Nucleic acids, vectors, host cells, and methods for the production of beta-fructofuranosidase from Aspergillus niger.

Abstract

The present invention provides nucleic acids, vectors, host cells, and methods for the production of beta-fructofuranosidase from Aspergillus niger. The present invention represents an advance in the field of genetic engineering and provides a method for obtaining high yields of novel recombinant beta-fructofuranosidase encoded by the fopA gene of Aspergillus niger as a secreted protein.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering. More particularly, the present invention is directed to obtaining an improved production of a novel recombinant β - fructofuranosidase encoded by the fopA gene of Aspergillus niger as a secreted protein.

Background Art

[0002] Fructose oligomers, also known as fructooligosaccharides (FOS), constitute a series of homologous oligosaccharides. Fructooligosaccharides are usually represented by the formula GF n and are mainly composed of 1 - kestose (GF2), nystose (GF3), and β - fructofuranosylnystose (GF4), where 2, 3, and 4 fructosyl units are linked at the β - 2,1 position of glucose.

[0003] Fructooligosaccharides (FOS) are characterized by many beneficial properties such as low sweetness intensity and usefulness as prebiotics. Due to their low sweetness intensity (about 1 / 3 - 2 / 3 compared to sucrose) and low calorie value (about 0 - 3 kcal / g), fructooligosaccharides can be used as sugar substitutes in various types of foods. Furthermore, as prebiotics, fructooligosaccharides have been reported to be used in the protection against colon cancer, enhancement of various parameters of the immune system, improvement of mineral adsorption, beneficial effects on serum lipid and cholesterol concentrations, and exertion of blood glucose control for the control of obesity and diabetes (Dominguez, Ana Luisa, et al. “An overview of the recent developments on fructooligosaccharide production and applications.” Food and bioprocess technology 7.2 (2014): 324 - 337.).

[0004] However, fructooligosaccharides are found only in trace amounts as natural components in fruits, vegetables, and honey. Due to such low concentrations, it is practically impossible to extract fructooligosaccharides from food.

[0005] Attempts have been made to produce fructooligosaccharides from sucrose via enzymatic synthesis using microbial enzymes with transfructosylation activity. However, major limitations in previous attempts have included low catalytic efficiency, glucose-induced enzyme feedback inhibition leading to lower FOS yields, and the need for longer periods of time for sucrose conversion by enzymes expressed in recombinant host systems. Furthermore, the industrial production of microbial enzymes exhibiting transfructosylation activity presents challenges due to additional limitations associated with large-scale enzyme expression, enzyme stability, fermentation, and purification processes.

[0006] The commercial-scale production of fructooligosaccharides requires the efficient identification and mass production of enzymes. Due to the aforementioned limitations, the production of microbial enzymes with efficient transfructosylation activity presents cost challenges, which in turn increase the production cost of fructooligosaccharides.

[0007] Therefore, there has long been a need to identify microbial enzymes with excellent transfructosylation activity that can reduce the cost of fructooligosaccharide production, and to provide efficient, inexpensive, and industrially scalable means for their production. [Overview of the project] [Problems that the invention aims to solve]

[0008] The technical problem to be solved in this invention is to identify a novel β-fructofuranosidase (UniProtKB:Q96VC5_ASPNG) from Aspergillus niger and to improve its yield. [Means for solving the problem]

[0009] The aforementioned problem was solved by overexpression of a novel β-fructofuranosidase from Aspergillus niger by manipulating the nucleic acid sequence, protein sequence, promoter, recombinant vector, host cell, and secretory signal peptide to achieve a high yield of the novel recombinant β-fructofuranosidase.

[0010] Additionally, by modifying the fermentation strategy, a high yield of recombinant β-fructofuranosidase of approximately 2-5 gm / L was obtained.

[0011] Summary of the Invention The present invention relates to nucleic acids, protein sequences, vectors, and host cells for the recombinant expression of novel β-fructofuranosidases. The present invention also relates to a precursor peptide containing a signal peptide fused to a novel β-fructofuranosidase enzyme, enabling the efficient production of a higher yield of the enzyme as a secreted protein.

[0012] The present invention also relates to a method for expressing a novel recombinant β-fructofuranosidase as a secreted protein. The β-fructofuranosidase concentration is found to be approximately 2-5 gm / L. The enzyme exhibits nearly 85% purity after filtration, eliminating the need for costly chromatographic procedures. [Brief explanation of the drawing]

[0013] The features of this disclosure will be fully apparent from the following description, to be interpreted in conjunction with the accompanying drawings. This disclosure is further described through the use of the accompanying drawings, with the understanding that the drawings merely illustrate some embodiments of this disclosure and should not be considered as limitations of its scope.

