Murugesan Sivananth, Iyyaswami Regupathi, Kumar Sahana Vijay, Surendran Arathi
Department of Chemical Engineering, National Institute of Technology Karnataka, Mangalore, 575025, India.
Bioprocess Biosyst Eng. 2017 Aug;40(8):1163-1171. doi: 10.1007/s00449-017-1777-z. Epub 2017 May 6.
L-Asparaginase synthesized by Azotobacter vinelandii via submerged fermentation in the presence of sucrose was successfully extracted using Reverse micellar extraction. Single step enzyme purification process was developed by varying the process variables which resulted in maximum specificity and extraction of L-asparaginase. The effect of different variables, including broth pH, addition of alcohol during the forward extraction and pH of the fresh stripping aqueous phase, addition of alcohol and electrolyte during backward extraction process were studied. Lower concentration of butanol resulted in maximum activity of the enzyme during forward extraction while enzyme activity was found to increase further with the addition of higher concentrations of ammonium sulphate during backward extraction. Chromatographic analysis of L-asparaginase peak at ~7.65 min was intense for the back extracted sample confirming the maximum purity of L-asparaginase obtained. Purity of L-asparaginase was increased to about 379.68 fold.
通过在蔗糖存在下进行深层发酵，由棕色固氮菌合成的L-天冬酰胺酶成功地采用反胶束萃取法进行了提取。通过改变工艺变量开发了单步酶纯化工艺，该工艺实现了L-天冬酰胺酶的最大特异性和提取。研究了不同变量的影响，包括发酵液pH值、正向萃取过程中乙醇的添加量以及新鲜反萃水相的pH值、反向萃取过程中乙醇和电解质的添加量。较低浓度的丁醇在正向萃取过程中使酶的活性达到最大，而在反向萃取过程中发现随着硫酸铵浓度的增加酶活性进一步提高。对反萃样品在约7.65分钟处L-天冬酰胺酶峰的色谱分析显示峰强度很高，证实了所获得的L-天冬酰胺酶的最大纯度。L-天冬酰胺酶的纯度提高到了约379.68倍。
