Preparation method and application of biological enzyme liquid

Abstract

The invention discloses a preparation method and application of biological enzyme liquid, and belongs to the technical field of biological enzyme. The formula of the biological enzyme liquid disclosed by the invention consists of the following raw materials in percentage by mass: 0.2% of a biological enzyme SEQ ID NO.1-Leu-Cys-His-Arg-Asp-Phe-Tyr, 0.2% of a biological enzyme SEQ ID NO.1-Leu-Cys-His-Arg-Asp-Phe-Tyr and the balance of water. And 0.15% of a biological enzyme SEQ ID NO.2-Thr-Glu-Lys-Val-Asp-Trp-Ser, and the pH value of the biological enzyme SEQ ID NO.2-Thr. 2% of a preservative; 14% of an anionic surfactant; 8% of a nonionic surfactant; 3% of a builder 1; 6% of a builder 2; 2% of a softener; and 0.4% of the perfume is natural flower fragrance essence. Results show that the enzyme liquid prepared by the invention has the best stability at neutral pH and low temperature, and the activity is reduced in a high-temperature or strong acid environment; the optimal conditions for catalyzing conversion of linalool are as follows: 37 DEG C, pH 7.5 and Mn < 2 + >.

Description

Technical Field

[0001] The invention belongs to the technical field of biological enzymes, and in particular relates to a preparation method and application of a biological enzyme solution. Background Art

[0002] In the context of the rapid development of biotechnology and modern industry, bioenzymes, as an efficient, specific and environmentally friendly biocatalyst, have been widely used in many fields such as food processing, pharmaceutical manufacturing, environmental protection, textile printing and dyeing, bioenergy, etc. For example, in the food industry, amylase is used to hydrolyze starch to produce syrup, and protease is used for meat tenderization and dairy processing; in the pharmaceutical field, lysozyme can be used as a natural antibacterial agent, and urokinase is used for thrombosis treatment. With the continuous improvement of product quality, production efficiency and green environmental protection requirements in various industries, the demand for bioenzymes is growing, and higher standards are put forward for the activity, stability, specificity and other properties of bioenzymes.

[0003] At present, the preparation methods of biological enzyme liquid mainly include microbial fermentation, animal and plant tissue extraction and chemical synthesis. Microbial fermentation is the most commonly used method. It screens excellent microbial strains and produces a large number of biological enzymes under suitable fermentation conditions. However, there are problems such as long fermentation cycle, enzyme activity is easily affected by fluctuations in fermentation conditions, and the separation and purification process is complicated and costly. The animal and plant tissue extraction method is limited by the source and seasonality of raw materials, and it is easy to introduce impurities during the extraction process, resulting in low enzyme purity and difficulty in large-scale industrial production. Although the chemical synthesis method can accurately control the structure of the enzyme, the synthesis process often requires the use of toxic and harmful chemical reagents, which not only pollutes the environment, but also may affect the biological activity and safety of the enzyme. In addition, the existing biological enzyme liquid has poor stability during storage and use, and is easily reduced or even inactivated due to factors such as temperature and pH value, which greatly limits the scope of application and effect of the biological enzyme. Therefore, it has become an important issue to be solved in the current biotechnology field to develop a method for preparing a biological enzyme liquid that is efficient, low-cost, green and environmentally friendly and can significantly improve the activity and stability of the biological enzyme, and expand its application in more fields. Summary of the invention

[0004] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a method for preparing a biological enzyme solution. Another technical problem to be solved by the present invention is to provide an application of the biological enzyme solution for improving the yield of osmanthus extract and the utilization rate of raw materials.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A biological enzyme solution, the formula of which consists of raw materials in the following mass percentages: 0.2% of biological enzyme SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr; 0.15% of biological enzyme SEQ ID NO.2 - Thr - Glu - Lys - Val - Asp - Trp - Ser; 2% of preservative; 14% of anionic surfactant; 8% of non - ionic surfactant; 3% of washing aid 1; 6% of washing aid 2; 2% of softener; 0.4% of natural floral fragrance essence.