[0014] [Figure 1] Figure 1 depicts the sequence alignment of the native fopA gene and the modified fopA gene encoding β-fructofuranosidase. [Figure 2] Figure 2 shows the construction scheme for the pPICZαA vector. [Figure 3]Figure 3 depicts the results of the restriction digestion analysis performed on the recombinant plasmid pPICZαA-fopA. [Figure 4] Figure 4 depicts the results of the colony PCR screening performed on the Pichia integrants. [Figure 5] Figure 5 depicts the expression of β-fructofuranosidase upon induction from the recombinant Pichia pastoris host cells. [Figure 6] Figure 6(a) depicts the SDS-PAGE analysis of samples collected at different time intervals during the fermentation of the Pichia pastoris KM71H strain expressing the recombinant β-fructofuranosidase enzyme. Figure 6(b) depicts the SDS-PAGE analysis of the purified recombinant β-fructofuranosidase enzyme. [Figure 7] Figure 7 depicts the glucose standard curve used for the estimation of the activity of the β-fructofuranosidase enzyme. [Figure 8] Figure 8 depicts the production of fructooligosaccharides (FOS) from sucrose and the recombinant β-fructofuranosidase enzyme. [Figure 9] Figure 9 depicts the HPLC analysis chromatogram of the FOS sample.

[0015] Brief Description of Sequences and Sequence Listing SEQ ID NO: 1 - Amino acid sequence of the novel β-fructofuranosidase (654 amino acids) SEQ ID NO: 2 - Modified nucleic acid sequence of the gene encoding the novel β-fructofuranosidase (1965 base pairs)

Table 1

[0016] In all the secretion signal peptide sequences, a stretch of 4 amino acids (LEKR) was added for efficient Kex2 processing of the preprotein.

[0017]

Table 2

[0018] Array number 23 - Native nucleic acid sequence (1965 base pairs) of the fopA gene encoding the secreted β - fructofuranosidase [Table 3]

[0019] Definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this method belongs. Any vectors, host cells, methods, and compositions similar or equivalent to those described herein may also be used in the practice or testing of the vectors, host cells, methods, and compositions, but representative examples are described herein.

[0020] When a range of values is provided, it is understood that each intervening value between the upper and lower limits of that range, as well as any other stated or intervening value within the stated range, is included in the method and composition. The upper and lower limits of these smaller ranges can independently be included in these smaller ranges, and these are also included in the method and composition, subject to any specifically excluded limits within the stated range. If the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the method and composition.

[0021] It is acknowledged that certain features of a method described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of a method and composition described in the context of a single embodiment for brevity may also be provided separately or in any preferred partial combination. It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context otherwise explicitly specifies. It should be further noted that claims may be written to exclude any optional elements. This statement is therefore intended to serve as a prerequisite for the use of exclusive terms such as “solely,” “only,” and “likewise,” or for the use of “negative” limitations in connection with the description of elements of a claim.

[0022] As will be apparent to those skilled in the art who have read this disclosure, each of the individual embodiments described and illustrated in this application has separate components and features that can be readily separated from or readily combined with features of any other embodiment without deviating from the scope or spirit of the method of the present invention. Any described method may be performed in the order of the described events, or in any other logically possible order.

[0023] The term “host cell” includes individual cells or cell cultures that may or may have been recipients for the target of an expression construct. Host cells include offspring of a single host cell. Host cells for the purposes of the present invention refer to any strain of Pichia pastris that can be suitably used for the purposes of the present invention. Examples of strains that can be used for the purposes of the present invention include wild-type, mut+, mut S, and mut- strains of Pichia, such as KM71H, KM71, SMD1168H, SMD1168, GS115, and X33.

[0024] The terms "recombinant strain" or "recombinant host cell" refer to host cells that have been transfected or transformed using the expression construct or vector of the present invention.

[0025] The term "expression vector" refers to any vector, plasmid, or medium designed to enable the expression of a nucleic acid sequence inserted after host transformation.

[0026] The term "promoter" refers to a DNA sequence that defines the site where gene transcription begins. Promoter sequences are typically located directly upstream of the transcription start site or at the 5' end. RNA polymerase and necessary transcription factors bind to the promoter sequence to initiate transcription. Promoters can be either constitutive or inductive. Constitutive promoters are promoters that enable the continuous transcription of their associated genes, and their expression is usually not conditioned by environmental and developmental factors. Constitutive promoters are very useful tools in genetic engineering because they drive gene expression under inductor-free conditions and often exhibit better characteristics than commonly used inductive promoters. Inductive promoters are promoters that are induced by the presence or absence of biological or abiotic and chemical or physical factors. Inductive promoters are very powerful tools in genetic engineering because they can turn the expression of genes operably linked to them on or off at a specific stage of development or growth of an organism, or in a specific tissue or cell type.

[0027] The term "operably linked" refers to an association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is regulated by the other. For example, a promoter is operably linked to a coding sequence if it can regulate the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

[0028] The term "transcription" refers to the process of creating RNA copies of a gene sequence. These copies, called messenger RNA (mRNA) molecules, leave the cell nucleus and enter the cytoplasm, where they instruct the synthesis of the proteins they encode.

