Xylanase variants and methods
A technology of xylanase and variants, applied in the field of xylanase variants and methods
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[0206] Synthetic gene construction requires the in vitro synthesis of a polynucleotide molecule designed to encode a polypeptide of interest. Gene synthesis can be performed using a variety of techniques, such as the multiplexed microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054) and similar techniques in which oligonucleotides are synthesized and placed on an optically programmable microfluidic chip. Assemble. A preferred technique is
[0207] i. Regulatory sequence
[0208] The invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the invention operably linked to one or more control sequences which direct Expression of the coding sequence in a suitable host cell.
[0209] Polynucleotides can be manipulated in a variety of ways to provide for expression of variants. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the...
Embodiment 1
[0300] A single colony of the recombinant xylanase-encoding gene from Saccharomyces cerevisiae INVSc1 strain (ThermoFisher Scientific, USA: catalog #V8251-20) was inoculated into 96-well plates in 350 μL of a synthetic minimal culture with 2% glucose and no uracil supplement. Base. After induction, the plates were incubated overnight at 30°C, 85% humidity while shaking at 200 rpm. Determination of OD of overnight cultures 600 , and all cultures were diluted to a final OD of 0.4 600 into a fresh 96-well plate containing a total volume of 300 µL of SC selective medium supplemented with 1% galactose and 0.5% raffinose. The induction plate was incubated at 30°C, 85% humidity while shaking at 200rpm for 72h, after which the plate cells were pelleted by centrifugation at 4000rpm for 15min while cooling to 4°C. The supernatant was transferred to a fresh 96-well plate, covered, and stored at -20 °C until further use.
[0301] XIII. Example 3: Enzyme assay to determine pH 5.5 or lo...
Embodiment 2
[0302] Plates containing xylanase WT and / or variants produced according to Example 2 were thawed. The supernatant was diluted 175-fold into 0.1 M sodium acetate buffer (pH 5.5) for the activity assay at pH 5.5. Dilute the supernatant 20-80 times (depending on the plate) into 0.1 M citrate-phosphate buffer (pH 3.0) for assays measuring xylanase activity at pH 3, or dilute to pH 2. 5 HCl solution (89mmol NaCl, 6.6mmol KCl).
[0303] By dissolving 1.00 g of xylan (corn cob, Shanghai Yuanye Biotechnology Co., Ltd., supplier #S25540) and 0.320 g of sodium hydroxide in 50 mL of deionized water, then heating to 100 °C while stirring until the xylan was completely dissolved To prepare a 1% xylan solution. The xylan substrate is then adjusted to the desired pH. Use acetic acid to pH5.5, or use HCl to pH3.0 and pH2.5, then use 0.1M sodium acetate buffer (pH5.5), 0.1M citric acid-phosphate buffer (pH3.0) or pH2. 5 HCl solutions were adjusted to volume to 100mL.
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