Method for producing acrylamide using microbial catalyst
A biocatalyst, acrylamide technology, applied in biochemical equipment and methods, biochemical cleaning devices, enzymology/microbiology devices, etc., can solve problems that have not been discussed
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Embodiment 1 and 2
[0080] [Examples 1 and 2]: Production of acrylamide using acrylonitrile stored at 20°C or 28°C
[0081] (storage of acrylonitrile)
[0082] Acrylonitrile (manufactured by DIA-NITRIX CO., LTD.) was placed in a 500 mL glass bottle, and stored in a thermostat adjusted to 20°C and 28°C for 7 days, respectively.
[0083] (Preparation of biocatalyst)
[0084] Utilize the culture medium (pH7.0) that contains glucose 2%, urea 1%, peptone 0.5%, yeast extract 0.3%, cobalt chloride 0.05% (all by weight %) to have nitrile hydratase activity under 30 ℃ Rhodococcus rhodochrous J1 (FERMBP-1478) was cultured aerobically. This was separated using a centrifuge and washed with 50 mM phosphate buffer (pH 7.0) to obtain a bacterial cell suspension (15% by weight of dried bacterial cells).
[0085] (reaction from acrylonitrile to acrylamide)
[0086] 664 g of deionized water was added to a 1 L detachable jacketed flask, and the water temperature was controlled at 18°C. After 30 minutes, 0.8 g of...
Embodiment 3
[0091] [Example 3]: Production of transformant with nitrile hydratase derived from Rhodococcus rhodochrous M8 strain
[0092] (1) Preparation of chromosomal DNA from Rhodococcus rhodochrous M8 strain (hereinafter referred to as M8 strain.)
[0093] The M8 strain (SU 1731814) can be obtained from the Russian strain center IBFM (VKPMS-926).
[0094] In 100ml of MYK (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K 2 HPO 4 , 0.2%KH 2 PO 4 ) culture medium (pH 7.0) at 30°C for 72 hours with shaking. The culture solution was centrifuged, and the collected bacteria were suspended in 4 ml of Saline-EDTA solution (0.1 MEDTA, 0.15 M NaCl (pH 8.0)). Add 8 mg of lysozyme to the suspension, shake it at 37°C for 1 to 2 hours, and then freeze it at -20°C.
[0095] Next, 10 ml of Tris-SD S solution (1% SDS, 0.1M NaCl, 0.1M Tris-HCl (pH 9.0)) was added to the suspension while shaking gently. Furthermore, proteinase K (Merck & Co.) (final concentration: 0.1 mg...
Embodiment 4
[0132] [Example 4]: Production of transformant having nitrile hydratase derived from Psedonocardia thermophila JCM3095 strain
[0133] (1) Preparation of nitrile hydratase gene from pPT-DB1 plasmid DNA using PCR
[0134] pPT-DB1 is a plasmid containing a nitrile hydratase gene derived from Psedonocardia thermophila JCM3095 strain (hereinafter referred to as JCM3095 strain) obtained in JP-A-9-275978.
[0135] The JCM3095 strain is described in Japanese Patent Application Laid-Open No. 9-275978, and the sequences of the β subunit, α subunit, and activator are shown in Table 2.
[0136] [Table 2]
[0137] JCM3095 strain
amino acid sequence
β-subunit
serial number 9
serial number 10
α-subunit
serial number 11
serial number 12
activator
serial number 13
serial number 14
[0138] Based on the above sequence information, primers PSN-1 and PSN-2 were synthesized, and PCR was performed using pPT-DB1 pl...
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