[0007] The anionic surfactant is sodium dodecylbenzenesulfonate.

[0008] The non - ionic surfactant is fatty alcohol polyoxyethylene ether.

[0009] The washing aid 1 is sodium citrate.

[0010] The washing aid 2 is zeolite.

[0011] The softener is polyquaternium - 7.

[0012] The preservative is 3 - (2 - aminoethyl) - 1,2 - benzisothiazolin - 3 - one.

[0013] The preparation method of the biological enzyme solution includes:

[0014] 1) Dissolve 0.2% of SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr in PBS buffer solution with pH 7; dissolve 0.15% of SEQ ID NO.2 - Thr - Glu - Lys - Val - Asp - Trp - Ser in PBS buffer solution with pH 7, and add 1% glycerol to improve stability;

[0015] 2) Add 60% deionized water into the reaction kettle and heat it to 50 °C; add successively: 14% sodium dodecylbenzenesulfonate solution and stir for 20 min; 8% fatty alcohol polyoxyethylene ether solution and stir for 15 min; finally cool down to 30 °C;

[0016] 3) Add 3% sodium citrate and 6% zeolite, and stir for 10 min; add 3 - (2 - aminoethyl) - 1,2 - benzisothiazolin - 3 - one (2%) and stir in the dark;

[0017] 4) Slowly add the pre - dissolved SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr and SEQ ID NO.2 - Thr - Glu - Lys - Val - Asp - Trp - Ser, and stir at low speed to maintain the overall pH of 6.5 - 7.5;

[0018] 5) Add 2% of polyquaternium-7 and stir for 10 min; finally, add 0.4% of natural floral fragrance essence and stir for 5 min; make up deionized water to 100%, homogenize at 2000 rpm for 5 min with a homogenizer to ensure no stratification, and obtain the bio-enzyme solution.

[0019] Use of the bio-enzyme solution in promoting the conversion of linalool.

[0020] Advantages of the present invention:

[0021] 1) The innovative preparation method of the present invention avoids many drawbacks of traditional methods. Compared with the microbial fermentation method, it gets rid of the trouble of long fermentation cycle, will not affect the enzyme activity due to fluctuations in fermentation conditions, simplifies the separation and purification process, and reduces costs; compared with the extraction method from animal and plant tissues, it is not restricted by the source and seasonality of raw materials, and avoids the introduction of miscellaneous proteins, which is more conducive to large-scale industrial production; compared with the chemical synthesis method, it does not need to use toxic and harmful chemical reagents, which is both environmentally friendly and ensures the biological activity and safety of the enzyme.

[0022] 2) During the preparation process of the present invention, by dissolving the bio-enzyme in PBS buffer with a specific pH and adding glycerol, and strictly controlling conditions such as temperature and pH in subsequent steps, the activity and stability of the bio-enzyme are effectively improved. From the test results of the enzyme activity stability, the enzyme solution has the best stability at neutral pH and low temperature. The activity retention rate reaches 98.5±1.2% after storage at 4°C for 30 days, and the activity retention rate is 95.4±1.9% after incubation in an environment with pH 7 for 24 h, which greatly expands the application scope and effect of the bio-enzyme solution.

[0023] 3) Each component in the bio-enzyme solution formula of the present invention cooperates with each other. The bio-enzymes SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr and SEQ ID NO.2 - Thr - Glu - Lys - Val - Asp - Trp - Ser play catalytic and co-catalytic roles. The anionic surfactant (such as sodium dodecylbenzenesulfonate) is responsible for decontamination and emulsification, the non-ionic surfactant (aliphatic alcohol polyoxyethylene ether) realizes solubilization and stabilization, the builders (sodium citrate and zeolite) chelate metal ions and adsorb stains respectively, the softener (polyquaternium-7) has the effects of antistatic and softening, the preservative (3-(2-aminoethyl)-1,2-benzisothiazolin-3-one) ensures the product quality, and the natural floral fragrance essence adds fragrance. Each component acts synergistically to endow the bio-enzyme solution with multiple functions.

