Beta-glucuronidase as well as gene and applications thereof

Abstract

The invention discloses beta-glucuronidase, as well as a gene, an expression vector and a transformant of beta-glucuronidase, and applications in preparing glycyrrhetinic acid monoglucuronide (GAMG). The gene sequence of the beta-glucuronidase is shown as SEQ ID No.1. After the gene is bonded to the expression vector, the obtained product is transformed into pichia pastoris, and thus a pichia pastoris engineering strain is obtained. When the beta-glucuronidase recombinase expressed by the engineering strain is used for preparing GAMG, the result shows that the enzyme has the advantages that the specificity of the substrate is high, no by-product, namely, glycyrrhetinic acid is generated, and the yield of GAMG is about 95%, which proves that beta-glucuronidase and the gene of the beta-glucuronidase have good application prospects in production of GAMG.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a beta-glucuronidase and its gene and application. Background technique [0002] β-glucuronidase (β-glucuronidase) is a kind of glycosidase that can catalyze the hydrolysis of β-glucuronidase, which widely exists in human body, animals, plants and microorganisms. β-glucuronidase was first discovered to exist in various tissues and body fluids of humans and animals, especially in the liver, spleen and adrenal glands. Early people found that this enzyme was related to tumor invasion and metastasis. Now It has become an important means of tumor diagnosis and treatment by detecting the level of this enzyme in a specific part. Bacterial-derived β-glucuronidases have been well utilized since their discovery. For example, β-glucuronidase derived from Escherichia coli can hydrolyze substrates to form fluorescent or colored substances, so this characteristic can ...

AI Technical Summary

Problems solved by technology

[0004] Animal-derived β-glucuronidase has poor substrate specificity. When glycyrrhizic acid is hydrolyzed into GAMG, it will be further used as a substrate to hydrolyze a glucuronic acid group (GlcA) to form glycyrrhetinic acid (Glycyrrhetinic acid, GA), such as figure 1 As shown, so the product is a mixture of GA and GAMG, and the preparation process of the enzyme is complicated and costly

The β-glucuronidase produced by most microorganisms also has the problem of poor substrate specificity, and the β-glucuronidase produced by wild bacteria has low enzymatic activity.

The use of genetic engineering recombinant protein expression technology can solve the problem of low enzyme activity in wild bacterial enzyme expression, but after the β-glucuronidase gene is recombinantly expressed in the model strain, it loses substrate specificity and it is still difficult to obtain a single GAMG product (Zou Shuping, PhD thesis of Tianjin University, 2010)

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