Use of aspergillus niger beta-glucosidase in the preparation of gentiobiose

Abstract

The application discloses an application of Aspergillus niger beta-glucosidase in preparation of gentiobiose, and belongs to the field of genetic engineering and enzyme engineering. Through single factor and orthogonal experiment optimization, a formula for efficiently preparing gentiobiose is finally obtained, that is, under the condition that the glucose substrate concentration is 850 g / L, the pH is 5.0, the temperature is 60 DEG C, and the enzyme addition amount is 105 U / g, fermentation is carried out for 48 h, 163.4 g / L gentiobiose can be obtained, the conversion rate is 19.22%, although the conversion rate is 0.14% lower than the conversion rate of gentiobiose prepared by using beta-glucosidase from Cellulomonas, the enzyme addition amount is only 1 / 4 of that of the beta-glucosidase, and the enzyme has great industrial potential. It can be seen that the beta-glucosidase from the food-safety strain Aspergillus niger is used for preparing gentiobiose, the cost and energy consumption required by the reaction can be reduced, and the conversion rate of gentiobiose can be improved.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to the application of the Aspergillus niger β-glucosidase gene AnBgl in the preparation of gentiobiose. Background Technology

[0002] β-glucosidase (Bgl, EC 3.2.1.21) is a glycoside hydrolysase (GH) that hydrolyzes the non-reducing terminal glycosidic bonds of specific substrates to release glucose and the corresponding functional group (Tiwariet et al., 2013). It is mainly distributed in animals, plants, and microorganisms. Most β-glucosidases originate from the GH3 and GH1 families, with a few from the GH5, 9, 30, and 116 families (Ketudat et al., 2015). β-glucosidases from the GH1 family are mainly distributed in animals, plants, and archaea, while those from the GH3 family are mainly distributed in bacteria and fungi (Gudmundsson et al., 2016). Compared to bacterial β-glucosidases, fungal β-glucosidases exhibit significantly higher enzyme activities (Ahmed et al., 2017). Among them, β-glucosidases derived from Aspergillus niger strains have the highest enzyme activity. As an FDA-approved safe strain, Aspergillus niger-derived β-glucosidase has been used as a commercial enzyme (Sorensen et al., 2013). After comparing the transglycosylation ability of GH3 and GH1 family β-glucosidases in synthesizing gentiobiose from cellobiose glycosidic bonds, Guo et al. found that the transglycosylation ability of the GH3 family is more specific than that of the GH1 family, making it more favorable for the preparation of gentiobiose (Guo et al., 2016; Wang et al., 2011). Therefore, it is speculated that GH3 family β-glucosidases also possess this characteristic when synthesizing gentiobiose using high concentrations of monosaccharides.

[0003] Gentiobiose is composed of two glucose molecules linked by a β-1,6 glycosidic bond. As a functional oligosaccharide, it cannot be digested and absorbed by digestive enzymes in the human intestine and stomach. Instead, it acts on beneficial intestinal bacteria such as Lactobacillus and Bifidobacterium, promoting their growth (Singh et al., 2017). This results in improved gut health and inhibition of pathogenic microorganisms (Pan et al., 2018), thus possessing prebiotic properties. Due to its low calorie and low-calorie content, it can also prevent tooth decay (Xing Xueyan, 2017), making it suitable for patients with diabetes and other special conditions (Rycroft et al., 2001). Furthermore, gentiobiose derivatives have anti-inflammatory and anti-tumor effects (Ispirli et al., 2019), making it a potential drug for disease treatment. Gentiobiose has a distinctive, stimulating bitter taste, which is widely used in chocolate and coffee, making it a high-value sugar with significant development potential.

[0004] There are two main methods for preparing gentiobiose: extraction and enzymatic conversion. Early methods involved extracting and isolating gentiobiose from the rhizomes of *Gentiana* or amygdalin (Tang et al., 2000). Another extraction method involved extracting it from the byproducts of starch acid hydrolysis (Zhang Lin, 2016). These traditional processes suffer from drawbacks such as the scarcity of raw materials, high energy consumption, high industrial costs, and the generation of numerous byproducts containing impurities like organic acids, hindering the separation and purification of single products (Zhu et al., 2001). Enzymatic conversion primarily utilizes β-glucosidase for glycosidization or reverse hydrolysis. Enzymatic methods yield higher yields of oligogentiobiose than extraction methods and are easier to separate and purify (Zou Hui et al., 2004). The reaction system is environmentally friendly and efficient, showing broad application prospects.

[0005] There are many strains available for preparing gentiobiose, but there are few reports on preparation using AnBgl. Furthermore, the amount of enzyme added for high-yield gentiobiose preparation by enzymatic methods is generally maintained at 400 U / g to 800 U / g glucose. Such a large amount of purified enzyme solution would be very costly in industrial applications. Therefore, it is practically necessary to find a low-energy-consumption and high-yield preparation method. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide an application of Aspergillus niger β-glucosidase in the preparation of gentiobiose. This β-glucosidase is derived from the food-safe strain Aspergillus niger, and this application method not only reduces the cost and energy consumption required for the reaction but also improves the conversion rate of gentiobiose.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides the application of Aspergillus niger β-glucosidase in the preparation of gentiobiose.

[0009] The application involves preparing gentiobiose using glucose as a substrate; the concentration of glucose is 250 g / L to 850 g / L, further 450 g / L to 850 g / L; even further 650 g / L to 850 g / L; even further 750 to 850 g / L; and still even further 850 g / L.

[0010] The amino acid sequence of the Aspergillus niger β-glucosidase is shown in GenBank: CBA02054.1 or in SEQ ID NO.2, amino acids 1 to 843aa. In particular, amino acids 843 to 848aa in SEQ ID NO.2 are 6×His-tagged amino acid sequences, which will be used for subsequent protein purification.

[0011] The nucleotide sequence of the gene encoding Aspergillus niger β-glucosidase is shown in GenBank: FN430671.1 or SEQ ID NO. 1, bp 1–2529. Among them, bp 2527–2544 of SEQ ID NO. 1 is the nucleotide sequence encoding a 6×His tag.

[0012] The dosage of Aspergillus niger β-glucosidase is 35 U / g to 140 U / g glucose; further, 70 U / g to 140 U / g glucose; and even further, 105 U / g glucose.

[0013] The method for preparing Aspergillus niger β-glucosidase includes the following steps:

[0014] The Aspergillus niger β-glucosidase gene (AnBgl) was cloned, an expression vector was constructed, and the gene was integrated into the Pichia pastoris genome to obtain an engineered Pichia pastoris strain. The protein was induced to be expressed and purified to obtain Aspergillus niger β-glucosidase.