[0024] 4) The bio-enzyme solution prepared by the present invention shows excellent performance in promoting the conversion of linalool. By optimizing the glycosylation reaction conditions, the optimal reaction conditions when using linalool as the substrate are determined to be 37°C, pH 7.5, Mn 2+, under these conditions, the conversion efficiency of linalool is the highest, and the relative yield can reach 100 ± 4.5%, providing a better choice for production in related fields and also offering the possibility for the application expansion of bio - enzyme solution in other fields. Detailed implementation manners

[0025] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well - known to those skilled in the art.

[0026] Example 1 Preparation of SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr

[0027] 1. Synthesis of peptide segments

[0028] Method: Fmoc solid - phase peptide synthesis (SPPS)

[0029] Resin: Rink Amide MBHA resin (C - terminal amidation).

[0030] Coupling sequence (C→N): Fmoc - Tyr(tBu) - OH→Fmoc - Phe - OH→Fmoc - Asp(OtBu) - OH→Fmoc - Arg(Pbf) - OH→Fmoc - His(Trt) - OH→Fmoc - Cys(Trt) - OH→Fmoc - Leu - OH.

[0031] Deprotection: 20% piperidine / DMF, 5 min×2 for each step.

[0032] Coupling: HBTU / HOBt / DIPEA (4eq AA, 4eq HBTU, 8eq DIPEA), DMF solvent, 1 h at room temperature.

[0033] Cleavage: TFA / TIS / H2O (95:2.5:2.5), 2 h→precipitation with cold ether→purification by HPLC (C18 column, acetonitrile / water gradient).

[0034] Verification by mass spectrometry (MALDI - TOF / ESI - MS): Calculated MW = 1029.2 Da (free peptide).

[0035] 2. Modify DNA (3' - thiol modification (introduce a thiol group at the 3' - end for maleimide coupling))

[0036] Reagent: 3'-Thiol modifier C3 S - S CPG (Glen Research) for solid - phase DNA synthesis.

[0037] Steps: Synthesize DNA (forward primer: 5'-CTCCCACAGACCTGTCTATAC-3', SEQ ID NO.1) and introduce -S-S- modification at the 3' end. Reduce with 50 mM TCEP (pH 7.0) for 30 min → desalt (NAP-5 column) to obtain -SH-DNA.

[0038] 3. Peptide-DNA Conjugation

[0039] Condition: React the peptide segment (containing N-terminal Cys) with 3'-SH-DNA.

[0040] Buffer: 0.1 M PBS (pH about 7.0), 1 mM EDTA.

[0041] Steps: Dissolve the peptide segment (1.2 eq) in PBS. Add 10 mM Maleimide-PEG2-NHS (Thermo Fisher) to activate the peptide segment (30 min, 4 °C). Add 3'-SH-DNA (1 eq) and react at room temperature for 2 h. Termination: Add 10 mM β-mercaptoethanol.

[0042] 4. Purification and Verification

[0043] Purification method: HPLC (ion exchange or reverse phase)

[0044] Column: Dionex DNAPac PA200 (ion exchange) or C18 (reverse phase).

[0045] Gradient: Acetonitrile / 0.1 M TEAA (pH 7.0).

[0046] Gel electrophoresis (PAGE): 15% denaturing PAGE, stained with SYBR Gold to observe the change in migration rate.

[0047] Verification methods:

[0048] Mass spectrometry (MALDI-TOF)

[0049] Calculate MW (DNA + peptide):

[0050] DNA (5'-CTCCCACAGACCTGTCTATAC-3'): ~6600 Da (sodium salt form).

[0051] Peptide: 1029.2 Da.

[0052] Conjugate: ~7630 Da.

[0053] Ultraviolet absorption (260 / 280 nm): Confirm the DNA / peptide ratio.

