaCl are dissolved in ddH2O. Hydrochloric acid is added to adjust the pH to 7.0 and the volume is brought up to 1 L.

[0093] (7) 10×YNB (containing ammonium sulfate): 13.4 g of YNB (Yeast Nitrogen Base) was dissolved in 100 mL of sterile water, sterilized by membrane filtration, and stored at 4℃.

[0094] (8) 500× Biotin: Dissolve 20 mg of biotin in 100 mL of sterile water, sterilize by membrane, and store at 4°C.

[0095] (9) 1 mol / L, pH 6.0 potassium phosphate buffer: Prepare 1 mol / L dipotassium hydrogen phosphate and 1 mol / L potassium dihydrogen phosphate, and mix them to adjust the pH to 6.0.

[0096] (10) BMGY medium: 1 g tryptone, 0.5 g yeast extract and 1 mL glycerol were dissolved in 40 mL sterile water and autoclaved at 121℃ for 20 min. Before use, 5 mL of the above-mentioned membrane-sterilized 10×YNB, 100 μL of 500×Biotin, and 5 mL of 1 mol / L, pH 6.0 potassium phosphate buffer were added to a clean bench.

[0097] (11) BMMY medium: Dissolve 1 g tryptone and 0.5 g yeast powder in 40 mL of sterile water and autoclave at 121℃ for 20 min. Before use, add 5 mL of the above-mentioned membrane-sterilized 10×YNB, 100 μL of 500×biotin, 5 mL of 1mol / L, pH 6.0 potassium phosphate buffer and 250 μL of methanol to a clean bench.

[0098] (12) YPD medium (Yeast Extract Peptone Dextrose): 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose (15 g / L agar powder added to solid medium), autoclaved at 115℃ for 20 min.

[0099] (13) MD medium (Minimal Dextrose, glucose basal medium): Dissolve 2 g glucose and 1.5 g agar powder in 90 mL sterile water and autoclave at 115℃ for 20 min. Before use, after dissolving the medium and cooling it to about 50℃, add 10 mL of the above-mentioned membrane-sterilized 10×YNB and 200 μL of 500×Biotin to a clean bench.

[0100] II. Obtaining the wild-type sucrose phosphorylase encoding gene sp

[0101] Using extracted Bifidobacterium adolescentis as a template, upstream primer F and downstream primer R were designed, and NcoI and XhoI restriction sites were introduced at the upstream and downstream ends, respectively. The amplification primers for the sp gene in this application are as follows:

[0102] F (SEQ ID NO.5): CATGCCATGGGC ATGAAAAACAAAGTGCAGCTGATTA;

[0103] R (SEQ ID NO.6): CCGCTCGAG CGCCACCACCGGC.

[0104] Using F and R as upstream and downstream primers, the extracted Bifidobacterium adolescentis genome was used as a template to amplify the sp gene. The amplification system is shown in Table 1.

[0105] Table 1. Amplification system of Bifidobacterium adolescentis sp gene

[0106]

[0107] The amplification program was as follows: 98℃ pre-denaturation for 30 min; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 5 s, for 30 cycles; 72℃ extension for 10 min. PCR products were subjected to 1% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit.

[0108] The recovered product (sp wild-type gene) was ligated into the pET-28a(+) vector using NcoI and XhoI. The ligation product was first transformed into the cloning host *E. coli* JM109, plated on LB agar, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. Successful transformations were sequenced to obtain the pET28a-sp wild-type plasmid. The correctly verified plasmid was then transformed into the expression host BL21, plated on LB agar, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. Successful transformants were sequenced to obtain the pET28a-sp wild-type recombinant strain.

[0109] The amino acid sequence of wild-type sucrose phosphorylase SP is shown in SEQ ID NO.1, and the nucleotide sequence of wild-type sucrose phosphorylase SP is shown in SEQ ID NO.2.

[0110] SEQ ID NO.1:

[0111] MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSSVFPNGATEEDLAGIYRPRPGLPFTHYKFAGKTRLVWVS F TPQQVDIDTSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFALPPLLLHALSTGHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGES QAATGAAASNLDLYQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVYYVGALAGKNDMELLRKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTDDDTSISFTWRGETSQATLTFEPKRGLGVDNTTPVAMLEWEDSAGDHRSDDLIANPPVVA.