[0029] The term "translation" refers to the process of translating the sequence of messenger RNA (mRNA) molecules into a sequence of amino acids during protein synthesis. The genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence it encodes. In the cytoplasm, ribosomes read the mRNA sequence in groups of three bases and assemble them into proteins.

[0030] The term "expression" refers to the biological production of the product encoded by a coding sequence. In most cases, a DNA sequence, including the coding sequence, is transcribed to form messenger RNA (mRNA). The messenger RNA is then translated to form polypeptide products with the relevant biological activity. The expression process may also involve further processing steps for the transcribed RNA product, such as splicing to remove introns, and / or post-translational processing of the polypeptide product.

[0031] The term "modified nucleic acid," as used herein, refers to a nucleic acid encoding β-fructofuranosidase fused to a signal peptide. In embodiments, the modified nucleic acid is represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or functionally equivalent variants thereof. Functional variants include any nucleic acid that has substantial or significant sequence identity or similarity to SEQ ID NOs. 13-22 and retains its biological activity.

[0032] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to two or more amino acid residues joined to one another by peptide bonds or modified peptide bonds. These terms apply to amino acid polymers, which are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers, those containing modified residues, and naturally occurring amino acid polymers. “Polypeptide” refers to both short chains commonly called peptides, oligopeptides, or oligomers, and longer chains commonly called proteins. Polypeptides may contain amino acids other than those encoded by 20 genes. Similarly, “protein” refers to at least two covalently bonded amino acids and includes proteins, polypeptides, oligopeptides, and peptides. Proteins may be made from naturally occurring amino acid and peptide bonds, or from synthetic peptide mimetic structures. Therefore, “amino acid” or “peptide residue” as used herein means both naturally occurring and synthetic amino acids. “Amino acid” includes imino acid residues, such as proline and hydroxyproline. The side chain may be in either the (R) configuration or the (S) configuration.

[0033] The terms “signal peptide” or “signal peptide sequence” are defined herein as a peptide sequence typically present at the N-terminus of a newly synthesized secretory or membrane polypeptide that directs the polypeptide across or into the cell membrane (plasma membrane in prokaryotes and endoplasmic reticulum membrane in eukaryotes). It is typically subsequently removed. In particular, the signal peptide may be capable of directing the polypeptide into the cell’s secretory pathway.

[0034] The term “precursor peptide,” as used herein, refers to a peptide comprising a signal peptide (also known as a leader sequence) operably linked to Aspergillus niger β-fructofuranosidase. The signal peptide is cleaved during post-translational modification inside the Pichia host cell, and the mature β-fructofuranosidase (SEQ ID NO: 1) is released into the culture medium.

[0035] When used herein in relation to precursor peptides / proteins, the term "variant" refers to a peptide having amino acid substitutions, additions, deletions, or modifications that do not substantially reduce the activity of the signal peptide or enzyme. Variants include both structural and functional variants. The term "variant" also includes the use of substituted amino acids in place of unsubstituted parent amino acids.

[0036] Amino acid substitution tables that provide functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that are considered variants of each other:

[0037] [Table 4] [Modes for carrying out the invention]

[0038] This invention discloses nucleic acids, vectors, and recombinant host cells for the efficient production of biologically active and soluble recombinant β-fructofuranosidase from Aspergillus niger as a secreted protein. Furthermore, this invention provides a method for the commercial-scale production of recombinant β-fructofuranosidase.

[0039] This invention envisions a multidimensional approach to achieving high yields of novel recombinant β-fructofuranosidases in heterologous hosts. The native gene for β-fructofuranosidase has been modified for expression in Pichia pastris. Furthermore, the modified gene is fused to one or more signal peptides.

[0040] In one embodiment, a modified nucleic acid encoding a novel β-fructofuranosidase of Aspergillus niger is represented by Sequence ID No. 2.

[0041] In another embodiment, the modified nucleic acid is fused to one or more signal peptides.

[0042] In another embodiment, the signal peptide is selected from S. cerevisiae alpha factor (FAK), S. cerevisiae whole alpha factor (FAKS), S. cerevisiae alpha factor T (AT), Aspergillus niger alpha-amylase (AA), Aspergillus awamori glucoamylase (GA), Kluyveromyces maxianus inulinase (IN), S. cerevisiae invertase (IV), S. cerevisiae killer protein (KP), Gallus gallus lysozyme (LZ), and Homo sapiens serum albumin (SA).

[0043] In another embodiment, the signal peptide is provided in Table 5 below.

[0044] [Table 5]

[0045] In another embodiment, the signal peptide is selected from a list of modified signal peptides, such as those listed in Table 1.

[0046] In another embodiment, the nucleic acid fused to one or more modified signal peptides is selected from the group including SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, SEQ ID NOs: 17, SEQ ID NOs: 18, SEQ ID NOs: 19, SEQ ID NOs: 20, SEQ ID NOs: 21, SEQ ID NOs: 22 and their variants.