[0054] Table 1 Purification and Verification Results

[0055] Step Product Purity (HPLC / PAGE) Yield Peptide synthesis Leu-Cys-His-Arg-Asp-Phe-Tyr >95％ 60-80％ DNA modification <![CDATA[3'-SH-DNA or 3'-NH2-DNA]]> >90％ >90％ Coupling reaction DNA-peptide conjugate 70-90％ 30-60％

[0056] 5. Anneal with the reverse primer (5'-cccattgcacaactttttctca-3', SEQ ID NO.2)

[0057] Mix the DNA-peptide conjugate (SEQ ID NO.1-Leu-Cys-His-Arg-Asp-Phe-Tyr) with the reverse primer (SEQ ID NO.2) (molar ratio 1:1). Heat to 95 °C and slowly cool to 25 °C (1 °C / min) over 5 min.

[0058] Verify annealing: Non-denaturing PAGE (10%), SYBR Gold staining. Mobility difference between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).

[0059] Example 2 Preparation of the bio-enzyme solution

[0060] Table 2 Formulation composition of the bio-enzyme solution (mass percentage)

[0061]

[0062] 1. Pretreatment of raw materials

[0063] 1) Dissolution of bio-enzyme

[0064] Dissolve SEQ ID NO.1-Leu-Cys-His-Arg-Asp-Phe-Tyr (0.2%) in PBS buffer at pH 7.0 (avoid direct addition to high-salt / strong acid environment).

[0065] SEQ ID NO.2-Thr-Glu-Lys-Val-Asp-Trp-Ser (0.15%): Dissolve in the same way and 1% glycerol can be added to improve stability.

[0066] 2) Premixing of surfactants

[0067] Sodium dodecylbenzenesulfonate (14%): Slowly add to warm water (40 - 50 °C) and stir until completely dissolved.

[0068] Alcohol polyoxyethylene ether (8%): Dissolve directly in deionized water and stir at room temperature.

[0069] 2. Main body preparation (stepwise mixing)

[0070] 1) Preparation of surfactant base solution

[0071] Add 60% deionized water to the reaction kettle and heat it to 50°C. Add successively: sodium dodecylbenzenesulfonate (14%), and stir for 20 min. Add fatty alcohol polyoxyethylene ether (8%) and stir for 15 min. Finally, cool down to 30°C.

[0072] 2) Add builders and preservatives

[0073] Add sodium citrate (3%) and zeolite (6%), and stir for 10 min. Add 3-(2-aminoethyl)-1,2-benzisothiazolin-3-one (2%), and stir in the dark.

[0074] 3) Add bioenzyme

[0075] Slowly add the pre-dissolved SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr and SEQ ID NO.2 - Thr - Glu - Lys - Val - Asp - Trp - Ser, and stir at low speed (to avoid enzyme inactivation). Maintain the overall pH at 6.5 - 7.5 (fine-tune with citric acid / NaOH).

[0076] 4) Add softener and fragrance

[0077] Add polyquaternium - 7 (2%), and stir for 10 min. Finally, add natural floral fragrance (0.4%), and stir for 5 min.

[0078] 5) Make up the volume and homogenize

[0079] Make up deionized water to 100%. Homogenize at 2000 rpm for 5 min to ensure no stratification.

[0080] 3. Post-treatment and quality control

[0081] 1) Filtration and filling

[0082] Filter through a 5 - μm filter membrane to remove undissolved particles. Fill into light - proof containers and avoid high - temperature storage.

[0083] 2) Quality inspection indicators

[0084] Table 3 Quality inspection indicators

[0085]

[0086]

[0087] 4. Enzyme activity stability test

[0088] Test conditions: Temperature stability: Store at 4°C, 25°C, and 40°C for 30 days.

[0089] pH stability: Incubate in buffers of pH 5.0, 7.0, and 9.0 for 24 h.

[0090] Detection method: Use a fluorescent substrate (MCA-peptide) to measure protease activity (Ex / Em = 380 / 460 nm). Evaluate the activity retention rate of the enzyme solution at different temperatures and pH values.

[0091] The results are shown in Table 4. The enzyme solution has the best stability at neutral pH and low temperature, and its activity decreases in high temperature or strong acid environment.

