Granular body contg. polydroxy alkoxide acid ester
A technology of polyhydroxyalkanoate and granules, which is applied in the field of slow-release microcapsules, hemoglobin, cosmetic ingredients or fertilizer ingredients, gas, and phase fields, and can solve the problem of not being able to fully satisfy and it is difficult to obtain a uniform solution containing a large number of bubbles microparticles etc.
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reference example 1
[0543] (Reference Example 1) Production of Transformants with pHB Synthetase Production Ability
[0544] The inventors have previously applied for a method for producing a transformant produced by the TB64 strain and capable of producing pHB synthetase, but specific examples thereof are listed here. The TB64 strain was cultured overnight at 30°C in 100ml of LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride, pH7.4), and then separated and recovered by the method of Ma-Ma- et al. Chromosomal DNA. The resulting chromosomal DNA was partially decomposed with restriction enzyme Sau3Al. For the preparation of the vector, pUC18 was used, cut with the restriction enzyme BamH1, dephosphorylated (Molecular Cloning, Vol. 1, p. 572, 1989, published by Cold Spring Harbor Laboratory), and DNA Ligation Kit Ver.11 (Takara Shuzo) was used to connect with the chromosome The Sou3Al part of the DNA breaks down the fragment junctions. Next, Escherichia coli (Escheichia Coli) HB101 ...
reference example 2
[0549] (Reference Example 2) Preparation of Transformants with GST Fusion pHB Synthetase Production Ability
[0550] Inoculate the PTB 64-pHB strain into 200ml of LB medium, culture it with shaking at 125 times / min at 37°C for 12 hours, recover the cells obtained in this way by centrifugation, and recover plasmid DNA by conventional methods.
[0551] For PTB 64-pHB, an oligonucleotide (SEQ ID NO: 1) for an upstream side primer and an oligonucleotide (SEQ ID NO: 2) for a downstream side primer were designed and synthesized (Amersham Pharmacia Biotech). The oligonucleotide was used as a primer, and PTB64-pHB was used as a template to carry out PCR, and the pHB synthetase gene with a Bam H1 restriction site upstream and a Xhol restriction site downstream was amplified to full length (LA, PCR kit; Baojiu Co., Ltd.) .
[0552] The refined PCR amplified product was digested with Bam H1 and Xhol, inserted into a site corresponding to the plasmid PGEX-6P-1 (manufactured by Amersham P...
reference example 3
[0553] (Reference Example 3) Preparation of pHB Synthetase
[0554] In 100 ml of 2XYT medium (ptone 16 g / L, yeast extract 10 g / L, Nacl 5 g / L, pH 7.0) supplemented with ampicillin (100 μg / L), pre-culture the obtained discovery strain at 30°C overnight.
[0555] This was added to 10 liters of 2XYT medium (peptone 16 g / L, yeast extract 10 g / L, Nacl 5 g / L, pH 7.0) supplemented with ampicillin (100 μg / L), and cultured at 30°C for 3 hours , adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and culturing at 30° C. for 3 hours.
[0556] At 4°C, the recovered culture solution was centrifuged at 78000m / S2 (=8000G) for 10 minutes, and after removing the supernatant, the bacterial cell particles were resuspended in 500ml of PBS solution at 4°C. Inject each 40ml of the bacterial liquid into a container pre-cooled to 4°C, pressurize to 216MPa (=2200kg / cm 2 ), discharge the bacterial liquid from the nozzle a small amount each time, and carry out the bacte...
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