[0015] Preferably, the Pichia pastoris is Pichia pastoris GS115;

[0016] Preferably, the starting vector of the expression vector is pPIC9K; the expression vector is the expression vector pPIC9K-AnBgl;

[0017] Preferably, the Pichia pastoris engineered strain is Pichia pastoris GS115 / pPIC9K-AnBgl.

[0018] The preparation process of gentiobiose using the purified Aspergillus niger β-glucosidase was carried out using single-factor experiments on substrate concentration, temperature, reaction pH, and enzyme dosage. Finally, the substrate concentration, temperature, and reaction pH were optimized using orthogonal conditions to obtain the optimal process for preparing gentiobiose.

[0019] The preparation conditions for the gentiobiose are as follows: fermentation for 24h to 72h under the following conditions: glucose substrate concentration of 250g / L to 850g / L, pH of 4.0 to 6.5, temperature of 35℃ to 60℃, and addition of Aspergillus niger β-glucosidase of 35U / g to 105U / g glucose.

[0020] Furthermore, the preparation conditions for the gentiobiose are as follows: fermentation for 36h to 54h under the following conditions: glucose substrate concentration of 450g / L to 850g / L, pH of 4.0 to 6.0, temperature of 40℃ to 60℃, and addition of Aspergillus niger β-glucosidase of 70U / g to 105U / g glucose.

[0021] Furthermore, the preparation conditions for the gentiobiose are as follows: fermentation for 48 hours at a glucose substrate concentration of 650 g / L to 850 g / L, a pH of 4.0 to 6.0, a temperature of 40°C to 60°C, and an addition of 70 U / g to 105 U / g glucose of Aspergillus niger β-glucosidase.

[0022] Preferably, the optimal preparation conditions for gentiobiose are: fermentation for 48 hours under the following conditions: glucose substrate concentration of 850 g / L, pH of 5.0, temperature of 60°C, and addition of Aspergillus niger β-glucosidase of 105 U / g glucose, to obtain gentiobiose of 163.4 g / L with a conversion rate of 19.22%.

[0023] The present invention has the following advantages and effects compared with the prior art:

[0024] The gene sequence AnBgl, cloned from Aspergillus niger in this invention, was compared with the gene sequence of gentiobiose synthesized by β-glucosidase from Aspergillus niger only by DNAMAN. The homology was found to be 92.61%, but the conversion rate of gentiobiose was only 6.25% (Liu Lingling, 2009). This invention establishes a fermentation system for efficient synthesis of gentiobiose. Through single-factor and orthogonal experiments, the final efficient formulation for preparing gentiobiose was obtained: fermentation for 48 hours under the conditions of glucose substrate concentration of 850 g / L, pH of 5.0, temperature of 60℃, and enzyme addition of 105 U / g yielded 163.4 g / L gentiobiose with a conversion rate of 19.22%. Although this is 0.14% lower than the conversion rate reported by β-glucosidase from cellulosic bacteria (Xu Xinghao, 2020) (where homology was only 44.80% through DNAMAN homology comparison), the enzyme addition amount is only 1 / 4 of that, showing great industrial potential. Attached Figure Description

[0025] Figure 1 This is a graph showing the total RNA analysis of Aspergillus niger; lanes 1-2 represent RNA samples.

[0026] Figure 2 This is an amplification diagram of the target gene of Aspergillus niger β-glucosidase; in it, lane M3: DNA Marker 3; lanes 1-3: bands of the target gene AnBgl.

[0027] Figure 3 This is the pPIC9K-AnBgl expression plasmid map.

[0028] Figure 4 This is a gel image of the engineered strain P. pastoris GS115 / pPIC9K-AnBgl containing the target gene; lane M3: DNA Marker 3; lane 1: P. pastoris GS115; lane 2: P. pastoris GS115 / pPIC9K-AnBgl.

[0029] Figure 5 This is a gel image of protein purified using AnBgl; lane M: protein marker; lanes 1-2 are PBS washing buffer; lane 3: first protein elution buffer; lane 4: second protein elution buffer; lane 5: third protein elution buffer.

[0030] Figure 6 This is a diagram showing the enzymatic properties of AnBgl at its optimal temperature.

[0031] Figure 7 This is a diagram illustrating the enzymatic properties of AnBgl's temperature stability.

[0032] Figure 8 This is a diagram showing the enzymatic properties of AnBgl at its optimal pH.

[0033] Figure 9 This is a diagram illustrating the enzymatic properties of AnBgl's pH stability.

[0034] Figure 10 This is the standard curve of gentiobiose.

[0035] Figure 11 This is a graph showing the effect of glucose substrate concentration on gentiobiose yield.

[0036] Figure 12 This is a graph showing the effect of temperature on gentiobiose yield.

[0037] Figure 13 This is a graph showing the effect of pH on gentiobiose yield.

[0038] Figure 14 The graph shows the effect of enzyme addition on gentiobiose yield. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0040] Unless otherwise specified, the test methods in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0041] Example 1: Construction of an engineered strain of P. pastoris GS115 / pPIC9K-AnBgl

[0042] 1. Materials and Methods

[0043] 1.1 Materials

[0044] 1.1.1 Strains and Plasmids

[0045] Aspergillus niger was identified as Aspergillus niger NRRL3135, which is disclosed in the reference (Chen et al., 2010); Escherichia coli DH5α strain was purchased from Beijing Qingke Biotechnology Co., Ltd. for cloning; pMD-19T cloning vector was purchased from TAKARA for cloning the Anbgl gene; pPIC9K was a Pichia pastoris expression vector, and Pichia pastoris GS115 was a commercially available strain for the expression of β-glucosidase (Bgl).

[0046] 1.1.2 Primer Design and Synthesis

[0047] Primer design for cloning and testing AnBgl fragments.

[0048] Table 1 Primers for β-glucosidase gene cloning

[0049]

[0050] Note: The underlined parts at the bottom of the bases are homologous arms.

[0051] 1.1.3 Enzymes and Kits

[0052] (1) Tool enzymes

[0053] 2×Rapid Taq Master Mix, Phanta Max Super-Fidelity DNA Polymerase, and Phanta flash Super-Fidelity DNA Polymerase were all purchased from Nanjing Novizan Biotechnology Co., Ltd.; QuickCut EcoRⅠ, NotⅠ, and BglⅡ were all purchased from TAKARA Biotechnology Co., Ltd.

