PRODUCTION, PURIFICATION AND ASSAY OF PECTINASE ENZYME FROM ASPERGILLUS NIGER

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Abstract

Production of microbial enzymes at the industrial scale and their commercialization has gained a lot of focus and importance. Use of Microbes as bioreactors was also started due to these developments. Some of the industrially important enzymes from microbial origin include Lipases, Amylases, proteases, Xylynases and Pectinases. The current work involved the use of mice\robes isolated from the decaying fruit peel for the extraction of the Pectinase enzyme. The organisms were isolated from the fruit peel and were cultured onto SDA agar Plate as the isolation is of Fungi. The organisms were screened for the production of the Pectinase enzyme using Pectin agar media. Microscopic identification and Morphology study revealed that the Fungi isolated was Aspergillus niger Pectinase Production media was later used for the Lab scale production of Pectinase enzyme by inoculating A. niger and incubating for 10 days. The enzyme thus produced was purified by several steps including Salt precipitation and Dialysis. The enzyme thus produced was assayed and compared with the normal fruit juice extraction production. Quantitative estimation of protein was done using Lowry’s method at660nm to know the concentration of protein. The results thus obtained can conclude that the use of Aspergillus niger in the industrial production of Pectinase is highly beneficial. The amount of juice extracted in the control samples was less compared to the samples extracted using Pectinase enzyme. The enzyme pectinase was then preserved using microencapsulation technique by Sodium Alginate and stored at4°C.

Introduction

The Pectinases are the enzymes that are capable of hydrolyzing the Pectin polysaccharide into smaller fragments. All the peels of the fruits are made of pectin layer which can be easily digested by the microbial pectinases so as to extract the juices of the
fruits. Therefore, pectinase enzymes are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juice.
At present, almost all the pectinolytic enzymes used for industrial applications are produced by the fungi, namelyAspergillus sp., Rhizopusstolonifer, Alternaria mali, Fusariumoxysporum, Neurosporacrassa, Penicillium italicum ACIM F-152, and many others. Orange peels are used as substrate for the growth of Aspergillus niger. The high cost used in the importation of industrial extracellular enzymes has led to the high cost of finished industrial products. Besides, orange peels cause waste disposal problems since they are being thrown around indiscriminately. This study is therefore aimed at the production of pectinase from A. niger, purifying enzyme and assaying the enzyme activity.

Conclusion

Most of the microorganisms are capable of producing various extracellular and intracellular enzymes using various economical substrates. The effect of microbial enzyme activity is higher than the plant and animal derived enzymes. Pectinase is an extracellular enzyme, which is produced from various organisms including bacteria, fungi, and some actinomycetes. In the current work Pectinase enzyme was produced by Aspergillus niger which in turn was obtained from the decaying Fruit peels. The Culture was allowed to grow on SDA media containing Pectinase to selectively allow only the Pectinase producers to grow. The mycelia thus obtained after 7 days was stained and observed microscopically. The mycelium was identified as Aspergillu niger. The culture was further used for the production of the enzyme using PSAM medium. The culture was incubated for 20 days for the complete production of the extracellular enzyme. The enzyme thus produced was purified by several techniques like Filtration, Centrifugation, Salt precipitation and Dialysis. The purified product was used for the Enzyme assay which showed that the activity of the enzyme produced was 0.043μmol/min/mg. Quantitative estimation of the enzyme was performed by Lowry’s Method which showed the concentration of the enzyme to be 24μg/ml. To confirm the applicability of the enzyme, it was used for the extraction of the apple juice in comparison with the normal juice extraction in the absence of the enzyme. The experiment revealed that the juice produced in the presence of enzyme was double the volume of the juice produced using regular protocol.
The work concludes that the enzyme Pectinase can be produced from Aspergillus niger and can be used for the industrial extraction of fruit juices to increase the yield.

Acknowledgements

The Authors would like to express their deep sense of gratitude to Mrs D. Jyothsna of Bioaxis DNA Research Centre (P) Ltd. for her overwhelming support and guidance throught the project. They extend their sincere thanks to Mrs. Zehra Samana, Mrs. Suchitra Naidu and their parents for their constant and unfailing support.

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