Production parameter optimization for laccases by Aspergillus niger

Aspergillus niger and incubated at optimized temperature for 7 days at different

pH such as 5, 7, 9 and 12.

Effect of incubation period

In order to find the optimal time of incubation for the maximum laccase production.

100 ml production medium was inoculated with 1 ml culture of Aspergillus

niger and incubated at optimized temperature. The sample was withdrawn at

different time intervals viz. 0, 24, 48, 72, 96 and 120, 144,168, 192 h each time.

Effect of carbon source

To analyse the effect of carbon sources on laccase production100 ml production

medium supplemented with 2% different carbon sources viz. glucose, sucrose,

mannitol, maltose was prepared in Erlenmeyer flask (250 ml) with optimized

pH and autoclaved. The production media was inoculated with 1 ml culture

of Aspergillus niger followed by incubation at optimized temperature at optimized

time interval.

Effect of nitrogen source

In order to find the suitable nitrogen sources for maximum laccase production,

100 ml production medium supplemented with optimized carbon source and

2% different nitrogen sources viz. peptone, ammonium chloride, sodium nitrate,

ammonium sulfate was prepared and inoculated with 1 ml culture of Aspergillus

niger followed by incubation at optimized temperature for optimized time interval.

Production and Partial purificationat optimized culture conditions

For laccase production, 100 ml Nutrient broth media was prepared. A loopful

culture of Aspergillus nigerwas inoculated using a sterile loop and incubated at

30ºC at 120 rpm. The culture was served as seed culture after 72 h for further

inoculation. A 100 ml sterile production media containing 2% optimized carbon

and nitrogen source was prepared with optimized pH according to the composition

given by Unyayar et al., (2005) sterlized and inoculated with 5% seed culture (v/v)

and was incubated at optimized temperature for optimized time interval.

Extraction of crude enzyme

After incubation the cells were harvested by centrifugation at 10,000 rpm at 4ºC

for 10 min. The clear supernatant obtained was used as crude enzyme and was

stored in vials for further use.

International Journal of Bioinformatics and Biological Sciences : v.2 n.3&4. Dec. 2014

181