DNA molecule for coding feruloyl esterase, recombinant expression vector, genetically engineered bacterium and application thereof in production of feruloyl esterase

Abstract

The invention provides a DNA molecule for coding feruloyl esterase, a recombinant expression vector, a genetically engineered bacterium and application of the genetically engineered bacterium in production of feruloyl esterase, and belongs to the technical field of microbial genetic engineering. The nucleotide sequence of the DNA molecule for coding the feruloyl esterase is as shown in SEQ ID No. 1, and the amino acid sequence correspondingly coded by the DNA molecule is as shown in SEQ ID No. 2. The invention also discloses a recombinant expression vector and a genetically engineered bacterium which comprise the DNA molecule for coding the feruloyl esterase. According to the feruloyl esterase BcFae4711, a strain extracted from soil is used for sequencing, then a corresponding expression vector and engineering bacteria are obtained through genetic engineering modification, and the produced feruloyl esterase has relatively high enzymatic activity and yield; by optimizing the selection of carriers and the optimization of induction conditions, large-scale fermentation and industrial production are realized, ferulic acid is released through enzymolysis, high-value utilization of wastes such as straws is promoted, and pollution is reduced.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering, and more particularly to DNA molecules encoding ferulic acid esterase, recombinant expression vectors, genetically engineered bacteria, and their application in the production of ferulic acid esterase. Background Technology

[0002] Agricultural waste, such as straw and fruit shells, is a major byproduct of agricultural production. Traditional disposal methods mainly involve open-air dumping, burning, or simple landfilling, leading to environmental problems such as soil degradation, water eutrophication, and greenhouse gas emissions, while also causing a serious waste of biomass resources. Promoting its resource utilization is the core path to achieving rural environmental governance and green, low-carbon agricultural development.

[0003] The complex structure of lignocellulose in plant cell walls, such as the high crystallinity of cellulose and the ester bond cross-linking between hemicellulose and lignin, is a key bottleneck for resource utilization, requiring the synergistic action of cellulase, hemicellulase, and coenzymes. Ferulic acid esterase (FAE), as a core coenzyme, can specifically hydrolyze the ferulic acid ester bonds between lignin and polysaccharides, breaking the cell wall cross-linking structure and releasing fermentable sugars and high-value-added ferulic acid. Although FAE is widely present in microorganisms, the enzyme production efficiency of wild-type strains is generally below 0.5 U / mL, which is insufficient to meet industrial requirements.

[0004] Therefore, a more efficient method for producing ferulic acid esterase is needed. Summary of the Invention

[0005] In view of the above, in order to at least partially solve at least one of the aforementioned technical problems, the present invention provides a DNA molecule encoding ferulic acid esterase, a recombinant expression vector, a genetically engineered bacterium, and its application in the production of ferulic acid esterase.

[0006] According to one aspect of the present invention, a DNA molecule encoding ferulic acid esterase is provided, wherein the nucleotide sequence of the DNA molecule is shown in SEQ ID No. 1, and the corresponding encoded amino acid sequence is shown in SEQ ID No. 2.

[0007] According to another aspect of the present invention, a recombinant expression vector is provided, comprising: the ferulic acid esterase BcFae4711 gene as shown in SEQ ID No. 1.

[0008] According to another aspect of the present invention, a genetically engineered bacterium is provided, comprising: the above-described recombinant expression vector.

[0009] According to another aspect of the present invention, the use of the above-described DNA molecule, the above-described recombinant expression vector, or the above-described genetically engineered bacteria in the production of ferulic acid esterase is provided.

[0010] According to embodiments of the present invention, the ferulic acid esterase BcFae4711 is obtained by sequencing a strain extracted from soil, followed by genetic engineering to obtain a corresponding expression vector and engineered bacteria. The resulting ferulic acid esterase exhibits high enzyme activity and yield; the highest activity of the obtained BcFae4711 can reach 6.17 U / mL. By optimizing the selection of the vector and the induction conditions, large-scale fermentation is achieved for industrial production. Through enzymatic decomposition, ferulic acid (antioxidant, pharmaceutical raw material, etc.) is released, promoting the high-value utilization of waste such as straw, replacing chemical methods for degrading lignocellulose, and reducing pollution. Attached Figure Description

[0011] The above and other objects, features and advantages of the present invention will become more apparent from the following description of embodiments of the invention with reference to the accompanying drawings, in which:

[0012] Figure 1 This is a multiple sequence alignment diagram between BcFae4711 and three known esterases in this embodiment of the invention;

[0013] Figure 2 The above are the SDS-PAGE results of ferulic acid esterase obtained by pGEX-4T-1 induction expression according to an embodiment of the present invention.

[0014] Figure 3 The graph shows the enzyme activity and pH value of ferulic acid esterase BcFae4711 in an embodiment of the present invention, where A is the optimal pH value test result and B is the pH stability test result.

[0015] Figure 4 The graphs show the enzyme activity and temperature results of ferulic acid esterase BcFae4711 in an embodiment of the present invention, where A is the optimal temperature test result and B is the temperature stability test result.

[0016] Figure 5 This is a graph showing the hydrolytic capacity test results of ferulic acid esterase BcFae4711 on four model substrates in an embodiment of the present invention;

[0017] Figure 6 This is a graph showing the effect of culture medium type on the activity of BcFae4711 enzyme in an embodiment of the present invention;

[0018] Figure 7 This is a graph showing the effect of lactose concentration on the activity of BcFae4711 enzyme in an embodiment of the present invention;

[0019] Figure 8 This is a graph showing the effect of the initial pH of the culture medium on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0020] Figure 9This is a graph showing the effect of induction timing on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0021] Figure 10 This is a graph showing the effect of induction temperature on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0022] Figure 11 This is a graph showing the effect of shaker speed on the activity of BcFae4711 enzyme in an embodiment of the present invention;

[0023] Figure 12 This is a graph showing the effect of liquid volume on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0024] Figure 13 This is a graph showing the effect of induction time on the activity of BcFae4711 enzyme in an embodiment of the present invention;

[0025] Figure 14 This is a graph showing the effect of surfactant type on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0026] Figure 15 This is a graph showing the effect of glycerol concentration on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0027] Figure 16 This is a graph showing the effect of inoculum size on the enzyme activity of BcFae4711 in an embodiment of the present invention.

[0028] Figure 17 The effects of the pairwise interactions of various factors on the activity of BcFae4711 in the embodiments of the present invention can be presented by 3D response surface plots: (a) surface plot of the effects of induction timing and glycerol concentration on the activity of BcFae4711; (b) surface plot of the effects of induction timing and inoculum amount on the activity of BcFae4711; (c) surface plot of the effects of glycerol concentration and inoculum amount on the activity of BcFae4711. Detailed Implementation

[0029] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0030] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0031] When using expressions such as "at least one of A, B, and C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). When using expressions such as "at least one of A, B, or C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, or C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.).

[0032] In this invention, the terms "BcFae4711 enzyme", "feruloesterase BcFae4711", and "BcFae4711" all refer to the feruloesterase corresponding to the DNA molecule of SEQ ID No. 1 or the amino acid sequence of SEQ ID No. 2.