[0112] SEQ ID NO.2:

[0113] ATGAAAAACAAAGTGCAGCTGATTACCTATGCGGATCGCCTGGGCGATGGCACCATTAAAAGCATGACCGATATTCTGCGCACCCGCTTTGATGGCGTGTATGATGGCGTGCATATTCTGCCGTTTTTTACCCCGTTTGATGGCGCGGATGCGGGCTTTGATCCGATTGATCATACCAAAGTGGATGAACGCCTGGGCAGCTGGGATGATGTGGCGGAACTGAGCAAAACCCATAACATTATGGTGGATGCGATTGTGAACCATATGAGCTGGGAAAGCAAACAGTTTCAAGATGTGCTGGCGAAAGGCGAGGAAAGTGAATATTATCCGATGTTTCTGACCATGAGCAGCGTGTTTCCGAACGGCGCGACCGAAGAAGATCTGGCGGGCATTTATCGCCCGCGCCCGGGCCTGCCGTTTACCCATTATAAATTTGCGGGCAAAACCCGCCTGGTGTGGGTGAGC TTT

[0114] II. Obtaining the gene encoding the mutant sucrose phosphorylase

[0115] The single-point mutation Phe156Gly was determined by bioinformatics prediction and constructed by overlap PCR to obtain the mutant F156G.

[0116] The mutant was constructed using overlap PCR, and the primers required for the mutation are as follows:

[0117] F (SEQ ID NO.7): CATGCCATGGGC ATGAAAAACAAAGTGCAGCTGATTA;

[0118] R (SEQ ID NO.8): CCGCTCGAG CGCCACCACCGGC;

[0119] f-F156G (SEQ ID NO.9): GGTTGGGGTGAGCGGTA;

[0120] r-F156G (SEQ ID NO. 10): TACCGCTCAC CCACACC.

[0121] In the first step of the overlap PCR reaction system, F and r-F156G were used as upstream and downstream primers, respectively, and f-F156G and R were used as upstream and downstream primers, respectively. Using pET28a-sp as a template, PCR 1 reaction was performed to obtain the upstream and downstream fragments. The reaction system is shown in Tables 2 and 3.

[0122] Table 2. Reaction system for upstream fragment amplification

[0123]

[0124] Table 3. Reaction system for downstream fragment amplification

[0125]

[0126] The amplification program was as follows: pre-denaturation at 98℃ for 30 min; denaturation at 98℃ for 10 s, annealing at 54℃ for 20 s, extension at 72℃ for 5 s, for 30 cycles; and extension at 72℃ for 10 min.

[0127] After gel extraction and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system is shown in Table 4.

[0128] Table 4. Reaction system for the second step of overlap PCR

[0129]

[0130] The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 8 s, for 5 cycles; 72℃ extension for 10 min.

[0131] After PCR 2, 2 μL each of primers F and R were added to the system for PCR 3. The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 10 s, for 30 cycles; 72℃ extension for 10 min. The PCR products were subjected to 1.2% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit.

[0132] The recovered product (sp mutant gene) was ligated into the pET-28a(+) vector using NcoI and XhoI. The ligation product was first transformed into the cloning host *E. coli* JM109, plated on LB agar, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. Successful transformants were sequenced to obtain the pET28a-F156G mutant plasmid. The correctly verified plasmid was then transformed into the expression host BL21, plated on LB agar, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. Successful transformants were sequenced to obtain the mutant recombinant strain.

[0133] The amino acid sequence of the mutant sucrose phosphorylase SP is shown in SEQ ID NO.3, and the nucleotide sequence of the mutant sucrose phosphorylase SP is shown in SEQ ID NO.4.

[0134] SEQ ID NO.3:

[0135] MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSSVFPNGATEEDLAGIYRPRPGLPFTHYKFAGKTRLVWVS GTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFALPPLLLHALSTGHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNLDLYQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVYYVGALAGKNDMELLRKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTDDDTSISFTWRGETSQATLTFEPKRGLGVDNTTPVAMLEWEDSAGDHRSDDLIANPPVVA。

[0136] SEQ ID NO.4:

[0137] ATGAAAAACAAAGTGCAGCTGATTACCTATGCGGATCGCCTGGGCGATGGCACCATTAAAAGCATGACCGATATTCTGCGCACCCGCTTTGATGGCGTGTATGATGGCGTGCATATTCTGCCGTTTTTTACCCCGTTTGATGGCGCGGATGCGGGCTTTGATCCGATTGATCATACCAAAGTGGATGAACGCCTGGGCAGCTGGGATGATGTGGCGGAACTGAGCAAAACCCATAACATTATGGTGGATGCGATTGTGAACCATATGAGCTGGGAAAGCAAACAGTTTCAAGATGTGCTGGCGAAAGGCGAGGAAAGTGAATATTATCCGATGTTTCTGACCATGAGCAGCGTGTTTCCGAACGGCGCGACCGAAGAAGATCTGGCGGGCATTTATCGCCCGCGCCCGGGCCTGCCGTTTACCCATTATAAATTTGCGGGCAAAACCCGCCTGGTGTGGGTGAGC GGT

[0138] III. Expression, purification, and enzyme activity assay of sucrose phosphorylase

[0139] 1. Three-zone streaking activated recombinant strains.

