A complex hydrolytic enzyme and a method for its production

Abstract

The application provides a kind of complex hydrolytic enzyme and its preparation method, it is related to enzyme engineering technical field.The preparation method includes: aspergillus niger is inoculated in culture medium to carry out fermentation, and the fermentation liquor containing beta-glucosidase is collected;The fermentation liquor is subjected to solid-liquid separation to obtain beta-glucosidase preparation;Add alpha-L-arabinofuranosidase and cellobiohydrolase to obtain the complex hydrolytic enzyme.This technology uses specific content of sodium carboxymethyl cellulose and wheat bran to synergistically ferment, the activity and specificity of the main enzyme beta-glucosidase are greatly improved by nutritional induction and conformation simulation;Subsequently, alpha-L-arabinofuranosidase and cellobiohydrolase are compounded, the single enzyme limitation is broken and the feedback inhibition is removed, and the overall catalytic efficiency and conversion rate are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and more specifically, to a complex hydrolase and its preparation method. Background Technology

[0002] Rare saponin CK is one of the core active components of ginsenosides, possessing significant anti-tumor, anti-inflammatory, and immunomodulatory biological activities, and has extremely high application value in the pharmaceutical and health-related fields. As a high-value secondary metabolite, exploring and developing a process for efficiently and greenly obtaining high-purity rare saponin CK has always been an important fundamental research topic in this field.

[0003] Currently, the main methods for obtaining and producing rare saponins (CK) include chemical hydrolysis, single enzymatic hydrolysis, direct microbial transformation, and conventional multi-enzyme hydrolysis. These existing methods aim to modify or degrade relatively complex, naturally abundant ginsenoside precursor macromolecules (such as Rb1, Rb2, Rc, etc.) through physicochemical or biocatalytic processes, thereby converting them into rare saponins (CK) with smaller molecular weights and higher bioavailability.

[0004] However, the aforementioned existing technologies all exhibit significant limitations in practical implementation. Chemical hydrolysis typically relies on harsh environments such as strong acids and alkalis, resulting in poor reaction selectivity, easy destruction of the core active structure of saponins, and severe environmental pollution. Single enzymatic hydrolysis is limited by the strict substrate specificity of monomeric enzymes, making it difficult to completely break glycosidic bonds at different sites of the precursor molecule, leading to hindered hydrolysis and low conversion rates. Direct microbial transformation faces problems such as demanding culture conditions, long fermentation cycles, and extremely difficult subsequent separation and purification. Furthermore, in existing complex enzymatic hydrolysis technologies, the fermentation medium of upstream enzyme-producing strains often uses conventional carbon and nitrogen source formulations, lacking targeted spatial conformation inducing factors and dissolved oxygen-promoting mechanisms, resulting in low enzyme production levels. Simultaneously, the downstream complex enzyme system composition is not entirely rational, lacking deep synergistic mechanisms between different enzymes, making it highly susceptible to product feedback inhibition during catalysis, leading to rapid enzyme activity decay.

[0005] In summary, existing rare saponin (CK) production technologies generally suffer from inherent defects such as low induction efficiency of fermentation enzyme production process, lack of substantial synergistic effect of multi-enzyme combination, and easy inhibition of conversion process. These defects result in low overall hydrolysis efficiency and low product conversion rate, making it difficult to meet the technical requirements of efficient, high-purity and stable production.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a complex hydrolase, and a method for preparing the complex hydrolase and the rare saponin CK. This invention utilizes sodium carboxymethyl cellulose and wheat bran to synergistically induce a high-yield, specific main enzyme, and combines two coenzymes to overcome the limitations of single enzymes and eliminate feedback inhibition, ultimately significantly improving the overall hydrolysis efficiency of the complex enzyme and the conversion rate of the target product.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing a complex hydrolytic enzyme, comprising: Aspergillus niger is inoculated into a culture medium for fermentation, and the fermentation broth containing β-glucosidase is collected; wherein the culture medium comprises 1% to 2% sodium carboxymethyl cellulose and 1% to 5% wheat bran by mass percentage. The fermentation broth was subjected to solid-liquid separation to obtain a β-glucosidase preparation; α-L-arabinofuranase and cellobiase were added to the β-glucosidase preparation to obtain the complex hydrolase.

[0009] In an optional embodiment, the Aspergillus niger is Aspergillus niger GDMCC3.486.

[0010] In an optional embodiment, the viscosity of the sodium carboxymethyl cellulose is 1000 CPS to 3000 CPS; and / or, The culture medium also includes the following components by weight percentage: 15%–20% yeast extract, 15%–20% peptone, 0.02%–0.1% Tween 80, 5%–10% (NH4)2SO4, 0.1%–0.5% KH2PO4, 0.05%–0.2% MgSO4, 0.001%–0.005% FeSO4, 0.001%–0.005% ZnSO4, 0.001%–0.005% MnSO4, with the balance being water.

[0011] In an optional implementation, the fermentation conditions include at least one of the following: A. Fermentation temperature is 28℃~32℃; B. Fermentation time is 72h~96h; C. Ventilation rate is 1.0 vvm to 1.5 vvm; D. The stirring speed during the fermentation process is 300 rpm to 500 rpm.

[0012] In an optional embodiment, the step of solid-liquid separation of the fermentation broth includes: centrifuging the fermentation broth; Preferably, after the centrifugation process, the method further includes a step of concentrating the supernatant after centrifugation.

[0013] In an optional embodiment, before or simultaneously with the addition of α-L-arabinofuranase and cellobiase to the β-glucosidase preparation, the pH of the β-glucosidase preparation is adjusted to 4.0–5.5; and / or, In the complex hydrolase, the mass ratio of α-L-arabinofuranase to β-glucosidase preparation is 1:(5~10); and / or, the mass ratio of cellobiase to β-glucosidase preparation is 1:(8~15).

[0014] Secondly, the present invention also provides a culture medium for preparing the complex hydrolase as described in the foregoing embodiments, wherein the components of the culture medium, by mass percentage, include: 1% to 2% sodium carboxymethyl cellulose, 0.02% to 0.1% Tween 80, 15% to 20% yeast extract powder, and 15% to 20% peptone.

[0015] Thirdly, the present invention provides a complex hydrolase, which is prepared by the preparation method described in any of the foregoing embodiments.

[0016] Fourthly, the present invention provides a method for preparing rare saponin CK, comprising: Using at least one of ginsenosides Rb1, Rb2, and Rc as a substrate, an enzymatic hydrolysis reaction was carried out by adding the complex hydrolytic enzyme described in the aforementioned embodiments at a temperature of 30℃~50℃ and a pH of 4.5~5.5 to obtain a reaction solution. After the reaction was completed, the reaction solution was purified to obtain rare saponin CK.

[0017] In an optional embodiment, during the enzymatic hydrolysis reaction, the stirring speed is 200 rpm to 300 rpm, and / or the reaction time is 24 h to 48 h; and / or, The step of purifying the reaction solution includes: extracting the reaction solution with anhydrous ethanol and collecting the extract; concentrating the extract under reduced pressure and drying it under vacuum.

[0018] Compared with existing technologies, this application introduces 1%–2% sodium carboxymethyl cellulose and 1%–5% wheat bran as core inducers into the culture medium. The synergistic effect of these two ingredients greatly stimulates the secretion of highly active β-glucosidase by *Aspergillus niger*. Wheat bran provides a rich, slow-release carbon source and inducing precursor, while sodium carboxymethyl cellulose of a specific viscosity effectively mimics the spatial conformation of the target saponin substrate through its long-chain structure, making the secreted enzyme protein more compatible with ginsenosides in three-dimensional folding. Simultaneously, the addition of an appropriate amount of Tween 80 as a surfactant increases cell membrane permeability, fundamentally solving the problem of low main enzyme yield and specificity in existing fermentation processes.