[0033] The term "homology" refers to the level of similarity or percentage identity between polynucleotide sequences in terms of percentage nucleotide positional similarity (i.e., sequence similarity or identity). As used here, homology also refers to the concept of similar functional properties between different polynucleotide molecules; for example, promoters with similar functions may have homologous cis elements. Polynucleotide molecules are homologous when they specifically hybridize under certain conditions to form a double-stranded molecule. Under these conditions (called stringent hybridization conditions), one polynucleotide molecule can be used as a probe or primer to identify another polynucleotide molecule sharing homology.

[0034] The term "promoter" refers to a polynucleotide molecule that, in its native state, is located upstream of or 5' of the translation start codon in the reading frame (or protein-coding region) and participates in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0035] The term "operably linked" refers to the linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribed polynucleotide molecule (e.g., a target gene), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule influences the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are portions of a single, consecutive polynucleotide molecule, and more preferably, they are adjacent. For example, if a promoter regulates or mediates the transcription of a target gene within the cell, then the promoter is operably linked to the target gene.

[0036] The term "recombinant expression vector" refers to one or more DNA vectors used to achieve transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, along with vectors containing helper plasmids, are commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: the cis-acting sequence required for T-DNA transfer, a selection marker engineered for expression in plant cells, and the heterologous DNA sequence to be transcribed.

[0037] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.

[0038] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in microbial cells.

[0039] The terms "recombinant host cell line" or "host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell, and may also be a monocotyledonous or dicotyledonous plant cell.

[0040] In realizing the concept of this invention, it was discovered that ferulic esterase can hydrolyze the ester bonds cross-linked with phenolic acids such as ferulic acid and hemicellulose, lignin, etc., breaking the dense network structure in plant cell walls, improving the degradation efficiency of lignocellulose, and releasing high-value-added ferulic acid or ferulic acid dimers. Strengthening research on ferulic esterase plays a positive role in the in-depth degradation of lignocellulose and the resource utilization of agricultural waste. Ferulic esterase can release ferulic acid while degrading biomass. Ferulic acid (FA), also known as 4-hydroxy-3-methoxycinnamic acid, possesses antioxidant, lipid-lowering, antibacterial, anti-inflammatory, and antitumor properties, and is widely used in food, health products, cosmetics, and pharmaceuticals, with broad market application prospects.

[0041] Obtaining heterologous expression strains using molecular biology techniques is an important means to improve the activity of ferulic acid esterase. Therefore, strengthening the screening of natural microorganisms and heterologous expression of superior ferulic acid esterase genes through molecular biology methods are effective ways to obtain ferulic acid esterase, which is of great significance for the industrial application of ferulic acid esterase.

[0042] Specifically, according to one aspect of the present invention, a DNA molecule encoding ferulic acid esterase is provided, wherein the nucleotide sequence of the DNA molecule is shown in SEQ ID No. 1, and the corresponding encoded amino acid sequence is shown in SEQ ID No. 2.

[0043] According to embodiments of the present invention, the ferulic acid esterase BcFae4711 is obtained by sequencing a strain extracted from soil, followed by genetic engineering to obtain a corresponding expression vector and engineered bacteria. The resulting ferulic acid esterase exhibits high enzyme activity and yield; the highest activity of the obtained BcFae4711 can reach 6.17 U / mL. By optimizing the selection of the vector and the induction conditions, large-scale fermentation is achieved for industrial production. Through enzymatic decomposition, ferulic acid (antioxidant, pharmaceutical raw material, etc.) is released, promoting the high-value utilization of waste such as straw, replacing chemical methods for degrading lignocellulose, and reducing pollution.

[0044] SEQ ID No.1 sequence:

[0045]

[0046] SEQ ID No.2 sequence:

[0047] .

[0048] According to embodiments of the present invention, a recombinant expression vector is also provided, comprising:

[0049] The ferulic acid esterase BcFae4711 gene, as shown in SEQ ID No. 1.

[0050] According to embodiments of the present invention, recombinant expression vectors can be constructed using conventional techniques in the art, such as operably linking corresponding promoters to adapt to different fermentation regulation requirements (e.g., temperature / chemical induction); resistance labeling elements, such as ampicillin / kanamycin, resistance-free labeling systems, etc., and other operably linked expression units.

[0051] According to an embodiment of the present invention, the recombinant expression vector is used to express ferulic acid esterase BcFae4711; the expression vector is an inducible expression system designed from eukaryotic or prokaryotic microorganisms.

[0052] Specifically, the present invention does not limit the types of recombinant expression vectors. For example, recombinant expression vectors can be obtained through adaptive design using Pichiapastoris system, Aspergillus system, Gram-positive bacteria system, lactic acid bacteria system and corresponding vectors.

[0053] According to embodiments of the present invention, the expression vector includes at least one of pGEX-4T-1, pCold-TF, or pET28a.

[0054] In some specific embodiments of the present invention, the expression of ferulic acid esterase BcFae4711 can be achieved by using pGEX-4T-1, pCold-TF or pET28a as expression vectors. Preferably, the pGEX-4T-1 vector can be used to produce ferulic acid esterase BcFae4711 with an activity of up to 6.17 U / mL.

[0055] According to another aspect of the present invention, a genetically engineered bacterium is also provided, comprising a recombinant expression vector having the ferulic acid esterase BcFae4711 gene as shown in SEQ ID No. 1.

[0056] According to embodiments of the present invention, a genetically engineered bacterium capable of producing ferulic acid esterase BcFae4711 can be obtained by transfecting a recombinant expression vector into a host cell. The cultivation of the genetically engineered bacterium can be performed by methods known to those skilled in the art using existing techniques, and the fermentation method can be optimized and improved through routine experiments. Fermentation can be carried out under fermentation conditions known in the art in a suitable culture medium. The culture medium may contain: a carbon source, a nitrogen source, trace elements, and combinations thereof. During cultivation, the pH of the culture can be adjusted. During cultivation, the temperature of the culture can be controlled. During cultivation, the rotation speed can be controlled. During cultivation, the fermentation time can be controlled. During cultivation, dissolved oxygen can be controlled.

[0057] Specifically, the host cell is Escherichia coli, specifically Escherichia coli BL21(DE3), which is a B-line Escherichia coli modified by DE3 lysogenization of λ phage. It is more suitable for recombinant proteins with good solubility and no cytotoxicity, i.e. enzymes, and can be compatible with pGEX-4T-1, pCold-TF or pET28a as expression vectors.

[0058] According to another aspect of the present invention, the application of the above-described recombinant expression vector or genetically engineered bacteria in the production of ferulic acid esterase is also provided.

[0059] Specifically, this includes adding lactose to genetically engineered bacteria to induce expression of ferulic acid esterase BcFae4711.

[0060] Specifically, the concentration of lactose is 0~10 g / L, preferably 1~3 g / L, more preferably 2 g / L. For example, the concentration of lactose can be 0 g / L, 0.01 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0061] According to an embodiment of the present invention, lactose is added at the 2nd to 10th hour after the start of culturing the recombinant engineered bacteria, preferably at the 4th to 8th hour.

