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CN-121975881-A - Biochemical process for preparing scopolamine

CN121975881ACN 121975881 ACN121975881 ACN 121975881ACN-121975881-A

Abstract

The invention relates to a biochemical method for preparing scopolamine, belonging to the technical field of bioengineering. The invention aims to realize the efficient conversion of scopolamine to scopolamine and improve the yield and industrial applicability of target products. The invention provides a biochemical method for preparing scopolamine, which comprises the step of adding scopolamine 6 beta hydroxylase into a culture medium containing L-scopolamine, wherein the amino acid sequence of the scopolamine 6 beta hydroxylase is shown as any one of SEQ ID NO. 1-5. Realizes the biochemical method of scopolamine high-efficiency and high-selectivity synthesis.

Inventors

  • QIN JIUFU
  • ZHU HONGLIN
  • ZHAO LV
  • WANG YINING
  • YANG YUFAN
  • JI TIANTIAN

Assignees

  • 四川大学

Dates

Publication Date
20260505
Application Date
20260204

Claims (10)

  1. 1. A biochemical method for preparing scopolamine is characterized in that scopolamine 6 beta hydroxylase is added into a culture medium containing L-scopolamine, and the amino acid sequence of the scopolamine 6 beta hydroxylase is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9.
  2. 2. The method of claim 1, wherein the concentration of scopolamine 6 beta hydroxylase is 0.5 mg/mL.
  3. 3. The method of claim 1, wherein the medium comprises 2-OG 1mM, feSO4 0.4mM, sodium ascorbate 4mM, catalase 2 mg/mL, L-hyoscyamine 100 mg/L, and hyoscyamine 6. Beta. Hydroxylase 0.5 mg/mL.
  4. 4. The process of claim 1, wherein the reaction is carried out at 30 ℃ from 2 to 24 h.
  5. 5. The application of the hyoscyamine 6 beta hydroxylase in preparing scopolamine is characterized in that the amino acid sequence of the hyoscyamine 6 beta hydroxylase is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9.
  6. 6. The application of a recombinant vector containing a gene for encoding scopolamine 6 beta hydroxylase or a recombinant microbial cell containing a gene for encoding scopolamine 6 beta hydroxylase in preparation of scopolamine is characterized in that the gene for encoding scopolamine 6 beta hydroxylase is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.10.
  7. 7. The hyoscyamine 6 beta hydroxylase is characterized in that the gene for encoding the hyoscyamine 6 beta hydroxylase is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.1.
  8. 8. A gene encoding the scopolamine 6 beta hydroxylase of claim 7.
  9. 9. A recombinant vector comprising the gene according to claim 8.
  10. 10. A recombinant microbial cell comprising the gene of claim 8.

Description

Biochemical process for preparing scopolamine Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to a biochemical method for preparing scopolamine. Background Scopolamine (Scopolamine) is an important tropane alkaloid, has remarkable pharmacological effects of anticholinergic, sedative, analgesic and central inhibitory effects, and can be widely applied to the clinical fields of parkinsonism treatment, anti-motion sickness, smooth muscle spasm relief, nerve agent poisoning adjuvant treatment and the like. Compared with precursor scopolamine, scopolamine has higher pharmacological activity, stronger central penetration capacity and smaller side effect, so that the clinical demand for scopolamine is increasing. At present, scopolamine supply mainly depends on extraction and acquisition of artificially cultivated solanaceae plants. However, natural plant extraction is limited by the problems of long growth period, low content, large environmental impact, complex extraction process, high ecological cost and the like, and is difficult to meet the increasing market demands. Although chemical total synthesis of scopolamine has been realized by existing researches, the synthesis path is long and the selectivity control is difficult due to the complex molecular structure and the large number of chiral centers, so that the economic and feasible large-scale preparation is difficult to realize. Therefore, a biochemical method capable of screening and applying high-performance H6H enzyme elements and realizing efficient and high-selectivity synthesis of scopolamine in an in-vitro pure enzyme system is needed to overcome the problems of low conversion rate, insufficient enzyme efficiency, limited industrial amplification potential and the like in the prior art. Disclosure of Invention The invention aims to provide a scopolamine biochemical preparation method based on high-activity H6H enzyme, which realizes the efficient conversion of scopolamine into scopolamine by systematically screening multi-source H6H and constructing an efficient in-vitro pure enzyme reaction system, and improves the yield and industrial applicability of target products. The invention provides a biochemical method for preparing scopolamine, which comprises the step of adding scopolamine 6 beta hydroxylase into a culture medium containing L-scopolamine, wherein the amino acid sequence of the scopolamine 6 beta hydroxylase is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9. Further defined, the concentration of hyoscyamine 6 beta hydroxylase is 0.5 mg/mL. Further defined, the medium is composed of 2-OG 1mM, feSO4 0.4mM, sodium ascorbate 4mM, catalase 2 mg/mL, L-hyoscyamine 100 mg/L and hyoscyamine 6. Beta. Hydroxylase 0.5 mg/mL. Further defined, the reaction is 2-24 h at 30 ℃. The invention provides an application of scopolamine 6 beta hydroxylase in preparation of scopolamine, wherein the amino acid sequence of the scopolamine 6 beta hydroxylase is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9. The invention provides application of a recombinant vector containing a gene for encoding scopolamine 6 beta hydroxylase or a recombinant microbial cell containing a gene for encoding scopolamine 6 beta hydroxylase in preparation of scopolamine, wherein the gene for encoding scopolamine 6 beta hydroxylase is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.10. The invention provides a hyoscyamine 6 beta hydroxylase, and the gene for encoding the hyoscyamine 6 beta hydroxylase is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.1. The invention provides a gene for encoding the scopolamine 6 beta hydroxylase. The present invention provides a recombinant vector comprising the above gene. The present invention provides a recombinant microbial cell comprising the above-described gene. The scopolamine biochemical preparation method based on the high-activity H6H enzyme has the beneficial effects that the scopolamine biochemical preparation method based on the high-activity H6H enzyme is used for realizing the high-efficiency conversion of scopolamine to scopolamine by systematically screening multi-source H6H and constructing a high-efficiency in-vitro pure enzyme reaction system, and the yield and industrial applicability of a target product are improved. Drawings FIG. 1 is an SDS-PAGE protein electrophoresis of five pure enzymes. Protein molecular weight standards (markers), abH6H, hnH6H, maH6H, dsH6H, aaH6H, are sequentially from left to right. All five recombinant proteins exhibited distinct specific bands at about 38 kDa; FIG. 2 shows the relative conversion of scopolamine produced from different sources of H6H over 2H. The relative catalytic fold of other enzymes was calculated using AaH H conversion to scopolamine under the same reaction conditions as the reference value (set to 1). Error bars represen