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Polylactic acid degrading enzyme with protein degrading activity

A technology for degrading active polylactic acid, which can be used in peptide/protein components, hydrolase, medical preparations containing active ingredients, etc., and can solve the problems of limited separation and purification and property research.

Inactive Publication Date: 2009-06-17
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, studies have found that some specific enzymes derived from microorganisms can degrade polylactic acid, but the isolation, purification and properties of these enzymes are currently very limited.

Method used

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  • Polylactic acid degrading enzyme with protein degrading activity
  • Polylactic acid degrading enzyme with protein degrading activity
  • Polylactic acid degrading enzyme with protein degrading activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Screening of degrading strains

[0041] Spot or spread activated Amycolatopsis orientalis strains (strain bank, commercially available) and environmental sample enrichment solution on PLA emulsified plates, culture them upside down in a 30°C incubator, and observe the strains for 15-30 days The growth and the formation of the transparent hydrolysis circle were selected, and the bacterial strains that could grow on the plate and form an obvious transparent hydrolysis circle were screened. To prevent moisture from evaporating, replenish water regularly and maintain a humid environment. Screening media components and production methods are as follows:

[0042] Basic medium: 250mg yeast extract, 1g (NH 4 ) 2 SO 4 , 100mg NaCl, 200mg MgSO 4 ·7H 2 O, 20mgCaCl 2 2H 2 O, 10mg FeSO 4 ·7H 2 O, 0.5 mg Na 2 MoO 4 2H 2 O, 0.5 mg Na 2 WO 4 , 0.5 mg MnSO 4 , 1.6g K 2 HPO 4 , 0.2g KH 2 PO 4 Distilled water to 1L, natural pH.

[0043] Preparation of PLA em...

Embodiment 2

[0045] Example 2 Purification of Enzymes

[0046] Amycolatopsis orientalis was fermented and cultured in a 3L GBCS-5 biological fermenter. The medium was 10g gelatin, 10g glucose, 2g yeast extract, 5g NaCl, 1.6g K2HPO4, 0.2g KH2PO4, 0.5g MgSO4 and dilute to 1L distilled water. The crude enzyme liquid obtained by centrifuging the supernatant from the fermentation broth was concentrated by ultrafiltration, and then separated and purified by CM SepharoseFast Flow cation exchange chromatography, TSK-gel phenyl-5PW hydrophobic chromatography, and Sephadex G-50 fine molecular sieve chromatography. Specifically, the concentrated crude enzyme solution was dialyzed with 20mM phosphate buffer containing 10mM NaCl, pH 7.0 to remove salt, and then subjected to CM Sepharose Fast Flow chromatography. The electrophoresis results showed that the obtained enzyme components were not yet purified, and the three elution peaks collected separately and proceed to the next step of separation. Ammon...

Embodiment 3

[0048] Example 3 Determination of Enzyme Characteristics

[0049] 1. Determination of Enzyme Molecular Weight

[0050] The purified degrading enzyme components were analyzed by 10% SDS-PAGE, and the molecular weights of polylactic acid degrading enzymes I, II and III were calculated as 24.0, 19.5 and 18.0 kDa by mobility calculation.

[0051] 2. Determination of isoelectric point

[0052] The isoelectric points of the three polylactic acid degrading enzymes were measured by isoelectric focusing electrophoresis. After electrophoresis, the gel was cut to measure the pH gradient, and the isoelectric point of the protein was estimated. At the same time, lysozyme with a pI of about 11 was used as a control. Based on the pH gradient measurement results of gel cutting and electrophoretic patterns, it can be speculated that the isoelectric points of the three isolated polylactic acid degrading enzymes I, II and III are all greater than 10, so these three enzymes are all basic protein...

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Abstract

The invention discloses a polylactic acid degrading enzyme with protein degrading activity prepared by fermentation and purification of strains of amycolatum sp. and amycolatopsis sp.; wherein the enzyme comprises a polylactic acid degrading enzyme I, a polylactic degrading enzyme II and a polylacic degrading enzyme III, said poly degrading enzyme I comprises a protein with an N-terminal amino acid sequence of IVGGGTAPTVSWGAQ or its mutant variation protein; the degrading enzyme II comprises a protein with an amino acid sequence shown as SEQ ID NO:3 or its mutant variation protein; the degrading enzyme III comprises a protein with an amino acid sequence shown as SEQ ID NO: 4 or its mutant variation protein. The degrading enzyme of the invention has a proteolysis activity, simultaneously has high-efficiency degradation capability for degradable plastics polylactic acid, not only applies to the traditional protease industries such as decontaminant, cosmetics, leather processing, but also has applications potential on the cyclic utilization of the polylactic acid producers and treatment of combined rubbish containing degradable plastics, hair and keratin or the like.

Description

technical field [0001] The invention relates to a class of functional polylactic acid degrading enzymes, in particular to a class of polylactic acid degrading enzymes with protein degradation activity, and belongs to the field of biotechnology. Background technique [0002] Polylactic acid is a degradable plastic that came into being in response to people's requirements for sustainable development of the environment and energy. At present, it has achieved a large number of applications in the medical, textile and packaging industries, and has become the most potential alternative to existing plastics. polymer materials. Although polylactic acid has degradable properties, its degradation process in nature is relatively slow, and it is difficult to quickly and completely degrade it. Therefore, while using polylactic acid products, it is necessary to develop polylactic acid-degrading strains and degrading enzymes to accelerate polylactic acid. Degradation and recycling of wast...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N15/57C11D3/386C14C1/00A61K38/48A61K8/66C12R1/01
Inventor 陈冠军刘巍峰李凡王莎
Owner SHANDONG UNIV
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