[0140] 2. Pick a single colony and inoculate it into 5 mL of LB medium containing Kan antibiotic (50 μg / mL), and incubate at 220 rpm and 37°C for 12 h.

[0141] 3. Transfer the seed culture to 250 mL LB medium (250 μg Kan stock solution) at an inoculation rate of 2%, and incubate at 220 rpm and 37℃ for 2-3 h until the OD600 reaches 0.6-0.8.

[0142] 4. After the OD600 reaches 0.6-0.8, add isopropyl thiogalactoside IPTG (final concentration 0.5 mM) to the fermentation broth and culture at 160 rpm and 16℃ for 16-20 h with shaking.

[0143] 5. Pour the bacterial culture into a 250 mL centrifuge cup and centrifuge at 8000 rpm for 10 min using a refrigerated centrifuge, retaining the bacterial precipitate.

[0144] 6. Add 25 mL of pH 7.0 lysis buffer to the collected bacterial pellet, mix thoroughly, and then transfer to a cell sonicator for disruption.

[0145] 7. Centrifuge the cell lysate at 4℃ and 12000 rpm for 30 min, collect the supernatant, and mix the crude enzyme solution with the pretreated Ni 2+ The resin mixture was thoroughly mixed and poured into a 100 mL beaker. The beaker was placed in an ice bath and stirred on a magnetic stirrer at 100 rpm for 1-2 h. The protein (with a 6×His tag at the N-terminus) bound to the resin was then passed through a column at 4 °C until Ni... 2+ All the resin was retained in the chromatography column. One column volume of Wash Buffer was added to the column to elute the Ni. 2+ Impurities bound to the resin. After washing away the impurities, add one column volume of Elution Buffer to the chromatography column, and use the Elution Buffer to remove Ni. 2+The enzyme was resuspended in resin and allowed to stand for 10 min. Finally, the filtrate containing the target protein was collected using an ultrafiltration tube. The ultrafiltration tube was placed at 4℃ and 4000 rpm for ultrafiltration concentration. After concentration to below 500 μL, 5 mL of Tris-HCl (pH 7.0) was added and the concentration was further reduced to about 1 mL. This process was repeated twice, and the enzyme solution was collected to obtain pure SP enzyme solution. The protein concentration of the pure SP enzyme solution was determined using a BCA protein concentration kit and stored at 4℃ for later use.

[0146] The enzyme activity of SP was determined using the DNS (3,5-dinitrosalicylic acid) method. SP catalyzes the conversion of sucrose and phosphate to fructose and glucose-1-phosphate; therefore, the enzyme activity was determined by measuring the concentration of the product fructose. The catalytic reaction was carried out in 50 mM phosphate buffer (pH 7.0). 20 μL of substrate sucrose solution (50 mM) and 170 μL of PBS buffer were added, and the mixture was incubated at 40°C for 2–3 min. 10 μL of SP was added to initiate the reaction, which was allowed to proceed precisely for 10 min. 400 μL of DNS reagent was added to terminate the reaction, and the mixture was immediately boiled in a water bath for 5 min for color development. After the reaction solution cooled to room temperature, 200 μL of the chromogenic solution was transferred to a 96-well microplate, and the absorbance was measured at 540 nm. The enzyme activity units were calculated based on the standard curve. The amount of enzyme required to produce 1 µmol of fructose per minute was defined as one unit (U) of SP enzyme activity.

[0147] The results showed that the specific activity of mutant F156G was 302.8 U / mg, which was 2.1 times that of WT (144.2 U / mg).

[0148] Example 2: Construction, expression and preparation of sucrose phosphorylase in Pichia pastoris

[0149] Using the pET28a-sp and pET28a-F156G plasmids extracted in Example 1 as templates, upstream primer F1 and downstream primer R1 were designed to introduce EcoRI and NotI restriction sites upstream and downstream of the sp and F156G genes, respectively. The required amplification primers are as follows:

[0150] F1 (SEQ ID NO.11): CCG GAATTCC ATGAAAAACAAAGTGCAGCTGATTA;

[0151] R1 (SEQ ID NO.12): CATG CCATGG CGCCACCACCGGC.

[0152] Using F1 and R1 as upstream and downstream primers, the extracted pET28a-sp and pET28a-F156G plasmids were used as templates to amplify the sp and F156G genes. The amplification system is shown in Table 5.

[0153] Table 5. Amplification systems of sp and F156G genes

[0154]

[0155] The amplification program was as follows: 98℃ pre-denaturation for 30 min; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 5 s, for 30 cycles; 72℃ extension for 10 min. PCR products were subjected to 1% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit.

