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我国是世界上抗生素生产、消费和出口大国,年产量约占世界总产量的70%[1-2]。在生产抗生素的过程中,会产生大量的抗生素菌渣。据估算,每生产1 t抗生素,约产生8~10 t的抗生素菌渣,我国每年产生的抗生素菌渣约为2×106 t,产量巨大[3-4]。抗生素菌渣主要由剩余培养基、残留的抗生素、微生物代谢产物以及发酵菌丝体组成。其中,残留的抗生素会对生态环境和人群健康造成潜在的危害[5-6]。根据我国2020年发布的《国家危险废物名录》[7],抗生素菌渣属于化学原料药生产过程中产生的废弃品及中间体,全过程须按危险废物进行管理,不可随意处置。抗生素菌渣处理技术有焚烧、填埋、堆肥等。这些处理技术存在不能完全消除菌渣危害、成本高、会造成二次污染等问题。抗生素菌渣中含有丰富的蛋白质,是一种潜在的生物质能,如能将其回收利用,可较好地实现菌渣中氮源的资源化[8-9]。
菌渣中蛋白质的回收主要包括菌渣中蛋白质溶出和菌渣水解液中蛋白质的分离纯化2个过程。由于菌渣的蛋白质主要存在于菌丝体中,为了获得更多的蛋白质,就需要对菌渣进行水解[10-11]。常用的菌渣水解方法有物理法(热水解、超声波、高压均质等)、化学法(酸碱处理、臭氧氧化处理等)和生物法(酶制剂和嗜热溶胞法)。与化学、物理水解技术相比,生物法中的嗜热溶胞法是利用优势嗜热溶胞菌分泌的胞外酶(蛋白酶和淀粉酶)进行生物溶胞作用,使菌渣中的有机物溶出。该方法具有操作简单、经济环保和水解效果好等优点[12-14]。菌渣经水解后,需要从混合溶液中进一步沉淀分离出蛋白质。常用的蛋白质沉淀分离方法有等电点沉淀法、盐析法、有机溶剂沉淀法、非离子多聚物沉淀法等[15-17]。
本实验采用嗜热溶胞法水解青霉素菌渣,以青霉素菌渣水解液为研究对象,采用等电点沉淀法、盐析法(硫酸铵)、有机溶剂沉淀法(乙醇、丙酮)、选择性变性沉淀法(TCA)和非离子多聚物沉淀法(聚乙二醇)对水解液沉淀分离,并通过比较不同沉淀法的蛋白质沉淀率,选出最优的蛋白质沉淀法;然后对最优沉淀法进行单因素实验,以确定蛋白质沉淀率的影响因素及取值范围;之后,用响应曲面法选取最优沉淀法分离蛋白质的最佳工艺条件;最后,用最优工艺条件对青霉素菌渣水解液进行蛋白质分离实验验证。本实验研究结果可为菌渣的资源化处理提供参考。
沉淀法分离青霉素菌渣中蛋白质工艺的优化
Proteins from penicillin residue were isolated by trichloroacetic acid precipitation method
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摘要: 为高效回收抗生素菌渣中的蛋白质,采用不同沉淀法分离青霉素菌渣中的蛋白质。首先,考察不同沉淀法对蛋白质的沉淀效果,选出最优蛋白质沉淀方法;然后,开展单因素实验并基于响应曲面法选择分离青霉素菌渣中蛋白质的最优工艺参数;最后,利用最优工艺条件对青霉素菌渣进行蛋白质分离实验验证。结果表明,三氯乙酸(TCA)沉淀法沉淀效果最好,其分离青霉素菌渣中蛋白质的最优工艺条件为:TCA的质量分数为20%、水解液pH为3.0、沉淀时间为10 h和离心转速为10 000 r·min−1;此条件下的预测蛋白质沉淀率为89.5%,实际蛋白质沉淀率为89%。该研究结果可为菌渣蛋白质高效回收提供参考。Abstract: In order to solve the problem of protein recovery from antibiotic residue, different precipitation methods were used to separate protein from penicillin residue. Firstly, the effects of different precipitation methods on protein precipitation were investigated, and the optimal protein precipitation method was selected. Then, the single factor experiment and response surface experiment were carried out to optimize the process parameters of the optimal precipitation method for the separation of proteins from penicillin residue. Finally, proteins were isolated from penicillin residue under optimum conditions. The results showed that the best precipitation effect was obtained by trichloroacetic acid (TCA) precipitation method. The optimal extraction conditions were: mass fraction of TCA was 20%, pH of hydrolysate was 3.0, precipitation time was 10 h and centrifugation rotation was 10 000 r·min−1. Under these conditions, the precipitation rate of protein was 89.5%. Under the optimal conditions, the protein precipitation rate is 89%, and the experimental value is in good agreement with the predicted value, so that the optimal protein precipitation method can be obtained. This study can provide reference for protein recovery from fungus residue.
