Application of standard addition for the determination of carboxypeptidase activity in Actinomucor elegans bran koji.

J Fu, L Li, X Q Yang, M J Zhu

College of Bioscience and Biotechnology, South China University of Technology, Guangzhou 510006, China.

Prikladnaia biokhimiia i mikrobiologiia 2011 Sep-Oct

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Leucine carboxypeptidase (EC 3.4.16) activity in Actinomucor elegans bran koji was investigated via absorbance at 507 nm after stained by Cd-nihydrin solution, with calibration curve A, which was made by a set of known concentration standard leucine, calibration B, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations inactive crude enzyme extract, and calibration C, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations crude enzyme extract. The results indicated that application of pure amino acid standard curve was not a suitable way to determine carboxypeptidase in complicate mixture, and it probably led to overestimated carboxypeptidase activity. It was found that addition of crude exact into pure amino acid standard curve had a significant difference from pure amino acid standard curve method (p < 0.05). There was no significant enzyme activity difference (p > 0.05) between addition of active crude exact and addition of inactive crude kind, when the proper dilute multiple was used. It was concluded that the addition of crude enzyme extract to the calibration was needed to eliminate the interference of free amino acids and related compounds presented in crude enzyme extract.

Citation

J Fu, L Li, X Q Yang, M J Zhu. Application of standard addition for the determination of carboxypeptidase activity in Actinomucor elegans bran koji. Prikladnaia biokhimiia i mikrobiologiia. 2011 Sep-Oct;47(5):609-14