Chitin enzyme SvChiAJ54 and coding gene and application thereof
A technology of chitinase and coding gene, which is applied in the field of functional enzymes, can solve problems such as lack, and achieve the effects of high degradation efficiency, good temperature stability, and good pH stability
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Embodiment 1
[0037] Example 1 Cloning of the gene encoding chitinase SvChiAJ54
[0038] Extract bacteria Streptomyces violascens The genomic DNA is used as a template, and PCR amplification is carried out to obtain the target gene fragment—the coding gene of chitinase SvChiAJ54, as shown in SEQ ID NO.2, comprising 726 bases, and the expressed chitinase SvChiAJ54 Contains 241 amino acids, as shown in SEQ ID NO.1.
[0039] When cloning the target gene, remove the self-signal peptide and use the carrier signal peptide. The specific primers used for PCR amplification are as follows:
[0040] Upstream primer: 5'-ctcagctgcaGATACCCGCGCCGCCGCCGACGACTTCATC-3', as shown in SEQ ID NO.3.
[0041] Downstream primer: 5'-catcatcagtggtggtggtggtggtgGCAGCTGAGGTTGTCGCCGGG-3', as shown in SEQID NO.4.
[0042] The reaction system for PCR amplification is: 2×PCR Buffer 25 μl, dNTP 10 μl, sterile water 10 μl, upstream and downstream primers 1.5 μl each, template 1 μl, KOD Fx enzyme 1 μl, total system 50 μl....
Embodiment 2
[0044] Example 2 Expression and purification of chitinase SvChiAJ54
[0045] The target gene fragment was seamlessly connected to the pP43NMK vector, and the recombinant plasmid was transferred into Bacillus subtilis Bacillus subtilis In WB800 competent cells, positive transformants were obtained by screening with LB medium solid plate containing kanamycin. After colony PCR verification, positive clones were picked and sequenced, and the correctly sequenced recombinant plasmid was extracted and named pP43NMK-SvChiAJ54.
[0046] containing recombinant plasmids Bacillus subtilis The WB800 strain was inoculated into LB liquid medium, cultivated at 37°C for 12 hours, then inserted into 50 mL of LB liquid medium at 1% of the inoculum, and cultured at 30°C and 220 r / min shaker for 45 hours to obtain a culture solution. . The culture medium was centrifuged at 4°C and 8000 r / min for 15 min, and the supernatant was collected, which was the crude enzyme solution.
Embodiment 3
[0048] Example 3 Enzymatic activity assay of chitinase SvChiAJ54
[0049] The Enzymatic Activity of Crude Chitinase SvChiAJ54 and the Specific Enzyme Activity of Pure Enzyme
[0050] Take 20 μL of crude enzyme solution and pure enzyme solution and add them to 180 μL of colloidal chitin solution (concentration of colloidal chitin is 0.01 g / ml) prepared with 50 mM phosphate buffer (pH 6.0). , react at 45°C for 10 min, boil for 5 min to terminate the reaction, add 300 μL of DNS reagent in a boiling water bath for 10 min for color development, cool on ice for 5 min, add 1 mL of deionized water, take 200 μL of it and measure its absorbance at OD540nm value. Enzyme activity was defined as the amount of enzyme required to produce 1 μmol of reducing sugar per min under standard conditions. The enzyme activity of the crude enzyme solution was determined to be 4700 U / L, and the enzyme activity of the pure enzyme solution was 184.31 U / mg.
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