A kind of recombinant chitosanase and its production method and application
A technology of chitosanase and chitosan, applied in coding gene and its production method and application, recombinant chitosanase and its production method and application, in the field of recombinant chitosanase, can solve the problem of poor application performance, enzyme It has the problems that the activity needs to be further improved and the cost of use is high, so as to achieve the effects of good thermal stability and pH stability, good industrial application prospects and mild reaction conditions.
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Embodiment 1
[0051] Embodiment 1, the acquisition of Csn46A gene and protein
[0052] Taking spoiled and degraded shrimp and crab shell waste as a sample, inoculate it into the enrichment medium with chitosan as the single carbon source (50 g of chitosan, 5 g of yeast extract, CaCl 2 0.3g, MgSO 4 .7H 2 O 0.3g, FeSO 4 0.3g, KH 2 PO 4 0.6g, Tween 80 2.5g, constant volume to 1L, pH 5.5), culture conditions: temperature 37°C, time 72h, rotation speed 150rpm, used to enrich environmental microorganisms that can degrade chitosan. Collect all precipitated components in the mixture by centrifugation, and extract the total DNA of the above samples. According to the conserved region of the chitosanase-related gene sequence of the glycoside hydrolase 46 family, degenerate primers were designed, and the total DNA of the above sample was used as a template for PCR amplification, and the obtained fragments were sequenced, and the potential potential was amplified by TAIL-PCR technology. Chitosa...
Embodiment 2
[0057] Expression and purification of embodiment 2, recombinant chitosanase (Csn46A)
[0058] The recombinant plasmid in Example 1 was transformed and expressed into the host Escherichia coli BL21 (DE3) to obtain the recombinant bacteria, which was inoculated into 1L LB liquid medium (containing 50 μg / mL kanamycin), at 37°C, 200rpm cultured to OD 600 Between 0.6-0.8, add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 1 mM, and induce overnight at 30°C. After the cells were collected by centrifugation, the cells were resuspended with buffer A (20mM Tris-Hcl washing solution, 0.5M NaCl, 20mM imidazole, pH 8.0) at a ratio of 1:10 (v / v), and then placed in an ice-water bath Medium sonication (200W, ultrasonic 3s, intermittent 4s, 120 times), and centrifugation to collect the supernatant is the crude enzyme solution, which contains the recombinant protein Csn46A, ie recombinant chitosanase (Csn46A).
[0059] Based on the sequence of encoding His-Tag tag protein ...
Embodiment 3
[0062] Embodiment 3, recombinant chitosanase (Csn46A) property detection and enzymatic properties
[0063] 1. Enzyme activity detection of Csn46A
[0064] The assay method of the enzymatic activity of chitosanase is as follows: add 350 μ L chitosan substrate (1.0%, W / V; pH6.0, 200 mM acetic acid-sodium acetate buffer) and 50 μ L appropriate The diluted enzyme solution was reacted at 50° C. for 10 minutes, and then the content of reducing sugar in the reaction solution was determined by DNS method. Enzyme activity unit (1U) is defined as: under the above reaction conditions, the amount of enzyme required to generate 1 μmol of glucosamine per minute. Wherein, the chitosan substrate preparation method: dissolve 1.0 g of chitosan with an appropriate amount of 100 mM acetic acid solution. The pH was then adjusted to 6.0 with 100 mM sodium acetate solution. After this treatment, chitosan forms a uniform and transparent solution.
[0065] 2. Determination of the optimum reaction ...
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