淀粉酶高产菌株的筛选、紫外诱变及产酶条件优化

Screening, UV mutagenesis, and production optimization of a strain with high yield of amylase

摘要:

【背景】提高淀粉酶的稳定性进而适应多变的工业生产条件，是淀粉酶开发的重要方向。【目的】淀粉酶在食品加工、布料退浆、酿酒制造、养殖等领域都有广泛的应用，但目前生产用的淀粉酶来源单一，在溶液中的稳定性较差，需要扩大淀粉酶来源，增强酶的稳定性，使其进一步适应复杂多变的工业生产环境。【方法】利用选择培养基在三门峡黄河湿地表层土样中筛选得到20株具有淀粉酶活性的菌株，采用杯碟法检测粗酶液的活性并对其中活性最高的3株菌进行鉴定，对其中酶活性最高的菌株运用紫外照射的方式进行诱变并测定致死率，选择突变后酶活最高的菌株，优化培养条件，并测定突变后淀粉酶的活性和作用条件范围。利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid，DNS)法测定诱变前后酶活并进行活性对比。【结果】在三门峡黄河湿地表层土壤中筛选得到3株淀粉酶活性较高的菌株并编号S03、S08和S17。根据16S rRNA基因序列比对、革兰氏染色形态观察结合生理生化分析显示，菌株S03、S08和S17分别为Aeromonas、Exiguobacterium和Bacillus，其淀粉酶最适NaCl浓度是10%−12%，最适作用温度为45℃(S03、S17)和25℃(S08)；发酵36 h后，菌株S03和S17的淀粉酶活性达到最大值，菌株S08最佳的发酵时间为42 h；淀粉酶最适pH为7.0−9.0。利用紫外诱变筛选到酶活性最高的菌株并编号S17M。在pH为8.0，温度为40℃时S17M生长情况较好；Mn²⁺、Ti⁴⁺和Mg²⁺对菌株的生长具有促进的作用，Cu²⁺、Fe²⁺、Fe³⁺和Na⁺会抑制菌株的生长；有机溶剂会抑制菌株生长，其中甲醛和冰乙酸的抑制作用明显；经紫外诱导筛选优化后，菌株S17M淀粉酶在NaCl浓度为8%−14%，pH 5.0−10.0，15−55℃的温度范围内都具有较高的酶活性，金属离子和有机溶剂的抑制作用减弱，最大酶活性可以达到195.62 U/mL，是原始菌株的3.19倍。【结论】利用紫外诱变的方式可以显著提高酶活性和稳定性，更适合环境条件变化剧烈的工业生产环境，具有应用于不同工业生产条件的潜力。

Abstract:

[Background] It is an important goal for amylase development to improve the stability under variable industrial production conditions. [Objective] Amylase is widely used in food processing, desizing of cloth, brewing, and breeding industry. However, current amylase in industrial production features a few sources available and poor stability in solution. Thus, efforts should be made to expand the sources of amylase and enhance the stability of the enzyme so that it can adapt to the complex and changeable production environment. [Methods] A total of 20 strains with amylase activity were screened out from the surface soil of the Yellow River Wetland in Sanmenxia with a selective medium. The activity of the crude enzyme solution was detected with the cylinder plate method and 3 strains with high amylase activity were preliminarily identified. Among them, the strain with highest amylase activity was mutagenized by ultraviolet (UV) and the lethality rate was determined. The strain with the highest enzyme activity after mutagenesis was selected. Then the culture conditions were optimized, and the activity of amylase after mutation and the ranges of action conditions were determined. 3,5-dinitrosalicylic acid (DNS) method was used to determine the enzyme activity before and after mutation for a comparison. [Results] The 3 strains with high amylase activity were numbered S03, S08, and S17. The 16S rRNA gene sequence alignment, Gram staining, and physiological and biochemical characterization revealed that strains S03, S08, and S17 are members of Aeromonas, Exiguobacterium, and Bacillus, respectively. The optimal NaCl concentration for the amylase was 10%−12%, and the optimal temperature was 45 °C (S03, S17) and 25 °C (S08). Moreover, the optimal pH was 7.0−9.0, and the optimal fermentation time was 36 h (S03 and S17) and 42 h (S08). After UV mutagenesis, the strain with the highest enzyme activity was screened out and marked as S17M. S17M grew well at pH of 8.0 and 40 °C. Mn²⁺, Ti⁴⁺, and Mg²⁺ promoted the growth of S17M, while Cu²⁺, Fe²⁺, Fe³⁺, Na⁺, and organic solvents (particularly formaldehyde and glacial acetic acid) inhibited it. S17M demonstrated high amylase activity in the presence of NaCl at 8%–14%, at pH of 5.0–10.0, and at 15–55 °C, and the inhibitory effect of metal ions and organic solvents was decreased. The maximum enzyme activity was up to 195.62 U/mL, 3.19 folds that of the original strain. [Conclusion] UV mutagenesis can significantly improve enzyme activity and stability, so that the resultant enzyme can adapt to the variable industrial production environments. Thus, it has the potential to be applied to different industrial production conditions.