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[Beta]-glucuronidase mutant with improved thermostability

A technology of aldolidase and thermal stability, applied in the field of bioengineering, can solve the problems of limited practical application and poor heat resistance

Active Publication Date: 2018-08-17
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the poor thermostability of this enzyme limits its practical application

Method used

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  • [Beta]-glucuronidase mutant with improved thermostability
  • [Beta]-glucuronidase mutant with improved thermostability
  • [Beta]-glucuronidase mutant with improved thermostability

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Experimental program
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Effect test

Embodiment 1

[0012] Embodiment 1: Construction of β-glucuronidase mutant engineered bacteria

[0013] By analyzing the three-dimensional structure of TPGUS-P and aligning with the homologous sequence of the same family of thermophilic glycosidases, it was determined that the 212th valine, 220th threonine, 238th cysteine ​​and 348th valine The acid was mutated to arginine, and 23 amino acids at the N-terminus of β-glucuronidase were truncated. Using the pGAPzα-tpgus plasmid containing the tpgus gene as a template, primers were designed, and site-directed mutation was carried out by PCR to obtain recombinant plasmid pGAPzα-tpgusM1 containing the mutated gene (PCR system is shown in Table 2). The primers used for site-directed mutation are as follows:

[0014] Table 1 Primers used for mutation

[0015]

[0016] Table 2 Plasmid cloning system

[0017]

[0018]

[0019] PCR reaction conditions: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 55°C for 30s...

Embodiment 2

[0024] Embodiment 2: Expression and purification of β-glucuronidase mutant

[0025] Pick positive monoclonal colonies and transfer to 30mL of YPD medium (2% peptone, 1% yeast extract, 2% glucose) containing 50 μg / mL bleomycin, cultivate overnight at 30°C at 200rpm to obtain seed liquid, and seed The solution was transferred to 300mL YPD medium (2% peptone, 1% yeast extract, 2% glucose) containing 50μg / mL thiobleomycin according to the inoculum amount of 1% and cultivated at 200rpm at 30°C, and then every 12 hours Glucose was added to a final concentration of 2%, and cultured continuously for 48 hours.

[0026] Under the condition of 12000 rpm at 4°C, centrifuge for 10 minutes to collect the supernatant, use -20°C pre-cooled acetone to mix an equal volume with the supernatant, and then immediately centrifuge at 12000 rpm at 4°C for 10 minutes. The supernatant after centrifugation was removed, and the precipitate was resuspended with buffer H (50 mM pH4.5 HAc-NaAc). The resuspe...

Embodiment 3

[0030] Embodiment 3: β-glucuronidase activity and thermostability assay

[0031] β-glucuronidase can catalyze the hydrolysis of glycosidic bonds of glycyrrhizic acid (GL) to generate 3-O-monoglucuronyl glycyrrhetinic acid (GAMG). This reaction is simple, rapid and easy to detect. This study The enzyme activities of β-glucuronidase and its mutants were determined by this reaction. Take 50μL concentration as 0.01g L -1 Add enzyme solution to 50μL containing GL concentration 2.5mmol·L -1 , in acetic acid-sodium acetate buffer solution with a pH of 4.5, reacted at 45°C for 10 minutes, then added 900 μL of methanol to terminate the reaction, and the sample liquid was detected for the content of GAMG by high-performance liquid chromatography. Definition of β-glucuronidase activity: Under the above conditions, the amount of β-glucuronidase required to convert 1 nmol GAMG per minute is an activity unit.

[0032] Take 100μL concentration as 0.01g L -1 The pure enzyme solution was h...

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Abstract

The invention discloses a [beta]-glucuronidase mutant with improved thermostability, and belongs to the field of bioengineering. [Beta]-glucuronidase, which is derived from talaromyces pinophilum Li-93 (preservation number is CGMCC No.11765), undergoes site-specific mutagenesis, so that the mutation of valine on the 212th site, threonine on the 220th site, cysteine on the 238th site and valine onthe 348th site, which are coded by the [beta]-glucuronidase, into arginine is promoted; and 23 amino acids at the N terminal of the enzyme ([beta]-glucuronidase) are cut off, and a recombinant plasmidof mutant gene is transformed in pichia pastoris GS115 and is expressed, so that the [beta]-glucuronidase mutant with improved thermostability is obtained. According to the [beta]-glucuronidase mutant provided by the invention, half-life periods under 55 DEG C and 70 DEG C are prolonged by 81min and 25min in comparison with wild type, namely 3 times and 2.3 times as much as that of the wild type.A process of pyrolytic conversion GL is established on the basis of the mutant. The [beta]-glucuronidase mutant provided by the invention has a broad industrial application prospect.

Description

technical field [0001] The invention relates to a beta-glucuronidase mutant with improved thermal stability, which belongs to the technical field of bioengineering. Background technique [0002] β-glucuronidase (β-glucuronidase, EC: 3.2.1.31) can recognize and catalyze various types of β-glucuronide glycosidic bonds, while releasing β-glucuronic acid and corresponding ligands or aglycones . Most of the enzymes belong to glycosidase family 1 (GH1) and glycosidase family 2 (GH2). In recent years, studies have found that the enzyme is also distributed in glycosidase family 79 (GH79). It has been found that mammals, fungi and bacteria can all secrete β-glucuronidase. β-glucuronidase has been widely cited in many fields, such as human drug analysis, prodrug enzyme-directed therapy, tumor pathology research, etc. In addition, because β-glucuronidase can produce color reaction after reacting with the substrate, it is mostly used as a marker gene to locate the expression of other...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12P33/20
CPCC12N9/2402C12P33/20C12Y302/01031
Inventor 李春贾锦彤冯旭东吕波
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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