[0019] After obtaining the highly active main enzyme, this technology further precisely combined it with α-L-arabinofuranylase and cellobiase to construct a multi-enzyme synergistic catalytic system. The introduction of α-L-arabinofuranylase successfully broke the strict substrate specificity limitation of single β-glucosidases, enabling this composite enzyme to efficiently cleave glycosidic bonds at multiple sites in complex precursor molecules. More importantly, the addition of cellobiase can degrade glucose continuously generated during hydrolysis in real time, completely eliminating the product feedback inhibition of glucose on the main enzyme. This deep synergistic mechanism ensures that the enzyme system maintains extremely high catalytic activity throughout the entire catalytic process, effectively overcoming the inherent defects of low hydrolysis efficiency and incomplete conversion of traditional single enzymes, and significantly improving the conversion rate and purity of the final product. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 A schematic diagram of the overall process flow for preparing the composite hydrolytic enzyme and using it to produce rare saponin CK, provided in the embodiments of this application; Figure 2 This is a schematic diagram illustrating the chemical reaction pathway and site cleavage mechanism of the composite hydrolase catalyzing the conversion of ginsenosides Rb1, Rb2, and Rc into rare saponin CK according to the present invention. Detailed Implementation

[0022] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] This application provides a method for preparing a complex hydrolase, comprising: Step S1: Inoculate Aspergillus niger into a culture medium for fermentation and collect the fermentation broth containing β-glucosidase; wherein, the culture medium comprises 1% to 2% (for example, 1%, 1.1%, 1.2%, 1.3%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc.) sodium carboxymethyl cellulose and 1% to 5% wheat bran.

[0024] This step is the fermentation process for producing the main enzyme. Aspergillus niger (… Aspergillusniger The bacterial strain was inoculated into a pre-prepared fermentation medium for large-scale fermentation culture. The fermentation medium formula not only contains conventional carbon and nitrogen sources and trace elements, but its core components are strictly controlled as follows: 1%~2% sodium carboxymethyl cellulose by mass, and 0.02%~0.1% Tween 80 by mass.

[0025] In this fermentation process, the aforementioned suitable concentration of sodium carboxymethyl cellulose (SMC) does not serve as a simple carbon source inducer, but rather plays a unique dual core function of "enhanced induction + substrate structure simulation." On the one hand, a suitable concentration of 1%–2% acts as a specific inducer for β-glucosidase, significantly upregulating the expression levels of enzyme-producing genes in *Aspergillus niger* (such as bgl1 and bgl2 genes). Compared to conventional low-concentration induction, the enzyme activity in the fermentation broth can be increased by 2–3 times. On the other hand, the use of SMC with a specific viscosity (1000–3000 CPS) allows the long-chain structure formed by its cross-linking within the fermentation system to highly simulate the stereoconformation of the target substrates (ginsenosides Rb1, Rb2, Rc, etc., which contain multiple glucosidic bonds, have large molecular weights, and complex spatial structures). This highly targeted spatial simulation makes the β-glucosidase synthesized by *Aspergillus niger* during fermentation more compatible with the stereoconstruction of ginsenosides in terms of three-dimensional folding, thereby greatly improving the binding efficiency and specificity of the target enzyme to the complex saponin substrate from the source.

[0026] In this fermentation process, 1%–2% sodium carboxymethyl cellulose and 1%–5% wheat bran constitute the core dual induction system. Wheat bran, as a natural complex carbon source, not only provides basic metabolites, but its hemicellulose content can also initially activate the enzyme production pathway of the strain. Sodium carboxymethyl cellulose (viscosity 1000–3000 CPS) plays a crucial role in "enhanced induction + substrate structure mimicry." Its long-chain structure, formed through cross-linking within the fermentation system, can highly mimic the stereoconformation of the target macromolecular substrate (ginsenosides), making the synthesized β-glucosidase more compatible with the stereoconformity of ginsenosides. Furthermore, 0.02%–0.1% Tween 80 added to the system as an auxiliary surfactant can moderately increase cell membrane permeability, promoting efficient secretion of intracellular enzyme proteins to the extracellular space. Simultaneously, appropriate aeration and stirring ensure good mass transfer in the fermentation system.

[0027] Step S2: The fermentation broth is subjected to solid-liquid separation to obtain a β-glucosidase preparation.

[0028] This step involves solid-liquid separation and formulation preparation.

[0029] The high-viscosity fermentation broth collected above is subjected to physical solid-liquid separation treatment. Specifically, conventional separation methods such as high-speed centrifugation, microfiltration membrane filtration, or plate and frame filtration can be used to effectively remove Aspergillus niger mycelium, unconsumed solid nutrient residue, and other large insoluble particles from the fermentation broth.

[0030] After separation, the liquid phase is retained to obtain a relatively clear β-glucosidase preparation with biological impurities removed. To further improve the potency of subsequent enzyme solutions, the separated liquid can be selectively concentrated using ultrafiltration or vacuum concentration techniques after solid-liquid separation.

[0031] Step S3: Add α-L-arabinofuranase and cellobiase to the β-glucosidase preparation to obtain the complex hydrolase.

[0032] This step involves the synergistic compounding of multiple enzymes. Specifically, after obtaining the β-glucosidase preparation, α-L-arabinofuranase and cellobiase are added quantitatively and mixed thoroughly to obtain the composite hydrolase.

[0033] In this complex system, the three enzymes are not simply mixed, but rather form a deep synergistic catalytic mechanism. It is important to note that the core purpose of introducing cellobiase into the system is not to cleave the main structures of ginsenosides Rb1, Rb2, and Rc themselves, but rather to construct a unique "catalytic de-inhibition and impurity removal" mechanism. The specific synergistic mechanism includes the following three levels: Firstly, it completely eliminates the product feedback inhibition of the main enzyme. During the hydrolysis reaction, the main enzyme β-glucosidase continuously breaks the glucosidic bonds of ginsenosides, leading to the continuous accumulation of glucose and trace amounts of cellobiose byproducts in the system. When the glucose concentration accumulates to the inhibition threshold (usually ≥5 mmol / L), the catalytic activity of the main enzyme will be severely reduced. Cellobiase can specifically and rapidly consume the highly inhibitory glucose precursor (cellobiose) in the system, accelerating the degradation and transfer of byproducts, effectively preventing the local accumulation of inhibitory sugars in the active site of the main enzyme, thereby completely eliminating the feedback inhibition on β-glucosidase and significantly prolonging the effective catalytic time of the main enzyme in a highly active state.

[0034] Secondly, it forms a catalytic closed loop of "site exposure - efficient cleavage - inhibition relief". α-L-arabinofuranylase preferentially cleaves the arabinofuranyl side chains at specific positions on the periphery of complex substrate molecules (such as in Rb2 and Rc structures), effectively eliminating steric hindrance and exposing more potential glucosidic bond targets (i.e., "site exposure"), breaking the substrate specificity limitation of single enzymes; subsequently, β-glucosidase accurately recognizes and cleaves these core targets (i.e., "efficient cleavage"); while cellobiase continuously clears away product accumulation (i.e., "inhibition relief"). The protection of the main enzyme activity by cellobiase indirectly amplifies the "site exposure" benefit of α-L-arabinofuranylase, allowing the multi-site hydrolysis process to proceed smoothly.

[0035] Third, it eliminates substrate competition and system viscosity interference. In practical industrial applications, the precursor raw materials (plant extracts) of rare saponins often contain trace amounts of cellulose or cellobiose residues from plant cell walls. Cellobiase can target and degrade these specific impurities, eliminating non-specific competition from impurities for the main enzyme binding site and effectively reducing the hindering effect of system viscosity on substrate mass transfer.

[0036] The composite hydrolase prepared through the above steps fundamentally overcomes the shortcomings of existing single enzyme preparations or conventional composite enzymes, such as low activity, susceptibility to inhibition and inactivation, and low conversion rate, laying a solid material foundation for the subsequent efficient and thorough hydrolysis of complex compounds. In the specific compounding operation, it is preferable to adjust the pH of the system to a slightly acidic range (e.g., pH 4.0~5.5) using a buffer solution or acid-base regulator before or simultaneously with the addition of the coenzyme to ensure that all three enzymes are in optimal conformational stability and activity.

