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Polyhydroxyalkanoate particle degrading enzyme as well as applications thereof and production method of (R)-3HB

A technology of polyhydroxyalkanoic acid and degrading enzymes, applied in the directions of hydrolase, fermentation, etc., can solve the problems of complex preparation process, difficult separation, not many and so on.

Inactive Publication Date: 2016-10-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such a preparation process is complicated, difficult to separate, and the yield is greatly affected
Moreover, chemical synthesis methods often need to use solvents, reactants or unfavorable by-products that are harmful to the human body or the environment. The production process is not environmentally friendly, and the final product will also contain a small amount of harmful components, which will affect its further application.
[0006] However, there are not many methods for producing (R)-3HB monomer through environmentally friendly biological methods, and there is even a lack of methods that can use microorganisms for industrial production.

Method used

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  • Polyhydroxyalkanoate particle degrading enzyme as well as applications thereof and production method of (R)-3HB
  • Polyhydroxyalkanoate particle degrading enzyme as well as applications thereof and production method of (R)-3HB
  • Polyhydroxyalkanoate particle degrading enzyme as well as applications thereof and production method of (R)-3HB

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of the bktB knockout strain H.mediterranei EPSΔbktB and the phaZh1 gene knockout strain H.mediterranei EPSΔphaZh1

[0035] Construction of H. mediterranei EPSΔbktB:

[0036] 1. Construction of the homologous integration vector pHFX-DbktB for the knockout gene bktB

[0037] Using the whole genome of the extreme halophilic archaea Haloferax mediterranei (strain deposit number: CGMCC 1.2087) as a template, the primer pair DbktB-F1-HindIII / DbktB-R1-BamHI and DbktB-F2-BamHI / DbktB-R2-KpnI were used to amplify A DNA fragment of 814 bp upstream (sequence 1) and 799 bp downstream (sequence 2) of gene bktB was added. PCR amplification program: pre-denaturation at 94°C for 5 minutes; then 30 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min; extension at 68°C for 10 min, and 25 μL of the amplification system. Use HindIII and BamHI to digest the upstream fragment, and BamHI and KpnI to digest the downstream fragment. The upstream and downstream fr...

Embodiment 2

[0051] Example 2 Cultivation of H.mediterranei EPSΔbktB and H.mediterranei EPSΔphaZh1 and extraction of PHA particles:

[0052] Medium:

[0053] AS-168 medium: each liter contains Bacto casamino acids (Difco) 5g, Bacto yeastextract (Oxoid) 5g, sodium glutamate 1g, trisodium citrate 3g, magnesium sulfate heptahydrate 20g, potassium chloride 2g, sodium chloride 200g, trace amount of iron sulfate, trace amount of manganese chloride, pH 7.0~7.2.

[0054] PA medium: per liter contains 110g of sodium chloride, 9.6g of magnesium chloride hexahydrate, 14.4g of magnesium sulfate heptahydrate, 1g of calcium chloride, 5g of potassium chloride, 2g of ammonium chloride, 0.0375g of potassium dihydrogen phosphate, 10g of glucose, Ferric ammonium citrate solution 1mL, trace element solution SL-61mL.

[0055] Trace element solution SL-6 contains: Zinc ions 2~4mM, Manganese ions 1~2mM, Borate 40~55mM, Cobalt ions 6~10mM, Copper ions 0.5~1.5mM, Nickel ions 1~2mM, Molybdate 1~2mM ; Adjust the ...

Embodiment 3

[0060] The self-degradation of embodiment 3 PHA particles

[0061] The two PHA particles prepared in Example 2 were resuspended in the solution to obtain the PHA particle suspension (2M KCl, 100mM Tris-HCl (pH 7.5), 5mM MgCl 2 and PHA particles, OD 650 ~1.0), the suspension was reacted at 45°C for more than 5 hours. The result is as figure 2 As shown, in the bktB-knockout recombinant strain H. mediterranei EPSΔbktB, the isolated PHB particles self-degraded; while in the phaZh1-knockout recombinant strain H. Therefore, the PHBV particles accumulated by the bacteria did not degrade in vitro. The results indicated that the protein PhaZh1 had the function of degrading PHA particles, and was attached to the PHA particles, so it could be separated together.

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PUM

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Abstract

The invention discloses a polyhydroxyalkanoate particle degrading enzyme as well as applications of the polyhydroxyalkanoate particle degrading enzyme and a production method of (R)-3HB. For the polyhydroxyalkanoate particle degrading enzyme PhaZh1 or polypeptide, the enzyme has the amino acid sequence shown as the sequence 8 in a sequence table, and the polypeptide has at least 70%, preferably 80%, further preferably 90%, more preferably 95%, further more preferably 98%, and the most preferably 99% of homology with the enzyme, and has the enzyme activity. The enzyme or polypeptide can degrade the polyhydroxyalkanoate particles formed by accumulation of microbial fermentation, and thus the chiral monomer (R)-3HB is prepared.

Description

technical field [0001] The present invention generally relates to an enzyme, in particular to a polyhydroxyalkanoic acid particle degrading enzyme and a method for producing (R)-3HB using the enzyme. Background technique [0002] Bacteria that grow in environments where carbon sources are abundant but other nutrients are deficient [1] or Archaea [2] Polyhydroxyalkanoate (PHA) is accumulated in cells, and PHA is stored as a carbon source or energy source. When there is a lack of carbon sources in the environment, these stored PHAs will be mobilized by cells to maintain the survival of bacteria [3] . The PHA produced by this microorganism is a fully biodegradable polymer with good biocompatibility. Currently there are methods for producing PHA by microbial fermentation. [0003] The extremely halophilic archaea Haloferax mediterranei (CGMCC 1.2087) can utilize a variety of carbon sources (such as glucose, starch and chitin, etc.) [2-5] A large amount of hydroxybutyric ac...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12P7/42
Inventor 向华刘桂明韩静赵大贺
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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