[0047] In another embodiment, the modified nucleic acid is cloned into an expression vector.

[0048] In another embodiment, the expression vector is configured for the secretory or intracellular expression of recombinant β-fructofuranosidase from Aspergillus niger.

[0049] In yet another embodiment, the expression vector is selected from the group including pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, pGAPZαC, pPIC3, pPIC3.5, pPIC3.5K, PAO815, pPIC9, pPIC9K, IL-D2, and pHIL-S1.

[0050] The expression of the modified β-fructofuranosidase (fopA) gene fused to the signal peptide is preferably driven by a constitutive or inductive promoter.

[0051] In another embodiment, the nucleic acid to be expressed is operably linked to a promoter.

[0052] In another embodiment, the constitutive or inducible promoter is selected from the group listed in Table 6.

[0053] [Table 6]

[0054] In another embodiment, the promoter is the AOX1 promoter, which is induced by methanol and inhibited by glucose.

[0055] In this embodiment, a suitable host is transformed by an expression vector containing the modified gene of interest (a β-fructofuranosidase gene fused to a nucleic acid encoding a signal peptide).

[0056] In another embodiment, yeast cells are transformed with an expression vector containing the gene of interest.

[0057] In another embodiment, the yeast cells are Pichia pastris.

[0058] In yet another embodiment, the Pichia pastris host cell is a mut+, mut S, or mut- strain. Mut+ represents a methanol-utilizing phenotype.

[0059] In yet another embodiment, the Pichia pastris host cell line is selected from the group including KM71H, KM71, SMD1168H, SMD1168, GS115, and X33.

[0060] In another embodiment, the present invention provides a β-fructofuranosidase precursor peptide in which β-fructofuranosidase of Aspergillus niger is fused to one or more signal peptides.

[0061] In another embodiment, the β-fructofuranosidase of Aspergillus niger has the amino acid sequence shown in SEQ ID NO: 1 and its functional variant. Functional variants include any protein sequence that has substantial or significant sequence identity or similarity to SEQ ID NO: 1 and / or has substantial or significant structural identity or similarity to SEQ ID NO: 1 and retains its biological activity.

[0062] In another embodiment, the signal peptide is selected from the group including whole alpha factor of S. cerevisiae (FAK) shown in SEQ ID NO: 3, whole alpha factor of S. cerevisiae (FAKS) shown in SEQ ID NO: 4, alpha factor T (AT) of S. cerevisiae shown in SEQ ID NO: 5, alpha-amylase of Aspergillus niger shown in SEQ ID NO: 6, glucoamylase of Aspergillus awamori shown in SEQ ID NO: 7, inulinase of Kluiveromyces maximanus shown in SEQ ID NO: 8, invertase of S. cerevisiae (IV) shown in SEQ ID NO: 9, killer protein of S. cerevisiae (KP) shown in SEQ ID NO: 10, lysozyme of the red junglefowl shown in SEQ ID NO: 11, serum albumin of Homo sapiens shown in SEQ ID NO: 12, and variants thereof.

[0063] In one embodiment, a method for producing recombinant β-fructofuranosidase of Aspergillus niger is provided.

[0064] Aspects of the present invention relate to the fermentation of recombinant Pichia pastris cells containing a modified recombinant β-fructofuranosidase (fopA) gene. After completion of fermentation, the fermentation broth is subjected to centrifugation and filtered using microfiltration to separate the recombinant enzyme. The recovered recombinant enzyme is concentrated using tangential flow ultrafiltration or evaporation, and the concentrated enzyme is formulated.

[0065] In one embodiment, a method for expressing Aspergillus niger β-fructofuranosidase at a high level is: a. A step of culturing recombinant host cells in a suitable fermentation medium to obtain recombinant β-fructofuranosidase enzyme secreted into fermentation broth. b. A step of collecting the supernatant from the fermentation broth, wherein the supernatant contains recombinant β-fructofuranosidase, and c. Step to purify recombinant β-fructofuranosidase. Includes.

[0066] In another embodiment, the fermentation medium is a basal salt medium as shown in Table 7.

[0067] In yet another embodiment, the supernatant from the fermentation broth is collected using centrifugation.

[0068] In one embodiment, the percentage of inoculum or starting culture used to initiate fermenter culture is in the range of 2.0% to 15.0% (v / v).

[0069] In another embodiment, the pH of the fermentation medium is maintained within the range of 4.0 to 7.5, and the secreted enzymes undergo proper folding and are biologically active within this pH range.

[0070] In yet another embodiment, the temperature during the fermentation process is in the range of 15°C to 40°C.

[0071] In another embodiment, the duration of the fermentation process is in the range of 50 to 150 hours.

[0072] In a further embodiment, the fermentation broth is centrifuged using continuous online centrifugation at a speed in the range of 2000 × g to 15000 × g.

[0073] The supernatant obtained after centrifugation is subjected to microfiltration for purification, and biologically active recombinant β-fructofuranosidase is recovered.