[0062] According to embodiments of the present invention, the lactose can be added at 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 hours after the start of culturing the recombinant engineered bacteria, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0063] According to embodiments of the present invention, if the inducer is added too early, lactose may be consumed prematurely as a carbon source nutrient. If induction is delayed, the early growth of the strain leads to the consumption of a large amount of nutrients in the culture medium, resulting in a lack of nutrients such as proteins required for enzyme synthesis and expression during induction. Furthermore, the growth phase may produce certain metabolites that inhibit the expression of exogenous proteins, leading to low enzyme activity. The optimal timing for lactose addition is during the logarithmic growth phase, when metabolic activity is strongest, T7 RNA polymerase expression is sufficient, and the response to lactose induction is highly efficient. When added early, lactose is hydrolyzed into glucose and galactose by β-galactosidase and consumed as a carbon source, reducing induction efficiency. When added in the mid-logarithmic phase, the carbon source is sufficient, and lactose mainly binds to the Lac repressor protein, initiating T7 RNA polymerase expression of the target gene.

[0064] According to embodiments of the present invention, the culture medium used in the induced expression culture process is at least one of LB medium, SOB medium, TB medium, LBBM medium, LBBMG medium, LBBNM medium, LBBSMMG medium, and MX medium, preferably at least one of LBBMG medium or SOB medium.

[0065] According to embodiments of the present invention, the pH value of the culture medium is 5 to 8, preferably 5.5 to 6. For example, the initial pH value of the culture medium can be 5, 5.5, 6, 6.5, 7, 7.5, or 8, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0066] According to embodiments of the present invention, Escherichia coli grows fastest at pH 6.0 to 7.5. pH regulates nutrient absorption efficiency and energy metabolism enzyme activity by affecting the transmembrane proton gradient. Non-extreme pH values ​​do not lead to abnormal protein charge distribution, do not increase the probability of inclusion body formation, and lactose inducers require β-galactosidase activation.

[0067] According to an embodiment of the present invention, the inoculum amount of the recombinant engineered bacteria is 0.1~3.5%v / v, preferably 0.2~3.2%v / v.

[0068] According to embodiments of the present invention, the inoculation amount of recombinant engineered bacteria can be 0.1% v / v, 0.2% v / v, 0.5% v / v, 1% v / v, 1.5% v / v, 2% v / v, 2.5% v / v, 3% v / v, or 3.2% v / v, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0069] According to embodiments of the present invention, the culture medium further includes a surfactant, preferably glycerol, wherein the concentration of glycerol is 0-8 g / L, preferably 0.01-1 g / L, and more preferably 0.5 g / L. The concentration of glycerol can be 0 g / L, 0.01 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 6 g / L, 7 g / L, or 8 g / L, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0070] According to embodiments of the present invention, surfactants are amphiphilic to cell membranes and can cause changes in cell membrane permeability, thereby altering the cell membrane's ability to transport and absorb nutrients. In addition, surfactants can also affect the activity of membrane proteins, thus influencing the metabolism of microbial cells.

[0071] Specifically, glycerol can serve as a carbon source for the growth of the strain. As the concentration of glycerol increases, the enzyme activity of BcFae4711 first increases and then decreases. Therefore, if the concentration of glycerol is too high, the osmotic pressure of the culture medium will increase sharply, causing cell dehydration and inhibiting metabolic activity. Excessive glycerol requires additional energy for conversion, resulting in an overload of the TCA cycle and weakening the ability to synthesize recombinant proteins. If the concentration of glycerol is too low, the energy supply will be insufficient, leading to a decrease in cell density. In addition, glycerol helps proteins fold correctly by maintaining the intracellular hydrophilic environment. At low concentrations, BcFae4711 is prone to forming inclusion bodies (such as the pET28a system), and the proportion of soluble enzymes will decrease.

[0072] According to embodiments of the present invention, the temperature for the culturing process of induced expression is 16~32℃, preferably 20~28℃, and more preferably 23~26℃. The temperature for the culturing process of induced expression can be 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, or 32℃, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0073] According to embodiments of the present invention, the induction temperature affects the induction rate and cell growth rate. Therefore, different exogenous proteins and expression systems will have different optimal induction temperatures. Generally, the optimal induction temperature is the induction temperature that can balance the induction rate and cell growth rate.

[0074] According to an embodiment of the present invention, as the induction time increases, the activity of BcFae4711 begins to decrease, which may be due to the degradation of the target protein caused by proteases released after cell death.

[0075] In some more preferred embodiments of the present invention, the SOB culture medium is preferably 25 mL / 250 mL, the initial pH is preferably 5.5, the induction time is preferably 5.04 h, the shaker speed is 160 rpm, the lactose is 1 g / L, the induction temperature is 24 °C, the inoculum size is 0.21% (v / v), the induction time is 18 h, and the glycerol content is 0.5 g / L. Under these conditions, the BcFae4711 activity can be achieved to 6.17 U / mL.

[0076] The present invention will be further explained below through specific embodiments. Unless otherwise specified, all reagents used are commercially available reagents and all experimental methods used are conventional experimental methods in the art. In the following experiments, each treatment was repeated three times in the data measurement, and the results are expressed as mean ± standard deviation.

[0077] The materials used in the following embodiments are as follows:

[0078] Strains: Burkholderia cepacia 20700 was isolated from soil. Escherichia coli DH5α and BL21(DE3) (Takara, Japan) were used for plasmid preparation and protein expression, respectively. Plasmids pET28a, pCold-TF, and pGEX-4T-1 were used to construct expression vectors.

[0079] Reagents: EZgene plasmid extraction kit and EZNA gel extraction kit were purchased from Biomiga (USA) and Omega Bio-tek (USA), respectively. Restriction enzymes and other reagents used for gene manipulation were purchased from NEB (New England Biolabs). Methyl ferulic acid and ferulic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade.

[0080] Culture medium: The preparation methods of the following culture media are mainly based on the reference [Go lot in, V.A., et al., Optimization of cold-adapted alpha-galactosidase expression in Escherichiacoli. Protein Expr Purif, 2016. 123: p. 14-8.].

