海洋源枯草芽孢杆菌生产壳聚糖酶的发酵工艺研究

Optimization of fermentation conditions for chitosanase production by Bacillus subtilis LC1-1

目的 对1株海洋源产非诱导型壳聚糖酶的枯草芽孢杆菌LC1-1的液体发酵产酶条件进行筛选优化,并在中试水平的50 L液体深层通气发酵实验中验证摇瓶实验结果的可行性与可放大性。方法 采用Minnimum-Run Equireplicated Res Ⅳ设计和中心复合设计(Central Composite Design)对培养基成分及发酵条件进行优化。结果 Minnimum-Run Equireplicated Res Ⅳ实验设计与统计学分析表明:蔗糖浓度和发酵温度是影响LC1-1产壳聚糖酶的2个显著性因素。以壳聚糖酶活力为响应值,对2个显著性因素进行中心复合设计,并经响应面法优化分析得到影响酶活性的二阶模型,确定了壳聚糖酶的最优产酶条件为:蔗糖浓度22.7 g/L,胰蛋白胨15.0 g/L,KHPO2.0 g/L,KHPO1.0 g/L,NaCl 5.0 g/L,MgSO·7 HO 0.7 g/L,酵母粉2.0 g/L,p H 5.5,发酵温度34℃,装液量20%,转速180 r/min,接种量2%,发酵周期20 h。在该条件下测得壳聚糖酶活力为(32.23±0.32)U/mL。在50 L发酵罐中对摇瓶的优化结果进行中试水平工程化验证,发酵17 h得到最高酶活力为39.9 U/mL,证实了摇瓶优化条件的可重现性及可放大性。结论 通过优化菌株LC1-1的发酵条件,提高了菌株产酶能力,并在50 L发酵罐中对摇瓶优化结果进行中试水平的验证,为进一步工程化生产壳聚糖酶提供了参考依据。

Objective To optimize the conditions for the submerged production of chitosanase by Bacillus subtilis LC1-1 and verify the feasibility and magnification of the shaking flask test results in the 50 L liquid deep aerated fermentation experiment at the pilot level. Methods The Minnimum-Run Equireplicres Ⅳ Design and Central Composite Design were used to optimize the medium composition and fermentation conditions. Results Sucrose concentration and fermentation temperature were the two most significant factors affecting chitosanase activity. To pinpoint optimal conditions for chitosanase production, followed analysis were performed by use of the central composite design together with the response surface methodology.The optimal conditions for chitosanase production were determined by using medium containing Sucrose concentration 22.7 g/L, Peptone 15.0 g/L, KHPO2.0 g/L, KHPO1.0 g/L, NaCl 5.0 g/L, MgSO·7 HO 0.7 g/L, yeast 2.0 g/L, pH 5.5, and with fermentation temperature at 34 ℃, filling ratio of 20%, rotation speed at 180 r/min, inoculation amount set at 2%, and fermentation conducted for 20 h, respectively. Under the optimized conditions, a decent level of chitosanase activity of(32.23 ± 0.32) U/mL was achieved. The set of optimized conditions were further verified by scaling up to pilot level through submerged fermentation by using 50 L fermenter, which resulted in a chitosanase activity amounting to 39.9 U/mL. These results exemplified the success of the optimized fermentation conditions and the scalability of the optimal conditions as aforementioned. Conclusion By optimizing the fermentation conditions of strain LC1-1, the enzyme production capacity of strain LC1-1 was improved, and the pilot test was carried out to verify the optimization results of shaking flask in a 50 L fermentation tank, which provided a reference for further engineering production of chitosanase.