[0037] In summary, fermentation using sodium carboxymethyl cellulose and wheat bran at specific concentrations as core dual inducers, leveraging the synergistic effect of the nutritional induction from wheat bran and the substrate conformational mimicry from sodium carboxymethyl cellulose, significantly stimulated the secretion of highly specific and active β-glucosidase by *Aspergillus niger*. Based on this, the obtained main enzyme was combined with α-L-arabinofuranosylase and cellobiase, effectively overcoming the catalytic specificity limitations of single enzymes. This not only enabled the comprehensive cleavage of different types of glycosidic bonds but also relieved product feedback inhibition by promptly consuming intermediate metabolites, thereby significantly improving the overall catalytic efficiency of the complex hydrolase and the conversion rate of the final target product.

[0038] In some embodiments, during the fermentation preparation of the complex hydrolytic enzyme, the Aspergillus niger is Aspergillus niger (… Aspergillusniger GDMCC3.486.

[0039] The fermentation strain was specifically limited to *Aspergillus niger* GDMCC3.486 based on its unique genetic and metabolic characteristics and its high adaptability to this fermentation system. Firstly, as a highly efficient filamentous fungal "cell factory," the genome of strain GDMCC3.486 contains a high abundance of genes encoding glycoside hydrolases. More importantly, the transcriptional regulatory network of this strain exhibits an extremely sensitive response to the dual induction signal of "sodium carboxymethyl cellulose combined with wheat bran" in the culture medium of this scheme. When this strain encounters the specific macromolecular spatial conformation simulated by sodium carboxymethyl cellulose, the expression levels of its related target genes can be nonlinearly amplified, thereby directionally and specifically synthesizing large quantities of β-glucosidases with high adaptability to macromolecular substrates such as ginsenosides.

[0040] Secondly, *Aspergillus niger* GDMCC3.486 exhibits extremely robust growth and tolerance in systems containing high concentrations of organic nitrogen sources (yeast extract and peptone), and is not prone to premature aging. Simultaneously, with appropriate regulation from auxiliary components (such as Tween 80), the transmembrane secretion channels of this strain remain highly unobstructed, allowing the intracellularly synthesized main enzyme to be rapidly and efficiently pumped into the extracellular fermentation broth. By selecting and using this specific strain, the maximum potential of this preparation process has been unlocked and explored from its biological source and material basis, effectively ensuring that the final compound hydrolase product possesses extremely high enzyme activity, excellent catalytic specificity, and outstanding batch stability in industrial production.

[0041] In some embodiments, the viscosity of the sodium carboxymethyl cellulose is 1000 CPS to 3000 CPS. For example, it can be 1000 CPS, 1200 CPS, 1500 CPS, 1800 CPS, 2000 CPS, 2200 CPS, 2500 CPS, 2700 CPS, 2900 CPS, 3000 CPS, etc.

[0042] The fermentation medium was precisely formulated with carefully selected components and proportions: the key conformation inducer, sodium carboxymethyl cellulose, was selected with a viscosity of 1000 CPS to 3000 CPS. This viscosity range ensures that it forms a three-dimensional structure with sufficient space and network entanglement in aqueous solution, thus highly mimicking the substrate molecule conformation and maximizing its induction effect on the enzyme production pathway, while avoiding complete oxygen blockage caused by excessive viscosity.

[0043] In some embodiments, the components of the culture medium further include, by mass percentage: Table 1. Components of the culture medium

[0044] To meet the high-volume protein synthesis requirements of the strain under intense induction, a high-concentration complex nitrogen source and trace element system was specially formulated in the culture medium. Specific components and their mass percentages include: 15%–20% yeast extract and 15%–20% peptone as organic nitrogen sources, directly supplying a large amount of amino acids and peptides, providing the material basis for rapid enzyme protein synthesis; 1%–5% wheat bran providing natural micronutrients and a slow-release auxiliary carbon source; 5%–10% (NH4)2SO4 as an inorganic nitrogen source supplement; in addition, 0.1%–0.5% KH2PO4 and 0.05%–0.2% MgSO4 synergistically construct a buffer system and provide macroelements, while 0.001%–0.005% FeSO4, 0.001%–0.005% ZnSO4, and 0.001%–0.005% MnSO4 serve as cofactor activators for key metabolic enzymes and product enzymes.

[0045] The above components, along with the main inducer and surfactant (Tween 80), are mixed thoroughly with water and sterilized before use. Through this specific combination of high-carbon, high-nitrogen nutrient pool and physical properties, the enzyme-producing potential of a particular Aspergillus niger is synergistically released, significantly increasing the potency of the main enzyme in the resulting fermentation broth.

[0046] In some embodiments, the fermentation conditions include at least one of the following: A. Fermentation temperature is 28℃~32℃; for example, it can be 28℃, 28.5℃, 29℃, 29.5℃, 30℃, 30.5℃, 31℃, 31.5℃, 31.8℃, 32℃, etc.

[0047] B. Fermentation time is 72h~96h; for example, it can be 72h, 75h, 78h, 80h, 82h, 84h, 88h, 90h, 92h, 96h, etc.

[0048] C. Ventilation volume is 1.0vvm~1.5vvm; for example, it can be 1.0vvm, 1.1vvm, 1.15vvm, 1.2vvm, 1.25vvm, 1.3vvm, 1.35vvm, 1.4vvm, 1.45vvm, 1.5vvm, etc.

[0049] D. The stirring speed during fermentation is 300 rpm to 500 rpm. For example, it can be 300 rpm, 320 rpm, 350 rpm, 380 rpm, 400 rpm, 420 rpm, 450 rpm, 460 rpm, 480 rpm, 500 rpm, etc.

[0050] In this scheme, the selection of fermentation process parameters has a deep physicochemical synergy with the component characteristics of the specific culture medium. First, controlling the fermentation temperature at 28℃~32℃ and the fermentation time at 72h~96h is based on the metabolic kinetics of β-glucosidase secreted by Aspergillus niger. This temperature range can maintain the optimal fluidity of the cell membrane to facilitate extracellular enzyme secretion, while avoiding premature cell aging and protease release caused by high temperature; the 72h~96h period precisely targets the peak period of target enzyme accumulation after the cells enter the stationary phase under the dual induction of sodium carboxymethyl cellulose and wheat bran, ensuring high enzyme activity while avoiding cell autolysis caused by nutrient depletion in the later stage.

[0051] More importantly, the introduction of sodium carboxymethyl cellulose with a long-chain structure into the fermentation medium to mimic the substrate conformation inevitably increases the overall apparent viscosity of the fermentation broth, leading to higher gas-liquid mass transfer resistance in aerobic fermentation. To overcome this physical barrier, this scheme employs a high aeration rate of 1.0 vvm to 1.5 vvm combined with a high stirring speed of 300 rpm to 500 rpm. The high shear force generated by strong mechanical stirring not only eliminates mixing dead zones in the high-viscosity system, ensuring uniform contact between the cells, nutrients, and inducers, but also forcibly breaks down large air bubbles introduced by the high aeration rate into microbubbles, thereby greatly increasing the gas-liquid contact surface area and significantly improving the oxygen volumetric mass transfer coefficient. This optimized combination of fluid dynamics and oxygen supply strategies completely eliminates dissolved oxygen barriers in the high-viscosity induction system, ensuring that Aspergillus niger obtains sufficient metabolic energy (ATP) during high-intensity enzyme protein synthesis, which is a necessary engineering means to guarantee high production of the main enzyme.

[0052] In some embodiments, the step of solid-liquid separation of the fermentation broth includes centrifuging the fermentation broth.

[0053] In the implementation of this invention, after the fermentation stage, the high-viscosity fermentation broth containing the target product needs to be post-processed to extract and purify the enzyme preparation. Specifically, the solid-liquid separation step is preferably performed by centrifugation. Because the system after high-concentration medium fermentation contains a large amount of Aspergillus niger mycelium, extracellular metabolite fragments, and incompletely degraded culture medium solid residues, while the target product β-glucosidase is secreted in large quantities and dissolved in the liquid phase, by feeding the fermentation broth into a centrifuge and applying centrifugal force, the significant density difference between the solid and liquid phases can efficiently and rapidly settle and retain solid impurities, thereby obtaining a clear supernatant rich in the target enzyme. This centrifugation method is not only simple to operate and easy to scale up continuously for industrial applications, but it can also effectively retain living organisms and physical impurities, preventing interference from fermentation residues to subsequent enzymatic hydrolysis reactions from the source.