[0081] LB medium (1 L): 5 g yeast extract, 10 g tryptone, 10 g NaCl;

[0082] SOB medium (1 L): 5 g yeast extract, 20 g tryptone, 0.5 g NaCl, 10 ml 0.25 mol / L KCl solution, 5 ml 2 mol / L MgCl2·6 H2O;

[0083] TB medium (1 L): 24 g yeast extract, 12 g tryptone, 4 mL glycerol, 100 mL 0.17 mol / L KH2PO4 and 0.72 mol / L K2HPO4·3 H2O buffer solution;

[0084] LBBM medium (1 L): 5 g yeast extract, 10 g tryptone, 10 g NaCl, 100 mL 0.17 mol / L KH2PO4 and 0.72 mol / L K2HPO4·3 H2O buffer solution, 5 mL 2 mol / L MgCl2·6 H2O;

[0085] LBBNM medium (1 L): 5 g yeast extract, 10 g tryptone, 30 g NaCl, 100 mL 0.17 mol / L KH2PO4 and 0.72 mol / L K2HPO4·3 H2O buffer solution, 5 mL 2 mol / L MgCl2·6 H2O;

[0086] LBBMG medium (1 L): 5 g yeast extract, 10 g tryptone, 10 g NaCl, 100 mL 0.17 mol / L KH2PO4 and 0.72 mol / L K2HPO4·3 H2O buffer solution, 5 mL 2 mol / L MgCl2·6 H2O, 4 mL glycerol;

[0087] LBBSMG medium (1 L): 5 g yeast extract, 10 g tryptone, 10 g NaCl, 100 mL 0.17 mol / L KH2PO4 and 0.72 mol / L K2HPO4·3 H2O buffer solution, 5 mL 2 mol / L MgCl2·6 H2O, 4 mL glycerol, 72 g sorbitol;

[0088] MX medium (1 L): 5 g yeast extract, 10 g tryptone, 100 mL 0.17 mol / L KH2PO4 and 0.72 mol / L K2HPO4·3 H2O buffer solution, 5 mL 2 mol / L MgCl2·6 H2O, 4 mL glycerol, 72 g sorbitol.

[0089] Example 1: BcFae4711 gene sequencing

[0090] Whole-genome sequencing and functional gene annotation were performed on strain B. cenocepacia 20700. Total DNA was extracted from this strain using a bacterial genome extraction kit. The theoretical molecular weight and isoelectric point of ferulic esterase BcFae4711 were predicted online; the presence of a signal peptide in protein BcFae4711 was predicted using Signal P analysis; and multiple sequence alignment was performed using MEGA X and ESPript 3.

[0091] Analysis revealed a 1722 bp open reading frame (ORF) encoding a hypothetical 574-amino acid ferulic acid esterase, named BcFae4711. Online prediction indicated a molecular weight of 59.27 kDa and a pI of 5.31. SignalP 4.1 analysis showed that BcFae4711 contains a 21-amino acid signal peptide. The molecular weight of BcFae4711 is larger than that of most bacterial ferulic acid esterases (27-45 kDa).

[0092] Figure 1This is a multiple sequence alignment diagram of BcFae4711 and three known esterases in this embodiment of the invention. The three esterases for sequence alignment are MHETase-6QZ1 from Ideonella sakaiensis; AoFae-6G21 from Aspergillus oryzae; and FoFaeC-8BHH from Fusarium oxysporum.

[0093] according to Figure 1 It has been observed that BcFae4711 possesses the highly conserved "Gly-x-Ser-x-Gly" motif among esterases, and that BcFae4711 exhibits a typical Ser-Asp-His catalytic triad structure (Ser 210, His 493, Asp 456). The conserved GXSXG motif and the catalytic triad (Ser 210, His 493, Asp 456) are indicated by boxes and stars, respectively.

[0094] Example 2: Cloning of the BcFae4711 gene

[0095] Primers F / R were designed based on the gene sequence encoding the BcFae4711 protein (as shown in Table 1 below) for cloning the target gene.

[0096] Using genomic DNA as a template, the target gene BcFae4711 was amplified. The PCR reaction system (50 μL) contained 5 μL 10×Q5 buffer, 4 μL dNTP (2.5 mM), 1 μL genomic DNA, 1 μL F primer (10 mM), 1 μL R primer (10 mM), 0.5 μL Q5 polymerase, and water added to a final volume of 50 μL.

[0097] Amplification conditions: 95℃ pre-denaturation for 3 min, 30 cycles: 94℃ denaturation for 30 s, 65℃ annealing for 1.5 min, 72℃ extension for 1 min; 72℃ further extension for 10 min. PCR amplification products were detected by 1% agarose gel electrophoresis. The PCR products were then ligated into the pMD-18T vector and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0098] The results showed that the BcFae4711 gene of B.cepacia 20700 was obtained by PCR amplification and successfully cloned into the vector pMD18-T. The target gene BcFae4711 was verified by gel electrophoresis.

[0099] Table 1

[0100]

[0101] Note: BamHI restriction sites are marked with solid lines, HindIII with short dashes, and NotI with dashed lines.

[0102] Example 3: Construction and Induction of Recombinant Expression Vector

[0103] Construction of recombinant expression vectors:

[0104] Using the recombinant plasmid pMD-18T-BcFae4711 containing the BcFae4711 gene prepared in Example 2 as a template, primers F1 / R1, F2 / R2 and F3 / R3 (Table 1) were designed, and the BcFae4711 gene containing restriction sites (BamHI and XhoI or BamHI and HindIII) and corresponding homologous fragments of the expression vector were amplified by PCR.

[0105] The purified gene and plasmid were double-digested with the corresponding restriction enzymes. The digestion system is shown in Table 2. The reaction conditions were 37℃ for 2 h. The digestion products were verified by 1% (m / v) agarose gel electrophoresis and purified using a gel extraction kit. Ligation was performed overnight at 4℃ using T4 DNA ligase. The PCR products were ligated into different expression vectors, and successfully cloned into the expression vectors pET-28a, pCold-TF, and pGEX-4T-1. The successfully constructed recombinant plasmids were transformed into *E. coli* BL21(DE3) cells. Positive clones were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced recombinant bacteria were deposited in glycerol tubes.

[0106] Table 2. Expression vector double enzyme digestion reaction system

[0107]

[0108] Induced expression and testing:

[0109] The recombinant strain preserved in glycerol tubes was inoculated at a rate of 0.5% into LB medium containing 50 μg / mL ampicillin and activated at 37°C and 200 rpm for 12 h. The activated seed culture was then inoculated at a rate of 0.2% into fresh LB liquid medium containing resistance (100 μg / mL ampicillin was added for plasmids pGEX-4T-1-BcFae4711 and pCold-TF-BcFae4711, while 40 μg / mL kanamycin was added for plasmid pET28a-BcFae4711), and cultured at 37°C and 220 rpm until OD500. 600The concentration was 0.6-0.8, and 0.1 mM IPTG was added for induction. The cells were then cultured for 20 h at 20℃ (pGEX-4T-1-BcFae4711 and pET28a-BcFae4711) and 15℃ (pCold-TF-BcFae4711), respectively.

[0110] The fermentation broth was centrifuged at 10,000 rpm for 5 min at 4°C to collect the bacterial cells, which were then resuspended in buffer (50 mM, pH 5.5 citrate buffer). The cells were then disrupted by sonication (100 W, 20 kHz: 2 s on, 3 s off; total time 15 min). The disrupted cell solution was centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatant was obtained as the crude enzyme solution.

[0111] Protein expression was detected by SDS-PAGE: 20 μL of sample was mixed with 5 μL of 5×SDS loading buffer, boiled, and then analyzed by SDS-PAGE. The stacking gel concentration was 4.5%, the separating gel concentration was 10%, and electrophoresis was performed at a constant voltage of 100 V.

