CAJ | 학술논문

通过RT—PCR方法克隆得到Candidatropicalis木糖醇脱氢酶基因xyl2，将该基因连入酵母表达载体pYES2的诱导型启动子GAL1下，构建表达质粒pYES2-xyl2；同时用从Pichiapastoris中克隆获取的甘油醛磷酸脱氢酶基因GAP换下GAL1基因，构建含组成型启动子GAP基因的表达质粒pYES2-GAP—xyl2；通过电转化法将其依次转入酿酒酵母S．cerevisiaeINVSc1，山梨醇培养基上筛选的转化子经木糖醇梯度驯化培养，筛选出1株耐木糖醇浓度为20％的酿酒酵母重组菌株ZCX4和1株在半乳糖诱导下耐木糖醇浓度为15％的重组菌株YDX2。酶活测定表明。重组菌株ZCX4比酶活0．621U／mg（蛋白），是YDX2比酶活的2．29倍。摇瓶发酵结果显示，重组菌株ZCX4木糖醇消耗76．46g/L，木糖醇消耗率为76．46％，是重组茵株YDX2木糖醇消耗率的1．63倍，说明木糖醇脱氢酶实现了高效表达。
통과RT—PCR방법극륭득도Candidatropicalis목당순탈경매기인xyl2，장해기인련입효모표체재체pYES2적유도형계동자GAL1하，구건표체질립pYES2-xyl2；동시용종Pichiapastoris중극륭획취적감유철린산탈경매기인GAP환하GAL1기인，구건함조성형계동자GAP기인적표체질립pYES2-GAP—xyl2；통과전전화법장기의차전입양주효모S．cerevisiaeINVSc1，산리순배양기상사선적전화자경목당순제도순화배양，사선출1주내목당순농도위20％적양주효모중조균주ZCX4화1주재반유당유도하내목당순농도위15％적중조균주YDX2。매활측정표명。중조균주ZCX4비매활0．621U／mg（단백），시YDX2비매활적2．29배。요병발효결과현시，중조균주ZCX4목당순소모76．46g/L，목당순소모솔위76．46％，시중조인주YDX2목당순소모솔적1．63배，설명목당순탈경매실현료고효표체。
Yeast expression vector pYES2-xyl2 was constructed by cloning xylitol dehydrogenase gene xyl2, which originated from Candida tropicalis and placed under the inducible promoter GALl of the vector. Meanwhile, the other yeast expression vector pYES2-GAP-xyI2 containing the constitutive strong promoter GAP gene instead ofGAL gene was constructed. The plasmids containing xyl2 gene were transformed into industrial strain of S.cerevisiae INVScl by electroporation. The recombinant transformants ZCX4 and YDX2 grew well on plates in condition of high-concentration xylitol. The xylitol dehydrogenase specific activity of recombinant strain ZCX4 was 0.621 U/mg protein, 2.39 times as much as the recombinant strain YDX2, In addition, flask-shaking fermentation results revealed that the consumption of xylitol for ZCX4 was 76.46 g/L, 1.63 times as much as the recombinant strain YDX2. The results demonstrated that the recombinant stain could utilize xylitol efficiently by xylulose fermentation.