A novel high-efficiency β-glucosidase csbgl and its coding gene and application
A technology of glucosidase and stevioside, which is applied in biology, beta-glucosidase CsBGL and its encoding gene and application field, can solve the problems of low efficiency, low enzyme yield, poor specificity, etc., and achieves operational production. The method is simple, the catalytic specificity is strong, and the effect of good industrialization prospects
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Embodiment 1
[0045] Example 1: Preparation of crude enzyme solution of β-glucosidase CsBGL
[0046] Chryseobacterium sp. 1433 was inoculated into LB antibiotic-free medium, cultured at 30° C. and 200 rpm for 24 h, and then centrifuged at 3,000-10,000 rpm for 2-10 min to collect bacterial pellets. With 50mM Na at pH 6.0-8.0 2 HPO 4 / NaH 2 PO 4 The cells were resuspended in the buffer, and then centrifuged at 3000-10000rpm for 2-10min to collect the cell pellet again, and then use 10-20mL pH 6.0-8.0 50mM Na 2 HPO 4 / NaH 2 PO 4 The cells were resuspended in the buffer, disrupted by ultrasonic waves, centrifuged at 12,000 rpm for 20 min, and the supernatant was collected as the crude β-glucosidase CsBGL enzyme solution.
Embodiment 2
[0047] Example 2: Native-PAGE electrophoresis of β-glucosidase CsBGL crude enzyme solution under acidic conditions
[0048] The β-glucosidase CsBGL crude enzyme solution obtained in Example 1 was subjected to Native-PAGE electrophoresis. The specific parameters of the electrophoresis were: the positive and negative electrodes were inverted, traced with methyl green (0.002%), and a constant current of 10 mA at 4°C was used. Electrophoresis 2 ~ 3h.
[0049] Native-PAGE gels for acidic conditions are formulated as follows:
[0050] 1) Separating gel: 0.06M KOH, 0.376M HAc, pH 4.3 (7.7% gel concentration, acrylamide:methacrylamide is 37.5:1);
[0051] 2) Stacking glue: 0.06M KOH, 0.063M HAc, pH 6.8 (3.125% glue concentration, acrylamide:methacrylamide is 3:1);
[0052] 3) Running buffer: 0.14M L-alanine, 0.35M glacial acetic acid, pH 4.5.
[0053] Cut out the corresponding lanes in the PAGE gel with a clean scalpel, use pH 7.4, 50 mM Na 2 HPO 4 / NaH 2 PO 4 The buffer was wa...
Embodiment 3
[0054] Example 3: Cloning of β-glucosidase CsBGL encoding gene, construction of recombinant vector and heterologous expression
[0055] 1. Extraction of genomic DNA from Chryseobacterium sp. 1433
[0056] Chryseobacterium sp. 1433 was inoculated into LB medium, cultured at 30°C and 200rpm for 24h, and then centrifuged at 3000-10000rpm for 2-10min to collect the bacterial pellet. Using the Tiangen genomic DNA extraction kit, DNA was extracted from the collected bacterial cell pellets according to the instructions to obtain the genomic DNA of Chryseobacterium sp. 1433.
[0057] 2. Identification of β-glucosidase CsBGL amino acid sequence and coding gene nucleotide sequence
[0058] The genomic DNA of Chryseobacterium sp. 1433 extracted in step 1 was sequenced.
[0059] The Native-PAGE electrophoresis activity staining band obtained in Example 2 was cut into 1mm 3 The nucleotide sequence of β-glucosidase CsBGL was determined, as shown in SEQ ID NO. .1, a total of 2271bp; the ...
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