A method for improving the xylobiose production of xylanase, mutants and applications
A technology of xylanase and xylobiose, which is applied in the field of agricultural biology and can solve problems such as differences in the ability to produce xylobiose
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Embodiment 1
[0033] Example 1 Preparation of xylanase mutant recombinant vector pET-28a(+)-xyna-N209H / M245W with improved xybiose yield.
[0034] The xylanase wild-type (before mutation) sequence fragment was cloned into the expression vector pET-28a(+), and the recombinant vector was named pET-28a(+)-xyna; the recombinant vector pET-28a(+)-xyna was used as the template , and amplify it by primers carrying the mutation site to obtain a recombinant vector carrying the mutant sequence. First, use primers N209H-F and N209H-R to construct mutant plasmid pET-28a(+)-xyna-N209H, after sequencing is correct, use this as template, use primers M245W-F and M245W-R to construct mutant plasmid pET-28a(+ )-xyna-N209H / M245W.
[0035] The specific primers for the xylanase mutant pET-28a(+)-xyna-N209H / M245W that improve xylobiose production are:
[0036] N209H-F: (SEQ ID NO:5),
[0037] N209H-R: (SEQ ID NO: 6);
[0038] M245W-F: (SEQ ID NO:7),
[0039] M245W-R: (SEQ ID NO:8).
[0040] Example 2 Prepa...
Embodiment 3
[0045] Example 3 Determination of enzymatic properties
[0046] (1) Determination of optimal temperature of xylanase mutant and wild type
[0047] The optimum temperatures of the mutants XynA-N209H / M245W and XynA were determined at different temperatures (15, 20, 25, 30, 35, 40) in a 100 mM citric acid-disodium hydrogen phosphate buffer (pH 6.5) buffer system. , 45, 50, 55, 60 °C) for the remaining enzyme activity determination. Define the highest enzyme activity as 100%, and calculate the relative enzyme activity at each temperature to determine the optimal temperature of the enzyme, such as figure 2 The optimum temperature for both the xylanase mutant and wild type is shown to be 40°C.
[0048] (2) Determination of optimal pH of xylanase mutant and wild type
[0049]Prepare the substrate solution with 100mM citric acid-disodium hydrogen phosphate buffer (pH2.5-7.0) and 50mM Tris-HCl (pH8.0-9.0), at 40℃, pH2.5-9.0 The enzyme activities of the xylanase mutant and the wild...
Embodiment 4
[0054] Example 4 The yield of xylobiose detected by HPAEC-PAD
[0055] Add 900 µL of 1% beech xylan and 100 µL of the purified wild-type or mutant enzyme solution obtained in Example 2 into a 2 mL EP tube (both in an amount of 50 U / mL), the reaction temperature is 37 ° C, pH 6. 5. The reaction was carried out in a water bath shaker at 150 rpm for 2 hours. After the reaction was completed, the enzyme was inactivated by boiling for 10 min, centrifuged at 12,000 rpm for 10 min, the supernatant was collected, and filtered with a 0.22 um filter. The obtained filtrate was used as a sample to measure the product by HPAEC-PAD. content.
[0056] The conditions of HPAEC-PAD chromatographic analysis and detection: chromatographic column Dionex CarboPac PA-100 (4 × 250-mm), mobile phase is 1M NaOH solution.
[0057] Test results such as Image 6 , Figure 7 As shown, the xybiose yield of the double point mutant was 6869 nm, and the xynA-N209H / M245W had a 15% increase in xylobiose yield...
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