[0074] In one embodiment, the supernatant obtained after centrifugation is concentrated using a tangential flow filtration-based ultrafiltration system.

[0075] The membrane cutoff size used in a tangential flow filtration (TFF) system, which can be used to remove impurities and concentrate the collected culture supernatant, may be in the range of 5 to 100 kDa.

[0076] In another embodiment, centrifugation is not required for the method due to the high yield and purity of the secreted enzyme.

[0077] In this invention, the β-fructofuranosidase concentration obtained is found to be in the range of 2 to 5 g / L, and the purity is approximately 85%. [Examples]

[0078] The following embodiments specifically describe the methods by which the present invention is carried out. However, the embodiments disclosed herein do not limit the scope of the present invention in any way.

[0079] Example 1: Modified nucleic acid for the expression of recombinant β-fructofuranosidase of Aspergillus niger in Pichia pastris The cDNA of the native β-fructofuranosidase (fopA) from Aspergillus niger is represented by SEQ ID NO: 23, and the amino acid sequence of the novel β-fructofuranosidase is represented by SEQ ID NO: 1.

[0080] The native cDNA was modified to maximize expression in Pichia pastris. The modified nucleic acid is represented by Sequence ID No. 2. The differences between the native and modified sequences are illustrated in Figure 1.

[0081] To maximize expression in Pichia pastris, the expression cassette encoding β-fructofuranosidase was modified. The modified open reading frame contains a modified nucleotide sequence (SEQ ID NO: 2) encoding β-fructofuranosidase fused to the signal peptide. The nucleic acid was designed so that the encoded signal peptide contains an additional 4-amino acid (LEKR) stretch for efficient Kex2 processing of the precursor peptide.

[0082] For expression in Pichia pastris, preferred codons were used in place of rare codons.

[0083] The nucleotide sequence of the modified open reading frame encoding β-fructofuranosidase fused with the modified signal peptide is given below: The alpha factor (FAK) of S. cerevisiae is represented by sequence number 13. The entire alpha factor (FAKS) of S. cerevisiae is represented by sequence number 14. The alpha factor T(AT) of S. cerevisiae is represented by sequence number 15. The alpha-amylase (AA) of Aspergillus niger is represented by sequence number 16. • The glucoamylase (GA) of Aspergillus awamori is represented by sequence number 17. The inulinase (IN) of *Cluiveromyces maximanus* is represented by sequence number 18. • Invertase (IV) of S. cerevisiae is represented by sequence number 19. The killer protein (KP) of S. cerevisiae is represented by Sequence ID No. 20. • Lysozyme (LZ) of the red junglefinch is represented by sequence number 21. • Serum albumin (SA) in Homo sapiens is represented by sequence number 22.

[0084] The nucleic acid sequence of Sequence ID No. 13 was chemically synthesized and cloned into the pPICZαA vector, while the remaining modified nucleic acid sequences were generated by overlap extension PCR using the expression cassette of Sequence ID No. 13 as a template.

[0085] Example 2: Polypeptide sequence of β-fructofuranosidase fused to a signal peptide Recombinant precursor proteins were obtained by translating the gene encoding β-fructofuranosidase from Aspergillus niger fused with a signal peptide.

[0086] The signal peptides used in the modified precursor peptides were S. cerevisiae alpha factor (FAK) represented by SEQ ID NO: 3, S. cerevisiae whole alpha factor (FAKS) represented by SEQ ID NO: 4, S. cerevisiae alpha factor T (AT) represented by SEQ ID NO: 5, Aspergillus niger alpha-amylase (AA) represented by SEQ ID NO: 6, Aspergillus awamori glucoamylase (GA) represented by SEQ ID NO: 7, Kluiveromyces maximanus inulinase (IN) represented by SEQ ID NO: 8, S. cerevisiae invertase (IV) represented by SEQ ID NO: 9, S. cerevisiae killer protein (KP) represented by SEQ ID NO: 10, Red Jungle Jungle lysozyme (LZ) represented by SEQ ID NO: 11, and Homo sapiens serum albumin (SA) represented by SEQ ID NO: 12. The modified signal peptide contains an additional stretch of 4 amino acids (LEKR) for efficient Kex2 processing of the precursor peptide.

[0087] The signal peptide is cleaved during post-translational modification inside the Pichia host cell, and mature recombinant β-fructofuranosidase containing the amino acid sequence of SEQ ID NO: 1 is released into the culture medium.

[0088] Example 3: Development of recombinant host cells by transformation with recombinant plasmids The vector used in the method was pPICZαA. The vector contained a modified open reading frame and inducible promoter, AOX1, as described in Example 1. A modified sequence encoding a recombinant protein was cloned into the pPICZαA vector.

