3 Biotech

, 9:365 | Cite as

Laccase: addressing the ambivalence associated with the calculation of enzyme activity

  • Komal Agrawal
  • Pradeep VermaEmail author
Original Article


Laccase (benzenediol: oxygen oxidoreductase) a unique multi-copper oxidase enzyme has been studied rigorously since its identification. However, there is ambivalence associated with various aspects of laccase, e.g., assay conditions and calculations. Our aim was to minimize its ambivalence, thus, total of five formulas (F1–F5) were used to determine laccase activity of white and blue laccase. In case of enzymatic profiling of blue laccase, its activity ranged from 0.04 to 464.3 U L−1 whereas in case of white laccase it ranged from 0.05 to 1404.7 U L−1. The affinity of laccase at various enzyme concentration (0.3–0.9 mg mL−1), time (5 and 10 min) along with various substrates, i.e., 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol (GCL), 2,6-dimethoxyphenol (DMP) and syringaldazine (SYZ), and its concentration (ABTS 0.5–1.5 mM, GCL 20–30 mM, DMP 1–5 mM, SYZ 10–30 mM) were inferred. The optimal substrate concentrations were 1.5 and 0.5 mM ABTS for blue and white laccase, respectively, with 30 mM GCL and 2 mM DMP being the common parameter. The optimal substrate concentrations were 0.5 mM ABTS, 20 mM GCL, 1 mM DMP and 30 mM SYZ for commercial laccase. It was observed that the optimal protein load and reaction time was 0.3 mg mL−1 and 5 min in all the cases, however, in case of white laccase it was 0.6 mg mL−1 at 10 min for DMP and in case of commercial laccase it was 0.9 mg mL−1 and 5 min for SYZ. In the present study, F1 was most appropriate among the five formula used as it incorporates all the significant factors and use of single formula will help reduce future ambiguity.


Laccase Ambivalence 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Guaiacol (GCL) 2,6-dimethoxyphenol (DMP) Syringaldazine (SYZ) 



The authors are thankful to DBT (Grant nos. BT/304/NE/TBP/2012 and BT/PR7333/PBD/26/373/2012). The authors would also like to acknowledge the financial support provided by CURAJ Ajmer, India.

Author contributions

KA performed the laboratory work, studied and prepared the MS, PV planned and supervised the whole work.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, Pradeep Verma states that there is no conflict of interest.


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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.Bioprocess and Bioenergy Laboratory, Department of MicrobiologyCentral University of RajasthanAjmerIndia

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