CAJ | 학술논문

从福建省永泰县温泉采集样品中筛选到1株产耐热木聚糖酶嗜热菌株TC-W7,并获得该木聚糖酶基因。在此基础上,采用易错PCR技术在木聚糖酶基因中引入突变,研究Mg2+浓度、Mn2+浓度、dTTP/dCTP浓度等条件对突变率的影响。通过形态特征、生理生化试验及16S rRNA序列相似性比对分析,初步鉴定菌株TC-W7为土壤芽胞杆菌(Geobacillus),菌株TC-W7在最适温度75℃和 pH 8.2条件下,其木聚糖酶活力为215.83 U/mL,Triton X-100和DDT能显著增强该酶的活性。在 Mg2+浓度为20μmol/L,Mn2+浓度为0.80μmol/L,dTTP/dCTP浓度为0.30 mmol/L的致突变条件下,碱基突变率为0.98%。 Geobacillus sp. TC-W7产木聚糖酶具有较好的耐热和耐碱等工业应用特性,对该酶易错PCR致突变条件优化结果,可用于后续木聚糖酶的耐热定向进化。
종복건성영태현온천채집양품중사선도1주산내열목취당매기열균주TC-W7,병획득해목취당매기인。재차기출상,채용역착PCR기술재목취당매기인중인입돌변,연구Mg2+농도、Mn2+농도、dTTP/dCTP농도등조건대돌변솔적영향。통과형태특정、생리생화시험급16S rRNA서렬상사성비대분석,초보감정균주TC-W7위토양아포간균(Geobacillus),균주TC-W7재최괄온도75℃화 pH 8.2조건하,기목취당매활력위215.83 U/mL,Triton X-100화DDT능현저증강해매적활성。재 Mg2+농도위20μmol/L,Mn2+농도위0.80μmol/L,dTTP/dCTP농도위0.30 mmol/L적치돌변조건하,감기돌변솔위0.98%。 Geobacillus sp. TC-W7산목취당매구유교호적내열화내감등공업응용특성,대해매역착PCR치돌변조건우화결과,가용우후속목취당매적내열정향진화。
A heat-resistant xylanase-producing bacterial strain TC-W7 from samples collected in a hot spring in Yong-tai County, Fujian Province was screened and obtained xylanase gene of the strain. Based on these an error-prone PCR ( Ep-PCR) technique was adopted to introduce mutation in the xylanase gene, to study the effects of the concentration such as Mg2+, Mn2+ and dTTP/dCTP and other conditions on the mutation rate. It was initially identified that strain TC-W7 belonged to Geobacillus through morphology features, physiological and biochemical tests as well as 16S rRNA sequence comparative analysis. Under the most suitable temperature 75℃ and pH 8. 2, the activity of xylanase was at 215. 83 U/mL, Triton X-100 and DDT could remarkably increase the activity of xylanase. The base mutation rate was at 0. 98% under the mutagenic conditions of 20. 0 μmol/L Mg2+, 0. 80 μmol/L Mn2+ and 0. 30 mmol/L dTTP/dCTP. The xylanase-producing Geobacillus sp. TC-W7 had a fine heat and alkali resistance and other industry appli-cable features. The results of Ep-PCR mutagenic conditions optimization of the enzyme can be used for the following-up heat-resistance oriented evolution of the xylanase.