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目的对产壳聚糖酶菌株Y116进行菌种鉴定,并提高菌株Y116壳聚糖酶的产量。方法对产壳聚糖酶菌株Y116进行了形态学观察、生理生化特征鉴定和16SrDNA序列同源性分析;采用单因素法和响应面法,对菌株Y116产酶的培养基成分和培养条件进行优化。结果及结论菌株Y116归属于芽孢杆菌属(Bacillus sp.)。单因素实验中,酶活力达到最高值时各因素的数值分别为:乳糖35g/L,豆饼粉30g/L,Mg~(2+)2mmol/L,NaCl 5g/L,初始pH 5.0,接种量5%,培养温度30℃和发酵时间96h。利用Plackett-Burman(PB)设计对影响壳聚糖酶产量的8个主要因素进行评价,确定了豆饼粉浓度、初始pH和Mg~(2+)浓度是影响菌株产酶量的3个主要因素;利用中心组合设计(CCD)及响应面分析,最终得到3个主要因素的最优值:豆饼粉浓度39.43g/L,pH 5.94,Mg~(2+)浓度2.05mmol/L,酶活力比优化前提高了5倍。复使用10次后,其相对酶活仍为42.3%,说明具有良好的批次操作稳定性。该酶经过固定化后热稳定性和pH稳定性均得到了提高。
Objective To identify the strain producing chitosanase Y116 and increase the yield of strain Y116 chitosanase. Methods The morphological observation, physiological and biochemical characteristics identification and 16S rDNA sequence homology analysis were carried out on the strain of chitosanase Y116. The composition and culture conditions of strain Y116 were optimized by single factor method and response surface methodology . Results and Conclusion The strain Y116 belongs to Bacillus sp. In the single factor experiment, the maximum values of enzyme activity were as follows: lactose 35g / L, soybean meal 30g / L, Mg 2+ 2mmol / L, NaCl 5g / L, initial pH 5.0, 5%, culture temperature 30 ℃ and fermentation time 96h. Eight major factors influencing the yield of chitosanase were evaluated by using Plackett-Burman (PB) design. The results showed that the concentration of soybean meal, the initial pH and the concentration of Mg 2+ were the three main factors affecting the yield of chitosanase The optimal combination of CCD and response surface analysis was used to obtain the optimal values of three main factors: soybean meal concentration 39.43g / L, pH 5.94, Mg 2+ concentration 2.05mmol / L, enzyme activity ratio Optimized before the increase of 5 times. After 10 cycles, the relative enzyme activity was still 42.3%, indicating good batch stability. The enzyme has been immobilized after the thermal stability and pH stability have been improved.