Glycosyl transferase mutant with improved thermal and organic solvent stability

A technology of glycosyltransferase and organic solvent, which is applied in the field of genetic engineering, can solve the problems of limited thermal stability and chemical stability of natural proteins, restricting the practical application of industrialization, etc., to improve the tolerance of organic solvents, enhance the application value, heat The effect of stability improvement

Active Publication Date: 2022-08-09
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a room temperature enzyme, UGT109A3 glycosyltransferase has limited thermal and chemical stability of its natural protein, which restricts its practical industrial application.

Method used

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  • Glycosyl transferase mutant with improved thermal and organic solvent stability
  • Glycosyl transferase mutant with improved thermal and organic solvent stability
  • Glycosyl transferase mutant with improved thermal and organic solvent stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Purify the wild-type glycosyltransferase protein recombinantly expressed in E.coli BL21 (DE3) with a 6xHis tag at the N-terminus, and use the Hampton commercial protein crystallization kit to perform extensive protein crystallization conditions screening to obtain initial protein crystals and optimize crystal growth. The pH, precipitant, salt concentration and other conditions were used to obtain UGT109A3 enzyme and donor substrate UDP-glucose complex crystals suitable for diffraction. The data were collected by X-ray diffraction at the Shanghai Light Source (SSRF) BL 19U1 line station. The diffraction data processing online station HKL3000 software is integrated to obtain the scale suffix file, and the resolution reaches The crystal structure of glycosyltransferase UGT109A3-UDP complex was solved by molecular replacement; the structure of UGT109A3-UDP is a typical GT-B fold, consisting of N-terminal domain (NTD) and C-terminal domain (C -terminal domain, CTD) consists...

Embodiment 2

[0070] Heterologous expression and purification of glycosyltransferase UGT109A3 mutant

[0071] Specific steps are as follows:

[0072] The transformants with correct sequencing were picked and transferred to LB liquid medium containing kanamycin (50 mg·L-1), cultured at 37°C and 220 rpm / min, and the overnight culture medium was inoculated with kanamycin-containing medium at a ratio of 1%. of autoinduction medium, cultivated to OD at 37°C at 220rpm 600 About 0.6-0.8, add isopropyl 1-β-D-thiogalactopyranoside (IPTG to the medium, the final concentration is 0.5mM L -1 )), and the induction culture was continued for 20 hours at 18°C.

[0073] The cultured bacterial solution was collected by centrifugation at 5000rpm for 15min at 4°C, and 10mL bindingbuffer (pH 8.0, 50mM Tris-HCl buffer, 500mM NaCl, 20mM imidazole) was added to the ratio of 1g wet bacteria to the bindingbuffer, resuspended at high pressure Broken in a homogenizer. The crushed solution was centrifuged at 4°C an...

Embodiment 3

[0075] Determination of Tm Value of Glycosyltransferase UGT109A3 Mutant

[0076] Specific steps are as follows:

[0077] Combine UGT109A3 glycosyltransferase mutant protein with SYPRO TM Orange fluorescent dye was combined, and the melting curve was measured in a real-time quantitative PCR instrument (Real-time qPCR). The reaction system is shown in Table 2.

[0078] Combining glycosyltransferase UGT109A3 mutant protein with SYPRO TM After the Orange protein gel stain was thoroughly mixed in the eight-connected tube, the eight-connected tube was placed in a fluorescence quantitative PCR instrument for measurement. Set the relevant measurement program, gradually increase the temperature from 25°C to 90°C, obtain the melting curve, and calculate the Tm value. The melting curve results are as follows: figure 1 shown.

[0079] Using differential scanning calorimetry (Differential Scanning Calorimetry, DSC), it was confirmed that the obtained glycosyltransferase UGT109A3 double...

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Abstract

The invention provides a glycosyl transferase mutant with improved comprehensive stability of heat and organic solvents. On the basis of enzyme structure analysis and protein stability calculation aided design, amino acids coded by a glycosyl transferase UGT109A3 wild type gene sequence are replaced with Ser203Cys, Asn304Trp, Ala45Leu, Cys127Tyr and Thr229Ile to obtain a glycosyl transferase UGT109A3m (S203C / N304W) double-site mutant and a UGT109A3m (A45L / C127Y / T229I) three-site mutant, the thermal stability Tm value of the obtained optimal mutant is increased by 7.1 DEG C, and in a 50% (v / v) DMSO reaction system, the thermal stability Tm value of the optimal mutant is increased by 7.1 DEG C, and the thermal stability Tm value of the optimal the relative catalytic activity is improved from 60% to 91%, and the actual application value of the glycosyl transferase is improved through the result.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular, to a glycosyltransferase mutant with improved thermal and organic solvent stability. Background technique [0002] Natural glycosides are an important source of innovative drugs. Glycosylation plays an important role in the pharmacodynamics, bioavailability and mechanism of action of natural products. Glycosyltransferase-mediated glycosylation is an important strategy for natural product glycosylation. UGT glycosyltransferase is an enzyme specifically responsible for catalyzing this glycosylation reaction, which transfers the glycosyl of active nucleoside sugars, such as from uridine diphosphate-glucose (UDP-glucose), to hormones, metabolites , pathogenic bacteria and a series of small molecular compound receptors such as plant and exogenous toxic substances. Some UGT glycosyltransferases catalyze reactions are also heterogeneous, that is, they can not only recognize st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/11
CPCC12N9/1051C12Y204/01017Y02A50/30Y02P20/55
Inventor 张勇高雪纯陈润莎刘慧侠吴金鸿延泉德
Owner SHANGHAI JIAO TONG UNIV
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