该研究旨在挖掘更符合工业化生产的木聚糖酶,提升木聚糖高值转化效率,推动生物能源化使用。先以木聚糖为唯一碳源的固体培养基筛选能产木聚糖酶的菌株,然后通过形态学和分子生物学鉴定木聚糖酶生产菌株的归属,进一步优化菌株的发酵产酶条件,最后对菌株所产木聚糖酶的酶学性质进行研究。以木聚糖酶活力最高的赖氨酸芽孢杆菌属XYL1菌株作为研究对象,研究发现最适碳源为细麸皮,最适氮源为(NH4)2SO4,最适发酵培养温度、pH和培养周期分别为35 ℃、8.0和48 h。最后研究了木聚糖酶的酶学性质,得出其最适催化温度和pH值为55 ℃和7.0。在30~45 ℃和pH 5.0~8.0时保温1 h仍保持较高的酶活力,分别达到96%和72%以上;金属离子Ni2+、Mg2+、Ca2+、Zn2+、Cu2+、Mn2+对酶反应都有一定的促进作用,而Fe2+、Fe3+、Co2+和十二烷基磺酸钠对酶有严重的抑制作用;乙二胺四乙酸对酶反应有轻微的抑制作用,表明该菌所产木聚糖酶可能不是金属酶。反应动力学研究表明,木聚糖酶催化木聚糖水解的Km为 27.23 mg/mL,Vmax为0.071 mg/(mL·min)。研究结果为XYL1菌株产木聚糖酶在工业生产中的应用奠定了基础。
The purpose of this study is to explore xylanase more in line with industrial production, improve the high-value conversion efficiency of xylan, and promote the use of bioenergy.Firstly, xylanase producing strains were screened in solid medium with xylan as the only carbon source.Then, the attribution of xylanase producing strain was identified by morphology and molecular biology, and the fermentation conditions of the strain were further optimized.Finally, the enzymatic properties of xylanase produced by the strain were studied.The XYL1 strain of Lysinibacillus with the highest xylanase activity was taken as the research object.It was found that the optimum carbon source was fine bran, the optimum nitrogen source was ammonium sulfate, and the optimum fermentation culture conditions of temperature, pH and culture period were 35 ℃, 8.0, and 48 h, respectively.Finally, the enzymatic properties of xylanase were studied.The optimal catalytic reaction conditions of temperature and pH were 55 ℃ and 7.0.The enzyme activity remained relatively high even after being incubated for 1 h within the range of 30~45 ℃ and pH 5.0~8.0, reaching over 96% and 72%, respectively.Metal ions Ni2+, Mg2+, Ca2+, Zn2+, Cu2+ and Mn2+ had a certain promoting effect on enzyme reactions, while Fe2+, Fe3+, Co2+ and sodium dodecyl sulfate(SDS) had a serious inhibitory effect.Ethylene diamine tetraacetie acid(EDTA) had a slight inhibitory effect on enzyme reactions, indicating that the xylanase produced by this bacterium may not be a metalloenzyme.Reaction kinetics studies had shown that the Km and Vmax of xylanase catalyzed hydrolysis of xylan were 27.23 mg/mL and 0.071 mg/(mL·min), respectively.The results lay a foundation for the application of xylanase produced by XYL1 strain in industrial production.
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