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Transaminase mutant derived from aspergillus terreus and application of transaminase mutant

A transaminase and mutant technology, applied in the field of biopharmaceuticals, can solve the problems of easy racemization, affecting the optical purity of products, etc., and achieve the effect of high yield

Active Publication Date: 2020-08-18
卡柔恩赛生物技术湖北有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this synthetic route, the chiral raw material is directly used as the starting point, and it is easy to racemize in the subsequent formation and non-cyclization reactions, which affects the optical purity of the product.

Method used

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  • Transaminase mutant derived from aspergillus terreus and application of transaminase mutant
  • Transaminase mutant derived from aspergillus terreus and application of transaminase mutant
  • Transaminase mutant derived from aspergillus terreus and application of transaminase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Molecular docking evaluation of candidate transaminases and reaction substrate N-BOC-3-pyridone

[0041]Homologous protein modeling and evaluation are performed on the proteins that have not been analyzed for the tertiary structure in the present invention, and the three-dimensional transaminase model required by the present invention is obtained. The reaction substrate N-BOC-3-pyridone was molecularly docked with candidate transaminases. The RMSD threshold was set at 0.5 angstroms to ensure that the docking conformation was as diverse as possible, and the docking method with the highest scoring function was selected. 12 candidate transaminases of the present invention were comprehensively evaluated, and 6 candidate transaminases including Aspergillus terreus NIH2624 transaminase were selected for subsequent transaminase activity tests.

Embodiment 2

[0042] Example 2: Activity testing of transaminases with higher evaluation scores based on molecular docking results

[0043] The six candidate transaminase mRNA sequences were codon-optimized and related regulatory sequences were added (with BglII and XhoI endonuclease gene fragments on both sides of the fragments) for gene synthesis.

[0044] Recombinant plasmid transfection:

[0045] 1. Take Escherichia coli competent cells BL21 from -80°C and thaw in an ice box at room temperature.

[0046] 2. Add 1 ul plasmid to the competent cells and place in a 4°C refrigerator or ice box for 30 minutes.

[0047] 3. Heat shock in a water bath at 42°C for 120s, and immediately place it on an ice box for 2 minutes.

[0048] 4. Add 900 ul of recovery medium to each tube, shake at 150 rpm, and incubate at 37°C for 45 minutes.

[0049] 5. Centrifuge at low speed to take 50ul of the transformation solution from the bottom of the tube and spread it on a petri dish containing kan at the work...

Embodiment 3

[0061] Example 3: Determining the amino acid mutation site by virtual mutation of a single amino acid around the active center of Aspergillus terreus NIH2624 transaminase

[0062] The present invention performs virtual mutation on Aspergillus terreus NIH2624 transaminase, uses molecular simulation software to rationally design Aspergillus terreus NIH2624 transaminase, and selects mutation sites, which can effectively save the time for mutation site screening and improve mutation efficiency.

[0063] The present invention uses the docking results in Example 1 to perform data analysis to determine the spatial distance and action relationship of each amino acid in the active region.

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Abstract

The present invention relates to a mutant polypeptide derived from aspergillus terreus NIH2624 transaminase (XP-001209325.1), a polynucleotide encoding the polypeptide, and an application of the polypeptide in the preparation of (R)-1-BOC-3-aminopiperidine. The transaminase is directionally modified through computer simulation, and modified amino acid sites are selected from one or more of Y60, V62, F115, W184, G216, F217, N218, L235, G237, V238, T239, C273, T274, T275 and A276. The mutant obtained by modification is applied to the production of (R)-1-BOC-3-aminopiperidine, has the advantagesof good stereoselectivity and catalytic activity, mild catalytic reaction, improvement of the activity by 61.9 times compared with wild transaminase under the same production conditions, obvious improvement of the chiral purity and yield of the product, and good application prospect.

Description

Technical field: [0001] The invention belongs to the field of biopharmaceuticals, in particular to a transaminase mutant derived from Aspergillus terreus NIH2624 and its application in the preparation of (R)-1-BOC-3-aminopiperidine. Background technique: [0002] Diabetes is mainly a metabolic disease characterized by hyperglycemia caused by insufficient insulin secretion or insulin action disorder. Insufficient insulin secretion is defined as type Ⅰ, and insulin action disorder is defined as type Ⅱ diabetes, and type Ⅱ diabetes patients account for more than 90% of diabetic patients. [0003] The current drugs for the treatment of type 2 diabetes are mainly divided into sulfonylureas, biguanides, α-glucosidase inhibitors, insulin sensitizers, thiazolidinediones, dipeptidyl peptidase IV (DPP-IV) inhibitors Among them, the most efficient and safe one is currently recognized as (DPP-IV) inhibitors, which can selectively bind to dipeptidyl peptidase IV reversibly to inhibit th...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P17/12C12R1/19C12R1/66
CPCC12N9/1096C12N15/70C12Y206/01C12P17/12
Inventor 冯俊唐圆圆熊艳艾忠良佘飞
Owner 卡柔恩赛生物技术湖北有限公司
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