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Aspergillus niger hydrolytic enzyme AnCu3, coding gene and application of aspergillus niger hydrolytic enzyme AnCu3

A technology of aspergillus niger and encoding gene, applied in the directions of hydrolase, application, genetic engineering, etc., can solve the problems of increasing the risk of breast cancer in women and harming the reproductive system of the baby boy.

Active Publication Date: 2019-12-20
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The aromatic components of many cosmetics also contain such substances, which will enter the body through the respiratory system and skin of women. If used too much, it will increase the risk of breast cancer in women and have a certain chance of harming the reproductive system of the male baby.

Method used

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  • Aspergillus niger hydrolytic enzyme AnCu3, coding gene and application of aspergillus niger hydrolytic enzyme AnCu3
  • Aspergillus niger hydrolytic enzyme AnCu3, coding gene and application of aspergillus niger hydrolytic enzyme AnCu3
  • Aspergillus niger hydrolytic enzyme AnCu3, coding gene and application of aspergillus niger hydrolytic enzyme AnCu3

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Cloning of the gene encoding the esterhydrolase AnCu3 of embodiment 1

[0019] 1. Aspergillus niger culture

[0020] Under aseptic conditions, inoculate Aspergillus niger into a 300mL Erlenmeyer flask containing 100mL of fermentation medium, and culture on a shaker at 30±1°C and 150±10r / min for 4-8d. The fermentation medium is composed of glucose 70g / L, soybean cake powder 10g / L, MgSO 4 0.20g / L, NaNO 3 2.0g / L, NaH 2 PO 4 1.0g / L, adjust the pH to 4.5, and sterilize at 115°C for 20min.

[0021] 2. Extraction of total RNA from Aspergillus niger

[0022] The above-mentioned Aspergillus niger cultured for 6 days was sampled, and the total RNA was extracted using the BIOMIGA fungal RNA extraction kit. Specific steps are as follows:

[0023] (1) Weigh 100mg of fungal culture into a 1.5mL or 2.0mL centrifuge tube, freeze in liquid nitrogen, grind the fungus into powder with a grinding pestle (if possible, put the tissue in a mortar, and grind it into powder with liqui...

Embodiment 2

[0067] Embodiment 2 constructs the escherichia coli engineering strain expressing ester hydrolase AnCu3

[0068] 1. Linearization of pET-28a(+) vector

[0069] Primers were designed to linearize the pET-28a(+) vector by PCR. Primers were designed as follows:

[0070] Forward primer: 5'-CTGAGATCCGGCTGCTAA-3',

[0071] Reverse primer: 5'-ACTTCCTCTTGGCACCAGGCCGCTGCT-3'

[0072] The PCR reaction system is shown in Table 4.

[0073] Table 4 PCR reaction system of linearized vector pET-28a(+)

[0074] Reagent Volume (μl) wxya 2 o

21.0 dNTPs (2.5mM each) 3.0 10×Ex Taq Buffer 3.0 Forward primer (10μM) 0.6 Reverse primer (10μM) 0.6 pET-28a(+) vector 0.8 Q5DNA Polymerase (5U / μl) 1.0 Total 30.0

[0075] PCR amplification cycle

[0076]

[0077]

[0078] The amplified DNA band is used for plasmid construction after DNA purification.

[0079] 2. Construction of pET-AnCu3 plasmid

[0080] pass II for plasmid...

Embodiment 3

[0086] Example 3 Esterhydrolase AnCu3 crude enzyme liquid aqueous phase system catalyzes the hydrolysis of DEHP

[0087] 1. Induced expression of esterhydrolase AnCu3

[0088] Pick the verified correct transformants, transfer them to LB liquid test tubes containing appropriate kanamycin sulfate antibiotics, culture overnight at 37±1°C, and then inoculate 300mL of LB medium containing 100mL with a 1% (v / v) inoculation volume Erlenmeyer flasks were cultured on a shaker at 37±1°C and 200±10rpm for 3h, then the inducer IPTG with a final concentration of 0.5mM was added, and cultured at 20±1°C and 200±10rpm for 20h.

[0089] 2. Preparation of crude enzyme solution of esterhydrolase AnCu3

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Abstract

The invention discloses an aspergillus niger hydrolytic enzyme AnCu3. The amino acid sequence of the aspergillus niger hydrolytic enzyme AnCu3 is shown as SEQID No.1. The invention further discloses an application of the aspergillus niger hydrolytic enzyme AnCu3 to catalyzing and hydrolyzing of plasticizer dimethyl glycol phthalate (2-ethylhexyl) ester (DEHP) in a water phase system. The inventionprovides an enzyme which can catalyze and hydrolyze the dimethyl glycol phthalate (2-ethylhexyl) ester, and a coding gene for the first time, and a new way is provided for hydrolyzing of the dimethylglycol phthalate ( 2-ethylhexyl) ester.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to an application of Aspergillus niger ester hydrolase AnCu3 in hydrolyzing bis(2-ethylhexyl) phthalate (DEHP). [0002] technical background [0003] Phthalic acid esters (also known as phthalic acid esters, phthalic acid esters, PAEs), is a general term for esters formed by phthalic acid, commonly known as plasticizers and plasticizers. PAEs are often added to polyvinyl chloride (also known as plastic, polyvinyl chloride, PVC) as the main functional component to increase the flexibility and durability of PVC. At present, the consumption of PAEs exceeds 8 million tons per year. PAEs can be used as additives for paints, adhesives, cosmetics and lubricants, and as the main additive component for the improvement of the flexibility of PVC materials. The modified PVC materials produced are widely used in Industries such as construction, automotive, medical pro...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/18C12N15/70
Inventor 李秀婷孙宝国徐友强王晓程
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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