Protease Enzyme Assay

Aim:

To determine the protease activity of given bacterial culture.

Introduction:

            Protease enzyme is naturally present in all organisms and it corresponds to 1-5% of total protein content Protease is the third largest group of industrial enzymes and has a worldwide sale of 60%. Proteases can hydrolyse peptide bonds in proteins and they are also called peptidase or proteinase or proteolytic enzymes . Proteases are classified into three groups based on their acid base behavior that is, acid, neutral, and alkaline proteases . Acid proteases have a pH range of about 2.0-5.0 and they are produced only by fungi. Neutral pH of protease ranges from 7.0-8.0 and they are mainly of plant origin and finally proteases with pH above 8 are said to be alkaline proteases. Proteolytic enzymes are ubiquitous in nature and they are found in all living organisms such as plants, animals and microbes.

            The protease producing bacterial strains are as Bacillus subtilis, Bacillus licheniformis, and Bacillus thuringiensis.

Materials required:

Casein 

Carbonate buffer

10 % TCA (Trichloro acetic acid solution)

Bacterial broth culture

Folin ciocalteau reagent

Sodium carbonate

Preparation of 0.44M Na2CO3

0.2 M solutions each of sodium carbonate anhydrous (21.2 g/L) and sodium hydrogen carbonate (16.8 g/L) were prepared. 50 ml of 0.2 M sodium carbonate solution was pipette into a 100 ml volumetric flask and made upto the mark with 0.2 M sodium hydrogen carbonates.

Tyrosine Stock solution:

200 mg of Tyrosine was dissolved in distilled water to make 100 ml in a volumetric flask, which resulted in 200 mg  µg/ml.

Estimation of Protease :

Casinase activity determination by using 2% casein  in 0.2 M carbonate buffer  (pH 10) as substrate. Casein solution (0.5 ml) with an equal volume of suitably diluted enzyme solution was incubated at 55°C. 

After 10 min, the reaction was terminated by the addition of 1 ml of 10% trichloroacetic acid. 

The mixture was centrifuged.

The supernatant was added 5 ml of 0.44 M Na2CO3 and 1ml of two-fold diluted Folin Ciocalteau reagent. 

After 30 min, the colour developed was read at 660 nm against a reagent blank prepared in the same manner.

Tyrosine served as the reference standard. The optical density of these solutions was measured in a Shimadzu (Japan) spectrophotometer.

One unit of enzyme activity was defined as the amount of enzyme that released one ug of tyrosine per ml per min.

Tyrosine Standard:

Into a series of 10 ml volumetric flasks 1,2, 3,4, and 5 ml of standard stock solution of tyrosine was taken and distilled water was added to make upto 10 ml mark in each volumetric flask. 

Mixed well and the optical density was measured at 660 nm after developing the colour as described above, against a reagent blank prepared in same manner.

A standard curve was constructed taking concentration of Tyrosine µg/ml on X-axis and corresponding optical density on Y-axis.

Result:

Amylase Enzyme Assay

Aim:

To determine the amylase activity of given bacterial culture.

Introduction:

            Starch is an abundant carbon source in nature. α-amylase (1, 4 α D-glucanohydrolase; EC 3.2.1.1) hydrolyses α-1, 4-glucosidic linkage in starch and related molecules. It is one of several enzymes involved in starch degradation. Amylases constitute one of the most important groups of industrial enzymes being extensively used in food, textiles, paper, brewing and distilling industries. Most of the available amylases produced commercially are of microbial origin. The enzyme is widespread among aerobes and anaerobes. Gram positive bacteria, particularly the genera Bacillus and Clostridia are prolific producers of amylases.

Principle:

The α -amylase activity is measured using a colorimetric method with 3,5-dinitrosalicylic acid (DNS) reagent. In this method, starch by α –amylase is converted into maltose. Maltose released from starch is measured by the reduction of 3,5-dinitrosalicylic acid.

                         

                        α –Amylase

Starch + H2O                            Maltose (reducing agent)

Maltose reduces the pale yellow coloured alkaline 3, 5-Dinitro salicylic acid (DNS) to the orange-red colored. The intensity of the color is proportional to the concentration of maltose present in the sample.

This intensity change in color is measured using a  spectrophotometer as the absorbance at 540nm wavelength. Wave length is set to 540 nm because it is the region where orange-red color absorbs.