[0112] The constructed pET28a-BcFae4711 recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. After expression induction with 0.5 mM IPTG, whole-cell lysates were obtained by sonication. Soluble and insoluble precipitates were separated by centrifugation at 12,000 × g. SDS-PAGE analysis showed a target protein-specific band at approximately 60 kDa, consistent with the expected molecular weight. This band was only present in the whole-cell lysate and insoluble precipitate, while no corresponding signal was detected in the soluble supernatant. The experimental results indicate that the recombinant target protein exists in inclusion body form during host bacterial expression, and renaturation is required to obtain the bioactive protein.

[0113] Enzyme activity assay: Using MFA as the substrate, the amount of ferulic acid produced by the hydrolysis of BcFae4711 was determined by HPLC, and the enzyme activity of BcFae4711 was calculated. 20 μL of 25 mM substrate solution was mixed with 430 μL of citrate buffer (50 mM, pH 5.5), preheated at 50 °C for 3 min, then 50 μL of appropriately diluted enzyme solution was added, and the reaction was carried out at 50 °C for 10 min. The reaction was then terminated by adding 500 μL of acetonitrile. An equal volume of buffer solution was used as a blank instead of the enzyme solution. The substrate and product were analyzed by HPLC using a UV detector and a ZORBAX Eclipse Plus C-18 column. The sample was filtered through a 0.22 μm filter before analysis. The mobile phase consisted of methanol:0.7% acetic acid (1:1, v / v), eluted at a constant flow rate of 0.5 mL / min. The detection wavelength was 320 nm, and the temperature was 30 °C. The activity unit (U) of ferulic acid esterase is defined as the amount of enzyme required to release 1 μmol of ferulic acid in 1 min at 50 °C and pH 5.5.

[0114] The enzyme activity assay results showed that only the total protein component and the supernatant had extremely low enzyme activity.

[0115] The constructed pCold-TF-BcFae4711 recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. After expression under 15°C cold shock induction conditions (0.5 mM IPTG), the theoretical molecular weight of the fusion protein TF-BcFae4711 was approximately 111 kDa. Fractional centrifugation experiments verified that, under the synergistic effect of the molecular chaperone TF and the cold shock promoter cspA, the target protein was distributed in a soluble form in the cytoplasm, achieving a significant optimization of the expression form compared to the inclusion body expression mode formed by the pET28a-BcFae4711 system. However, enzyme activity assays showed that although the enzyme activity detected in the soluble fraction was improved compared to the pET28a system, it was still lower. Further verification using enterokinase-specific cleavage experiments confirmed that the soluble characteristics of the target protein were completely preserved after removing the TF fusion tag, but enzyme activity did not show substantial improvement. Experimental data show that although soluble expression of the target protein was achieved through vector engineering strategies, its spatial conformation suffered from critical misfolding, preventing the catalytic center from forming an effective functional conformation. This finding provides important experimental evidence for subsequent targeted modification of protein folding mechanisms and optimization of expression conditions. Figure 2 The SDS-PAGE results of ferulic acid esterase obtained by induction expression of vector pGEX-4T-1 in an embodiment of the present invention are shown.

[0116] according to Figure 2As can be seen, the successfully constructed pGEX-4T-1-BcFae4711 recombinant plasmid was transformed into *E. coli* BL21(DE3) cells and expression was induced. The theoretical molecular weight of the fusion protein GST-BcFae4711 is approximately 85 kDa, but SDS-PAGE results showed no protein band at this location, while a band resembling the target protein appeared at around 60 kDa. This is consistent with the size of the target protein without the GST tag, and most of the target protein was expressed in a soluble manner. The reason for this phenomenon may be that the GST fusion tag was hydrolyzed or cleaved by other mechanisms during expression. Enzyme activity assay results showed that the crude enzyme activity was 1.54 U / mL.

[0117] Example 4: Purification of recombinant BcFae4711 for enzymatic property studies

[0118] 1. Purification of recombinant BcFae4711

[0119] Because the N-terminal GST tag of the fusion protein GST-BcFae4711 was destroyed during the induction expression process of the expression vector pGEX-4T-1-BcFae4711, a recombinant plasmid with a 6*His tag added to the C-terminus of the target protein was constructed for induction expression.

[0120] Using the recombinant plasmid pMD-18T-BcFae4711 containing the BcFae4711 gene as a template, primers were designed.

[0121] His-S (SEQ ID No. 11):

[0122] 5'-GGTTCCGCGTGGATCCTTGAACAGAAAATCTGCATTC -3'.

[0123] His-A (SEQ ID No. 12):

[0124] 5'-CGATGCGGCCGCTCAATGATGATGATGATGATGGCGGCAACTGAAGCT-3'.

[0125] The BcFae4711 gene, containing restriction enzyme sites (Not I and BamHI), a C-terminal 6*His tag, and a homologous fragment to the expression vector pGEX-4T-1, was amplified by PCR. The plasmid pGEX-4T-1 was double-digested with enzymes. The PCR product was ligated into the expression vector pGEX-4T-1 using the NovoRecplus one-step PCR cloning kit (Novoprotein, Shanghai).

[0126] The successfully sequenced recombinant plasmid was transformed into the expression host *E. coli* BL21. The recombinant strain was activated using LB medium and then inoculated into 100 mL of SOB liquid medium containing 100 μg / mL ampicillin at an initial pH of 5.0. The culture was incubated at 37°C for 4 h, followed by induction with 0.05 mM IPTG at 240 rpm for 24 h at 26°C. The culture was centrifuged at 9400 × g for 10 min at 4°C, and the cells were collected. The cells were resuspended in lysis buffer (50 mM potassium phosphate, pH 7.0). Cells were lysed by sonication (100 W, 20 kHz: 2 s on, 3 s off; total time: 15 min). The cell lysate was centrifuged at 15,000 × g for 15 min, and the supernatant was the crude enzyme solution of BcFae4711.

[0127] First, the Ni-HisTrapHP column (GE Healthcare, USA) and AKTA FPLC protein purification system were equilibrated using equilibration buffer (20 mM sodium phosphate, 0.3 M NaCl, 20 mM imidazole, pH 8.0). The recombinant protein BcFae4711 was eluted using a linear elution method with the elution buffer consisting of 20 mM sodium phosphate, 0.3 M NaCl, 500 mM imidazole, pH 8.0. The purified protein was dialyzed to remove imidazole. Protein purity was analyzed by SDS-PAGE. Protein concentration was determined using a BCA assay kit. The purified BcFae4711 was used for enzymatic property studies.

[0128] 2. Optimal pH and pH stability of BcFae4711

[0129] Using methyl ferulic acid as a substrate, the optimal reaction pH for BcFae4711 was investigated. Buffers (50 mM) with pH ranges of 2.0–12.0 were selected: Gly-HCl buffer (pH 2.0–3.5), citrate buffer (pH 3.0–6.5), phosphate buffer (pH 6.0–8.0), barbiturate buffer (pH 6.0–7.5), Gly-NaOH buffer (pH 9.0–10.5), and Tris-HCl buffer (pH 8.0–8.5). Enzyme activity was measured using the method described above, and the results are as follows: Figure 3 As shown in Figure A.

[0130] To investigate the pH stability of BcFae4711, it was incubated in buffer solutions of different pH values ​​at 37°C for 30 min, and then cooled in an ice-water mixture for 30 min. The residual ferulic acid esterase activity was then measured, and the results are as follows: Figure 3 As shown in B.

