石斛碱是传统植物金钗石斛(Dendrobium nobile)的特征药用活性成分,结构上属于石斛型生物碱(dendrobine-type total alkaloids,DTTAs)。石斛碱的积累受周期和环境的影响,仅从植物中获取难以满足需求。该研究的对象Trichoderma longibrachiatum UN32是实验室前期通过复合诱变获得的产DTTAs的正突变株。为提高UN32的生物碱产量,使产物表达水平趋于稳定,该研究从UN32发酵培养基中无机盐的添加量出发,利用响应面法确定金属离子的最优配比。根据Plackett-Burman法设计筛选出对响应结果影响程度最显著的3个因素分别为Zn2+、Cu2+、Fe2+,其最佳添加时间和添加浓度分别是Cu2+ 0.5 mmol/L(第6天)、Fe2+ 0.4 mmol/L(第6天)和Zn2+ 0.7 mmol/L(第3天)。响应面统计的结果显示,当模型响应值生物碱量达最大值时,Cu2+、Zn2+和Fe2+的浓度分别为0.54、0.69、0.39 mmol/L,此时生物碱产量达到(317.36±6.48) μg,比初始培养基提高了72.68%。综上,金属离子及其不同组合对菌株UN32生物碱的积累具有促进作用,该方法具有较好的开发价值和应用前景。
Dendrobine is the characteristic ingredient of the traditional plant Dendrobium nobile with medicinal activity, structurally belonging to the dendrobine-type total alkaloids (DTTAs).The accumulation of dendrobine is influenced by the cycle and the environment, and it is difficult to obtain from plants alone to meet the demand.Trichoderma longibrachiatum UN32, the subject of this study, is a DTTAs-producing positive mutant obtained by compound mutagenesis in the previous study. To improve the DTTAs yield of UN32 and stabilize the expression level of the product, this study determined the optimal ratio of metal ions from adding inorganic salts in the fermentation medium of UN32 using response surface methodology.The three factors that had the most significant effect on the response results were Zn2+, Cu2+and Fe2+, and the optimum addition times and concentrations of Cu2+, Fe2+, and Zn2+ were 0.5 mmol/L on the 6th day, 0.4 mmol/L on the 6th day and 0.7 mmol/L on the 3rd day, respectively.The results of the response surface statistics showed that the alkaloid production reached (317.36±6.48) μg when the model response value alkaloid amount reached its maximum at concentrations of 0.54, 0.69, and 0.39 mmol/L for Cu2+, Zn2+, and Fe2+, respectively, which was 72.68% higher than the initial medium.In summary, the metal ions and their different combinations have promoted the accumulation of alkaloids in strain UN32, and the method has good development value and application prospects.
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