产纤维素酶新菌株的筛选及其产酶特性研究

Breeding of a Novel Cellulase-producing Bacterial Strain and Characters Analysis of Its Cellulase

关键词:  梭形芽孢杆菌  纤维素酶  新菌株  CMC酶活力

Key words:  Lysinibacillus fusiformis  cellulase  novel strain  CMCase activity

摘要:

[目的]筛选分离新的纤维素酶产酶菌株,并对其生长特性和产酶特性进行研究。[方法]从堆肥样品中分离得到一株产纤维素酶菌株,定名为CP1,利用16S rRNA序列对比和Biolog微生物鉴定系统进行鉴定。通过测定菌株生长速率确定其最适生长条件。采用CMC发酵培养基进行纤维素酶的研究,确定其产酶曲线。测定粗酶液最适作用温度和pH值,热稳定性和金属离子对其影响。[结果]经鉴定该菌株为梭形芽孢杆菌(Lysinibacillus fusiformis),其最适生长温度为30℃,最适生长pH值为6。在含CMC-Na的培养基培养,该菌株的生长与产酶同步进行,培养48h菌株生长量达到最大,培养液的CMC酶活力同时达到最大值,为0.46U/mL。该菌株所产的纤维素酶既有酸性CMC酶活力,又有碱性CMC酶活力,并以碱性CMC酶为主,酸性CMC酶的活力只有碱性CMC酶的71.4%。其酸性CMC酶的最适作用pH值为6.0,碱性CMC酶的最适作用pH值为8.0。另外,该菌株所产碱性CMC酶活的最适作用温度为40~50℃,而且0.5%的Cu²⁺使其酶活降低45%。[结论]菌株CP1为首次报道的梭形芽孢杆菌产纤维素酶新菌株,具有独特的酸碱性环境下的纤维素酶活力。

Abstract:

[Objective] Screening and isolating novel cellulase producing strains, and studying on grwoth property and characters analysis of the cellulase.[Method] A novel endoglucanase-producing strain, defined as CP1, was isolated from composite heaps samples collected in rural area of Nanning, Guangxi Province, China. The strain was identified by 16S rRNA alignment and Biolog microbial identification system.Optimal growth conditions were determined by growth rate.Studies on the cellulase were carried out by fermentation in CMC fermentation medium and CMCase activities were determined by time.Optimal pH and temperature of enzyme activity, thermol resistance as well as influences of metal ions on the crude CMCase activities were assayed.[Result] The strain was identified as Lysinibacillus fusiformis.The optimized temperature and pH for the growth of strain CP1 were 30℃ and pH6.0, respectively. The growth and endoglucanase-production of strain CP1 were synchronous that the maximum growth with absorption at OD600 reaching to 1.29 and the maximum CMCase activity of 0.46U/mL were simultaneously occurred at culturing for 48h. The endoglucanase produced by strain CP1 exhibited CMCase activity both in acid range and in alkaline range. The alkaline CMCase was the major endoglucanase. The activity of acid CMCase was only 71.4% of the alkaline CMCase. The optimal pH was pH6 for acid CMCase and pH8.0 for alkaline CMCase. Furthermore, the alkaline CMCase activity of endoglucanase produced by strain CP1 was decreased by 45% in addition of 0.5% Cu²⁺.[Conclusion] The strain was a novel cellulase producing strain first reported from Lysinibacillus fusiformis, and its cellulase had CMCase activities at wide pH range across acidic to alkaline conditions.