[0092] Table 4 Activity retention rate of the enzyme solution at different temperatures and pH values

[0093] Condition Activity retention rate (%) 4°C, 30 days 98.5±1.2 25°C, 30 days 92.3±2.1 40°C, 30 days 75.6±3.5 pH 5, 24 h 60.2±4.8 pH 7, 24 h 95.4±1.9 pH 9, 24 h 82.7±3.2

[0094] 5. Optimization of glycosylation reaction (using linalool as substrate)

[0095] Substrates: Linalool (1 mM), UDPG (2 mM).

[0096] Experimental conditions: Temperature (25 °C, 37 °C, 50 °C), pH (6.0, 7.5, 9.0), metal ions (Mn 2+ , Mg 2+ , Ca 2+ , 10 mM). Detection: Quantify the product (linalool-UDP) by HPLC (C18 column, acetonitrile / water gradient).

[0097] The results are shown in Table 5. The optimal conditions are 37 °C, pH 7.5, and Mn 2+ .

[0098] Table 5 Conversion efficiency of linalool under different reaction conditions

[0099]

[0100]

[0101] The above description is illustrative rather than restrictive to the present invention. Those of ordinary skill in the art understand that many modifications, variations, or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. A biological enzyme solution, characterized in that, The formulation consists of raw materials in the following mass percentages: 0.2% of the bio-enzyme SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr; 0.15% of the bio-enzyme SEQ ID NO.2 - Thr - Glu - Lys - Val - Asp - Trp - Ser; 2% of a preservative; 14% of an anionic surfactant; 8% of a non-ionic surfactant; 3% of builder 1; 6% of builder 2; 2% of a softener; 0.4% of a fragrance, natural floral fragrance essence.

2. The biological enzyme solution according to claim 1, characterized in that, The anionic surfactant is sodium dodecylbenzenesulfonate.

3. The bio-enzyme liquid according to claim 1, characterized in that, The non-ionic surfactant is fatty alcohol polyoxyethylene ether.

4. The bio-enzyme solution according to claim 1, wherein, The builder 1 is sodium citrate.

5. The bio-enzyme liquid according to claim 1, wherein The builder 2 is zeolite.

6. The bio-enzyme liquid according to claim 1, characterized in that, The softener is polyquaternium-7.

7. The bio-enzyme solution according to claim 1, characterized in that, The preservative is 3-(2-aminoethyl)-1,2-benzisothiazolin-3-one.

8. The preparation method of the bio-enzyme solution according to any one of claims 1-7, characterized in that, Including: 1) Dissolve 0.2% of SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr in PBS buffer at pH 7; dissolve 0.15% of SEQ ID NO.2 - Thr - Glu - Lys - Val - Asp - Trp - Ser in PBS buffer at pH 7, and add 1% glycerol to improve stability. 2) Add 60% deionized water to the reaction kettle and heat to 50°C; sequentially add: 14% sodium dodecylbenzenesulfonate solution and stir for 20 min; 8% fatty alcohol polyoxyethylene ether solution and stir for 15 min; finally cool down to 30°C. 3) Add 3% sodium citrate and 6% zeolite and stir for 10 min; add 3-(2-aminoethyl)-1,2-benzisothiazolin-3-one (2%) and stir in the dark. 4) Slowly add the pre-dissolved SEQ ID NO.1 - Leu - Cys - His - Arg - Asp - Phe - Tyr and SEQ ID NO.2 - Thr - Glu - Lys - Val - Asp - Trp - Ser, and stir at low speed to maintain the overall pH at 6.5 - 7.

5. 5) Add 2% polyquaternium-7 and stir for 10 min; finally add 0.4% natural floral fragrance essence and stir for 5 min; make up deionized water to 100%, homogenize at 2000 rpm for 5 min with a homogenizer to ensure no stratification, and obtain the bio-enzyme solution.

9. Use of the bio-enzyme solution according to claim 1 in promoting the conversion of linalool.

Images