[0131] Figure 3 The graphs show the enzyme activity and pH results of ferulic acid esterase BcFae4711 in an embodiment of the present invention, where A is the optimal pH test result and B is the pH stability test result.

[0132] according to Figure 3 It can be seen that the optimal reaction pH for BcFae4711 is 5.0, and more than 80% of the enzyme activity is retained when the pH is 4.0-5.5. Furthermore, pH stability measurements show that BcFae4711 is more stable under slightly acidic conditions. When the pH is 5.0-6.5, BcFae4711 can retain approximately 60% of its enzyme activity.

[0133] 3. Optimal reaction temperature and temperature stability of BcFae4711

[0134] Using methyl ferulic acid ester as a substrate and citrate buffer (50 mM, pH 5.0) as the reaction buffer, the effect of different reaction temperatures (30-70℃) on the activity of BcFae4711 enzyme was investigated. To examine the thermostability of the enzyme, recombinant BcFae4711 was incubated at different temperatures (30-70℃) for 30 min, then cooled on ice for 30 min. The residual ferulic acid esterase activity was measured under standard assay conditions. The results are as follows: Figure 4 As shown in Figure A.

[0135] To determine the half-life, the target protein BcFae4711 was incubated at 45°C for 1.5 h and then at 50°C for 1 h. Samples were taken at each time point, and residual enzyme activity was determined according to standard methods. Relative enzyme activity is the percentage of residual enzyme activity relative to the initial enzyme activity. The results are shown below. Figure 4 As shown in B.

[0136] Figure 4 The graphs show the enzyme activity and temperature results of ferulic acid esterase BcFae4711 in an embodiment of the present invention, where A is the optimal temperature test result and B is the temperature stability test result.

[0137] according to Figure 4 It can be seen that enzyme activity begins to decline rapidly at temperatures above 60℃. Temperature stability results show that at pH 5.0, incubation at 45℃ for 30 min resulted in only a 25% loss of activity. However, the thermostability of BcFae4711 decreased rapidly at temperatures above 50℃. Furthermore, the half-life of BcFae4711 at 45℃ and 50℃ was investigated. The results show that the half-life of BcFae4711 was 72.6 min at 45℃, while it decreased to 37.5 min at 50℃.

[0138] 4. Substrate specificity and kinetic constants of BcFae4711

[0139] BcFae4711 substrate specificity: Prepare 25 mM solutions of different substrates, including MFA, MpCA, MCA, and MSA. Determine BcFae4711 enzyme activity under optimal conditions. Define the highest enzyme activity as 100%, and calculate the remaining relative enzyme activities.

[0140] Determination of BcFae4711 kinetic constants: The hydrolysis kinetic parameters of BcFae4711 on MFA, MpCA, MCA, and MSA were determined. Substrate solutions of MFA, MpCA, MCA, and MSA at different concentration gradients were prepared, and the enzyme activity of BcFae4711 was measured at the optimal temperature and pH for a reaction time of 5 min. The maximum reaction rate V of BcFae4711 was calculated using data analysis and plotting software. max and the Mi constant K m The result is as follows Figure 5 As shown.

[0141] Figure 5 The graph shows the hydrolytic capacity test results of ferulic acid esterase BcFae4711 on four model substrates in an embodiment of the present invention.

[0142] according to Figure 5 It can be seen that BcFae4711 has the strongest hydrolytic ability for MCA (defined as 100% enzyme activity), and also has a strong hydrolytic ability for MpCA and MFA, with relative enzyme activities of 96.8% and 72.9%, respectively. It has the lowest specificity for MSA, with a relative enzyme activity of only 0.9%.

[0143] Furthermore, the kinetic parameters of BcFae4711 were determined. The maximum reaction rate V of BcFae4711 was calculated using Graphpad Prism 5 by measuring the hydrolytic activity of BcFae4711 for different concentrations of different substrates. max and the Mi constant K m The results are shown in Table 3 below.

[0144] Table 3. Determination of enzyme kinetic parameters of BcFae4711

[0145]

[0146] As shown in Table 3, BcFae4711 exhibits the highest catalytic efficiency for the substrate MCA, V max It was 111.2 U / mg. K mK is one of the characteristic constants of an enzyme, used to represent the affinity between the enzyme and its substrate; a larger value indicates a smaller affinity. Table 3 shows that BcFae4711 has the highest substrate affinity for MCA, K... m It is 0.22 mM.

[0147] Therefore, the enzymatic properties of this enzyme indicate that BcFae4711 has an optimal pH of 5.0 and remains stable under slightly acidic conditions; the optimal temperature for BcFae4711 is 50℃. In conclusion, BcFae4711 is a novel ferulic acid esterase with significant research value and application potential.

[0148] Example 5: Optimization of conditions for the production of ferulic acid esterase BcFae4711

[0149] 1. Optimization of lactose-induced enzyme production conditions

[0150] Fermentation conditions for BcFae4711 production by recombinant strain pGEX-4T-1-BcFae4711 were optimized using lactose as an inducer, aiming to improve the expression level of BcFae4711. The optimizations mainly focused on factors such as culture medium type, initial pH, lactose concentration, inoculum size, induction timing, induction temperature, shaking speed, and induction time, as shown in Table 4 below.

[0151] Table 4. Factors and levels for optimizing lactose-induced enzyme production conditions.

[0152]

[0153] Figure 6 The figure shows the effect of culture medium type on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0154] according to Figure 6 It can be seen that the composition of the culture medium can significantly affect the expression of recombinant proteins. To select a suitable culture medium for BcFae4711-induced expression, eight commonly used media were compared: MX, LBBSMG, LBBM, LB, LBBMG, TB, LBBNM, and SOB. Figure 6 As shown, the recombinant protein BcFae4711 exhibited the highest enzyme activities of 1.67 and 1.62 U / mL in SOB and LBBMG media, respectively. Since SOB medium has a simpler composition than LBBMG, it was chosen for induction of expression.

[0155] Figure 7 The figure shows the effect of lactose concentration on the enzyme activity of BcFae4711 in an embodiment of the present invention.

[0156] according to Figure 7It can be seen that lactose concentration is also an important factor affecting the activity of BcFae4711. In this study, six different lactose concentrations (0, 2, 4, 6, 8, and 10 g / L) were set. When the lactose concentration was low (2 g / L), the activity of BcFae4711 was high, while at higher lactose concentrations, the activity of BcFae4711 decreased. The highest BcFae4711 activity was 1.86 U / mL when the lactose concentration was 2 g / L.

[0157] Figure 8 The figure shows the effect of the initial pH of the culture medium on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0158] according to Figure 8 It can be seen that the optimal pH value for BcFae4711 expression was investigated within the pH range of 5 to 8. The activity of BcFae4711 first increased and then decreased with increasing pH value, and the activity of BcFae4711 was highest at pH value of 5.5, reaching 1.75 U / mL.

[0159] Figure 9 The figure shows the effect of induction timing on the activity of BcFae4711 enzyme in the embodiments of the present invention.