[0089] Using standard molecular biological procedures, the modified nucleic acid of Sequence ID No. 2, which encodes the β-fructofuranosidase (fopA) gene, was cloned between the XhoI / SacII restriction sites present in the MCS of the pPICZαA vector and in-framed with the S. cerevisiae signaling sequence alpha factor (FAK) to create the expression cassette for Sequence ID No. 13. The vector map of pPICZαA is shown in Figure 2.

[0090] Putative recombinant plasmids were selected on low-salt LB medium containing 25 μg / ml zeosin and screened by XhoI / SacII restriction digestion analysis.

[0091] XhoI / SacII restriction digestion analysis of the recombinant plasmid pPICZαA-fopA resulted in the release of a 1980 bp fragment. The results of the restriction digestion analysis are shown in Figure 3.

[0092] Subsequently, linear recombinant pPICZαA-fopA DNA was electroporated into Pichia pastrius KM71H cells. Pichia incorporates were selected on yeast extract peptone dextrose sorbitol agar (YPDSA) containing 100 μg / ml zeosin.

[0093] Integration was screened by colony PCR (cPCR). Templates were prepared from each pichia integration site for cPCR using alkaline lysis. The results of the colony PCR screening are shown in Figure 4.

[0094] Pichia integraters were grown in BMD1 medium for 48 hours and then further induced first in BMM2 medium, followed by continuous induction in BMM10 medium, resulting in a final methanol concentration of 0.5% in the culture medium. At the end of the 96-hour induction period, culture supernatants were collected from different clones. Total protein from each collected supernatant was precipitated with 20% TCA and analyzed by SDS-PAGE.

[0095] Upon induction, the β-fructofuranosidase protein band was observed to be approximately 110 kDa in size, as depicted in Figure 5.

[0096] The calculated molecular weight was approximately 70.85 kDa. Glycosylation may have contributed to the increase in molecular weight.

[0097] Example 4: Fermentation of recombinant Pichia pastris expressing β-fructofuranosidase from Aspergillus niger. Recombinant Pichia pastris cells containing the modified β-fructofuranosidase (fopA) gene as described in Example 1 were fermented in a 50 L fermenter. Fermentation was carried out in a basal salt medium as described herein. The selected recombinant host was KM71H, which is a mut S strain that metabolizes methanol in a slow manner.

[0098] Preparation of pre-seed and seed inoculum: Reserve seeds were generated by inoculating 25 mL of sterile YEPG medium from glycerol stock and growing overnight at 30°C in a temperature-controlled orbital shaker. 15–25 ODs were used to generate the seeds. 600 The inoculum was grown in a basal salt medium in a shake flask with controlled flow at 30°C in a temperature-controlled orbital shaker until it reached [a certain stage].

[0099] Fermentation process The entire fermentation process, from inoculation of the seed culture into the fermenter to final harvesting, took approximately 130 hours. A basal salt medium was prepared and sterilized in situ within the fermenter.

[0100] Table 7 provides the compositions of basal salt media optimized for the fermentation process.

[0101] [Table 7]

[0102] Pichia Trace Minerals (PTM) salt solutions were prepared as described in Table 8. The PTM salts were dissolved in a 1 L volume and sterilized by filter. The PTM salt solution was added to the basal salt medium at a ratio of 4 ml per liter of initial medium volume after sterilization.

[0103] [Table 8]

[0104] Proliferation phase: The growth phase was initiated by inoculating 5% of the seed culture into a basal salt medium in a 50L fermenter and continued for approximately 24 hours. Dissolved oxygen (DO) levels were continuously monitored and never fell below 40%.

[0105] After 18 hours, a DO spike was observed, indicating the depletion of the carbon source (glycerol). 600 The glycerol-fed batch was initiated by supplying 50% glycerol (containing 12 ml of PTM salt per liter of supply) for approximately 6 hours until the volume reached 200.

[0106] Induction period: Once sufficient biomass was generated, the induction phase was initiated by discontinuing glycerol supply and starting methanol supply. Methanol (supplemented with 12 ml of PTM salt per liter of supply) was supplied at a rate of 0.5 g to 3 g per liter of initial fermentation volume. The DO was maintained at 40%, and the methanol supply was adjusted accordingly.

[0107] The induction of the β-fructofuranosidase (fopA) gene was regularly monitored by analyzing the culture supernatant using an enzyme activity assay. 600 The induction period was continued for approximately 100 hours until the amount reached 600 and the wet biomass reached approximately 560 grams per liter of culture broth.

[0108] Fermentation was stopped after 130 hours, and the enzyme activity in the fermenter broth at the end of fermentation was determined to be 10,573 units by the DNS method (Miller, 1959). One unit is defined as the amount of enzyme required to release 1 micromolar reducing sugar (glucose equivalent) from a 10% sucrose solution in 100 mM citrate buffer at 55°C and pH 5.5. The total amount of recombinant β-fructofuranosidase in the culture broth was estimated by the Bradford assay.

[0109] Fermentation conditions: The fermentation parameters considered are given in Table 9. These essential parameters were monitored during the fermentation process.