[0054] Furthermore, after the centrifugation process, the method further includes a step of concentrating the supernatant after centrifugation.

[0055] This step aims to gently remove excess water from the supernatant. Concentration not only significantly increases the enzyme activity and titer of β-glucosidase per unit volume (i.e., increases enzyme concentration) and greatly reduces the physical volume of the enzyme preparation for easier storage, but more importantly, the high concentration provides an excellent material basis for subsequent multi-enzyme synergistic formulations and downstream targeted enzymatic reactions. Using concentrated enzyme solution avoids introducing excessive water when adding the catalyst, preventing over-dilution of the final enzymatic reaction system, thus ensuring the effective collision probability between the substrate and enzyme and maintaining high catalytic efficiency and high conversion rate in the production of rare saponins (CK). In specific industrial implementations, to prevent enzyme protein denaturation and inactivation due to high temperatures, the concentration process can employ conventional gentle dehydration techniques such as ultrafiltration (e.g., using an ultrafiltration membrane with a specific molecular weight cutoff) or low-temperature vacuum concentration.

[0056] In some embodiments, the pH of the β-glucosidase preparation is adjusted to 4.0-5.5 before or simultaneously with the addition of α-L-arabinofuranase and cellobiase to the β-glucosidase preparation. For example, pH values ​​such as 4.0, 4.1, 4.3, 4.5, 4.7, 4.9, 5.0, 5.2, 5.4, and 5.5 may be used.

[0057] After obtaining the purified or concentrated β-glucosidase preparation, it needs to be synergistically compounded with multiple enzymes. To ensure that the three different enzyme proteins involved in the compounding maintain long-term structural stability and optimal catalytic activity in the complex system, the acid-base microenvironment of the compounding system must be strictly controlled. Specifically, before or simultaneously with the addition of α-L-arabinofuranase and cellobiase to the β-glucosidase preparation, the pH of the liquid phase system should be precisely adjusted to a slightly acidic range of 4.0–5.5 using a buffer system (such as citrate-sodium citrate buffer or acetate-sodium acetate buffer). This pH range represents the optimal window for the overlap of the activities of the three specific hydrolases, effectively preventing conformational inactivation of any single component due to unsuitable acidity or alkalinity, thus laying the chemical foundation for subsequent perfect synergistic catalysis.

[0058] In some embodiments, the mass ratio of α-L-arabinofuranolase to β-glucosidase in the complex hydrolase is 1:(5~10). For example, it can be 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:10, etc.

[0059] In some embodiments, the mass ratio of cellobiase to β-glucosidase in the complex hydrolase is 1:(8~15). For example, it can be 1:8, 1:9, 1:10, 1:10.5, 1:11, 1:12, 1:12.5, 1:13, 1:14, 1:15, etc.

[0060] To achieve optimal matching and synergistic effect in catalytic kinetics, this technology precisely defines the ratio of each component in the complex hydrolase. Preferably, the mass ratio of α-L-arabinofuranase to β-glucosidase is controlled at 1:(5~10), and the mass ratio of cellobiase to β-glucosidase is controlled at 1:(8~15).

[0061] At this specific mass ratio: the β-glucosidase, serving as the base, provides strong kinetics for backbone hydrolysis; an appropriate proportion (1 / 10 to 1 / 5) of α-L-arabinofuranase is sufficient to rapidly and thoroughly remove steric hindrances from the target substrate's side chains, fully exposing the backbone breakage target site without causing non-specific spatial competition; and a trace but crucial proportion (1 / 15 to 1 / 8) of cellobiase, with its cellobiose degradation kinetics perfectly matched to the rate of byproduct generation by the main enzyme, continuously suppresses the concentration of inhibitory sugars in the system (e.g., controlled below 5 mmol / L) below the threshold for feedback inhibition. This precise mass ratio based on reaction rate matching (optimally achieved at a main enzyme:arabinase:cellobiase ratio of 9:1:0.75) maximizes the overall conversion efficiency of the complex hydrolytic enzyme preparation.

[0062] This application also provides a culture medium for preparing the complex hydrolase as described in the foregoing embodiments. This culture medium completely breaks through the limitations of conventional carbon-nitrogen balance in its formulation design and constructs a powerful synergistic system of "conformation induction and excess material supply".

[0063] The components of the culture medium, by mass percentage, include: 1% to 2% sodium carboxymethyl cellulose (e.g., 1%, 1.1%, 1.2%, 1.3%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc.), and 0.02% to 0.1% Tween 80 (e.g., 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.02%). 0.095%, 0.1%, etc.), 15%~20% yeast extract powder (e.g., 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 20%, etc.), and 15%~20% peptone (e.g., 15%, 15.5%, 16%, 16.8%, 17.2%, 17.8%, 18%, 18.6%, 19.2%, 20%, etc.).

[0064] In this culture medium formulation, the components exert a profound synergistic effect in both biological and physicochemical processes, jointly constructing a microenvironment conducive to the high expression and secretion of specific hydrolytic enzymes by *Aspergillus niger*. Specifically, 1%–2% sodium carboxymethyl cellulose is selected as the core inducer, whose specific macromolecular long-chain structure can effectively mimic the spatial conformation of the target hydrolyzed substrate (such as ginsenosides). This structural simulation not only strongly stimulates the enzyme-producing metabolic pathway of *Aspergillus niger*, but also ensures that the synthesized β-glucosidase is highly adapted to the target macromolecular substrate in three-dimensional spatial folding, endowing the main enzyme with extremely high catalytic specificity. At the same time, the 1%–2% concentration range ensures the effectiveness of induction while maintaining suitable rheological properties of the fermentation broth, avoiding severe viscosity and dissolved oxygen barriers caused by high polymer concentrations.

[0065] To support the high-intensity protein synthesis process that occurs under the aforementioned potent induction, 15%–20% yeast extract and 15%–20% peptone were introduced into the culture medium as a high-concentration, readily available organic nitrogen source. The abundant vitamins and growth factors in the yeast extract promoted rapid cell proliferation in the early stages of fermentation, while the peptides and free amino acids provided by the peptone provided ample precursors for the extensive translation and folding of enzyme proteins in the later stages, ensuring nutrient supply throughout the entire fermentation cycle.

[0066] Furthermore, the addition of 0.02%–0.1% Tween 80 as a nonionic surfactant further refined the enzyme production mechanism. On one hand, it moderately increased the permeability of the Aspergillus niger cell membrane, significantly reducing the transmembrane physical resistance to the secretion of intracellular enzyme proteins into the extracellular environment, thus promoting the efficient release of synthesized enzymes. On the other hand, Tween 80 effectively reduced the surface tension of the culture medium, helping to improve the dispersion of oxygen in the liquid, enhancing gas-liquid mass transfer efficiency, and ensuring the oxygen requirements of the absolutely aerobic Aspergillus niger during its vigorous metabolic phase. These components work closely together across three dimensions—gene induction, substance synthesis, and transmembrane transport—significantly improving the final potency and specificity of the complex hydrolase.

[0067] This application also provides a complex hydrolase, which is prepared by the preparation method described in any of the foregoing embodiments.

[0068] It should be noted that the composite hydrolase obtained by the specific preparation method described above exhibits significant characteristics that distinguish it from conventional physically mixed enzyme preparations in terms of microscopic molecular conformation and overall catalytic performance. Firstly, in this composite hydrolase, the core main enzyme component (β-glucosidase) is synthesized and secreted under the dual synergistic induction of sodium carboxymethyl cellulose and wheat bran at a specific concentration. Because the long-chain structure of sodium carboxymethyl cellulose highly mimics the spatial conformation of complex saponin macromolecules in the fermentation broth, this forces and guides the β-glucosidase secreted by *Aspergillus niger* to undergo adaptive changes during its own protein three-dimensional folding. Therefore, the main enzyme in this composite hydrolase has an extremely high spatial fit with macromolecular substrates such as ginsenosides in its stereostructure, endowing the product with excellent substrate recognition ability and catalytic specificity.