[0054] (2) Reagent kit

[0055] Agarose gel extraction kit: HiPure PCR Pure Micro Kit, purchased from Meiji Biotechnology Co., Ltd.;

[0056] Plasmid extraction kit: HiPure Plasmid Micro Kit, purchased from Meiji Biotechnology Co., Ltd.;

[0057] Reverse transcription reagent: TransScript One-Step gDNA Removal and cDNA SynthesisSuperMix, purchased from Beijing TransGen Biotech Co., Ltd.

[0058] Cloning vector kit: pMD TM The 19-T Vector Cloning Kit was purchased from TAKARA Biotechnology Co., Ltd.

[0059] BCA Protein Assay Kit: Purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0060] 1.1.4 Culture Media and Reagents

[0061] (1) Culture medium

[0062] LB medium: For the culture of Escherichia coli DH5α, weigh 10g tryptone, 5g yeast extract, and 10g NaCl, add distilled water to a final volume of 1L, and sterilize at 121℃ for 30min. LB solid medium is prepared by adding 2% agar powder to this mixture.

[0063] YPD medium: For the culture of Pichia pastoris GS115, weigh 10g peptone, 5g yeast extract, and 10g glucose, add distilled water to a final volume of 1L, and sterilize at 115℃ for 15min. YPD solid medium is prepared by adding 2% agar powder to this mixture.

[0064] MD medium: used for screening Pichia pastoris GS115 transformants. Weigh 2g of agar and add it to 80mL of distilled water. Sterilize at 121℃ for 30min and cool until it is not hot to the touch. Then add 10mL of 10× glucose and 10mL of 10× YNB.

[0065] BMGY medium: Weigh 10g of peptone and 5g of yeast extract, add distilled water to a final volume of 350mL, sterilize at 121℃ for 30min, and cool until it is no longer hot to the touch. Add 50mL of 10×YNB, 50mL of 10×glycerol, 50mL of 1M phosphate buffer, and 500μL of 500×Biotin.

[0066] BMMY medium: Weigh 10g of peptone and 5g of yeast extract, add distilled water to a final volume of 400mL, sterilize at 121℃ for 30min, and cool until it is no longer hot to the touch. Add 50mL of 10×YNB, 50mL of 1M phosphate buffer, 2% filtered sterilized methanol, and 500μL of 500×Biotin.

[0067] (2) Reagents

[0068] Gentianobiose standard was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.; analytical grade gentiobiose and ampicillin were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; all components of the culture medium were purchased from Beijing Solarbio Science & Technology Co., Ltd.; and the nucleic acid dye GoldView was used. TM The reagents were purchased from Beijing Saibaisheng Gene Technology Co., Ltd.; the reagents for protein electrophoresis and nucleic acid electrophoresis were purchased from Dongsheng Biotechnology Co., Ltd. DNA Marker 3 and protein Marker were purchased from Guangzhou Dongsheng Biotechnology Co., Ltd., and Ni-NTA was purchased from Shanghai Sangon Biotech Co., Ltd.

[0069] 1.2 Methods

[0070] 1.2.1 Obtaining the target gene

[0071] RNA was extracted from Aspergillus niger using the STE method, and cDNA was synthesized using a reverse transcription kit. The cDNA was then amplified using primers 9K-Bgl-F and 9K-Bgl-R to obtain fragments with homologous arms. The DNA polymerase used to clone the target gene was Phanta Max Super-Fidelity DNA Polymerase from Novizan.

[0072] (1) Add the following components to the PCR tube:

[0073] Table 2 Target gene amplification system

[0074]

[0075] (2) The PCR reaction system is as follows: pre-denaturation (95℃, 3min), denaturation (95℃, 15s), annealing (55℃, 15s), extension (72℃, 15s / kb) and final extension (72℃, 5min), with a total of 35 cycles of denaturation, annealing and extension.

[0076] (3) Take an appropriate amount of PCR product and detect it by 1% agarose gel electrophoresis. Then, use the gel recovery kit from MGI to recover the product. Finally, use Nano-Drop to detect the concentration of the recovered nucleic acid.

[0077] 1.2.2. Enzyme digestion of the empty vector pPIC9K for expression

[0078] The expression vector in this experiment was constructed using homologous recombination. This required linearization of the pPIC9K expression vector through double digestion. The enzyme used was QuickCut from TAKARA, with EcoRI and NotRI restriction sites. The digestion system is as follows:

[0079] Table 3 Enzyme digestion system for expression vectors

[0080] Expression empty carrier 1μg EcoRI (rapid enzyme) 1μL Fast Enzyme NotI 1μL 10×QuickCut Green Buffer 2μL <![CDATA[ddH2O]]> Add to 20 μL

[0081] (1) After mixing the enzyme digestion system, incubate it in a metal bath (37℃, 60min). After incubation, inactivate it in a metal bath (85℃, 60min).

[0082] (2) Take an appropriate amount of enzyme digestion product and perform 1% agarose gel electrophoresis to detect the enzyme digestion. Compare the size of the fragments before and after enzyme digestion to determine whether the enzyme digestion was successful. Then, the fragments are recovered.

[0083] 1.2.3 Homologous Recombination to Construct Vectors

[0084] In this experiment, the homologous recombination enzyme used was the Uniclone One Step Seamless Cloning Kit (SC612) from Jinsha Biotechnology Co., Ltd. The fragment following the homologous arm in 1.2.1 was ligated to the empty vector in 1.2.2 using the enzyme from this product. The specific method is as follows:

[0085] (1) Mix the following mixture in a PCR tube:

[0086] Table 4. Vectors constructed by homologous recombination

[0087] Linearized empty carrier (0.02 × number of bases) ng fragment containing homologous arms (0.04 × number of bases)ng 2×Uniclone Seamless Cloning Mix 5μL <![CDATA[ddH2O]]> Add to 10 μL

[0088] (2) The cells were then incubated in a PCR instrument (50℃, 30 min). After incubation, the cells were transferred into E. coli DH5α and cultured for 12 h. The transformants were identified by selecting pPIC9K-AnBgl and sent for testing. Transformants with correct sequencing were preserved.

[0089] 1.2.4 Enzyme digestion of expression vector pPIC9K-AnBgl

[0090] The expression vector pPIC9K-AnBgl plasmid was extracted using a plasmid extraction kit from MGI Tech. Subsequently, pPIC9K-AnBgl was digested with BglII from TAKARA's QuickCut series to obtain a linearized fragment for transformation. The specific steps are as follows:

[0091] (1) The pPIC9K-AnBgl enzyme digestion system is shown in Table 5; plasmid

[0092] Table 5. Enzyme digestion system of pPIC9K-AnBgl

[0093] pPIC9K-AnBgl 20μg Rapid enzyme BglII 10μL 10×QuickCut Green Buffer 10μL <![CDATA[ddH2O]]> Add to 100 μL

[0094] (2) After mixing the enzyme digestion system, incubate it in a metal bath (37℃, 60min). After incubation, inactivate it in a metal bath (85℃, 60min).