[0110] [Table 9]

[0111] Example 5: Cell collection and purification The enzyme was collected by continuous centrifugation at 8000 RPM. The clear supernatant obtained after centrifugation was subjected to microfiltration using a spiral TFF membrane with a 0.1 micron cutoff. The filtrate was further subjected to ultrafiltration and diafiltration using a spiral TFF membrane with a 10 kDa cutoff, and was sufficiently concentrated to achieve the desired activity. The enzyme was formulated by adding 35-50% glycerol and food-grade preservatives to the final preparation. The final purity of the enzyme was observed to be 85% as determined by SDS-PAGE analysis.

[0112] Figure 6(a) depicts the SDS-PAGE analysis of samples collected at different time intervals during fermentation of Pichia pastris KM71H strain expressing recombinant β-fructofuranosidase enzyme. Figure 6(b) depicts the SDS-PAGE analysis of the purified recombinant β-fructofuranosidase enzyme.

[0113] The β-fructofuranosidase concentration was found to be approximately 2.4 g / L. In most batches, the concentration was between 2 and 5 g / L. The purity of recombinant β-fructofuranosidase was observed to be approximately 85%.

[0114] Example 6: Estimation of β-fructofuranosidase activity A study was conducted to estimate the activity of β-fructofuranosidase. For this estimation study, the amount of reducing sugar produced due to the action of the β-fructofuranosidase enzyme was calculated using the DNS (3,5-dinitrosalicylic acid) method (GL Miller, “Use of dinitrosalicylic acid reagent for determination of reducing sugar”, Anal. Chem., 1959, 31, 426-428).

[0115] To perform the enzyme activity assay, 10% sucrose (dissolved in 100 mM citrate buffer) was used as a substrate. β-fructofuranosidase was recovered from the fermentation broth and processed by ultrafiltration. The ultrafiltered sample was then diluted to 25,000X by serial dilution in 100 mM citrate buffer and used. The reaction volume was 2.5 mL. The reaction was continued for 15 minutes while maintaining the pH at 5.5.

[0116] After incubation, 3 mL of DNS (3,5-dinitrosalicylic acid) was added to each reaction mixture, boiled for 10 minutes, cooled, and the absorbance was read spectrophotometrically at 540 nm.

[0117] The ODs of glucose at different concentrations were measured as shown in Table 10 and illustrated in Figure 7. Subsequently, enzyme activity was calculated based on absorbance measurements after the reaction, as shown in Table 11. Figure 7 illustrates the glucose standard curve used to estimate the activity of the β-fructofuranosidase enzyme.

[0118] [Table 10]

[0119] [Table 11]

[0120] Example 7: Production of fructooligosaccharides (FOS) from sucrose and recombinant β-fructofuranosidase enzyme A study was conducted to understand the enzymatic activity in the formation of fructooligosaccharides. A 100 mL solution of 90% (w / v) sucrose was prepared in 150 mM sodium citrate buffer (pH 5.5). To this, 96.7 μL of β-fructofuranosidase enzyme with an activity of 51692 units / ml (equivalent to a total of 5000 units of enzyme) was added.

[0121] The reaction mixture was prepared in a 250 mL conical flask and incubated at 65°C and 220 rpm. Samples were taken at regular time intervals and analyzed on thin-layer chromatography (TLC) plates.

[0122] Glucose, sucrose, fructose, and FOS (containing kestose, nystose, and fructofuranosylnistose) were used as standards for thin-layer chromatography analysis. The mobile phase used was n-butanol:glacial acetic acid:water (4:2:2 v / v), and the developer / staining solution used was urea phosphate.

[0123] Figure 8 depicts the TLC analysis performed for the production of fructooligosaccharides (FOS) from sucrose and recombinant β-fructofuranosidase enzymes.

[0124] For quantitative estimation of fructooligosaccharide production, the samples were further subjected to high-performance liquid chromatography (HPLC). HPLC analysis was performed using an amine column (Zorbax NH2 column, Agilent Technologies) with dimensions of 4.6 (ID) × 150 mm (length) and 5 μm (particle size). Standard solutions of glucose, fructose, kestose, nystose, fructofuranosylnistose, and sucrose at different concentrations were run to generate standard curves.

[0125] Figure 9 depicts the HPLC chromatogram of the FOS sample. Table 12 shows the percentage of fructooligosaccharide (FOS) formation and the recovered glucose, fructose, and sucrose at the end of the 60-minute reaction time.

[0126] [Table 12]

[0127] To convert sucrose to FOS, 100 ml of 90% (w / v) sucrose solution was reacted with β-fructofuranosidase enzyme. After terminating the reaction by heat at the end of 60 minutes, the amounts of FOS, sucrose, glucose, and fructose recovered from the reaction were measured and presented with 90% and 100% sucrose as baseline.

[0128] The study demonstrated that the purified enzyme can effectively convert a very large amount of sugar into fructooligosaccharides.