[0160] according to Figure 9 It can be seen that the optimal induction time is 6 hours, at which point the cells are in mid-logarithmic growth phase.

[0161] Figure 10 The figure shows the effect of induction temperature on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0162] according to Figure 10 It can be seen that the induction temperature is a key factor affecting the secretion and solubility of exogenous proteins, protein yield and enzyme activity. Lowering the induction temperature can promote the soluble expression of the target protein. In the study of this invention, the optimal induction temperature for BcFae4711 expression is 24℃, while in related technologies, the optimal induction temperature for heterologous protein expression in E. coli cells is 20~25℃.

[0163] Figure 11 This is a graph showing the effect of shaker speed on the activity of BcFae4711 enzyme in an embodiment of the present invention; Figure 12 The figure shows the effect of liquid volume on the enzyme activity of BcFae4711 in an embodiment of the present invention.

[0164] according to Figure 11 and Figure 12It can be seen that dissolved oxygen content affects the growth of *E. coli* and plays an important role in the expression of exogenous proteins. The effects of dissolved oxygen level on BcFae4711 expression were investigated by examining two factors: shaker speed and liquid volume. The results showed that the activity of BcFae4711 first increased and then decreased with increasing shaker speed and liquid volume.

[0165] Figure 13 The figure shows the effect of induction time on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0166] according to Figure 13 It can be seen that induction time is also an important factor affecting the soluble expression of proteins. Although prolonged induction usually increases the activity of the target protein, the optimal induction time varies for different target proteins. With prolonged induction time, the activity of BcFae4711 continuously increases, reaching a maximum at 24 h. With further extension of induction time, the activity of BcFae4711 begins to decrease, which may be due to the degradation of the target protein caused by proteases released after cell death.

[0167] Figure 14 This is a graph showing the effect of surfactant type on the activity of BcFae4711 enzyme in an embodiment of the present invention. Figure 15 The figure shows the effect of glycerol concentration on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0168] according to Figure 14 and Figure 15 It can be seen that surfactants are amphiphilic to cell membranes, altering cell membrane permeability and thus changing the cell membrane's ability to transport and absorb nutrients. Furthermore, surfactants also affect the activity of membrane proteins, influencing microbial cell metabolism. Experimental results show that different types of surfactants have different effects on the activity of ferulic acid esterase BcFae4711. While glycerol increased the activity of BcFae4711, Tween-20 and Tween-80 had no significant effect, while the remaining surfactants inhibited the enzyme's activity. This may be due to the different effects of different surfactants on the cell membrane. Studies on glycerol concentrations revealed that compared to the untreated group, the addition of 0.5–2 g / L of glycerol increased the activity of BcFae4711, reaching its highest value at a concentration of 0.5 g / L.

[0169] Figure 16 The figure shows the effect of inoculum size on the activity of BcFae4711 enzyme in an embodiment of the present invention.

[0170] according to Figure 16It can be seen that during the induction of recombinant bacterial expression of BcFae4711, the inoculum size affects the biomass in the fermentation broth to some extent, and biomass is usually related to the amount of heterologous protein produced by the recombinant bacteria. When the inoculum size is higher than 0.1%, different inoculum sizes (0.2-3.2% (v / v)) have no significant effect on the activity of BcFae4711. The highest activity of BcFae4711, reaching 1.55 U / mL, is achieved when the inoculum size is 0.2%.

[0171] Example 5: Induction conditions for the production of ferulic acid esterase BcFae4711 - Plackett-Burman experiment

[0172] Based on the results of the single-factor experiments, six factors were selected for the Plackett-Burman (PB) experimental design, including induction temperature (X1), rotation speed (X2), inoculum size (X3), lactose concentration (X4), induction timing (X5), induction time (X6), and glycerol concentration (X7). High (1) and low (-1) levels were selected for each factor, with BcFae4711 enzyme activity as the response value. The PB experiment was designed using statistical analysis software, and a regression model was established based on the experimental data.

[0173] Seven significant factors were selected based on the results of single-factor experiments. A Plackett-Burman (PB) experimental design was then used to analyze these six factors using statistical analysis software. The results are shown in Table 5. The activity of BcFae4711 ranged from 1.03 to 5.46 U / mL. The significance level of each factor was determined by the P-value; a P-value < 0.05 indicated that the factor was significant. Table 6 shows that the factors significantly affecting BcFae4711 activity were induction timing (X5), glycerol concentration (X7), and inoculum size (X4), while temperature (X1), rotation speed (X2), lactose concentration (X3), and induction time (X6) had no significant effect on BcFae4711 activity. Therefore, the above three significant factors should be further studied in subsequent experiments, while the two non-significant factors can be ignored. In subsequent experiments, the initial temperature (X1), rotation speed (X2), lactose concentration (X3), and induction time (X6) were set to 24 ℃, 160 rpm, 2 g / L, and 18 h, respectively, based on the optimal results of the single factors.

[0174] Table 5 Factor Levels and Statistical Analysis of PB Experimental Design

[0175]

[0176] Note: "*" indicates significance at the 5% level (P<0.05); "**" indicates significance at the 1% level (P<0.01).

[0177] Table 6 PB Experimental Design and Results

[0178]

[0179] 3. Steepest Slope Climbing Experiment

[0180] Based on the regression model obtained from the PB experiment, three significant factors were selected. The direction and step size of these factors' effects were then determined according to their relative magnitudes, leading to the design of a steepest ramp-up experiment. The experiment proceeded along the steepest upward path, incorporating practical experience, until BcFae4711 activity ceased to increase. Through this steepest ramp-up design, the point of highest BcFae4711 activity would be close to the optimum; therefore, the point of highest BcFae4711 activity was used as the center point of the RSM for subsequent experiments.

[0181] To determine the optimal regions for the five significant factors, a steepest ramp design was employed. Regression analysis of the PB experiment results determined the direction of change for each factor. To obtain the maximum BcFae4711 activity, the value of induction timing (X5) should be reduced, while the values ​​of glycerol concentration (X7) and inoculum size (X4) should be increased (Table 7). The results showed that the fourth group of experiments exhibited the highest BcFae4711 activity, reaching 5.43 U / mL. Therefore, the fourth group of experiments was used as the center point for subsequent response surface methodology experiments.

[0182] Table 7. Experimental Design and Results for the Steepest Climb

[0183]

[0184] 4. Response Surface Analysis

[0185] After approximating the region of highest BcFae4711 activity through the steepest ramp experiment, the Box-Behnken experimental design from the response surface methodology was employed. Optimization software was used to further investigate the three key factors (induction temperature (A), induction time (B), and shaker speed (C)) and the center point determined by the PB experiment and the steepest ramp experiment, aiming to enhance the activity of BcFae4711. Each factor was assigned three levels, coded with -1, 0, and 1 respectively.

[0186] Based on the results of the PB experiment and the steepest ascent experiment, a Box-Behnken experimental design was used to conduct a three-factor (induction temperature, induction time, and shaker speed) three-level response surface methodology experiment, with the BcFae4711 activity Y as the response value. Each factor had three levels, coded as -1, 0, and 1, respectively, for a total of 15 experiments. Table 8 shows the experimental design and results. As can be seen from the table, the BcFae4711 activity varied considerably, depending on different culture conditions. The highest BcFae4711 activity (6.06 U / mL) was observed in group 13, while the lowest activity (2.66 U / mL) was observed in group 10.