[0095] (3) Take an appropriate amount of enzyme digestion product and perform 1% agarose electrophoresis to detect it. Compare the size of the fragments before and after enzyme digestion to determine whether the enzyme digestion was successful.

[0096] (4) The bands after successful enzyme digestion were recovered by column chromatography. The reaction solution was recovered by column chromatography using the kit from MGI Recovery according to the instructions.

[0097] (5) Finally, add 20 μL of ultrapure water to dissolve the nucleic acid and use Nano-Drop to detect the nucleic acid concentration.

[0098] 1.2.5 Transformation of Pichia pastoris

[0099] The Pichia pastoris strain used in this study was GS115, and the engineered strain was constructed using the PEG chemical transformation method: P. pastoris GS115 / pPIC9K-AnBgl.

[0100] The specific methods for preparing Pichia pastoris competent cells and their transformation are as follows:

[0101] (1) Pichia pastoris GS115 was activated by streaking on YPD plates and cultured upside down at 30℃ for 2 days;

[0102] (2) Pick a single colony from the plate and put it into a 5 mL YPD tube for overnight incubation (30℃, 200 rpm);

[0103] (3) Take 1 mL of the overnight culture and transfer it to a fresh 50 mL YPD medium for incubation (30℃, 200 rpm). Stop shaking the culture when the OD600 of the culture reaches 0.8 to 1.2.

[0104] (4) Pour 10 mL of bacterial culture into a 50 mL centrifuge tube, centrifuge (3,000 g, 3 min, 25 °C), and remove the supernatant;

[0105] (5) Add 10 mL of 1×TE (about 9 mL of ddH2O + 1 mL of 10×TE) to the precipitate, and mix it with a pipette. Centrifuge (3,000 g, 3 min, 25 °C) to remove the supernatant.

[0106] (6) Add 1 mL of conversion solution (50 μL 10×TE + 100 μL 1MLiAc + 850 μL ddH2O) to the precipitate, and transfer all the bacterial culture from the large centrifuge tubes to 1.5 mL centrifuge tubes. Incubate in a metal bath at 30 °C for 10 min.

[0107] (7) Centrifuge (3,000g, 3min, 25℃), remove the supernatant with a pipette, and add 100μL of conversion solution 2 (100μL 10×TE + 100μL 1MLiCl + 800μL ddH2O) to the precipitate to obtain Pichia pastoris competent cells;

[0108] (8) Add the recovered linearized fragment to 100 μL of competent cells and incubate in a 30°C metal bath for 10 min;

[0109] (9) Add 700 μL of heat shock solution (70 μL 10×TE + 70 μL 1MLiCl + 560 μL 50% PEG3350) to the mixture, mix gently, and incubate in a 30℃ metal bath for 20 min.

[0110] (10) Heat shock in a 42℃ metal bath for 20 min;

[0111] (11) After heat shock, immediately place in an ice bath for 5 min, then centrifuge (3,000 g, 1 min, 25 °C), and pipette off the supernatant;

[0112] (12) Add 1 mL of YPD culture medium to the precipitate and transfer it to a 2 mL centrifuge tube. Incubate on a shaker (30℃, 200 rpm, 2 h). Place the centrifuge tube vertically or at an angle upwards. Do not place it horizontally, as ethanol will be produced during the recovery period and will open the cap.

[0113] (13) Centrifuge the revived bacterial culture (3,000g, 1min, 25℃), discard the supernatant until only 50-100μL of liquid remains, add the precipitate, mix by pipetting, and then spread it on MD screening plates.

[0114] (14) Invert the plate and incubate it in a 30℃ incubator. After 2 days, colonies can be picked for PCR identification.

[0115] 1.2.6 Identification of Pichia pastoris transformants

[0116] The host used in this study was Pichia pastoris. Because it contains a cell wall, colony identification of transformants is more difficult than with Escherichia coli. To ensure strong amplification, the DNA polymerases used in the past for identifying yeast transformants were relatively expensive, high-fidelity enzymes with good amplification performance, such as KOD-Fx. This study used a simpler lysis method: the template was treated with 10mM NaOH before PCR, and then PCR identification was performed using Novizan 2×Rapid Taq Master Mix, which can save costs. The specific steps are as follows:

[0117] (1) Using a sterile pipette tip or a sterilized toothpick, pick up the Pichia pastoris transformant into a PCR tube containing 10 μL of 10 mM NaOH solution, mix well, and place it in a PCR instrument at 95°C for 12 min to lyse. After cooling, it can be used as a template.

[0118] (2) The PCR system for colony identification is shown in Table 6:

[0119] Table 6. Pichia pastoris colony PCR system

[0120] template 1μL 3AOX1 1μL 5AOX1 1μL 2×Rapid Taq Master Mix 5μL <![CDATA[ddH2O]]> 2μL

[0121] (3) The PCR program conditions are as follows: pre-denaturation (94℃, 10min), denaturation (94℃, 15s), annealing (55℃, 15s), extension (72℃, 15s / kb) and final extension (72℃, 5min), with a total of 30 cycles of denaturation, annealing and extension.

[0122] (4) After PCR, the product was subjected to 1% agarose gel electrophoresis. The electrophoresis results were observed. The correct transformants were selected and cultured in fresh YPD liquid medium. The transformants were then preserved with glycerol and stored at -80℃.

[0123] 1.3 Results and Analysis

[0124] 1.3.1 RNA extraction from Aspergillus niger

[0125] like Figure 1 As shown, the RNA in lane 1 is of excellent quality and can be used for subsequent reverse transcription for gene amplification.

[0126] 1.3.2 Amplification of AnBgl

[0127] like Figure 2 As shown, there is a clear band between 2000 and 3000 bp, which is preliminarily identified as the AnBgl target gene with a homologous arm.

[0128] 1.3.3 Construction of expression vector pPIC9K-AnBgl

[0129] The AnBgl target gene with homologous arms was ligated into the expression vector pPIC9K and then sequenced. Sequencing results showed that the gene sequence was correct. The pPIC9K-AnBgl expression plasmid map is shown below. Figure 3 As shown.

