A high-yielding specific functional oligopeptide enzyme derived from Aspergillus niger and its engineering bacteria
A technology of engineering bacteria and oligopeptides, applied in the field of genetic engineering, can solve problems such as cumbersome processing procedures, high purification costs, and large limitations, and achieve the effects of reducing production costs, reducing environmental pollution, and improving efficiency
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Embodiment 1
[0022] Example 1 Construction of M00988 CPA mutant
[0023] 1. Cloning of Aspergillus niger M00988 CPA and construction of expression vector
[0024] According to the carboxypeptidase gene sequence of NCBI Aspergillus niger, two pairs of primers CPA-F / CPA-R were designed, and the sequences are shown in Table 1.
[0025] Table 1 The sequences of primers for carboxypeptidase gene cloning
[0026] The DNA of Aspergillus niger M00988 was extracted as a template, the sequence is shown in SEQ ID NO: 6, and the target gene fragment CPA was obtained by PCR amplification with CPA-F / CPA-R primers. The PCR amplification conditions are: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 68°C for 1 minute, extension at 72°C for 1 minute, 35 cycles, and extension at 72°C for 10 minutes to obtain a fragment with a size of about 1500 bp, which was detected by agarose electrophoresis Such as figure 1 shown.
[0027] Use the obtained target gen...
Embodiment 2
[0039] Example 2 Preparation and application of high F value oligopeptides
[0040] 1. Chlorella protein extraction
[0041] Accurately weigh a certain amount of chlorella powder, add deionized water at a solid-liquid ratio of 1:48 (m / V), stir and swell at room temperature for 12 hours. Add 5.37% sodium hydroxide solid to the chlorella suspension, pay attention to adding slowly during the addition process, and keep stirring. After completion, sonicate for 60 min at 43 °C. Next, use 0.1mol / L sodium hydroxide solution and hydrochloric acid solution to adjust the pH=7, centrifuge at 5000rpm 25°C for 10min, take the supernatant, add 95% alcohol at 4°C at a ratio of 1:4 (V / V) solution, stirred for 1 min, and mixed thoroughly. Centrifuge again at 5000 rpm at 25° C. for 10 minutes, and discard the supernatant. Wash the obtained precipitate with a small amount of deionized water, remove the surface alcohol, and then dissolve the precipitate with 0.1mol / L PBS solution with pH=6.
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