Application of heat-resistant alpha-glucuronidase polypeptide fusion body in preparation of glucuronic acid

A technology of glucuronidase and glucuronic acid, applied in the field of glucuronic acid, can solve problems such as waste of resources and environmental pollution, and achieve the effects of less reaction by-products, large amount of products and improved activity

Active Publication Date: 2021-04-02
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

China is a big agricultural country, and the annual output of straw is not less than 1 billion tons, accounting for 25%-30% of the world's total straw output. If it is only burned for fertilizer, it will waste resources and cause environmental pollution

Method used

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  • Application of heat-resistant alpha-glucuronidase polypeptide fusion body in preparation of glucuronic acid
  • Application of heat-resistant alpha-glucuronidase polypeptide fusion body in preparation of glucuronic acid
  • Application of heat-resistant alpha-glucuronidase polypeptide fusion body in preparation of glucuronic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] According to the α-glucuronidase gene sequence (the accession number of the GenBank database of Ncbid: GeneID:896879) of the family 67 of Thermotoga (Thermotiga), namely the nucleotide sequence design primer of coding wild-type TmAguA, specifically:

[0039] upstream primer

[0040] F: 5'CATG CCATGG ACTACAGGATGTGCTGGCT-3' (SEQ ID NO.3)

[0041] Downstream primers (four types)

[0042] R1: 5'CCG CTCGAG CGGATATATCTTTCTTTCCCTT-3' (SEQ ID NO.4)

[0043] R2: 5'CCG CTCGAG TGCATGAAAGAATGCCTGATGCGGATATATCTTCTTCCC-3' (SEQ ID NO. 5)

[0044] R3: 5'CCG CTCGAG GCCGCTGCCGCTGCCCGCCGCCGGATATATCTTCTTCCC-3' (SEQ ID NO. 6)

[0045] R4: 5'CCG CTCGAG GCCGCCGCTGCCGCCGCCGCCCGGATATATCTTCTTCCC-3' (SEQ ID NO. 7)

[0046] The recombinant plasmid pET-28a-xynB-Hspr-aguA (J.Carbohydrate Chemistry,2018,37(4)210-224.) containing the wild-type α-glucuronidase gene of Thermotoga maritima was selected as a template, respectively Four pairs of primers F and R1 (wild-type TmAguA), F and R2 ...

Embodiment 2

[0048] Using the same method as in Example 1 to obtain purified TmAguA and TmAguAPL1, further compare the effects of pH and temperature on the enzyme activity of TmAguA and TmAguAPL1:

[0049] (1) Optimum reaction pH: place the recombinant enzymes TmAguA and TmAguAPL1 at 75°C to determine the effect of different pH on the enzyme activity, take the highest enzyme activity as 100%, calculate the relative activity of the enzyme, and make a relative activity-pH curve. The enzyme reaction was carried out in 50 mM o-benzyme buffer (pH 5.5-10.0), and the pH of the buffer was adjusted at room temperature. The above determination was repeated three times, and the relative standard deviation was less than 10%.

[0050] (2) pH stability: place the recombinant enzymes TmAguA and TmAguAPL1 in a buffer solution with a pH value of 5.5-10 and incubate at 37°C for 1 hour, then measure the residual activity of the enzyme. Relative activity, for pH stability curve. The buffer used was 50 mM o-be...

Embodiment 3

[0058] The method is the same as in Example 2, except that equimolar amounts of TmAguA and TmAguAPL1 are further selected to evaluate the effect of enzymatically hydrolyzing xylan to prepare glucuronic acid. The specific method:

[0059] Beech wood xylan was dissolved in phosphate buffer solution to prepare a beech wood xylan solution with a concentration of 10 mg / mL, and 20 U / g (substrate) of β-1,4-endoxylanase was added, and then Add 1.82 μmol / L of purified TmAguA and incubate at 85°C and pH7.5 for 2 hours as a control group; add purified TmAguAPL1 with equimolar concentration (1.82μmol / L) and incubate at 75°C and pH8.5 for 2 hours, Results (see image 3 ) shows that: compared to the control group (wild-type enzyme), the sample group (TmAguAPL1) converts beech wood xylan to produce glucuronic acid production increased by 48.6-60.3%, and the gained TmAguAPL1 is hydrolyzed 4-O-methylglucuronic acid xylan The sugar releases the highest glucuronic acid, its nucleic acid sequenc...

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Abstract

The invention discloses application of a heat-resistant alpha-glucuronidase polypeptide fusion body in preparation of glucuronic acid. An amino acid sequence of the fusion body is as shown in SEQ ID NO.1. According to the invention, seven amino acid residues are fused at the C end of heat-resistant alpha-glucuronidase protein to form a brand-new polypeptide fusion body, the specific activity of the obtained polypeptide fusion body is improved by 297% compared with that of wild type enzyme TmAguA, the Kcat and Kcat/Km of a corresponding hydrolyzed 4-O-methyl glucuronic acid xylo-oligosaccharidesubstrate are improved by 356% and 363% respectively, and the stability is improved by 21.1%-65.7% compared with that of the wild type enzyme; the fusion body is used for preparing the glucuronic acid, and compared with the wild type enzyme, the fusion body has the advantage that the yield of the glucuronic acid prepared by converting beechwood xylan through the polypeptide fusion body is increased by 21.6-60.3%; and the fusion body has the advantages of high conversion efficiency, few reaction byproducts, high yield and purity, easiness in separation and purification, less chemical pollutionand the like.

Description

technical field [0001] The invention belongs to glucuronic acid in the fields of food, medicine, chemical industry and clean production, and specifically relates to the application of a heat-resistant α-glucuronidase polypeptide fusion body in the preparation of glucuronic acid. Background technique [0002] Glucuronic acid is a compound formed by the oxidation of the primary alcoholic hydroxyl group of glucose into a carboxyl group. It has highly reactive aldehyde and carboxyl groups, and can bind toxic substances containing hydroxyl, sulfhydryl, carboxyl, and amino groups to enhance the water solubility of toxic substances. Quickly excrete the kidneys to achieve detoxification. At the same time, glucuronic acid can combine with the active group phenolic hydroxyl group on the conjugated molecule to reduce the biological functions of related hormones and drugs. Glucuronic acid is an important medicinal chemical intermediate; amide and R-phenylhydrazide derivatives of glucuro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12P19/02C12P19/14
CPCC12N9/2402C12P19/02C12P19/14
Inventor 薛业敏谢方赵红阳薛梦柯
Owner NANJING NORMAL UNIVERSITY
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