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Sunday, August 5, 2012

Production and Estimation of Pectinase by Aspergillus niger


Purified pectinase is a multicomponent preparation highly effective in de-polymerising plant pectins with varying degree of esterification. Important enzymatic activities include pectin lyase and activities on polygalacturonic acid. Pectinase are utilized to eliminate pectin and pectin like colloids in fruit juices to clarify the juice and a means to prevent jelling of the juices during concentration steps of fruit juice processing. pectinase is also used in plant protoplast studies, pectinase is produced by various fungi although commercial production utilizes the species of aspergillus and pencillium.

Principle of Pectinase Production
Pectinase production by fungi is stimulated by the presence of pectin containing compounds in the fermentation medium, the enzyme is recovered from both the sources. Thus at harvest mycelium is dried and ground. The commercial preparation contains at-least two types of pectinase differing in the extent to which they degrade pectin.
Pectinase yield is more in solid state fermentation than in the submerged fermentation.media acidity plays an important role in the production of solid state fermentation.A low pH of 5.0 also maintains asepsis during culture. wheat bran, coffee pulp, citrus waste & apple pomace are few substrate used for pectinase production by solid state fermentation.

Pectinase Production Medium
Components
Weight in gms
NaNO3
2.0
KCl
0.5
MgSO4.7H2O
0.5
KH2PO4
1.0
FeSO4
0.01
Pectin
10.0
Water
1000 ml
pH
6.0
Wheat Bran / Coffee Pulp
 25.0

Solid State Fermentation of Aspergillus niger

  1. Aspergillus niger was inoculated on an agar slant of pectin medium and allowed to grow for 4days at 25oC.
  2. 25.0 gm of wheat bran was mixed with phosphate buffer at pH 5.0 to get required consistency & was autoclaved.
  3. The spores from agar slant was scraped and suspended in 5.0ml of buffer and this was added to the wheat bran. the flask was incubated at 25oC for 5 days.
  4. 50ml of phosphate buffer was added to the flask and kept in a shaker 37oC to extract the enzyme.
  5. The extract was centrifuged at 5000 rpm & supernatant was used for the assay of the enzyme.
Assay of Pectinase / Estimation of Pectinase
One unit of pectinase liberates 1 micro-mole of D-galactouronic acid from polygalactouronic acid per minute at 37oC, pH 5.0.

Reagents Required:
  1. 0.1M  citric acid or phosphate buffer, pH 5.0.
  2. Colour Reagent A: Dissolve 40g of anhydrous sodium carbonate in 600ml distilled water. add 16g of glycine & stir until it dissolves, make up the volume to 1L using distilled water.
  3. Colour Reagent B: Dissolve 12g of neoprene hclin 1L distilled water and store at 4oC in brown bottle.
  4. D-galactouronic acid standard - 1mg/ml
  5. 0.5% polygalactouronic acid substrate: Heat 500ml assay buffer on a hot plate, heat and still well until it dissolves. it will be slightly viscous and opaque. don't boil, cool and make up the volume. if required store at 4oC.
Estimation of Pectinase Procedure
  1. Take 6ml of the substrate in a tube and add 1ml of the enzyme solution.
  2. Incubate the tube at 37oC for 1 hour.
  3. Similarly, prepare a blank by denaturing the enzyme by boiling for 10min, After incubation transfer the tube to ice.
  4. Take 100µl of the sample & blank in two separate tubes& add 2ml of colour reagent A and colour reagent B.
  5. Mix well and keep in a boiling water bath for 15mins.
  6. Add 2ml of water and note the absorbance at 450nm.
  7. Prepare a standard D-galactouronic acid using 10µg - 125µg of it.
  8. Plot graph of absorbance against the amount of standard & find the amount of product releassed by  the enzyme.