[0130] 1.3.4 Construction of engineered strain P. pastorris GS115 / pPIC9K-AnBgl

[0131] like Figure 4 As shown, the band of about 2100 bp in lane 1 is the original band of the wild-type Pichia pastoris GS115 strain; the band of nearly 3000 bp in lane 2 is the band after AnBgl replaced the AOX1 alcoholase gene and was integrated into the Pichia pastoris GS115 genome.

[0132] Example 2 Enzymatic properties of recombinant β-glucosidase

[0133] 2 Materials and Methods

[0134] 2.1 Materials (Refer to the materials in 1.1)

[0135] 2.2 Methods

[0136] 2.2.1 Expression of β-glucosidase

[0137] In this experiment, the expression of β-glucosidase was mainly achieved by first enriching bacterial cells in a culture medium with glycerol as the main carbon source, and then inducing the expression of the methanol oxidase promoter protein using methanol. The specific procedures are as follows:

[0138] (1) Inoculate the correctly identified transformants into 5 mL of YPD liquid medium and incubate overnight (30°C, 200 rpm);

[0139] (2) Take 1 mL of liquid and inoculate it into 50 mL of BMGY medium. Place it on a shaker (30℃, 200 rpm) and culture it until the logarithmic phase. Then centrifuge it (10000 g, 5 min, 25℃), discard the supernatant, collect the cells, resuspend them in ultrapure water, and inoculate them into 50 mL of BMMY medium for fermentation for a period of time. Add 1 mL of methanol every day to induce expression.

[0140] 2.2.2 Purification of β-glucosidase

[0141] When constructing the vector, a His tag was added to the 3' end of the AnBgl target gene. Therefore, the AnBgl protein can be purified using a Ni-NTA affinity chromatography column. The specific steps are as follows:

[0142] (1) Collect the BMMY fermentation broth and centrifuge it (10000g, 12min, 25℃). Filter the supernatant (crude enzyme solution) through a 0.22μm aqueous filter membrane and place it in a beaker.

[0143] (2) Add 2 mL of Ni-NTA packing material to the supernatant (crude enzyme solution) and stir with a magnetic stirrer (4℃, 100 rpm) for 12 h;

[0144] (3) Add the supernatant (crude enzyme solution) and packing material into the chromatography column, unscrew the stopper at the bottom of the chromatography column and let the liquid flow out naturally;

[0145] (4) After the chromatography column is filled with only the packing material, tighten the stopper at the bottom of the chromatography column, add 2 mL of pH 8.0 PBS buffer to the packing material, shake horizontally for 5 min, let it settle naturally for 5 min, then unscrew the stopper at the bottom to let the liquid flow out naturally, collect the washing solution, and repeat this step 3 times.

[0146] (5) Add 1 mL of wash buffer to the packing material, let it settle naturally for 5 min and then collect the wash solution. This step is to wash away impurities and proteins. Repeat this step twice.

[0147] (6) Add 1 mL of elution buffer to the packing material, allow it to settle naturally for 5 min, and collect the eluent. This step is to elute the target protein. Repeat this step 10 times until the target protein is completely eluted.

[0148] (7) Add 20% ethanol / PBS solution to the packing material and store the packing material at 4°C;

[0149] (8) The collected eluent was dialyzed overnight with citrate-disodium hydrogen phosphate buffer at pH 5.0, followed by ultrafiltration. The collected enzyme solution was stored at 4°C for later use.

[0150] 2.2.3 Determination of enzyme activity

[0151] The specific steps for determining β-glucosidase activity using pNPG as a substrate are as follows:

[0152] (1) Add 0.9 mL of 50 mM citrate-disodium hydrogen phosphate buffer (pH 4.4) to the colorimetric tube, then add 100 μL of enzyme solution diluted by a certain factor, and place it in a 50°C water bath to preheat for 10 min with 5 mM pNPG (p-nitrophenol-β-D-glucoside). The blank group (CK) uses enzyme solution that has been boiled in a 100°C water bath for 10 min. The rest of the operation is the same.

[0153] (2) After preheating, add 1 mL of pNPG to the colorimetric tube and time for 10 min. After 10 min, immediately add 1 mL of 1 M Na2CO3. After cooling to room temperature, dilute to 25 mL with ultrapure water.

[0154] (3) Measure the absorbance at a wavelength of 410 nm using an ultraviolet spectrophotometer, and calculate the enzyme activity by substituting it into the standard curve of p-nitrophenol.

[0155]

[0156] Where Y represents the activity of β-glucosidase, U / mL; A represents the amount of p-nitrophenol obtained from the standard curve, μmol; and N represents the dilution factor of the enzyme solution.

[0157] Add 0.025 mL, 0.050 mL, 0.075 mL, 0.100 mL, 0.125 mL, and 0.150 mL of 10 mM p-nitrophenol stock solution to six colorimetric tubes, respectively, then add 1 mL of 1 M Na2CO3, and dilute to 25 mL with ultrapure water. Measure the absorbance using a UV spectrophotometer at a wavelength of 410 nm.

[0158] 2.2.4 Definition of Enzyme Activity

[0159] One enzyme activity unit is defined as the enzyme activity that hydrolyzes p-nitrophenol-β-D-glucoside (pNPG) in 1 mL of enzyme solution to produce 1 μmol of p-nitrophenol within 1 min.

[0160] 2.2.5 Determination of the optimal temperature and temperature stability of β-glucosidase

[0161] Take the purified enzyme solution diluted by a certain factor and place it in a citrate-disodium hydrogen phosphate buffer solution at pH 4.4. Measure the enzyme activity at 30, 40, 50, 60, 70, and 80°C. Define the highest enzyme activity as 100%, and use this as a control to calculate the optimal temperature for the remaining enzyme activities.

[0162] To study its temperature stability, the enzyme solution diluted by a certain factor was incubated at the above temperature for 2 hours, and its enzyme activity was measured. The initial enzyme activity (the highest enzyme activity without incubation) was defined as 100%, and the remaining enzyme activities were used as controls. The method for measuring enzyme activity is as described in 2.2.3.

[0163] 2.2.6 Determination of the optimal pH and pH stability of β-glucosidase

[0164] Take the purified enzyme solution diluted by a certain factor, place it at the optimal temperature, and measure the enzyme activity under pH conditions of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 (50 mM citrate-disodium hydrogen phosphate). Define the highest enzyme activity as 100%, and use this as a control to calculate the optimal pH.

[0165] To study its pH stability, the enzyme solution diluted by a certain factor was incubated at the pH conditions described above for 2 hours, and then the enzyme activity was measured. The initial enzyme activity (the highest enzyme activity without incubation) was defined as 100%, and the remaining enzyme activities were used as controls. The method for measuring enzyme activity is as described in 2.2.3.