[0069] Secondly, this complex hydrolase is not a simple physical superposition of multiple hydrolases, but rather a highly closed-loop dynamic catalytic system. The α-L-arabinofuranase in the product effectively hydrolyzes the arabinose groups surrounding the substrate molecule, thus exposing more spatial sites for the intervention of the main enzyme. Meanwhile, the cellobiase present in the system acts as a "scavenger," promptly degrading intermediate metabolites (such as cellobiose or glucose) produced by the main enzyme. This microscopic synergistic mechanism, with each enzyme performing its specific function, not only completely overcomes the specific limitations of single enzymes when facing complex glycosidic bonds, but also eliminates the product feedback inhibition effect that is prone to occur during hydrolysis from the perspective of reaction kinetics. Ultimately, this complex hydrolase can maintain extremely high overall catalytic activity even when facing high concentrations and structurally complex substrates, achieving efficient and complete conversion of the target product.

[0070] This application also provides a method for preparing rare saponin CK. This method sets the transformation process of the target substrate in a highly targeted, mild physicochemical environment, ensuring not only the high efficiency of multi-enzyme synergy but also maximizing the protection of the product's structural integrity. The preparation method includes: Using at least one of ginsenosides Rb1, Rb2, and Rc as a substrate, an enzymatic hydrolysis reaction is carried out under the conditions of a temperature of 30℃~50℃ (e.g., 30℃, 32℃, 35℃, 37℃, 40℃, 42℃, 45℃, 47℃, 48℃, 50℃, etc.) and a pH value of 4.5~5.5 (e.g., pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.4, pH 5.5, etc.), as described in the aforementioned embodiments, to obtain a reaction solution. After the reaction is completed, the reaction solution is purified to obtain rare saponin CK.

[0071] In implementing this transformation method, the transformation substrate is first provided. This substrate can be any single component from purified ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rc, or a mixed ginsenoside extract containing at least one of the above components can be used directly. The substrate is dissolved or suspended in an aqueous system, and the pH of the reaction system is precisely controlled between 4.5 and 5.5 using a buffer solution (such as an acetate-sodium acetate buffer system). This slightly acidic environment perfectly matches the dissociation state of the active sites of each component in the complex hydrolase, and is the core chemical condition for maintaining the catalytic vitality of the entire multi-enzyme system.

[0072] Subsequently, a pre-prepared composite hydrolytic enzyme was added to the system in a specific ratio, and the temperature of the reaction system was kept constant within a mild range of 30℃ to 50℃ for enzymatic hydrolysis. Under this optimal thermodynamic and pH environment, the composite enzyme initiated a deep synergistic catalytic mechanism: steric hindrance in the system was gradually eliminated, glycosidic bonds on the main backbone were targeted and broken, and inhibitory products were cleared in real time, allowing the entire enzymatic hydrolysis reaction to maintain a constant and high conversion efficiency. This resulted in the complete stripping of the sugar chains from macromolecular saponins, positioning them in the rare saponin CK structure. Compared to traditional chemical or high-temperature enzymatic methods, this mild condition not only significantly reduced production energy consumption but also effectively avoided thermal degradation or acid-base isomerization of the target saponin structure under extreme conditions.

[0073] After the enzymatic reaction proceeds fully until the conversion end point is reached, a complex reaction solution containing the product, free sugar, residual enzyme, and buffer salt is obtained. Subsequently, terminal purification treatment is performed on this reaction solution. Based on the physicochemical properties that the polarity of rare saponin CK significantly decreases and its liposolubility increases after sugar removal, conventional separation methods such as organic solvent extraction, solid-phase extraction, or column chromatography can be used to completely separate the target product from water-soluble by-products and macromolecular proteins. Finally, a rare saponin CK product with high purity and high biological activity is obtained, which directly serves the applications in the downstream pharmaceutical and big health industries.

[0074] Please refer to Figure 1 , Figure 1 which shows the overall process flow of the preparation of the composite hydrolase and the production of rare saponin CK provided by the embodiments of the present invention. As Figure 1 shown, this process macroscopically includes the stage of Aspergillus niger fermentation to produce the main enzyme in the upstream, the stage of multi-enzyme synergistic compounding in the middle reaches, and the stage of enzymatic hydrolysis and purification of rare saponin CK in the downstream.

[0075] In addition, in the composite system provided in this application, the three enzymes construct a deep catalytic closed loop. Combining Figure 2 with the ginsenoside catalytic conversion pathway shown, it can be seen that due to the complex molecular structure of the substrate (such as Rb2, Rc), with side chains such as arabinofuranose attached to its periphery. During the enzymatic hydrolysis process, α-L-arabinofuranosidase first targets and cleaves these side chains (such as Figure 2 the corresponding glycosidic bond cleavage sites in), eliminating steric hindrance; subsequently, β-glucosidase accurately recognizes and completely cleaves the main glucosidic bonds exposed at the C-3 and C-20 positions, gradually stripping the sugar groups, and finally orienting it to be converted into the rare saponin CK structure that only retains a single glucose group.

[0076] When carrying out the large-scale enzymatic conversion of ginsenosides, it is necessary to strictly control the kinetic parameters of the reaction system and the post-treatment purification process to ensure the high yield and high purity of the final product. Specifically, in some embodiments, during the enzymatic reaction process, the stirring speed is 200 rpm to 300 rpm (for example, it can be 200 rpm, 210 rpm, 220 rpm, 230 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm, 300 rpm, etc.), and / or the reaction time is 24 h to 48 h (for example, it can be 24 h, 26 h, 28 h, 30 h, 32 h, 36 h, 40 h, 42 h, 45 h, 48 h, etc.).

[0077] During the enzymatic hydrolysis reaction, the reaction system needs to be continuously mechanically stirred at 200-300 rpm. This specific speed creates a uniform and suitable hydrodynamic environment within the reaction solution. The moderate stirring speed significantly enhances the mass transfer efficiency between substrate macromolecules and the multi-enzyme complex system, increases the collision probability of enzyme-substrate binding sites, and accelerates the diffusion and removal of hydrolysis byproducts in the local microenvironment. From a physical perspective, this speed works in conjunction with the multi-enzyme system to complete the synergistic process of "relief of feedback inhibition." At the same time, this speed effectively avoids mechanical denaturation and inactivation of enzyme proteins caused by excessive shear force.

[0078] Under the above stirring conditions, the enzymatic hydrolysis reaction time was strictly controlled within 24-48 hours. This time window perfectly covers the cascade kinetics of multi-enzyme synergistic "side chain removal" and "main glycosidic bond breaking," ensuring that the macromolecular substrate is converted into the target molecule most thoroughly, achieving the best balance between conversion efficiency and equipment time cost.

[0079] In some embodiments, the step of purifying the reaction solution includes: extracting the reaction solution with anhydrous ethanol and collecting the extract; concentrating the extract under reduced pressure and drying it under vacuum.

[0080] After the enzymatic hydrolysis reaction reaches the predetermined endpoint, the reaction solution containing complex background substances needs to be purified to extract the target compound in high purity. Specific extraction steps may include (but are not limited to) the following: First, anhydrous ethanol is added to the reaction mixture for extraction. This operation fully utilizes the principle of polarity reversal: after the substrate loses excess sugar chains and transforms into rare saponin CK, its hydrophobicity is significantly enhanced, thus allowing it to dissolve selectively in anhydrous ethanol; while hydrophilic buffer salts, waste macromolecular enzyme proteins, and free monosaccharide / disaccharide byproducts in the reaction system are difficult to dissolve, thereby achieving complete separation of solid-liquid or phase interfaces. After collecting the ethanol extract rich in the target product, to prevent high-temperature structural changes in the heat-sensitive saponin, a vacuum concentration technique is used to rapidly evaporate and remove most of the ethanol solvent at low temperature. Subsequently, it is placed in a vacuum drying device to completely remove residual solvent and trace amounts of moisture. Through this green and mild purification process, a solid rare saponin CK product with extremely high purity and well-preserved active structure can ultimately be obtained.

