Costa-Silva Tales A, Flores-Santos Juan Carlos, Freire Rominne K B, Vitolo Michele, Pessoa-Jr Adalberto
a Department of Pharmaceutical and Biochemical Technology, School of Pharmaceutical Sciences , University of São Paulo , São Paulo , Brazil.
Prep Biochem Biotechnol. 2018;48(8):707-717. doi: 10.1080/10826068.2018.1487850. Epub 2018 Jul 11.
The efficacy of a simple laboratory method for cell disruption based on the glass bead stirring, sonication, osmotic shock, freezing and grinding, or use of solvents and detergents was assessed in this study, via measurements of the release of total protein and L-asparaginase activity. Three different microbial sources of L-asparaginase were used: Escherichia coli BL21 (DE3), Leucosporidium muscorum, and Aspergillus terreus (CCT 7693). This study adjusted and identified the best procedure for each kind of microorganism. Sonication and glass bead stirring led to obtaining filamentous fungus cell-free extracts containing high concentrations of soluble proteins and specific activity; however, sonication was the best since it obtained 4.61 ± 0.12 IU mg after 3 min of operation time. Mechanical methods were also the most effective for yeast cell disruption, but sonication was the technique which yielded a higher efficiency releasing 7.3 IUtotal compared to glass bead stirring releasing 2.7 IUtotal at the same operation time. For bacterium, sonication proved to be the best procedure due to getting the highest specific activity (9.01 IU mg) and total enzyme activity (61.7 IU). The data presented lead to conclude that the mechanical methods appeared to be the most effective for the disintegration of the all microbial cells studies. This is the first report related to the experimental comparison of L-ASNase extraction procedures from different microorganisms, which can also be used for extracting periplasm located enzymes from other organisms.
本研究通过测量总蛋白释放量和L-天冬酰胺酶活性，评估了一种基于玻璃珠搅拌、超声处理、渗透休克、冷冻研磨或使用溶剂和去污剂的简单细胞破碎实验室方法的效果。使用了三种不同的L-天冬酰胺酶微生物来源：大肠杆菌BL21(DE3)、苔藓丝孢酵母和土曲霉(CCT 7693)。本研究调整并确定了每种微生物的最佳方法。超声处理和玻璃珠搅拌可获得含有高浓度可溶性蛋白和比活性的丝状真菌无细胞提取物；然而，超声处理是最佳方法，因为在3分钟的操作时间后，它获得了4.61±0.12IU mg。机械方法对酵母细胞破碎也最有效，但超声处理是效率更高的技术，在相同操作时间下，超声处理释放的总酶量为7.3IU，而玻璃珠搅拌释放的总酶量为2.7IU。对于细菌，超声处理被证明是最佳方法，因为它具有最高的比活性(9.01IU mg)和总酶活性(61.7IU)。所呈现的数据表明，机械方法似乎对所有研究的微生物细胞破碎最有效。这是第一份关于不同微生物L-天冬酰胺酶提取方法实验比较的报告，该方法也可用于从其他生物体中提取周质定位的酶。