[0156] The gene was ligated to plasmid pPIC9K via two restriction enzyme sites, EcoRI and NotI. The ligation product was first transformed into the cloning host E. coli JM109, plated on LB solid medium, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. Successful verification was followed by sequencing to obtain pPIC9K-sp and pPIC9K-F156G plasmids. Plasmids pPIC9K-sp and pPIC9K-F156G were linearized with restriction endonuclease SalI and purified. After purification, they were electroporated into Pichia pastoris GS115 and plated on MD plates. After 2-3 days, transformants were picked and plated on G418 (Geneticin) resistant YPD solid medium to obtain wild-type and mutant recombinant strains.

[0157] Wild-type and mutant transformants were incubated separately for 16–18 h in 200 mL BMGY medium containing glycerol at 30 °C and 200 r / min. Afterward, all were transferred to 200 mL BMMY medium and induced with methanol (0.5% v / v). Fermentation was carried out at 30 °C and 200 r / min for 6 days, with 0.5% v / v methanol added daily. After fermentation, the fermentation broth was centrifuged at 12,000 r / min for 30 min at 4 °C. The supernatant was filtered through a 0.45 µm filter to obtain crude enzyme solutions of SP and F156G. The fermentation activity of SP and F156G mutants was determined using the DNS method described above.

[0158] The results showed that the fermentation activity of the F156G mutant was 944.7 U / mL, which was 2.34 times that of the wild-type SP (403.8 U / mL).

[0159] In summary, this application obtained an enhanced sucrose phosphorylase by molecularly modifying sucrose phosphorylase derived from Bifidobacterium adolescentis. Subsequently, an enhanced sucrose phosphorylase mutant was prepared by expression using a heterologous expression system.

[0160] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0161] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A sucrose phosphorylase mutant, characterized in that, Compared to wild-type sucrose phosphorylase, the sucrose phosphorylase mutant has an F156G amino acid substitution.

2. The sucrose phosphorylase mutant according to claim 1, characterized in that, The wild-type sucrose phosphorylase is derived from Bifidobacterium adolescentis; Optionally, the amino acid sequence of the wild-type sucrose phosphorylase is shown in SEQ ID NO.1; Optionally, the amino acid sequence of the sucrose phosphorylase mutant is shown in SEQ ID NO.

3.

3. The sucrose phosphorylase mutant according to claim 1 or 2, characterized in that, The N-terminus of the sucrose phosphorylase mutant is attached with a purification tag. Optionally, the purification tag includes a His tag.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a sucrose phosphorylase mutant as described in any one of claims 1 to 3; Optionally, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

5. A carrier, characterized in that, The carrier comprises the nucleic acid molecule according to claim 4; Optionally, the vector includes a plasmid.

6. A cell, characterized in that, The cell expresses the sucrose phosphorylase mutant according to any one of claims 1 to 3, comprising the nucleic acid molecule according to claim 4 or the vector according to claim 5; Optionally, the cells may include microorganisms.

7. The method for preparing the sucrose phosphorylase mutant according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: The cells of claim 6 are fermented, and an enzyme solution containing the sucrose phosphorylase mutant is separated from the fermentation product.

8. The method for preparing the sucrose phosphorylase mutant according to claim 7, characterized in that, Fermentation includes the steps of expanding and culturing the cells and inducing the expression of the sucrose phosphorylase mutant; Optionally, the fermentation satisfies one or more of the conditions shown in (A1) to (A4) below, or one or more of the conditions shown in (B1) to (B4) below: (A1) The amplification culture temperature is 35℃~39℃; (A2) Under the condition that the OD600 of the culture system is 0.6~0.8, an inducer is added to induce expression. The final concentration of the inducer in the initial system is 0.1 mmol / L~1 mmol / L. (A3) The induction temperature is 12℃~18℃, and the induction time is 16h~20h; (A4) The culture medium used for fermentation contains 8 g / L to 12 g / L tryptone, 8 g / L to 12 g / L sodium chloride and 4 g / L to 6 g / L yeast extract; (B1) The amplification culture temperature is 28℃~30℃, and the amplification culture time is 16h~18h; (B2) The final concentration of the inducer in the initial system for induced expression is 0.3%–3%; (B3) The induction temperature is 28℃~30℃, and the induction time is 4d~7d; (B4) The culture medium used for fermentation contains 18 g / L~22 g / L tryptone, 8 g / L~12 g / L yeast extract, 18 mL / L~22 mL / L glycerol, 5 g / L~8 g / L YNB, 0.3 mg / L~0.5 mg / L biotin and 0.05 M~0.15 M potassium phosphate buffer.

9. An enzyme preparation, characterized in that, The enzyme preparation comprises a sucrose phosphorylase mutant as described in any one of claims 1 to 3 or an enzyme solution as defined in any one of claims 7 to 8.

10. A method for preparing glucose-1-phosphate, characterized in that, Glucose-1-phosphate is prepared by catalyzing sucrose and phosphate using the sucrose phosphorylase mutant of any one of claims 1 to 3 or the enzyme solution defined in any one of claims 7 to 8.

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