[0187] Table 8 Response Surface Design and Results

[0188]

[0189] Through multiple regression analysis of 15 sets of experimental data, and after fitting the regression equation, the influence of each factor on the response value can be represented by the following function:

[0190] Y=-37.23+6.30×A+32.78×B+187.46×C+0.28×AB-10.14×AC+12.76×BC-0.43×A²-36.29×B²-348.31×C² (1).

[0191] In equation (1), Y is the predicted value (BcFae4711 activity); the analysis of variance and model reliability analysis of the regression equation are shown in Table 9. Table 9 shows that the experiment has a low coefficient of variation (CV). The lower the CV value, the higher the reliability of the experiment. In this experiment, CV = 7.61%, indicating that the experimental results are reliable. The correlation coefficient R of this equation is... 2 =0.9693, indicating that the model can explain the changes in BcFae4711 activity, showing a good fit of the equation. In the regression equation, the p-values ​​of the first-order terms A (induction timing) and C (inoculum size) are both less than 0.05, indicating that these two factors have a significant linear relationship with the expression of ferulic acid esterase BcFae4711 in *E. coli*; the interaction term AC is significant, indicating that the interaction of these three factors has a small effect on the expression of ferulic acid esterase BcFae4711 in *E. coli*; in the quadratic term, B... 2 and C 2 The results were significant (P<0.05), indicating a significant surface effect between glycerol concentration and inoculum size and ferulic acid esterase BcFae4711 activity.

[0192] Table 9. Regression coefficient analysis and significance analysis in the response surface methodology results.

[0193]

[0194] Note: "*" indicates significance at the 5% level (P<0.05); "**" indicates significance at the 1% level (P<0.01).

[0195] Figure 17 The effects of the pairwise interactions of various factors on the activity of BcFae4711 in the embodiments of the present invention can be presented by 3D response surface plots: (a) surface plot of the effects of induction timing and glycerol concentration on the activity of BcFae4711; (b) surface plot of the effects of induction timing and inoculum amount on the activity of BcFae4711; (c) surface plot of the effects of glycerol concentration and inoculum amount on the activity of BcFae4711.

[0196] Figure 17 The results in (a) indicate that the activity of BcFae4711 gradually increases with decreasing induction time, and this phenomenon is more pronounced when the glycerol concentration is set at a lower level. This suggests that, at an appropriate glycerol concentration, shortening the induction time will benefit the expression of BcFae4711. With increasing glycerol concentration, the activity of BcFae4711 first increases and then decreases, with less impact when the induction time is shorter. Figure 17 Analysis in (a) showed that the optimal induction time and glycerol concentration range for inducing BcFae4711 expression were 5-6 h and 0.42-0.62 g / L, respectively; the effects of A (induction time) and C (inoculum size) on BcFae4711 activity were as follows: Figure 17 As shown in (b), the activity ranged from 2.89 to 6.06 U / mL. When the inoculum size was low, the timing of induction had a smaller impact on enzyme activity. This is because at a higher inoculum size, the strain's growth time was too long, which would reduce the nutrients in the culture medium, thus affecting the strain during the induction phase. The balance between cell number, cell activity, exogenous protein expression rate, and correct protein folding is the key to high protein expression. Inoculum size and timing of induction are key factors that determine the number of cells during induction. Figure 17 (c) shows the effect of B (glycerol concentration) and C (inoculation amount) on the activity of BcFae4711. The activity of BcFae4711 first increased and then decreased with the increase of glycerol concentration and inoculation amount.

[0197] Using analytical software, the optimal activity of BcFae4711 was predicted with A (induction timing) = 5.04 h, B (glycerol concentration) = 0.51 g / L, and C (inoculum size) = 0.21% as critical values. The maximum predicted value of Y (BcFae4711 activity) was 6.17 U / mL. To verify the accuracy of the model, a validation experiment was conducted under the optimal fermentation conditions based on the above optimization results. Specifically, the experiment was repeated under the following conditions: lactose 1 g / L, pH 5.5, induction timing 5.04 h, induction temperature 24℃, shaker speed 160 rpm, liquid volume 25 mL / 250 mL, inoculum size 0.21% (v / v), induction time 18 h, and 0.5 g / L glycerol. The average value obtained was compared with the predicted value. The maximum BcFae4711 activity was 6.17 U / mL, which is close to the predicted value, proving that the model is relatively accurate and effective. The results showed that the activity of BcFae4711 was 4 times that of the unoptimized version.

[0198] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The DNA molecule encoding ferulic acid esterase, wherein, The nucleotide sequence of the DNA molecule is shown in SEQ ID No. 1, and its corresponding encoded amino acid sequence is shown in SEQ ID No.

2.

2. A recombinant expression vector, comprising: The ferulic acid esterase BcFae4711 gene, as shown in SEQ ID No.

1.

3. The recombinant expression vector according to claim 2, wherein, The recombinant expression vector is used to express ferulic acid esterase BcFae4711; The expression vector is an inducible expression system designed from eukaryotic or prokaryotic microorganisms.

4. The recombinant expression vector according to claim 2 or 3, wherein, The expression vector includes at least one of pET-28a, pCold-TF, or pGEX-4T-1.

5. A genetically engineered bacterium, comprising: The recombinant expression vector according to any one of claims 2 to 4.

6. The genetically engineered bacteria according to claim 5, wherein, The hosts of the genetically engineered bacteria include Escherichia coli DH5α, Escherichia coli BL21(DE3), Pichia pastoris, Bacillus, and Aspergillus niger, preferably Escherichia coli BL21(DE3).

7. The use of a DNA molecule according to claim 1, an expression vector according to any one of claims 2 to 4, or a genetically engineered bacterium according to claim 5 or 6 in the production of ferulic acid esterase.

8. The application according to claim 7, comprising: Lactose was added to the genetically engineered bacteria of claim 5 or 6 to induce expression of ferulic acid esterase BcFae4711.

9. The application according to claim 8, wherein, The culture medium used in the induction expression culture process is at least one of LB medium, SOB medium, TB medium, LBBM medium, LBBMG medium, LBBNM medium, LBBSMG medium, and MX medium, preferably at least one of LBBMG medium or SOB medium; The culture medium further includes a surfactant, preferably glycerol, wherein the concentration of glycerol is 0~8 g / L, preferably 0.01~1 g / L, and more preferably 0.5 g / L; The lactose is added between 2 and 10 hours after the start of culturing the recombinant engineered bacteria, preferably between 4 and 8 hours. The inoculation amount of the recombinant engineered bacteria is 0.1~3.5%v / v, preferably 0.2~3.2%v / v.

10. The application according to claim 9, wherein, The concentration of lactose is 0~10g / L, preferably 1~3g / L, and more preferably 2g / L. The pH value of the culture medium is 5-8, preferably 5.5-6; The temperature for the culture process of induced expression is 16~32℃, preferably 20~28℃, and more preferably 23~26℃.

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