Microbial Enzyme production and its characterization

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BACKGROUND

Enzymes are among the most essential products obtained for human needs from plants, animals, and microorganisms. At the moment, the use of enzymes in manufacturing is increasing as a result of the expansion of industries, particularly those in the beverage, food, textile, paper, and leather sectors.

Using microorganisms is considered for many purposes, including heavy metal absorption, gene engineering, digestion, production of novel anti-microbes, and particularly for producing industrial enzymes, as opposed to chemical methods that require harsh conditions such as high pressure and temperature. Innovative techniques to enhance industrial items like protease inhibitors and amylase, which are often used in industry and medical science, are developed in response to the demand for high-quality manufacturing.

The enzymes that digest starch or glycogen are called amylases. Amylases can originate from a variety of sources, including bacteria, animals, and plants. The fundamental advantage of employing microbes to produce amylases in large quantities at a reasonable cost is that they are easy to manage to produce enzyme with desired properties.

Members of the genus Escherichia have been some of the mainstays of enzyme production for decades, primarily due to their ability to overproduce amylase. Other microorganisms producing significant amounts of diverse amounts of amylase enzyme are decades old. Amylases have been produced by numerous yeasts, bacteria, fungi, and actinomycetes. Bacillus species such B. licheniformis, B. amyloliquefaciens, B. subtilis, and B. stearothermophilus, as well as Escherichia coli, Pseudomonas spp., Serratia spp., Micrococcus spp., and Proteus spp. Aspergillus-related fungus have traditionally been used to produce amylase, however other molds such as Mucor, Penicillium, Candida, Cephalosporium, and Neurospora can also be used to produce enzymes through solid-state fermentation (SSF), which is a more efficient method of production.

REQUIREMENTS

Soil sample
Nutrient agar medium
Starch agar medium
Production medium
Sterile distilled water
1% iodine solution
0.2 m Citrate buffer (pH 6.5)
Dinitrosalicylic acid
Ammonium sulphate
Water bath
Magnetic stirrer
Dialysis membrane
Centrifuge

PROCEDURE

  1. Weigh 1 gm of soil sample, add to 9 ml sterile distilled water.[1]
  2. Take a loopful of the soil suspension and spread it on a plate with nutrient agar medium. Incubate for 24 to 48 hours at 37C.
  3. Select isolated colonies for father procedure, add loopful to the sterile distilled water and shake well.
  4. Add 1 ml of the culture suspension to the centre of the starch agar plate, and incubate for 24 hr at 37C.
  5. Pour 1% iodine solution on the plate and observe for the substrate utilization zone.
  6. Choose the microorganisms that show the substrate-using zone and characterize and identify the organisms using Bergey’s manual.[3]
  7. Prepare enzyme production medium (Starch 1g/L, peptone 6g/L, MgSO4.7H2O 0.5g/L, KCl2.4H2O 0.5g/L) sterilized it, too.[2]
  8. Add selected strain of isolated organism to the production medium aseptically, and incubate at 37C for 24-48h in shaker incubator.
  9. Centrifuge the medium at 5000 rpm for 20 min, and collect the supernatant, use it as crude enzyme.
  10. Add ammonium sulphate in different ratio (20,40,60 and 80%) to the supernatant, to achieve optimum ratio add salt gradually to the enzyme in magnetic stirrer and cold condition.[1]
  11. Collect the precipitate, for further purification dialyse it against the 0.2 M citrate buffer (pH 6.5).
  12. Take 1 ml of enzyme and 1 ml 1% soluble starch in citrate buffer incubate at 40C for 30 min., stop the reaction by adding 2ml of dinitrosalicylic acid (DNS) reagent.
  13. The mixture heated for 5 min in the boiling water bath and absorbance was recorded at 540 nm.
Microbial enzyme production
Microbial enzyme production

CONCLUSION

We isolate the enzyme-producing bacterium from the process above, characterize it with Bergey’s manual, and purify it using salt precipitation, dialysis, and an enzyme assay with dinitrosalicylic acid reagent.

REFERENCES

  1. Akansha Karnwal, Varsha Nigam, “PRODUCTION OF AMYLASE ENZYME BY ISOLATED MICROORGANISMS AND IT’S APPLICATION”, International Journal of Pharmacy and Biological Sciences, Vol. 3 (4), 2013.
  2. Baydaa A. Hassan, Nibras yahya alsalami, Mohammed A. Jebor, “Amylase production, purification and characterization from Escherichia coli”, Journal of Pharmaceutical Science and Research, Vol. 10 (7), 2018.
  3. Koki Horikoshi, “Production of Alkaline Enzymes by Alkalophilic Microorganisms”, Agr. Biol. Chem., Vol. 35 (11), 1971.
  4. Babak Elyasi Far, Yassin Ahmadi, Ahmad Yari Khosroushahi, Azita Dilmaghani, “Microbial Alpha-Amylase Production: Progress, Challenges and Perspectives”, Advanced Pharmaceutical Bulletin, Vol. 10 (3), 2020.

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FAQs

Which technique is typically utilized for intracellular enzyme isolation?

It is a common approach for high pressure homogenization.

When ought I to employ a desalting technique?

Before, during, or after purification, desalination is an option.

Which enzymes are employed in the sector to metabolize proteins?

These include papain, chymosin (rennin), trypsin, chymotrypsin, bacterial and fungal proteases, as well as chymosin (rennin).