Materials Required:

DNS Reagents:

 DNS reagent contained DNS (1%), potassium sodium tartarate (Rochelle salt, (1 M) and NaOH (0.4 M) and ddH2O. 3.6.1.1. Procedure for 100 mL Reagent Dissolve by stirring at room temperature (RT) 1g DNS in 50 mL ddH2O, then added 20 mL 2 M NaOH and 28.2 g Rochelle salt, finally made up to 100 mL by ddH2O. The reagent was stored at RT. 3.6.2. Other solutions

Buffer:

0.02 M sodium phosphate buffer (pH, 6.9) with 0.006 M sodium chloride.

Starch solution:

1.0% starch solution was prepared fresh by dissolving 1.0 g soluble starch in 100 mL 0.02 M sodium phosphate buffer (pH, 6.9).

Maltose stock solution:

Dissolve 100 mg maltose in 100 ml of dist. water.

Maltose Working Standard:

Add 10 ml of maltose stock solution in 100 ml of of dist. water.(Concentration 100 µg/ml)

Procedure:

Ø Pipette out 0.5 mL enzyme solution and incubate tubes at 25^oC for 3 min.

Ø Add 0.5 mL starch solution and incubate for 5 min (RT).

Ø Stop the reaction by adding 1 mL DNS reagent.

Ø Heat the solution in a boiling water bath for 5 min.

Ø Cool it in running tap water.
Ø Make up the volume to 10.0 mL by the addition of ddH2O.

Ø Read the absorbance at 540 nm using UV-Vis Spectrophotometer

Ø Blank is prepared without enzyme.

Ø Prepare a standard graph with 0 -100 μg maltose.

Result:

Production of Biopesticide - Trichoderma viride

Aim:

To isolate, identify and Mass production of Trichoderma viride.

Introduction:

            Trichoderma viride is very promising method against soil borne plant parasitic fungi. The fungal pathogens play a major role in the development of diseases on many important field and horticultural crops; resulting in severe plant yield losses. Intensified used of fungicides has resulted in accumulation of toxic compound potentially hazardous to human and environment an also in the buildup of resistance of the pathogens. Large scale production, along with shelf life and establishment of bioagents in targeted niche, determine the success of biological control. Therefore cost effective large scale production, shelf life of formulation, establishment of bioagent in to targeted niche and consistency in disease control are the primary concern with augmentative biological control. Adaptation of technology in the biocontrol arsenal needs to be investigated. Development of acceptable easily prepared and cost effective formulations for delivery should be major goal.

Materials Required:

PDA Medium

Grains (Rice, Wheat, Pulses)

Procedure:

Isolation and Identification of T. viride

Fungal species Trichoderma viride was isolated from soil samples by using potato dextrose agar (PDA) mediumGreen conidia forming fungal bodies were selected and microscopic observation was identified to be Trichoderma viride. The culture was maintained on PDA slants.

Grain Medium

 Three Grains viz rice, wheat and pulses were used for estimating the biomass of Trichoderma viride at 26 ˚C. 20 g of each grain was washed well and boiled in distilled water for 1 hr. and then mesh properly and filter it, now makeup 1 liter with distilled water. Now these grain mediums were packed separately in individual 500 ml conical flask for Trichoderma viride. They were plugged with cotton wool and autoclaved at 15 psi for 1hr at 121˚C. After cooling, 1 gm of the fungal culture was inoculated into each flask, separately. All these action were done under laminar air flow chamber. They were incubated in BOD incubator at 26˚C for 3 weeks. To avoid clumping, after 7 days of inoculation, the flasks were shaken vigorously to separate the culture and to break the mycelial mat. After 14 days of incubation, the mycelial mat appeared in flasks. Now it was grow well for 21 days. The flasks were shaken in mechanical shaker for 10 min. The suspension was filtered through double layered muslin cloth and then taken biomass in each grain medium.

Result:

Among the grain media, pulses medium produced significantly higher of biomass production was recorded in Trichoderma viride. Abundance of minerals in the pulses medium may enhance the growth of fungi. Rice and wheat medium also supported the growth of both the tested fungi.

Ref:

Mridula et.al., 2012. Isolation, characterization & biomass production of Trichoderma viride using various agro products- A biocontrol agent. Advances in Applied Science Research, 2012, 3 (6):3950-3955.

