Using Site-Directed Mutagenesis Techniques To Improve Xylanase's Thermostability

This research is using site-directed mutagenesis techniques to improve xylanase thermosability. The site-directed mutagenesis is generallyuses in the molecular biology and the biochemistry effective method. Along with the PCR technology development, mutagenesis opened a new way for the site-directed mutagenesis. PCR lies between site-directed mutagenesis which leads mutagenesis with the traditional site-directed mutagenesis to compare, does not need to prepare the M13 single chain bacteriophage carrier, Reduces time which the experiment needs, and may make the mutantmassively to expand increases, thus enhances mutagenesis the efficiency. If must suddenly dislodge the spot to be located the DNA beginnings andends, may straight take over the use of the sudden change to directthe thing to carry on PCR, obtains the mutant; If must suddenlydislodge not in the DNA beginnings and ends, then uses overlap extendsthe PCR law to carry mutagenesisThe xylanase can degrade the Xylan to become the xylose or the oligoxylose. It has the wide spread use in the feed, the paper making, food as well as in the energy industry. To the enzyme member transformation, for several dozens years work all focuses on two aspects, one is based on the sequence rationalization design proposal, like chemical modification and so on; Two, use gene feasibility, simulation nature evolved advancement non-reasonable design proposal, like directional evolution and so on. Increases the protein with the non-own two sulfur bridges the stable viewpoint first to propose by villifranca ettt.al that, latter has many protein to introduce two sulfur bridges. Increases the protein with the non-own two sulfur bridges the stable viewpoint first to propose by villifranca ettt.al that, latter has many protein to introduce two sulfur bridges. The xylanase core region is extremely stable, in nearness invariable alpha screw C end construction disulphide group, possibly can enhance the xylanase member the thermostability. Through in the protein N terminal nearby remnant base replacement and the embedment change, introduces two sulfur bridges, enable the xylanase the stability to obtain the very big enhancement, simultaneously also may obtain the expansion through the sudden change under the alkalinity pH active scope.This laboratory clone obtains xylanase gene DQ147775 with xylanasegene AY536638 which already reported only has 3 nucleotides to bedifferent, the two homology reaches as high as above 99%, But DQ147775 and AY536638 compare in 3 ends few five alkali base butis incomplete. In carries on protein Blast, DNA Blast and in themassive reference analysis foundation to xylanase gene DQ147775. Designed 6 sudden changes through Primer Premier5.0 and Oligo6.0 to direct the thing.In the premer A₀ to join five alkali bases, makes up the uneven632 - 636, 5 alkali base, increased serine and a termination. The second pair premer: S₁ in original protein sequence 3dand 5 (position increase cysteine (3C/5C). A₁ increases cysteine inoriginal protein sequence 212th (212C). The third pair premer: In original protein sequence 39th glycine replace for cysteine (G39 C). Upstream premer and the downriver premer to have 20 alkali bases is overlap.Through OE-PCR obtains 1 to have 141alkali bases, the piece 2 has 525 alkali bases.Through the combination, expands with OE-PCR and PCR increases obtains8 mutants gene. The mutant gene cuts, the enzyme company, the transformation after the enzyme. Swims the appraisal after the electricity, the mutant gene truly clones to material particle pYES6on. Will include the mutant gene the reorganization material particleto transform into in the Saccharomyces cerevisiae.Carries on the induction expression in the Saccharomyces cerevisiae. Take the spatial material particle as the comparison, take the complete enzymeas the standard, in take does not lose the enzyme to live screens the thermostble best mutant as the premise under. Screens mutant 8(3C/5C/G39C/212C).In Saccharomyces cerevisiae the highly effective expressionreorganization enzyme optimum temperature is 55℃, most suitable pHis 4.0, in 55℃half-life time is 15Min. In 60℃half-lifetime was 5Min, the optimum temperature enhanced 5℃. The mostsuitable pH change is not big, the thermostability had theenhancement, the xylanase enzyme vigor with makes up Qi Qianbi to enhance 9%.