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表 1 青霉素菌渣理化性质
Table 1. Physical and chemical properties of penicillin bacteria residue
pH 含水率/% SCOD/(g·L−1) TCOD/(g·L−1) TSS/(g·L−1) VSS/(g·L−1) 氨氮/(g·L−1) 蛋白质/(g·L−1) 4.62 91.61 40.87 118.40 60.73 57.57 7.47 22.96 表 2 实验设计及结果
Table 2. Experimental design and results
实验号 A B C D 蛋白质沉淀率/% 1 25 3 10 12 000 84.2 2 15 3 12 10 000 78.1 3 20 4 10 8 000 84.9 4 20 4 8 10 000 83.4 5 20 3 8 12 000 86.3 6 25 3 8 10 000 81.8 7 15 4 10 10 000 73.7 8 20 3 10 10 000 87.9 9 20 2 12 10 000 83.7 10 20 3 10 10 000 90.5 11 15 3 10 8 000 75.9 12 25 3 10 8 000 84.9 13 20 4 12 10 000 83.3 14 20 3 10 10 000 89.5 15 15 3 8 10 000 71.3 16 20 2 10 8 000 83.1 17 15 3 10 12 000 77.2 18 20 3 12 12 000 87.4 19 20 2 8 10 000 77.8 20 25 4 10 10 000 82.3 21 20 3 10 10 000 89.8 22 15 2 10 10 000 70.1 23 20 3 12 8 000 86.3 24 20 2 10 12 000 82.9 25 25 3 12 10 000 84.7 26 20 3 10 10 000 89.8 27 20 4 10 12 000 85.7 28 20 3 8 8 000 85.3 29 25 2 10 10 000 79.1 表 3 蛋白质沉淀率回归方程模型的方差分析
Table 3. Analysis of variance of regression equation model of protein precipitation rate
方差来源 偏差平方和 自由度 均方 F P 模型 809.10 14 57.79 63.86 < 0.000 1 A 214.21 1 214.21 236.71 < 0.000 1 B 22.96 1 22.96 25.38 0.000 2 C 25.81 1 25.81 28.52 0.000 1 D 0.91 1 0.91 1.00 0.333 6 AB 0.040 1 0.040 0.044 0.8365 AC 3.80 1 3.80 4.20 0.059 6 0AD 1.00 1 1.00 1.11 0.3110 0 BC 9.00 1 9.00 9.95 0.007 0 BD 0.25 1 0.25 0.28 0.607 4 CD 0.002 5 1 0.002 5 0.002 763 0.958 8 A2 439.26 1 439.26 485.40 < 0.000 1 B2 155.21 1 155.21 71.52 < 0.000 1 C2 39.47 1 39.47 43.61 < 0.000 1 D2 2.57 1 2.57 2.84 0.114 2 残差 12.67 14 0.90 失拟项 8.93 10 0.89 0.95 0.569 4 纯误差 3.74 4 0.93 63.86 总计 821.77 28 -
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