[0166] 2.2.7 Kinetic determination of β-glucosidase

[0167] Using pNPG as a substrate, the K+ of β-glucosidase was measured. m and K cat Values ​​were determined by dissolving the substrate in 50 mM citrate-disodium hydrogen phosphate buffer to prepare pNPG solutions of 0.1 mM, 0.2 mM, 0.4 mM, 1.0 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM, and measuring the values ​​at pH 4.5 and 60 °C, following the method described in section 2.2.3. m and K cat The value was calculated using GraphPad Prism 8.0.

[0168] 2.3 Results and Analysis

[0169] 2.3.1 Results of protein purification

[0170] The total enzyme activity of the enzyme solution purified by Ni-NTA was measured to be 6520U. The total protein content was 33mg according to the BCA protein assay. Therefore, the specific enzyme activity of β-glucosidase was 198U / mg.

[0171] like Figure 5 As shown, lanes 1-2 are the washing solution of PBS, and lanes 3-5 are the protein gel images of different collection batches after purification. It can be seen that the purified protein bands are single and of excellent quality.

[0172] 2.3.2 Effect of temperature on enzyme activity

[0173] The enzyme was subjected to a catalytic reaction at 30℃~80℃, and the experimental results are as follows: Figure 6 As shown in the figure, the optimal temperature for this β-glucosidase is 60℃. Between 50℃ and 60℃, the enzyme activity retains over 70%, but when the temperature exceeds 60℃, the enzyme activity rapidly decreases, reaching only 15% at 70℃. The enzyme's stability was measured after incubation at 30℃–70℃ for 2 hours at each temperature. Figure 7 It can be seen that when the temperature is 50℃, the enzyme activity is still maintained at about 90%, but at the optimal temperature of 60℃, the enzyme activity is only 54%.

[0174] 2.3.3 Effect of pH on enzyme activity

[0175] The enzyme solution was placed in a citrate-disodium hydrogen phosphate buffer system with a pH of 3.5–7, and the enzyme activity at each pH was measured as follows: Figure 8 As shown. By Figure 8 It is known that this β-glucosidase is an acidophilic enzyme, exhibiting no activity under neutral or alkaline conditions. Its optimal pH is 4.5, and its activity decreases rapidly above pH 5. The enzyme activity was measured after placing the enzyme solution in buffer systems of different pH values ​​for 2 hours; the results are as follows. Figure 9 As shown, the activity of this β-glucosidase can still be maintained at over 98% under pH 4.5 conditions, and at pH 4-5 conditions, the activity can still be maintained at over 80%, indicating that this recombinant enzyme has good pH stability.

[0176] 2.3.4 Results of kinetic parameter measurement

[0177] The enzyme was subjected to reaction under optimal temperature and pH conditions, and its activity was measured. The reaction rate of different concentrations of pNPG was nonlinearly fitted using the Michaelis-Menten equation in GraphPadPrism 8.0 to calculate V. max K m K cat and K cat / K m The values ​​and results are shown in the table below.

[0178] pNPG 2.349 104.9 2449 44.66 198U / mg

[0179] K m The value is the Michaelis constant of the enzyme, representing the affinity between the enzyme and its substrate. When K... m A higher K value indicates a weaker affinity between the substrate and the enzyme, and vice versa. cat / K m The value is the catalytic constant, representing the catalytic efficiency of the enzyme. When K... cat / K m A higher value indicates a higher catalytic efficiency of the enzyme. The above results show that, after purification by protein affinity chromatography, the specific activity of β-glucosidase is as high as 198 U / mg. β-glucosidase has a high affinity for the substrate pNPG, and the enzyme also has high catalytic efficiency. Since β-glucosidase transfers pNPG glucosides during its reaction with pNPG, the specific activity can be determined by enzyme activity, K... m and K cat / K m Preliminary assessment of the β-glucosidase's transglycosylation ability indicates that β-glucosidase has a strong transglycosylation ability.

[0180] Example 3: Investigation of conditions for enzymatic preparation of gentiobiose

[0181] 3 Materials and Methods

[0182] 3.1 Materials (Refer to the materials in 1.1)

[0183] 3.2 Methods

[0184] 3.2.1 Optimization of substrate concentration prepared by enzymatic method

[0185] Pure glucose was dissolved in a citrate-disodium hydrogen phosphate buffer solution at pH 5.0 to prepare reaction substrates of different concentrations (250 g / L, 350 g / L, 450 g / L, 550 g / L, 650 g / L, 750 g / L, 850 g / L). 35 U / g of purified enzyme solution was added to prepare a 5 mL fermentation system, which was then fermented at 50 °C for 150 rpm on a shaker for 48 h. Each gradient was performed in triplicate. After the reaction, 1 mL of sample was transferred to a 1.5 mL centrifuge tube and inactivated in a 100 °C metal bath for 10 min. The sample was diluted through a 0.22 μm membrane and then analyzed by liquid chromatography.

[0186] 3.2.2 Temperature Optimization for Enzymatic Preparation

[0187] Pure glucose was dissolved in citrate-disodium hydrogen phosphate buffer (pH 5.0) to prepare a 450 g / L substrate concentration. A 5 mL fermentation system was prepared by adding 35 U / g of purified enzyme solution and placing it in a shaker at 150 rpm for 48 h at different temperatures (35℃, 40℃, 45℃, 50℃, 55℃, 60℃). Each gradient was performed in triplicate. After the reaction, 1 mL of sample was transferred to a 1.5 mL centrifuge tube and inactivated in a 100℃ metal bath for 10 min. The sample was diluted through a 0.22 μm membrane and analyzed by liquid chromatography.

[0188] 3.2.3 pH Optimization for Enzymatic Preparation

[0189] Pure glucose was dissolved in citrate-disodium hydrogen phosphate buffer at different pH values ​​(pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5) to prepare a reaction substrate with a concentration of 450 g / L. 35 U / g of purified enzyme solution was added to prepare a 5 mL fermentation system, which was then placed in a shaker at 50 °C and 150 rpm for 48 h. Each gradient was performed in triplicate. After the reaction was completed, 1 mL of sample was transferred to a 1.5 mL centrifuge tube and inactivated in a 100 °C metal bath for 10 min. The sample was diluted through a 0.22 μm membrane and then detected by liquid chromatography.

