SOIL ENZYMES AND THEIR ACTIVITY

SOIL ENZYES AND THEIR ACTIVITY

1. Soil Enzymes, Their importance and T heir activity Soil microbiology(MICRO -504) S peaker:- Aishwarya Chauhan MSc. Microbiology 1 st Year (F-2017-27-M)

2. What is soil? It is the region where most of the physical, biological, and biochemical reactions related to decomposition of organic wheathering of parent rock take place. What is enzyme? A substance which is used to increase the rate of reaction is known as enzymes.

3. What are soil enzymes? Soil enzymes increase the reaction rate at which plant residues decompose and release plant available nutrients. The substance acted upon by a soil enzyme is called the substrate.

4. For Example Glucosidase (soil enzyme) cleaves glucose from glucoside (substrate), a compound common in plants. Enzymes are specific to a substrate and have active sites that bind with the substrate to form a temporary complex.

5. Origin of Soil Enzymes Although the general origins of soil enzymes are:- (a ) microorganisms-living and dead , ( b) plant roots and plant residues and ( c) soil animals a. b. c.

6. Importances of Soil Enzymes Release of nutrients into the soil by means of organic matter degradation:-

7. Identification of microbial activity:- Soil microorganisms occurs in huge number and display an enormous diversity of forms and functions. M ajor microbial group in soil are bacteria( including actinomycetes), fungi, algae ( including cynobacteria) and protozoa .

8. Kind of Soil Enzymes Constitutive Always present in nearly constant amounts in a cell ( Pyrophosphatase) Inducible Present only in trace amounts or not at all, but quickly increases in concentration when its substrate is present . ( Amidase) Both types of enzymes are present in the soil.

9. Soil enzyme activities (1) are often closely related to soil organic matter, soil physical properties and microbial activity or biomass ( 2) changes much sooner ( in or after a short time) than other parameters, thus providing early indications of changes in soil health, and ( 3) involve simple procedures. In addition, soil enzyme activities can be used to measures microbial activity , soil productivity, and inhibiting effects of pollutants.

10. Amylase The starch hydrolyzing enzyme amylase is known to be constituted by α - amylase and β- amylase This enzyme is widely distributed in plants and soils so it plays a significant role in the breakdown of starch , which converts starch like substrates to glucose and/or oligosaccharides and β -amylase , which converts starch to maltose .

11. The α -amylases are synthesized by plants, animals, and microorganisms. Mode of Action

12. β -amylase is synthesized mainly by plants. Mode of Action

13. Amylase Screening Test Procedure:- Prepare the starch agar medium in the flask and autoclave the medium. After autoclaving, allow the mixture to cool down at 42-45 ˚C temp. Pour the starch agar medium in the sterile petriplate and rotate the plate gently for uniform distribution. Allow the mixture to solidify. Using sterile technique, make a single streak inoculation of each organism into the centre of the plates. Incubate the plate at 37˚C for 48 hours in inverted position. Flood the surface of plates with iodine solution for 30 sec. Pour of the excess of iodine solution. Observe the zone formed around bacteria. Composition of starch agar medium

14. Observations When the iodine solution was added to the starch agar plate, a clear zone was formed around the bacteria which shows that bacteria inoculated has the ability to solubilize starch by production of amylase enzyme and if there was no formation of the zone, then bacteria was not able to synthesize enzyme amylase.

15. Catalase Most of the aerobic bacteria utilize oxygen and produce hydrogen peroxide, which is toxic to their own enzyme system. Their survival in the presence of this antimetabolite is possible because they produce an enzyme called catalase which converts the hydrogen peroxide to water and oxygen.

16. Mode of action

17. Procedure 1)Take a clean slide and put a drop of H2O2 on it. 2)Pick up a small amount of culture from nutrient agar using a clean sterile platinum loop or a thin glass rod and add it to the drop of 2H2O2 taken on the slide.

18. Observations Positive:- Effervescence in the mixture on slide. Negative:- No effervescence in the mixture on slide.

19. Proteases Proteases in the soil play a significant role in N mineralization. This enzyme in the soil is generally associated with inorganic and organic colloids. There is a need to study the properties and factors affecting naturally occurring enzyme complexes such as those involving protease enzymes in the soil ecosystem as they may reveal some unknown role in maintaining soil health and fertility.

20. Protease test Gelatin agar media was prepared according to the composition . It was then autoclaved at 15lbs at 121 °C for 15 minutes. Sterile Gelatin agar plates were prepared. The plates were inoculated with the culture on surface by spot inoculation or streaking. They were incubated for 24 hrs at room temperature. 10% HgCl2 was then added to the plates and the clear zone formation around growing colony was considered as positive .

21. Urease Urease enzyme is responsible for the hydrolysis of urea fertilizers applied to the soil into NH3 and CO2 with the rise in soil pH . Soil urease originates mainly from plants and microorganisms found as both intra- and extra-cellular enzymes . On the other hand, urease extracted from plants or microorganisms is rapidly degraded in soil by proteolytic (breakdown of proteins into smaller polypeptides or amino acids) enzymes . Urease activity in soils is influenced by many factors. These include cropping history, organic matter content of the soil, soil depth, heavy metals, and environmental factors such as temperatures.

22. Mode of Action Ammonium hydroxide

23. Urease Screening Test Procedure Inoculate the slants with culture to be tested. Incubate the slants at 37 ˚C. Examine the tubes after 4 hours, one day (routine test) and 4 days (standard test).

24. Observations. Rapid positive- Intense red violet colour in 4 hours ( Proteus sp .) Positive – Red violet in 1-4 days ( Klebsiella sp.) N egative –Yellow or orange colour ( E. coli , P seudomonas sp .)

25. Application of Soil Enzymes Correlation with soil fertility: Difficult to show correlation especially fertility with enzyme activity . Correlation with microbial activity : Used as index of soil microbial population Correlation with biochemical cycling of various elements in soil (C, N, S ): has met with only limited success . Forensic purposes: Can determine where soil comes from by looking at activity, value of sample size.

26. Conclusions It is very essential to understand the possible roles of soil enzymes in order to maintain soil health and its fertility management in ecosystems. These enzymes, usually found in the soil, may have significant effects on soil biology, environmental management, growth and nutrient uptake in plants growing in ecosystems .