响应面优化硝普钠法测定发酵液中的蛋氨酸含量

为了优化硝普钠法(sodium nitroprusside method,SNP)测定发酵液中的蛋氨酸(Met)含量的实验条件,利用紫外可见分光光度计分析不同氨基酸中加入各SNP反应试剂、反应不同时间对反应液吸收峰的影响。在甘氨酸(Gly)添加量、NaOH加入量、SNP加入量、H₃PO₄加入量、各试剂加入后的反应时间等单因素实验的结果上,利用响应面法优化SNP法。结果表明:与SNP试剂反应后,除了组氨酸(His)外,其它氨基酸不会对Met的测定产生影响。反应试剂H₃PO₄的加入对513 nm处吸收峰的形成起决定性作用;发酵液中各成分SNP反应后,最大吸收峰无明显变化。利用单因素和响应面法优化SNP法,得出较佳测定方案:发酵液1500×g离心15 min;5 mL上清液中加入0.74 mL 5 mol/L NaOH和0.15 mL 10% SNP溶液,摇匀试管,放置30 min;将2 mL H₃PO₄逐滴加到混合物中,振荡试管,反应5 min,在513 nm处测吸收值。当Met浓度为1.0 g/L时,最佳测定条件下吸收值为1.952。通过光谱分析确认Gly的加入会降低Met和His反应的吸收峰值,可通过分析最大吸收峰波长是否在513 nm处,判断样品中是否有组氨酸影响测定。实验获得的SNP法可用于迅速筛选蛋氨酸高产菌。

Response Surface Methodology for Optimization of Determination of Methionine in the Fermentation Broth by Sodium Nitroprusside Method

The aim of the experiments was to optimize the determination of methionine(Met)in the fermentation broth by the sodium nitroprusside(SNP) method. The ultraviolet visible spectrophotometer was used to analyze the absorbance peak of different reaction,including different amino acids with SNP and Met with different reagents for different time.The optimal colorimetric procedure of SNP method was obtained with single factor tests,including the volume of glycine(Gly),the volume of NaOH,the amount of SNP added and the volume of concentrated phosphoric acid added,and response surface methodology was used to optimize the SNP method.The results showed that other amino acid had no affection on the determination of Met except for histidine(His)after reaction with SNP.The absorption peak was at 513 nm decided by the addition of phosphoric acid.The components of the broth had no new adsorption peak after reaction with SNP.The optimum determination method of Met with SNP was obtained by single-factor experiments and response surface method.The fermentation broth was centrifuged at 1500 × g for 15 min,5 mL the supernatant was added 0.74 mL 5 mol/L NaOH and 0.15 mL 10% SNP solution;the tube was shaken and allowed to stand 30 min;2 mL concentrated phosphoric acid was added to the mixture drop by drop.Then the tube was reacted shakingly for 5 min,and measured at 513 nm The value of absorption of 1 g/L Met was 1.952 with optimized SNP method.It was confirmed by spectral analysis that the addition of Gly could reduce the peak absorption of Met and His reaction with SNP method.It was possible to determine whether His affected the determination of Met by analyzing whether the maximum absorption wavelength was at 513 nm.The SNP method could be used to screen methionine producing bacteria quickly.