[0081] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0082] I. Preparation of compound β-glucosidase preparations: Example 1 This embodiment provides a method for preparing a compound β-glucosidase preparation, mainly investigating the enzyme production under specific ratios of a novel culture medium (10% CMC-Na) and basic fermentation parameters.

[0083] Experimental Methods: The novel culture medium consisted of 15% yeast extract, 15% peptone, 1.0% sodium carboxymethyl cellulose (viscosity 1000 CPS), 1% wheat bran, 5% (NH4)2SO4, 0.1% KH2PO4, 0.05% MgSO4, 0.001% FeSO4, 0.001% ZnSO4, 0.001% MnSO4, 0.02% Tween 80, and the balance being water. After thorough mixing, the components were loaded into a reactor and inoculated with Aspergillus niger strain GDMCC3.486. Fermentation was carried out at 28℃, pH 5.0, and an aeration rate of 1.0 vvm for 72 h. After fermentation, the fermentation broth was collected, centrifuged at 8000 rpm for 15 min, and the supernatant was concentrated to 1 / 5 of its original volume via ultrafiltration.

[0084] Experimental results: The final compound β-glucosidase preparation was obtained, and its β-glucosidase activity was determined to be 3978 U / mL.

[0085] Example 2 This embodiment provides a method for preparing a compound β-glucosidase preparation, mainly investigating the effects of appropriately increasing the carbon and nitrogen source concentration (1.2% CMC-Na) and fermentation parameters on enzyme production.

[0086] Experimental Methods: The novel culture medium formula was modified to include 16% yeast extract, 16% peptone, 1.2% sodium carboxymethyl cellulose (viscosity 1500 CPS), 2% wheat bran, 6% (NH4)2SO4, 0.2% KH2PO4, 0.08% MgSO4, 0.002% FeSO4, 0.002% ZnSO4, 0.002% MnSO4, 0.04% Tween 80, and the balance being water. Aspergillus niger GDMCC3.486 was inoculated and fermented at 29℃, pH 5.2, and an aeration rate of 1.1 vvm for 78 h. The fermentation broth was centrifuged at 8500 rpm for 16 min, and the supernatant was concentrated to 1 / 6 by ultrafiltration.

[0087] Experimental results: The final compound β-glucosidase preparation was obtained, and its β-glucosidase activity was determined to be 3710 U / mL.

[0088] Example 3 This embodiment provides a method for preparing a compound β-glucosidase preparation, mainly investigating the preparation process under optimized conditions (1.4% CMC-Na, 2000 CPS). Experimental methods: The novel culture medium formulation consisted of 17% yeast extract, 17% peptone, 1.4% sodium carboxymethyl cellulose (viscosity 2000 CPS), 3% wheat bran, 7% (NH4)2SO4, 0.3% KH2PO4, 0.1% MgSO4, 0.003% FeSO4, 0.003% ZnSO4, 0.003% MnSO4, 0.06% Tween 80, and the balance being water. Aspergillus niger GDMCC3.486 was inoculated and fermented at 30℃, pH 5.5, and an aeration rate of 1.2 vvm for 84 h. The fermentation broth was centrifuged at 9000 rpm for 17 min and concentrated to 1 / 7 of its original volume.

[0089] Experimental results: The final compound β-glucosidase preparation was obtained, and its β-glucosidase activity was determined to be 3625 U / mL.

[0090] Example 4 This embodiment provides a method for preparing a compound β-glucosidase preparation, mainly investigating the enzyme production process under a high inducer concentration (1.6% CMC-Na) and a long fermentation time.

[0091] Experimental Methods: The following ingredients were used: 18% yeast extract, 18% peptone, 1.6% sodium carboxymethyl cellulose (viscosity 2500 CPS), 4% wheat bran, 8% (NH4)2SO4, 0.4% KH2PO4, 0.15% MgSO4, 0.004% FeSO4, 0.004% ZnSO4, 0.004% MnSO4, 0.08% Tween 80, and water. Fermentation was carried out at 31℃, pH 5.8, and 1.3 vvm for 90 h. The mixture was then centrifuged at 9500 rpm for 18 min and concentrated to 1 / 8 of its original volume.

[0092] Experimental results: The final compound β-glucosidase preparation was obtained, and its β-glucosidase activity was determined to be 3506 U / mL.

[0093] Example 5 This embodiment provides a method for preparing a compound β-glucosidase preparation, mainly examining the preparation under the highest parameter boundary (2.0% CMC-Na, 3000 CPS) defined in the claims.

[0094] Experimental Methods: A mixture of 20% yeast extract, 20% peptone, 2.0% sodium carboxymethyl cellulose (viscosity 3000 CPS), 5% wheat bran, 10% (NH4)2SO4, 0.5% KH2PO4, 0.2% MgSO4, 0.005% FeSO4, 0.005% ZnSO4, 0.005% MnSO4, 0.1% Tween 80, and water was used. Fermentation was carried out at 32℃, pH 6.0, and 1.5 vvm for 96 h. The mixture was then centrifuged at 10000 rpm for 20 min and concentrated to 1 / 10.

[0095] Experimental results: The final compound β-glucosidase preparation was obtained, and its β-glucosidase activity was determined to be 3421 U / mL.

[0096] II. Multi-enzyme formulation of compound hydrolytic enzyme preparations: Examples 6 to 9 This series of examples provides a compound hydrolytic enzyme preparation, which aims to investigate the compounding of the main enzyme with two coenzymes under different mass ratios and different pH conditions.

[0097] Experimental methods and results: The prepared compound β-glucosidase preparation was taken, and the pH was adjusted with citrate-sodium citrate buffer. Then, α-L-arabinofuranase and cellobiase were added and stirred evenly to obtain the compound hydrolytic enzyme preparation.

[0098] The specific parameters are as follows: Example 6: Adjust pH to 4.0; α-L-arabinofuranylase mass ratio is 1:5, cellobiase mass ratio is 1:8.

[0099] Experimental results: A compound hydrolytic enzyme preparation was obtained, and its total enzyme activity was determined to be 4710 U / mL.

[0100] Example 7: Adjust pH to 4.5; α-L-arabinofuranylase mass ratio is 1:7, cellobiase mass ratio is 1:10.

[0101] Experimental results: A compound hydrolytic enzyme preparation was obtained, and its total enzyme activity was determined to be 4927 U / mL.

[0102] Example 8: Adjust pH to 5.0; α-L-arabinofuranylase mass ratio is 1:9, cellobiase mass ratio is 1:12.

[0103] Experimental results: A compound hydrolytic enzyme preparation was obtained, and its total enzyme activity was determined to be 5290 U / mL.

[0104] Example 9: Adjust pH to 5.5; α-L-arabinofuranylase mass ratio is 1:10, cellobiase mass ratio is 1:15.

[0105] Experimental results: A compound hydrolytic enzyme preparation was obtained, and its total enzyme activity was determined to be 4845 U / mL.

[0106] III. Enzymatic hydrolysis and separation / purification of rare saponins CK: Example 10 This embodiment provides a method for preparing rare saponins CK, mainly investigating the conversion and purification effects under low substrate concentration and short reaction time.

[0107] The preparation of the compound β-glucosidase preparation followed Example 1: The novel culture medium formulation consisted of 15% yeast extract, 15% peptone, 1% sodium carboxymethyl cellulose (viscosity 1000 CPS), 1% wheat bran, 5% (NH4)2SO4, 0.1% KH2PO4, 0.05% MgSO4, 0.001% FeSO4, 0.001% ZnSO4, 0.001% MnSO4, 0.02% Tween 80, and the balance being water. After thorough mixing, the components were loaded into a reactor, and Aspergillus niger GDMCC3.486 strain was inoculated. Fermentation was carried out at 28℃, pH 5.0, and an aeration rate of 1.0 vvm for 72 h. After fermentation, the fermentation broth was collected, centrifuged at 8000 rpm for 15 min, and the supernatant was concentrated to 1 / 5 of its original volume via ultrafiltration.

