Industrial production of Pectinase

Introduction:

Pectin is a polysaccharide ( heteropolysaccharides ) found in the cell wall of the mainly terrestrial plant . Pectin is a structural protein which is the main constituent of a primary cell wall of the plant. Pectin can also be produced and used in food as a gelling agent, filling ,medicines,sweets, and stabilizer in juice and drinks which are mainly extracted from citrus fruits. It is a jelly-like a matrix that helps to cement plant cell together.

Pectinase is an enzyme that hydrolyzed or breakdown the structural polysaccharide ie Pectin found in the cell wall of the terrestrial plant. Pectinase is also known as pectic enzyme and includes many enzymes like pectozyme,pectolyase, polygalacturonase etc but the most rapidly producing industrially and a most studied enzyme is pectinase . It is already mentioned in above paragraph that pectin is a jelly matrix that attaches cells together so pectinase is useful to degrade the pectin from the cell wall during the extraction of fruits juice from fruits. It is also useful in the production of fruits flavor and today's pectinase is used in the production of wine in which it helps in the breakdown of plant material that helps in extraction of flavor. Also, pectinase clarifies and breakdown the cloudiness of wine after extraction. Pectinase enzymes have its optimum temperature and pH as all enzyme have. A commercial pectinase may be activated at 45-55° C and works well in pH range 3-6.5 . Pectinase can be mainly isolated or extracted from the fungi ( Aspergillus niger).

As we already know that enzyme pectinase is mainly extracted from fungus ie ( Aspergillus Oryza) . Among the two fermentation process ie solid state and submerged state fermentation ,solid state fermentation is mainly used in the production of pectinase enzyme but submerged state fermentation can also be used. In solid state fermentation microbial growth and product formation both occur on the solid substrate without the involvement of water molecule .So, due to this reason fungi are most commonly used as the production organisms. As a solid substrate, we can use rice bran,wheat bran, sugarcane bagasse etc.

Production process:

The increasing demand of energy due to growing population, many renewable resources mostly agricultural and forest residues are being utilized as an alternative source of energy. They include starch,pectin,lignin,xylan,cellulose etc. Since they abundantly occur in nature so they are the main source of energy and alternative feedstock. Microorganisms are the biodegrading agent that use these substances as carbon or energy source ultimately producing the high amount of enzyme in varying environmental condition. Pectinase is also a one of the most producing enzyme and its product occupies about 10% of the overall manufacturing of enzyme.Pectinase production is commercially carried out using fungal strain using either solid state fermentation or submerged state fermentation. The production process of pectinase is described as follows;

1) Organisms involved;

In the production of pectinase, most of the fungal strains are used .Some of them are;

Aspergillus niger

Aspergillus went

Penicillium italicium

Penicillium chrysogen um

By screening the best organisms and measuring the glucose and pectinase activity Aspergillus niger was best-known heat tolerant filamentous fungi for the production of pectinase.

2) Culture media;

For the production of enzyme pectinase, solid state fermentation is the best-known process due to its simplicity, high productivity and more concentrated product than submerged state fermentation process . For the optimum growth condition and maximum enzyme production, some parameter should be controlled and all the substrate and inoculum be prepared carefully using substances and strains in suitable proportion and maintaining controlled temperature,concentration,pH etc. For this process . For solid state fermentation, wheat bran or rice husk is used as a substrate . The fungal strain Aspergillus niger is inoculated PDA media for 4 days at 30°C to produce high fungal and matured spores. After this process thus formed matured strain is then mixed with sterile water and scrubbed with loop and it is gently shaken to make it homogenous. The final suspension was prepared and ready for the inoculation in the substrate media . Thus formed suspension was then inoculated into the solid medium and then incubated at 30-50° C . So wheat bran is used for SSF however for SMSF media containing 2% sucrose and 2% pectin are used .Other components of media include tryptone, yeast extract , MgSO₄.5H₂O, FeSO₄ ,KCl, and others.

3) Fermenters and fermentation process :

a)Solid-state fermentation:

It is an alternative manufacturing technology of biomolecules for the culture of microorganisms to liquid fermentation and used widely in the industrial production of biomolecules and enzymes. Solid state fermentation uses solid substrates like rice or wheat bran and after inoculation of a microbial or fungal strain, it is left at controlled room temperature. The tank where this process is carried out is called fermenter which is about 1000 to 2500 square meter. This process uses culture substrate with low water content which is more appropriate for fungi . Compared to submerged state fermentation it is more cost -effective, smaller vessel,low energy consumption . In this process tray, chamber or drums are used as fermenters.

b) Submerged state fermentation:

This process is taking place in a liquid medium containing enough water for growth which is best for the growth of microorganisms like bacteria. It is high energy intensive rather than labour. It is the modern process and has befitted of low contamination rate .Comparing to solid state fermentation wide range of organisms and substrate can be utilised so the variety of products can be obtained . And it is fast than SSF. For this process stirred tank fermenter and other automated fermenters are used .

4) Fermentation process;

The media is sterilized and inoculum is added into the sterilized fermenter containing the media.

a) For pectinase production by Aspergillus niger

1. mode of operation; Fed-batch
2. pH - It ranges from 3-4
3. Temperature - The best growth obtains at temperature 30-40° C .
4. Duration - 4- 5 days
5. Agitation and aeration are done at regular interval.

b) For pectinase production by Penicillium SPS

1. mode of operation - fed batch
2. pH - ranges from 6-8
3. Temperature - 30° C
4. Duration - 5 days
5. Inoculum size - 10% ( at least 10⁶ spores per ml of inoculum

5) Product recovery and Purification of products;

After the completion of fermentation, the product pectinase is recovered . The Solid and liquid mass are separated. The liquid mass is further concentrated and purified by using following flow chart;

Extraction of the whole substrate insolvent

( for solid state fermentation only )

↓

Filtration/ Centrifugation

↓

Filtrate / supernatant ( crude enzyme )

↓

Addition of stabilizer

↓

Enzyme precipitation with cold ethanol or other organic solvent

↓

Drying

↓

Dry enzyme

Fig; Product recovery of pectinase enzyme

Enzyme Assay;

1) The reaction mixture is prepared and the reaction mixture consists of substrate buffer ( 1.25 gm of pectin is dissolved in 25ml of 0.25 molar sodium citrate buffer) and 0.5ml of enzyme solution ie pectinase

2) The mixture is incubated at 37° C for 30nmins . Then it is heated in boiling water bath for 5 mins .

3) The reaction is stopped by using 3% DNS .

4) A blank containig inactivated enzyme and other reagents in test is prepared .

5) The absorbance is taken at 750 nm.