Rapid screening of enzyme inhibitors using Profiling of Enzyme-Metabolite Assay by HPLC (PREMA-HPLC)

Figures & data

Table 1.  Results of the screening of fungal isolates against free enzyme tyrosinase.

S. No	Culture number	% Inhibition	S. No	Culture number	% Inhibition	S. No	Culture number	% Inhibition
1	CFRP1	Nil	18	CFRP25	24	35	CFR151	Nil
2	CFRP2	Nil	19	CFRP26	Nil	36	CFR161	Nil
3	CFRP3	Nil	20	CFR1	Nil	37	CFR171	Nil
4	CFRP4	Nil	21	CFR2	Nil	38	CFR181	Nil
5	CFRP5	7.4	22	CFR 3	Nil	39	CFR191	49
6	CFRP6	Nil	23	CFR4	Nil	40	CFR201	Nil
7	CFRP8	4.07	24	CFR5	Nil	41	CFR212	Nil
8	CFRP9	8.9	25	CFR 6	27	42	CFR222	Nil
9	CFRP10	Nil	26	CFR7	Nil	43	CFR232	Nil
10	CFRP11	Nil	27	CFR8	Nil	44	CFR242	Nil
11	CFRP13	Nil	28	CFR9	Nil	45	CFR252	Nil
12	CFRP14	Nil	29	CFR10	Nil	46	CFR262	46
13	CFRP15	Nil	30	CFR101	72	47	CFR272	Nil
14	CFRP17	6	31	CFR111	Nil	48	CFR282	Nil
15	CFRP19	Nil	32	CFR121	Nil	49	CFR292	Nil
16	CFRP20	Nil	33	CFR131	54	50	CFR302	Nil
17	CFRP22	Nil	34	CFR141	Nil

Figure 1.  HPLC profiles of metabolites from CFR 101 (A) HPLC of crude before incubation with enzyme. (B) HPLC of crude after incubation with enzyme and (C) HPLC of purified inhibitor. HPLC, high-performance liquid chromatography.

Table 2.  Reduction in peak area of metabolites of CFR 101 before and after the enzyme assay.

Peak no	Time (min)	HPLC peak area, units		% Reduction in peak area
		Before incubation	After incubation
1	3.982	570,760	541,557	5.11
2	8.023	2,882,711	2,642,631	8.31
3	8.432	437,654	413,756	5.4
4	10.678	3986721	3,798,783	4.72
5	13.952	7,591,112	2,549,430	66.41

Figure 2.  Dose-dependent inhibition of CFR101 (open circle) and kojic acid (filled diamond).

Figure 3.  Stability of immobilised and free tyrosinase. Free tyrosinase (open bar), immobilised tyrosinase (closed bar).

Figure 4.  Study of reusability of immobilised tyrosinase.