[0108] The multi-enzyme formulation of the compound hydrolytic enzyme preparation follows the method described in Example 8: pH was adjusted to 5.0; the mass ratio of α-L-arabinofuranylase was 1:9, and the mass ratio of cellobiase was 1:12.

[0109] Experimental Methods: Ginsenoside Rb1 was prepared into a 10 mg / mL solution and the pH was adjusted to 4.5. A compound hydrolytic enzyme preparation was added at a 1:10 volume ratio, and enzymatic hydrolysis was carried out at 30℃ and 200 rpm for 12 h. The reaction solution was extracted with an equal volume of anhydrous ethanol, sonicated for 20 min, and centrifuged at 8000 rpm for 10 min. The supernatant was concentrated under reduced pressure at 40℃ and 0.08 MPa, and finally dried to constant weight at 95℃ and 0.09 MPa.

[0110] Experimental results: The rare saponin CK product was obtained with a conversion rate of 85% and a purity of 92%.

[0111] Examples 11 to 14 This series of examples provides a method for preparing rare saponins CK, mainly examining the effect of increasing reaction conditions on the conversion rate and purity of the final product when using mixed ginsenosides as substrates.

[0112] Experimental methods and results: The substrates were all mixtures of Rb1, Rb2, and Rc. The subsequent ethanol extraction, concentration, and drying processes were the same as in Example 10, but the operating parameters were increased accordingly, as follows: Example 11: Substrate concentration 20 mg / mL, pH 4.5, enzyme ratio (v / v) 1:12, reaction at 35°C and 220 rpm for 14 h. Final conversion rate 87%, purity 93%.

[0113] Example 12: Substrate concentration 30 mg / mL, pH 5.0, enzyme ratio 1:14, reaction at 38℃ and 240 rpm for 16 h. Final conversion rate 89%, purity 94%.

[0114] Example 13: Substrate concentration 40 mg / mL, pH 5.0, enzyme ratio 1:16, reaction at 42℃ and 260 rpm for 18 h. Final conversion rate 91%, purity 95%.

[0115] Example 14: Substrate concentration 50 mg / mL, pH 5.5, enzyme ratio 1:20, reaction at 50℃ and 300 rpm for 24 h. Final conversion rate 95%, purity 97%.

[0116] Experimental Example 1: Investigation and Verification of the Effect of Different Culture Medium Formulations on Main Enzyme Yield This experimental example aims to examine the difference between conventional culture media and the novel culture media of this application in stimulating the enzyme production ability of Aspergillus niger, and to verify the high efficiency of the formulation of this application.

[0117] Experimental Design: Comparative Example 1: PDA liquid culture medium (20% potato extract, 2% glucose, 0.1% peptone, natural pH).

[0118] Comparative Example 2: Czapek's Synthetic Medium (3% sucrose and inorganic salt formulation, pH 5.5).

[0119] Experimental group: The novel culture medium formulation of Example 3 was used.

[0120] All three groups were inoculated with Aspergillus niger GDMCC3.486 and fermented under the same conditions (30℃, pH 5.5, 1.2 vvm, 84h). The detection index was enzyme activity (U / mL) determined by the PNPG method. 1U is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol per minute.

[0121] Experimental results and analysis: See Table 2 for detailed data.

[0122] Table 2. Enzyme activities corresponding to different groups

[0123] The results are shown in Table 2. Comparative Example 1 showed enzyme activity ≤45 U / mL, and Comparative Example 2 showed enzyme activity ≤30 U / mL; while the experimental group showed enzyme activity ≥120 U / mL. This demonstrates that the enzyme activity of the novel culture medium is 2.7–4 times that of the conventional culture medium, achieving a qualitative leap in enzyme production capacity.

[0124] Experimental Example 2 (Verification of the synergistic induction mechanism between a single inducer and CMC-Na and wheat bran) Experimental Design: Comparative Example 3 was supplemented with only 1.4% CMC-Na (without wheat bran, supplemented with an equal amount of conventional carbon source); Comparative Example 4 was supplemented with only 3% wheat bran (without CMC-Na); The experimental group used the synergistic induction formulation of this application (1.4% CMC-Na + 3% wheat bran, same as Example 3).

[0125] Experimental Results and Analysis: Comparative Example 3 (CMC-Na only) showed enzyme specificity ≈78% and enzyme activity approximately 1800 U / mL; Comparative Example 4 (wheat bran only) showed enzyme activity approximately 1500 U / mL; while the experimental group (synergistic formulation) showed enzyme specificity ≥90% and total enzyme activity increased to 3625 U / mL. This demonstrates that the long-chain conformational mimicry of CMC-Na and the nutritional induction by wheat bran produced a significant synergistic effect, which is not a simple superposition of two conventional carbon sources.

[0126] Experimental Example 3 (Single main enzyme vs. three-enzyme compound preparation) The study verified the difference between single β-glucosidase and three-enzyme complex formulations in hydrolyzing complex substrates, demonstrating the necessity of multi-enzyme complex formulations.

[0127] Experimental Design: Comparative Example 4 used only the prepared compound β-glucosidase preparation; the experimental group used the three-enzyme system (mass ratio 9:1:0.75) containing α-L-arabinofuranase and cellobiase as described in this application. The total enzyme activity of the two groups was the same, and the enzymatic hydrolysis parameters were the same as in Example 12.

[0128] Experimental Results and Analysis: Comparative Example 4: Conversion rate ≈ 40%, residual substrate ≈ 55%, purity ≈ 82%; Experimental Group: Conversion rate ≥ 89%, residual substrate ≤ 5%, purity ≥ 94%. This demonstrates that a single master enzyme cannot efficiently cleave multi-site glycosidic bonds and relieve product inhibition.

[0129] Experiment Example 4 (Two-enzyme system VS three-enzyme system, verification of the core mechanism of cellobiase) This experimental example aims to verify the deep synergistic mechanism of introducing cellobiase into the system in relieving product feedback inhibition.

[0130] Experimental design: Comparative Example 5 used a two-enzyme combination (β-glucosidase + α-L-arabinofuranosaccharidase); the experimental group used a three-enzyme combination (with added cellobiase). The total enzyme amount was the same, and the reaction conditions were the same as in Example 12. The main focus was on monitoring the dynamic changes in glucose concentration in the reaction system.

[0131] Experimental Results and Analysis: In Comparative Example 5, the conversion rate was approximately 70%, and the glucose concentration was approximately 8 mmol / L (reaching the threshold of 5 mmol / L for feedback inhibition). In the experimental group, the conversion rate was ≥92%, and the glucose concentration was effectively suppressed to approximately 4 mmol / L. This demonstrates that cellobiase indirectly reduced the instantaneous glucose concentration by consuming its precursor, thus completely relieving the feedback inhibition of the main enzyme.

[0132] Experimental Example 5 (Disassembly of the synergistic effect mechanism of three enzyme combinations) This experimental example aims to disassemble and quantify whether the three different enzyme components in the composite hydrolase system of this application produce a nonlinear synergistic effect in catalytic closure.

[0133] Experimental Design: For clear comparison, in this experimental example, β-glucosidase is designated as main enzyme A, α-L-arabinofuranase as coenzyme B, and cellobiase as coenzyme C.

[0134] The experiment consisted of 4 parallel groups: Comparative Examples 6 (single enzyme system: main enzyme A only), 7 (dual enzyme system: main enzyme A + coenzyme B), 8 (dual enzyme system: main enzyme A + coenzyme C), and the experimental group of this application (triple enzyme complex system: main enzyme A + coenzyme B + coenzyme C). The enzymatic reaction parameters and total enzyme ratio of each group are the same as those in Example 12 above.

[0135] Experimental Results and Analysis: Table 3. Results of Synergistic Effect Evaluation

[0136] Data Explanation and Mechanism Verification: As shown in Table 3, under the same conditions, the basic conversion rate using only β-glucosidase (main enzyme A) is only 40%. If we extrapolate from the theoretical linear additive effect: the improvement brought by adding coenzyme B alone is 30%, and the improvement brought by adding coenzyme C alone is 10%, then the theoretical predicted conversion rate of the three-enzyme mixture should be 80% (40%+30%+10%).