Vermicomposting

Aim

To prepare vermicomposting using earthworms and other biodegradable wastes.

Introduction :

The process of vermicomposting involves the dumping of all wastes like- kitchen wastes, cow dung, jute mats, etc. Earthworms are naturally used in a vermicomposting. African earthworm (Eudrillus engenial), Red worms (Eisenia foetida) and composting worm (Peronyx excavatus) are promising worms used for vermicompost production. All the three worms can be mixed together for vermicompost production.  The microorganism in the guts of the earthworm eats the organic wastes and breaks them into simpler parts. This procedure produces a fiber-rich carbon containing humus. The gut of the earthworm also provides optimum temperature, PH, oxygen and other favorable conditions which are required for the efficient growth of a microorganism that carries out the degradation of wastes.

Principle

This process is mainly prepared to add nutrients to the soil. Compost is a natural fertilizer and it allows for easy flow of water and air for growing the plants. The earthworms are mainly used in this process as they eat the organic matter and produce castings, or “worm poop” through their digestive systems. The nutrients profile of vermicomposts are

1. 1.6 percent of Nitrogen.
2. 0.7 percent of Phosphorus.
3. 0.8 percent of Potassium.
4. 0.5 percent of Calcium.
5. 0.2 percent of Magnesium.
6. 1.75 percent of Iron.
7. 96.5 percent of Manganese.
8. 24.5 percent of Zinc.

Materials required

• Water.
• Cow dung.
• Soil or Sand.
• Earthworms .
• Weed biomass
• A large bin (plastic or cemented tank).
• Dry straw and leaves collected from paddy fields.
• Biodegradable wastes collected from fields and kitchen.

Procedure

1. To prepare a compost, either a plastic or a concrete tank can be used. The size of the tank depends upon the availability of raw materials.
2. Collect the weed biomass and place them under the sun for about 8-12 days. Now chop them to the required smaller size using the cutter.
3. Prepare a cow dung slurry and sprinkle it on the heap for quick decomposition.
4. Add a layer (2 – 3 inch) of soil or sand at the bottom of the tank.
   Now prepare a fine bedding by adding partially decomposed cow dung, dried leaves and other biodegradable wastes collected from fields and kitchen. Distribute evenly on the sand layer.
5. Continue adding both the chopped bio-waste and partially decomposed cow dung layer-wise into the tank up to a depth of 0.5-1.0 ft.
6. Once, after adding all the bio-wastes, release the earthworm species over the mixture and cover the compost mixture with dry straw or gunny bags.
7. Sprinkle water on the regular basis to maintain the moisture content of the compost.
8. Cover the tank with a thatch roof to prevent the entry of ants, lizards, mouse, snakes, etc. and protect the compost from rainwater and direct sunshine.
9. Have a frequent check to avoid the compost from overheating. Maintain a proper moisture and temperature by turnings and subsequent staking.

Result

After the 24th day, around 4000 to 5000 new worms are introduced and the entire raw material is turned into the vermicompost in the form of worm excreta.

Conclusion

Vermicomposting is the scientific method of making compost, by using of earthworms which are commonly found living in soil, feeding on biomass and excreting it in digested form. The complete process is prepared either in pit, tank or heap method. The main benefits of Vermicomposting are:

1. Develops root growth of plants.
2. Improves the physical structure of the soil.
3. Enhances the soil quality with microorganisms.
4. Increases the fertility and water resisting in the soil.
5. Helps with germination, plant growth, and crop yield.
6. Nurtures soil with plant growth hormones such as auxins, gibberellic acid, etc.

Courtesy:

https://byjus.com/biology/vermicomposting/

Cultivation of Single Cell Protein – Spirulina

Aim:

To cultivate and mass production of spirulina

Introduction:

Spirulina is a blue green algae having unique combination of protein, vitamins, minerals. It is richest and cheapest source of nutrients to the human being. It is the richest source of protein (55-71%), minerals  like  iron, calcium, vitamins like beta-Carotene (Vitamin B12. It is used in the diets of malnourished children, adults and sportsmen.Sufficient quantity of iron has proved boom for anemic women and children. Due to its antioxidant nature, scavenges free radicals, prevent diseases like diabetes, cataract, arteriosclerosis, arthritis, aging and cancer are caused by free radicals. It contain gamma linolinic acid which prevent the accumulation of fats and cholesterol and reduces blood pressure. It is rich in protein, beta carotene and vitamin E is being used in the preparation of anti wrinkle cream, anti pimple lotion and various cosmetic products.

