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为解决现今纤维素酶生产的低效率和高成本问题,以及实现利用木质纤维素废弃物生产纤维素酶的目的,本研究结合羧甲基纤维素钠(CMC)水解圈法和胞外酶活测定法,从嗜热菌资源丰富的温泉区土壤中筛选出一株具有产羧甲基纤维素酶(CMCase)的嗜热地芽孢杆菌HTA426(Geobacillus kaustophilus HTA426).对菌株生产的酶进行酶学性质分析并将菌株应用于以木质纤维素为替代碳源产纤维素酶.采用硫酸铵沉淀法和离子交换层析法对粗酶液进行二级纯化,回收率和纯化倍数分别为10.14%和5.12,纯化后的CMCase的分子量约为40 k Da.其CMCase活性在温度为60℃,pH为7.0下达到最高.培育5 h后,酶的活性在温度为50~70℃下也能保持相对稳定.HAT426菌株能够在已经经过碱处理的甘蔗渣、稻杆及水葫芦为碳源的培养液中生长和生产CMCase.以甘蔗渣为碳源的培养液最适合于生产CMCase,其活性为103.67U·m L~(-1).菌株HTA426是首次报道的具有产纤维素酶能力的嗜热地芽孢杆菌,经纯化的纤维素酶具有广泛的pH适用性(pH 5~8)和良好的热稳定性(50~70℃),在运用于木质纤维素能源化中具有较大潜力.
In order to solve the problems of low efficiency and high cost of cellulase production, and to achieve the purpose of producing cellulase using lignocellulose waste, this study combined the carboxymethylcellulose sodium (CMC) hydrolysis cycle method and extracellular enzyme activity , A strain of Geobacillus kaustophilus HTA426 with CMCase activity was screened from the soil of the hot spring zone where the thermophilic bacterium is rich in enzyme. The nature of the strain was analyzed and the strain was applied to produce cellulase with lignocellulose instead of carbon source.The crude enzyme solution was secondary purified by ammonium sulfate precipitation method and ion exchange chromatography.The recoveries and purification times were 10.14% and 5.12, the molecular weight of the purified CMCase is about 40 kDa.The activity of CMCase reached the highest at pH 7.0 and pH 6.0 at 5 ℃, and the activity of CMCase remained relatively at 50-70 ℃ HAT426 strain can grow and produce CMCase in alkali-treated bagasse, rice straw and water hyacinth as carbon source.The CMCase is the most suitable culture medium with bagasse as carbon source and its activity is 103.67 U · m L ~ (-1). Strain HTA426 was the first reported Bacillus caldera with cellulase-producing activity. The purified cellulase had a wide range of pH applicability (pH 5-8) and good thermal stability (50-70 ℃) In the application of lignocellulose energy has greater potential.