[0190] 3.2.4 Optimization of the dosage of enzymes prepared by enzymatic method

[0191] Pure glucose was dissolved in a citrate-disodium hydrogen phosphate buffer solution at pH 5.0 to prepare a reaction substrate with a concentration of 650 g / L. Different enzyme concentrations (35 U / g glucose, 70 U / g glucose, 105 U / g glucose, and 140 U / g glucose) were added to prepare a 5 mL fermentation system, which was then placed in a shaker at 50 °C and 150 rpm for 48 h. Each gradient was performed in triplicate. After the reaction was completed, 1 mL of sample was transferred to a 1.5 mL centrifuge tube and inactivated in a metal bath at 100 °C for 10 min. The sample was diluted through a 0.22 μm membrane and then analyzed by liquid chromatography.

[0192] 3.2.5 Orthogonal optimization of enzymatic preparation of gentiobiose

[0193] Based on the exploration of single-factor experiments, the amount of enzyme added that yielded the highest output in the fermentation system was determined. Orthogonal optimization of the fermentation system for the enzymatic synthesis of gentiobiose was carried out by selecting temperature, pH, and substrate concentration. Each gradient was performed in triplicate. After the reaction, 1 mL of sample was taken into a 1.5 mL centrifuge tube and inactivated in a 100℃ metal bath for 10 min. The sample was diluted through a 0.22 μm membrane and then detected by liquid chromatography. Orthogonal experiments and data processing were carried out using SPSS.

[0194] 3.2.6 Validation of orthogonal results for the enzymatic preparation of gentiobiose

[0195] Based on the data processing results of the orthogonal experiment, the yield of the optimal combination scheme was verified. Three parallel groups were made. After 48 hours, the samples were placed in a 100℃ metal bath for 10 minutes to inactivate them. The samples were diluted through a 0.22μm membrane and then detected by liquid chromatography. The conversion rate of gentiobiose was calculated.

[0196]

[0197] 3.2.7 HPLC Detection Conditions and Methods for Gentianobiose

[0198] Detection instruments and equipment: The RID-2A differential detector of the Shimadzu LC-2030 high performance liquid chromatograph was used for detection. The column specifications were Hypersil APS-2 (250×4.6mm, 5μm).

[0199] Detection method: 10 μL injection, column temperature: 30℃; mobile phase ratio: 18% water / 82% acetonitrile; flow rate: 1.0 mL / min.

[0200] 3.2.8 Preparation of the gentiobiose standard curve

[0201] Prepare gentiobiose standard solutions at concentrations of 6, 12, 18, 24, and 30 g / L. Plot a standard curve with gentiobiose concentration on the x-axis and peak area detected by HPLC on the y-axis.

[0202] 3.3 Results and Analysis

[0203] 3.3.1 The results of the preparation of the gentiobiose standard curve are as follows: Figure 10 As shown, R 2 The value of 0.9999 indicates that the linear relationship is highly reliable, and the amount of gentiobiose produced can be calculated.

[0204] 3.3.2 Effect of substrate concentration on gentiobiose synthesis

[0205] like Figure 11 As shown, the yield of gentiobiose increases with increasing substrate concentration. The yield of gentiobiose varies significantly under different glucose substrate concentrations. The yield of gentiobiose is the highest at a glucose substrate concentration of 750 g / L, which is 47.94 g / L. Therefore, the optimal glucose substrate concentration is selected as 750 g / L.

[0206] 3.3.3 Effect of temperature on gentiobiose synthesis

[0207] Depend on Figure 12As shown, the yield of gentiobiose reached its highest value at 55℃, with a yield of 61.63 g / L, showing a significant difference compared to other temperatures. With increasing temperature, the yield initially increased and then decreased. This experimental result is consistent with the temperature-dependent enzymatic properties of β-glucosidase measured above. Since the enzymatic reaction of gentiobiose requires 48 hours, enzyme stability is crucial. The experiments investigating the optimal temperature and temperature stability of the enzyme revealed that the enzyme exhibited the best stability at 50℃. However, the optimal temperature is 60℃. Therefore, considering both optimal temperature and temperature stability, the highest yield of gentiobiose at 55℃ is consistent with the above experimental results. Thus, the optimal reaction temperature is determined to be 55℃.

[0208] 3.3.4 Effect of pH on gentiobiose synthesis

[0209] Depend on Figure 13 It can be seen that, with the change of pH, the yield of gentiobiose reached its highest level of 106.3 g / L at pH 4.5, which was significantly different from other groups. This is consistent with the above results of the optimal pH and the effect of pH on enzyme stability. Therefore, the optimal pH for preparing gentiobiose was determined to be 4.5.

[0210] 3.3.5 Effect of enzyme addition amount on gentiobiose synthesis

[0211] Depend on Figure 14 It can be seen that as the amount of enzyme solution added increases, the yield of gentiobiose also increases and eventually tends to plateau. There is no significant difference in the yield of gentiobiose when the enzyme solution addition amount is between 105 and 140 U / g glucose. Although the yield of gentiobiose at an enzyme solution addition amount of 140 U / g glucose is slightly higher than that at an enzyme solution addition amount of 105 U / g glucose, considering factors such as industrial cost, the optimal enzyme addition amount is determined to be 105 U / g glucose.

[0212] 3.3.6 Orthogonal experiments were conducted to explore the optimal formulation for the preparation of gentiobiose.

[0213] Based on the single-factor experimental results of the above enzymatic conversion for gentiobiose preparation, the optimal enzyme dosage for the highest yield in the fermentation system was determined to be 105 U / g glucose. An orthogonal experiment was conducted using glucose substrate concentration, pH, and temperature as three factors. Based on the experimental data, the optimal formulation for gentiobiose preparation was determined to be: temperature 60℃, substrate concentration 850 g / L, pH 5, and enzyme dosage 105 U / g. Experimental verification showed that under these conditions, the yield of gentiobiose was 163.4 g / L, with a conversion rate of 19.22%, only 0.14% different from the highest conversion rate currently achieved in my country, but the enzyme dosage was only one-quarter of the highest yield.

[0214] References

[0215] Ahmed A, Batool K, Bibi A. Microbial β-glucosidase: sources, production and applications[J]. Journal of Applied&Environmental Microbiology, 2017.

[0216] Ispirli H,Colquhoun IJ,Sahin E,et al.Preparation of gentiobiose-derived oligosaccharides by glucansucrase e81 and determination of prebioticand immune-modulatory functions[J].Carbohydrate Research,2019,486.

[0217] Chen Jun, Yang Zhifan, Liu Xiaoli, et al. Cloning and sequence analysis of the NRRL3135 bgl gene of Aspergillus niger [J]. Biotechnology Bulletin, 2010(07):208-215. Tang Chuanhe, Zhu Gaoxiang. Functional food additive - gentian oligosaccharide [J]. Jiangsu Food and Fermentation, 2000(01):29-31.

