CN-121975749-A - Scopolamine 6 beta hydroxylase and application thereof

Abstract

The invention discloses scopolamine 6 beta hydroxylase and application thereof, and belongs to the technical field of bioengineering. The invention aims to provide a hyoscyamine 6 beta hydroxylase with high activity. The invention provides a scopolamine 6 beta hydroxylase, the amino acid sequence of which is shown as SEQ ID NO. 4. The novel H6H enzyme with higher difunctional catalytic efficiency is discovered, and the key technical requirement of the scopolamine biological manufacturing capability is improved.

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. The hyoscyamine 6 beta hydroxylase is characterized in that the amino acid sequence of the hyoscyamine 6 beta hydroxylase is shown as SEQ ID No. 4.
2. 2. A gene encoding the scopolamine 6 beta hydroxylase of claim 1.
3. 3. The gene according to claim 2, wherein the gene sequence is shown in SEQ ID NO. 3.
4. 4. A recombinant vector comprising the gene of claim 2.
5. 5. A recombinant microbial cell comprising the gene of claim 2.
6. 6. The recombinant microbial cell of claim 5, wherein the microbial cell is a prokaryotic microbial cell or a eukaryotic microbial cell.
7. 7. Use of a hyoscyamine 6β hydroxylase of claim 1, a gene of claim 2 or 3, a recombinant vector of claim 4, or a recombinant microbial cell of claim 5 or 6 for the production of scopolamine.
8. 8. A method for producing scopolamine, characterized in that the scopolamine 6 beta hydroxylase of claim 1 is added to a medium containing L-scopolamine.
9. 9. The method of claim 8, 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 crude enzyme 958 ul.
10. 10. The method of claim 8, wherein the reaction conditions are a constant temperature of 30 ℃ of at least 24 h.

Description

Scopolamine 6 beta hydroxylase and application thereof Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to scopolamine 6 beta hydroxylase and application thereof. 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. In the plant biosynthetic pathway, hyoscyamine 6 beta-hydroxylase (hyoscyamine beta-hydroxylase, H6H) is the key rate-limiting enzyme that determines scopolamine production. The enzyme belongs to alpha-ketoglutarate/ferrous ion dependent dioxygenase family, and has two catalytic functions of catalyzing anisodamine to generate anisodamine through 6-position hydroxylation and catalyzing anisodamine to generate scopolamine through 6-position epoxidation and 7-position epoxidation. However, the existing source H6H enzyme has the problem of extremely low conversion rate of the second step of epoxidation reaction, so that the accumulation level of scopolamine is limited, and the large-scale application of scopolamine in the biological manufacturing of scopolamine is restricted. Therefore, the development of a novel H6H enzyme with higher bifunctional catalytic efficiency is a key technical requirement for improving the biological production capacity of scopolamine. Disclosure of Invention The invention aims to provide a hyoscyamine 6 beta hydroxylase with high activity. The invention provides a scopolamine 6 beta hydroxylase, the amino acid sequence of which is shown as SEQ ID NO. 4. The invention provides a gene for encoding the scopolamine 6 beta hydroxylase. Further defined, the gene sequence is shown in SEQ ID NO. 3. The invention provides a recombinant vector containing the gene. The present invention provides a recombinant microbial cell containing the above gene. Further defined, the microbial cells are prokaryotic or eukaryotic microbial cells. The invention provides an application of the scopolamine 6 beta hydroxylase, the gene, the recombinant vector or the recombinant microbial cell in scopolamine production. The invention provides a method for producing scopolamine, which comprises the step of adding scopolamine 6 beta hydroxylase into a culture medium containing L-scopolamine. 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 crude enzyme solution 958 ul. 10. The method of claim 8, wherein the reaction conditions are a constant temperature of 30 ℃ of at least 24 h. The equine vesicle (Przemalskia tangutica) is used as a specific alpine medicinal plant of Qinghai-Tibet plateau, has a higher proportion of scopolamine, and indicates that an H6H variant with high-efficiency catalytic activity can exist in the human body. Based on the above, the invention digs and obtains a novel H6H gene (PtH H) from the equine urinary bladder, and takes anisodamine source H6H (AtH H) with higher activity in the existing research as a reference object to evaluate the performance. The result shows that the catalytic efficiency of PtH H in scopolamine generation reaction is obviously superior to that of the existing enzyme source, and the catalytic efficiency of PtH H has the potential of being used as a high-efficiency biocatalysis element for industrial production. Drawings FIG. 1 shows SDS-PAGE of crude enzyme solutions expressed in E.coli by PtH H and AtH H. The protein molecular weight standard (Marker), the empty control (pET 28 a), atH 6. 6H, ptH6H and the Marker are arranged from left to right. The framed portion of the figure is the target protein band, atH H presents two bands, and modifications may occur. Both recombinant proteins showed distinct specific bands at about 38 kDa, indicating that PtH H was able to be stably expressed in the host, providing a reliable protein basis for subsequent in vitro catalytic activity analysis; FIG. 2 shows the relative catalytic efficiencies of PtH H and AtH H. The relative catalytic fold of PtH H was calculated using the amount of scopolamine converted by AtH H, which has been reported to have superior catalytic performance, u