[0137] However, the actual conversion rate of the three-enzyme complex experimental group in this application reached 92±1.8%, far exceeding the theoretical linear superposition value. This objective data strongly proves that the three enzymes in this application are not simply a physical mixture, but are actually fused through the mechanism of "exposed coenzyme B site + efficient cleavage of main enzyme A + release of coenzyme C inhibition" to form a highly closed-loop dynamic catalytic system, achieving a qualitative leap in hydrolysis efficiency and product purity.

[0138] Experiment Example 6 (Conventional Enzymatic Hydrolysis Parameters vs. Optimized Process Parameters) This experimental example aims to verify the advantages of the optimization of pH, temperature, rotation speed and reaction time in this application in terms of low consumption and low by-products.

[0139] Experimental design: Comparative Example 7 used conventional parameters (pH 6.0, 55℃, 150rpm, 36h); the experimental group used the parameters of this application (pH 5.0, 38℃, 240rpm, 16h).

[0140] Experimental Results and Analysis: Comparative Example 7 had a conversion rate of ≈65%, byproducts of ≈10%, and energy consumption that was 2.25 times that of the experimental group; the experimental group had a conversion rate of ≥89% and byproducts of ≤3%.

[0141] This demonstrates that the proposed scheme, based on a kinetic mechanism and mild conditions, effectively reduces energy consumption and avoids the generation of byproducts.

[0142] Experiment 7 (Substrate concentration tolerance verification) This experimental example aims to verify the adaptability of the process of this application to industrial high-concentration substrates.

[0143] Experimental design: Comparative Example 8 used a conventional low substrate concentration of 5 mg / mL; the experimental group used a high substrate concentration of 30 mg / mL as in Example 3.

[0144] Experimental Results and Analysis: Although the experimental group had an extremely high substrate concentration, its conversion rate (≥89%) and purity (≥94%) were superior to those of the low-concentration control group 8, and its yield per unit volume was as high as 26.7 mg / mL, breaking through the bottleneck of mass transfer obstruction in high-concentration substrates.

[0145] Experiment Example 8 (Verification of the Advanced Nature of the Extraction Process) This experimental example aims to verify the advantages of using anhydrous ethanol instead of conventional n-butanol in terms of extraction and purification efficiency.

[0146] Experimental design: Comparative Example 9 was extracted with n-butanol; the experimental group was extracted with anhydrous ethanol.

[0147] Experimental Results and Analysis: Comparative Example 9 showed an extraction rate of approximately 80%, with a light brown product; the experimental group showed an extraction rate of ≥95%, with a light yellow product, and the purification cycle was shortened by half (approximately 4 hours). Anhydrous ethanol demonstrated extremely high selectivity and operational advantages.

[0148] Experimental Example 9 (Comparison of Concentration and Drying Processes) This experiment aims to verify the protective effect of the combination of "reduced pressure concentration + vacuum drying" on the activity and morphology of heat-sensitive products.

[0149] Experimental design: Comparative Example 10 was concentrated by heating at atmospheric pressure (70°C) and dried by forced air (100°C). The experimental group used 44℃ vacuum concentration and 100℃ vacuum drying.

[0150] Experimental Results and Analysis: The product of Comparative Example 10 was prone to agglomeration, and its purity decreased to 88%; the product of the experimental group was a loose powder with a purity of ≥94% and a moisture content of ≤1%, proving that the mild desolventizing process reduced the high-temperature degradation of the target product.

[0151] Experimental Example 10 (Mild Enzymatic Hydrolysis of This Application vs. Traditional Chemical Hydrolysis) This experimental example aims to verify the disruptive advantages of the core mild bio-enzymatic method of this patent application in terms of product quality and energy consumption compared to the traditional strong acid / high temperature chemical hydrolysis process in the background art.

[0152] Experimental Design and Analysis: Traditional chemical hydrolysis typically requires extreme conditions (such as high temperature of 80°C and strong acidity of pH 1.0) to forcibly break down saponin molecules. If the enzyme preparation of this application is placed in such an environment, the enzyme conformation will be completely inactivated (conversion rate <1%). However, this application achieves complete hydrolysis under extremely mild conditions (such as 50°C and pH 5.0) through multi-enzyme complex.

[0153] Parallel comparisons demonstrate that the byproduct content of the mild enzymatic method in this application is reduced by more than 60% compared to the chemical method, the product appearance is significantly improved (from dark brown to light yellow), and the production energy consumption is only 1 / 3 of that of the chemical method.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a complex hydrolytic enzyme, characterized in that, include: Aspergillus niger is inoculated into a culture medium for fermentation, and the fermentation broth containing β-glucosidase is collected; wherein the culture medium comprises 1% to 2% sodium carboxymethyl cellulose and 1% to 5% wheat bran by mass percentage. The fermentation broth was subjected to solid-liquid separation to obtain a β-glucosidase preparation; α-L-arabinofuranase and cellobiase were added to the β-glucosidase preparation to obtain the complex hydrolase.

2. The method for preparing the complex hydrolase as described in claim 1, characterized in that, The Aspergillus niger is Aspergillus niger GDMCC3.

486.

3. The method for preparing the complex hydrolase as described in claim 1, characterized in that, The viscosity of the sodium carboxymethyl cellulose is 1000 CPS to 3000 CPS; and / or, The culture medium also includes the following components by weight percentage: 15%–20% yeast extract, 15%–20% peptone, 0.02%–0.1% Tween 80, 5%–10% (NH4)2SO4, 0.1%–0.5% KH2PO4, 0.05%–0.2% MgSO4, 0.001%–0.005% FeSO4, 0.001%–0.005% ZnSO4, 0.001%–0.005% MnSO4, with the balance being water.

4. The method for preparing the complex hydrolase as described in claim 1, characterized in that, The fermentation conditions include at least one of the following: A. Fermentation temperature is 28℃~32℃; B. Fermentation time is 72h~96h; C. Ventilation rate is 1.0 vvm to 1.5 vvm; D. The stirring speed during the fermentation process is 300 rpm to 500 rpm.

5. The method for preparing the complex hydrolase as described in claim 1, characterized in that, The step of solid-liquid separation of the fermentation broth includes: centrifuging the fermentation broth; Preferably, after the centrifugation process, the method further includes a step of concentrating the supernatant after centrifugation.

6. The method for preparing the complex hydrolase as described in claim 1, characterized in that, Before or simultaneously with the addition of α-L-arabinofuranase and cellobiase to the β-glucosidase preparation, the pH of the β-glucosidase preparation is adjusted to 4.0–5.5; and / or, In the complex hydrolase, the mass ratio of α-L-arabinofuranase to β-glucosidase preparation is 1:(5~10); and / or, the mass ratio of cellobiase to β-glucosidase preparation is 1:(8~15).

7. A culture medium for preparing the complex hydrolase as described in claim 1, characterized in that, The components of the culture medium, by mass percentage, include: 1% to 2% sodium carboxymethyl cellulose, 0.02% to 0.1% Tween 80, 15% to 20% yeast extract powder, and 15% to 20% peptone.

8. A complex hydrolytic enzyme, characterized in that, It is prepared by the preparation method according to any one of claims 1-6.

9. A method for preparing a rare saponin CK, characterized in that, include: Using at least one of ginsenosides Rb1, Rb2, and Rc as a substrate, an enzymatic hydrolysis reaction was carried out by adding the complex hydrolytic enzyme as described in claim 8 at a temperature of 30℃~50℃ and a pH of 4.5~5.5 to obtain a reaction solution. After the reaction was completed, the reaction solution was purified to obtain rare saponin CK.

10. The method for preparing rare saponin CK as described in claim 9, characterized in that, During the enzymatic hydrolysis reaction, the stirring speed is 200 rpm to 300 rpm, and / or the reaction time is 24 h to 48 h; and / or, The step of purifying the reaction solution includes: extracting the reaction solution with anhydrous ethanol and collecting the extract; concentrating the extract under reduced pressure and drying it under vacuum.

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