 Material Required

Spirulina strain

Zarrouk’s Medium  (pH 9.5) .

NaHCO₃                       16.0

NaNO₃                        2.5

 NaCl                           1.0

K₂SO₄                         1.0

K 2HPO₄                     0.5

MgSO4·7H₂O            0.2  

FeSO4·7H₂O             0.01

 Na-EDTA                  2.5

 Trace minerals

Conical Flasks

Apart from these you require thermometer, pH sensors, air compressors etc for checking various growing conditional parameters.

Procedure:

Strain maintenance:

For Spirulina cultivation, Zarrouk’s Medium was Prepared. Care was taken that the pH of medium is 9.5 after autoclaving. To achieve this, pH of the medium was adjusted to 8.5 which resulted in a pH of 9.5, after autoclaving and "recovery" of the medium. Growth and maintenance of culture is done at 30 ± 2 °C under 12/12 hour light-dark cycles.

Growth Conditions:

For suitable growth, 1 ml of Spirulina culture was inoculated in flasks containing 100 ml growth medium. These flasks were placed under light chamber at 30^oC to 35^oC.

Growth rate was measured by taking O.D (optical density) at 560nm. Agitation was done manually, four to five times a day, after regular interval of time/ or aeration by aquarium pump.  During cultivation, pH was monitored and controlled within values ranging from 8.5 to 10. The yield of cultures is expressed as total dry weight after 15 days of growth. The total dry weight is determined by harvesting the cells and drying it.

Ref:

1. Joshi et.al., 2014. To evaluate Lab scale Cultivation of Spirulina by using different substrates and to Evaluate its Chlorophyll and Protein content. Int. Res. J. Biological Sci. Vol. 3(1), 22-30, January (2014)

2. http://www.akmindia.in/spirulina-cultivation/

3. Indira Priyadarshani et.al., 2014. Influence of aeration and light on biomass production and protein content of four species of marine Cyanobacteria. Int.J.Curr.Microbiol.App.Sci (2014) 3(12): 173-182

Mass production of Brewer’s Yeast

Aim:

To cultivate and mass production of Brewer’s Yeast.

Introduction:

Saccharomyces belongs to the heterogeneous group of yeasts. They were first described by Anton van Leeuwenhoek in 1680 and were characterized by Luis Pasteur in 19th century as the agents responsible for alcoholic fermentation. Yeasts ferment carbohydrates, hence the name Saccharomyces (Gr. Saccharon = Sugar + myce = fungi). Most yeasts used industrially belong to the genus Saccharomyces especially the diploid and polyploid races. Bakers and brewers employ S. cerevisiae in their industries.

Brewers Yeast  which is used in the brewing of top-fermenting alcoholic bverages and is also eaten as a source of vitamin B.

            Many species of Saccharomyces used in the production of different alcoholic beverages are

01. Saccharomyces cerevisiae var. uvarum for production of Malt Beer

02. S. Cerevisiae, S. carlsbergensis for production Rum

03. S. Cerevisiae var. ellipsoideus for Production Wine

04. S. cerevisiae for production Whisky

Materials Required:

YPD (Yeast Extract-Peptone-Dextrose) Medium / Potato Dextrose Broth

Yeast Strain (Saccharomyces cerevisiae var. ellipsoideus)

Sugarcane Molasses

Procedure:

Strain Preparation/ Seed Culture Preparation:

            The seed culture was developed by inoculating a single colony of S. cerevisiae Saccharomyces cerevisiae var. ellipsoideus into a growth medium. The culture was incubated at 37°C for 24 h- 48 h at 150 rpm after adjusting the pH to 7.0. 

Batch culture method

Shake-flask cultures Batch cultures in 250 mL Erlenmeyer flasks were applied with 100 mL malt or molasses-based medium, inoculated with 2 mL pre-seed culture. The initial pH was adjusted to 4.5 and the flasks were incubated at 30 C with shaking at 150 rpm. Growth was monitored by measuring the absorbance at 600 nm through using a spectrophotometer by various time intervals.

Result:

The mass production of yeast in batch culture method was observed and measured the cell mass by UV spectrophotometer.