A heterologous expression engineering strain of alkaline protease from extreme environment and its application
An extreme environment, heterologous expression technology, applied in applications, genetic engineering, enzymes, etc.
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Embodiment 1
[0036] Example 1: Alkaline protease activity analysis of Microbacterium oxydans HSL10 wild-type strain.
[0037] (1) The culture characteristics of HSL10 and the effect of the addition of metal ions in the medium on the enzymatic activity of HSL10 protease ( figure 1 ).
[0038] The specific steps are: culturing Microbacillus oxydans HSL10 at low temperature in the medium, adding three common metal ions Ca in different medium 2+ , Mg 2+ and K + , using dialysis, ammonium sulfate salting out and molecular sieve to separate the protease to obtain the enzyme liquid, and detect the activity.
[0039] As a result, it was found that HSL10 has cold resistance characteristics ( figure 1 A), using Casein as the only C source medium can produce protease hydrolysis circle ( figure 1 B) 【3】 ; found that in SYN liquid medium (skim milk powder 1.0g, 0.5% yeast extract and 1% sodium chloride. Add deionized water to 100ml, adjust pH to 7.0) to cultivate HSL10, the protease obtained has ...
Embodiment 2
[0055] Example 2: Construction and expression of Microbacterium oxydans HSL10 protease gene ap1-HSL10 expression plasmid
[0056] (1) Genome sequencing of Microbacterium oxydans HSL10 was performed to analyze the possible homology of alkaline serine protease AP1 on its genome ( Figure 4 ).
[0057] The gene coding sequence (3210bp base) of alkaline protease AP1 is shown in SEQ ID No.1, and the amino acid sequence (1070 amino acids) is shown in SEQ ID No.2, and the encoded protein AP1 contains a serine protease domain and a catalytic Active triplet residues.
[0058] (2) PCR amplification of Microbacterium oxydans HSL10 protease gene ap1-HSL10 ( Figure 5 A)
[0059] The specific steps are: extracting the Microbacterium oxydans HSL10 genome, performing PCR amplification on the genome by designing primers F&R-AP1 to obtain the Microbacterium oxydans HSL10 protease gene, two segments of the target gene with the restriction sites of NdeI and HindIII, cutting the gel to recover...
Embodiment 3
[0068] Example 3: The engineering strain E.coli BL21(DE3) / pHSL-AP1 of the present invention was applied to the expression of alkaline protease AP1
[0069] In the culture of engineering bacteria, IPTG with different gradient concentrations of 0.0 to 1.2 mmol / L was used to induce expression in SYN medium, and after culturing at 37 °C for 24 h, it was found that the colony had a weak hydrolysis circle at the bottom of the corresponding medium, but there was no obvious hydrolysis circle at the periphery ( Image 6 ). Therefore, the applicant tried to induce expression by adding 1% glucose on the basis of SYN medium, and IPTG with different gradient concentrations of 0.0 to 1.2 mmol / L. The results showed that the heterologous expression host not only grew well, but also produced clear-edged transparent circles. Among them, the induction effect was the best when the IPTG concentration was 0.4 mmol / L ( Image 6 ).
[0070] Based on the above-mentioned experimental basis, the appl...
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