[0129] Example 8: Characterization of recombinant β-fructofuranosidase from Aspergillus niger We characterized the β-fructofuranosidase isolated from Aspergillus niger and identified its bioactive fragments. The following bioactive fragments of β-fructofuranosidase were found to be conserved and explain its catalytic activity:

[0130] [Table 13]

[0131] The following amino acid residues in Aspergillus niger β-fructofuranosidase have been further found to be involved in the formation of a hydrogen bond network around the catalytic triad. This hydrogen bond network is crucial for the stable stereochemistry around the catalytic triad: Arg-190 ·Tyr-369 Glu-318 ·His-332 · Asp-191 ·Thr-293 · Asp-119 ·His-144

[0132] The following hydrophobic residues in the β-fructofuranosidase of Aspergillus niger were also found to be involved in the formation of a negatively charged pocket around the active site: Leu-78 · Phe-118 · Ala-370 ·Trp-398 · Ile-143

[0133] Furthermore, the following key residues of Aspergillus niger β-fructofuranosidase involved in interactions at the entry point of the active pocket were identified: Glu-405 ·His-332 ·Tyr-404

Claims

1. The modified polypeptide is a β-fructofuranosidase of Aspergillus niger containing the amino acid sequence of SEQ ID NO: 1, fused to a signal peptide selected from the group including FAK containing the amino acid sequence of SEQ ID NO: 3, FAKS containing the amino acid sequence of SEQ ID NO: 4, AT containing the amino acid sequence of SEQ ID NO: 5, AA containing the amino acid sequence of SEQ ID NO: 6, GA containing the amino acid sequence of SEQ ID NO: 7, IN containing the amino acid sequence of SEQ ID NO: 8, IV containing the amino acid sequence of SEQ ID NO: 9, KP containing the amino acid sequence of SEQ ID NO: 10, LZ containing the amino acid sequence of SEQ ID NO: 11, and SA containing the amino acid sequence of SEQ ID NO:

12.

2. The signal peptide enables the extracellular secretion of a polypeptide containing the amino acid sequence of SEQ ID NO:

1. The modified polypeptide according to claim 1.

3. A nucleic acid encoding the polypeptide described in claim 1.

4. The nucleic acid according to claim 3, selected from the group including SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:

22.

5. An expression vector comprising the nucleic acid according to claim 3, operably linked to a promoter.

6. The expression vector according to claim 5, wherein the promoter of the β-fructofuranosidase gene is selected from the group comprising AOX1, ADH3, DAS, FLD1, LRA3, THI11, GAP, YPT1, TEF1, GCw14, and PGK1.

7. The expression vector according to claim 5, wherein the vector is selected from the group comprising pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, pGAPZαC, pPIC3, pPIC3.5, pPIC3.5K, PAO815, pPIC9, pPIC9K, IL-D2, pHIL-S1, and expression vectors configured for the secretory or intracellular expression of β-fructofuranosidase from Aspergillus niger shown in Sequence ID No.

1.

8. Recombinant Pichia pastris host cells comprising the expression vector described in claim 5.

9. Recombinant Pichia Pastris host cell according to claim 8, wherein the host cell is selected from the group comprising Pichia Pastris Mut+, Mut S, Mut-, Pichia Pastris KM71H, Pichia Pastris KM71, Pichia Pastris SMD1168H, Pichia Pastris SMD1168, Pichia Pastris X33, Pichia Pastris GS115, or any other Pichia Pastris host strain.

10. A method for producing recombinant Pichia pastris host cells capable of expressing the Aspergillus niger β-fructofuranosidase shown in Sequence ID No. 1, a. A step of synthesizing a modified nucleic acid selected from sequence numbers 13 to 22, b. The step of constructing a vector containing the modified nucleic acid, and c. Step (b) to transform Pichia pastris host cells using the vector described above to obtain recombinant Pichia pastris host cells. The method, including the method described above.

11. A method for expressing Aspergillus niger β-fructofuranosidase, as shown in Sequence ID No. 1, a. Obtaining a fermented broth by culturing recombinant Pichia pastris host cells capable of expressing the Aspergillus niger β-fructofuranosidase shown in Sequence ID No. 1 in a suitable fermentation medium, wherein the recombinant Pichia pastris host cells are host cells transformed with a vector containing a modified nucleic acid selected from Sequence ID Nos. 13 to 22, b. Collecting the supernatant from the fermentation broth, wherein the supernatant contains recombinant β-fructofuranosidase, and c. Purify recombinant β-fructofuranosidase. The method, including the method described above.

12. The method according to claim 11, wherein the fermentation medium is a basic salt medium.

13. The method according to claim 11, wherein the pH of the fermented broth is maintained within the range of 4.0 to 7.

5.

14. The method according to claim 11, wherein the temperature of the fermentation broth is maintained within the range of 15°C to 45°C.

15. Use of the modified polypeptide according to claim 1 for use in the production of fructooligosaccharides.

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