[0218] Xing Xueyan. High-efficiency expression and enzymatic properties analysis of β-glucosidase from filamentous fungi [D]. Tianjin University of Science and Technology, 2017.

[0219] Zhang Lin. Preparation, separation and purification of gentiobiose [D]. Nanjing Forestry University, 2016.

[0220] Zou Hui, Li Xiliu, Luo Mingji. Enzymatic production technology of oligogentanose [J]. China Food Additives, 2004(03):97-101.

[0221] Gudmundsson M, Hansson H, Karkehabadi S, et al. Structural and functional studies of the glycoside hydrolase family

[0222] 3 beta-glucosidase cel3a from the moderately thermophilic fungusrasamsonia emersonii[J].Acta Crystallogr D Struct Biol,2016,72(Pt 7):860-870.

[0223] Guo B,Sato N,Biely P,et al.Comparison of catalytic properties ofmultiple beta-glucosidases of trichoderma reesei[J].Appl MicrobiolBiotechnol,2016,100(11):4959-4968.

[0224] Ketudat C J,Mahong B,Baiya S,et al.Beta-glucosidases:multitasking,moonlighting or simply misunderstood?[J].Plant Science,2015,241:246-259.

[0225] Pan L,Farouk M H,Qin G,et al.The influences of soybean agglutinin andfunctional oligosaccharides on the intestinal tract of monogastric animals[J].International Journal of Molecular Sciences,2018,19(2).

[0226] Rycroft C E,Jones M R,Gibson G R,et al.Fermentation properties ofgentio-oligosaccharides[J].Letters in Applied Microbiology,2001,32(3):156-161.

[0227] Singh S P,Jadaun J S,Narnoliya L K,et al.Prebiotic oligosaccharides:special focus on fructooligosaccharides,its biosynthesis and bioactivity[J].Appl Biochem Biotechnol,2017,183(2):613-635.

[0228] Sorensen A,Lubeck M,Lubeck P S,et al.Fungal beta-glucosidases:abottleneck in industrial use of lignocellulosic materials[J].Biomolecules,2013,3(3):612-631.

[0229] Tiwari P,Misra B N,Sangwan N S.Beta-glucosidases from the fungustrichoderma:an efficient cellulase machinery in biotechnological applications[J].Biomed Research International,2013,2013:203735.

[0230] Wang Y,Li J,Xu Y.Characterization of novel beta-glucosidases withtransglycosylation properties from trichosporon asahii[J].J Agric Food Chem,2011,59(20):11219-11227.

[0231] Zhu Y,Kong FA facile and effective synthesis of alpha-(1-->6)-linkedmannose di-,tri-,tetra-,hexa-,octa-,and dodecasaccharides,and beta-(1-->6)-linked glucose di-,tri-,tetra-,hexa-,and octasaccharides using sugartrichloroacetimidates as the donors and unprotected or partially protected glycosides as the acceptors[J].Carbohydr Res,2001,332(1):1-21.

[0232] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of Aspergillus niger β-glucosidase in the preparation of gentiobiose, characterized in that: The amino acid sequence of the Aspergillus niger β-glucosidase is shown in GenBank: CBA02054.1 or SEQ ID NO.2, from line 1 to 843aa, and gentiobiose is prepared using glucose as a substrate.

2. The application according to claim 1, characterized in that: The dosage of Aspergillus niger β-glucosidase is 35 U / g to 140 U / g glucose.

3. The application according to claim 2, characterized in that: The dosage of Aspergillus niger β-glucosidase is 70 U / g to 140 U / g glucose.

4. The application according to claim 3, characterized in that: The dosage of Aspergillus niger β-glucosidase is 105 U / g glucose.

5. The application according to any one of claims 1 to 4, characterized in that: The concentration of glucose is 250 g / L to 850 g / L.

6. The application according to claim 5, characterized in that: The concentration of glucose is 450 g / L to 850 g / L.

7. The application according to claim 6, characterized in that: The concentration of glucose is 650 g / L to 850 g / L.

8. The application according to claim 7, characterized in that: The glucose concentration is 750–850 g / L.

9. The application according to claim 8, characterized in that: The glucose concentration is 850 g / L.

10. The application according to claim 1, characterized in that: The preparation conditions for the gentiobiose are as follows: fermentation for 24 h to 72 h under the following conditions: glucose substrate concentration of 250 g / L to 850 g / L, pH of 4.0 to 6.5, temperature of 35℃ to 60℃, and addition of Aspergillus niger β-glucosidase of 35 U / g to 105 U / g glucose.

11. The application according to claim 10, characterized in that: The preparation conditions for the gentiobiose are as follows: fermentation for 36 h to 54 h at a glucose substrate concentration of 450 g / L to 850 g / L, a pH of 4.0 to 6.0, a temperature of 40℃ to 60℃, and an addition of 70 U / g to 105 U / g glucose of Aspergillus niger β-glucosidase.

12. The application according to claim 11, characterized in that: The preparation conditions for the gentiobiose are as follows: fermentation for 48 h at a glucose substrate concentration of 650 g / L to 850 g / L, a pH of 4.0 to 6.0, a temperature of 40℃ to 60℃, and an addition of 70 U / g to 105 U / g glucose of Aspergillus niger β-glucosidase.

13. The application according to claim 12, characterized in that: The preparation conditions of the gentiobiose are as follows: fermentation for 48 h under the conditions of glucose substrate concentration of 850 g / L, pH of 5.0, temperature of 60℃, and addition of Aspergillus niger β-glucosidase of 105 U / g glucose, the amount of gentiobiose obtained is 163.4 g / L, and the conversion rate is 19.22%.

14. The application according to any one of claims 1 to 4, characterized in that: The method for preparing Aspergillus niger β-glucosidase includes the following steps: Cloning the β-glucosidase gene of Aspergillus niger AnBgl An expression vector was constructed and integrated into the Pichia pastoris genome to obtain an engineered Pichia pastoris strain. The protein was induced to be expressed and purified to obtain Aspergillus niger β-glucosidase.

15. The application according to claim 14, characterized in that: The Pichia pastoris is Pichia pastoris ( P. pastoris GS115; The starting vector of the expression vector is pPIC9K; the expression vector is the expression vector pPIC9K- AnBgl ; The Pichia pastoris engineered strain is a Pichia pastoris engineered strain. P. pastoris GS115 / pPIC9K- AnBgl .

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