^ Top

ENZYMES

INTRODUCTION

The student of Biology or Biochemistry will already have learned of the Universal association of enzymes with living matter and the important part played by them in all types of vital activity.

Enzymes are proteinous in nature. It acts with different substrate to produce small molecules to smallest molecule according to the reaction parameters like pH, temp, pressure and physical condition of the substrate.

All the enzymes are not capable to react alone with substrate to convert product. Sometimes there is requirement of presence of heavy metals like iron as iron ion, cobalt as cobalt ion or organic molecules, which are called co-factors.

There are two types of enzymes according to their sphere of action:

Exo-enzymes (2) Endo-enzymes

Exo-enzymes are such enzymes, which stimulate outside of the cells and react with substrate to convert product.

Endo-enzymes are such enzymes, which stimulate inside the cells and convert substrate to product, inside the cells.

Basically enzymes are like organic catalyst produced by living cells. Live catalyst enzymes are capable of accelerating chemical reaction by millions of times under normal room temperature in ‘soft’ solvents. They have another quite unique ability-strict selectiveness in the presence of definite enzyme only one, the only one, of all possible reactions will, as a rule proceed.

MANUFACTURING PROCESS

Basically major enzymes are produced by microbial fermentation process. Microbes may be bacteria, yeast or mold, it may be some times plant origin.

Production of a-Amylase (By Bacterial use) :-

Medium Composition:

The above composition ingredients dissolve in 25 times of water and sterilized at 115°C for 15 minutes. A solution of 10% Sodium Carbonate in water was also sterilized at 115°C for 15 minutes. These two solutions were mixed to prepare a culture medium 250 ml in 1 litre shake flask. Use inocculum of Bacillus species NO-38-2 (A.T.C.C.21783). Make medium pH-10 and shake culture at 37°C for 48 Hours. The cells were removed from the culture by a centrifuge and at pH – 9, 1 ml. of this culture filtrate contained 2500 units of alkaline amylase.

This culture broth thoroughly cooled and 3 volumes of acetone were added by which enzyme precipitated quantitatively. This precipitate was thoroughly washed with acetone and dried in air. About 11 grams brown powder were obtained from the 1 litre of the culture broth. The sample obtained thereby has an optimum pH around 9.0 and it was found to be an amylase.

Manufacturing Process of Fungal a-amylase:

The mold is cultivated on a nutrient medium containing the following composition

Preparation of Seed Culture:

Commercial viable strain of Aspergillus oryzae is grown in a test tube containing nutrient media 7 ml (previously sterile). Growth of strain in test tube is washed by sterile water and transfer to a shake flask containing a litre media of composition.

Starch – 60 gm, NaNO3 9 gm, MgSO4 1.0 gm, KH2PO4 1gm, KCI 0.5 gm, FeSO4 0.03 gm, Mg (NO3) 0.2 gm, Mg (H2PO4)

gm, and 10% extract of malt sprouts 10% by volume.

The seeding culture is grown for 30 Hours on a shaker reciprocating at a speed of 180 R.P.M. at a temperature of 30°C.

Commercial Process:

Now produce seed culture (0.5%) is transferred in sterile condition into a fermentation tank containing sterile media of composition.

And 20% extract of Malt sprouts 20% by volume.

There will be foam in the fermentor, which can be controlled by antifoam dip like sperm oil or any other suitable antifoam oil.

After completion of fermentation mycelium in the fermented broth be separated by filtration or centrifugal separation.

The activity of in the culture filtrate 20 units/ml. The filtrate is dialyzed with 0.03 M Phosphate bufter at pH 7.05 for 12 Hours. The obtained dialyzed is divided into several equal portions containing 1000 mg of protein each and each having the activity of a-amylase of 10,000 units. Each portion of the dialyzed is passed through an individual column packed with 10 gm of diethyl amine ethyl cellulose. The diameter of the column is 35 M.M., the height 250 M. M. During this operation a-amylase and other proteins are absorbed on the column packing.

Next the a-amylase is eluted from the sorbert. The process is carried out in two steps. The first elution is effect with 0.06 M phosphate buffer at pH – 7.15. During this operation only the accompanying proteins are eluted where as the a-amylase remains on the column packing.

a-Amylase is eluted during the 2^nd step when 0.11 M phosphate buffer at pH-7.15 containing 0.001 M solution of calcium chloride is passed through the column. The buffer completely elutes the fraction containing a-amylase. The field of a-amylase is 300 mg. having total activity of 10,000 units that is the yield of a-amylase after elution is 100%.

Dialysis, adsorption and elution of protein is carried out at a temperature of +8°C. The specific activity of a-amylase obtained according to this process was almost twice as great compared with the activity of a-amylase obtained by the known method.

The activity of the product is expressed in international units (I.U.). This is expressed in I.U/CC when the product is in solution and in I.U./gm when the product is solid.

Stabilization of a-amylase:

Bacterial a-amylase can be stabilize in presence of Sodium and Calcium salts in solution (especially Calcium Acetate).

b- Amylase:

b- Amylase the saccharifying enzymes produces maltose from starch, glycogen dextrins by selectivity removing maltose units at the non reducing ends of the molecule chains, b- Amylase activity also stops at the a -D (1 6) branching linkages of amylopectins leaving polysaccharides termed limited dextrin.

Manufacturing Process by Using Bacterial Culture

A medium containing 5% milk case in, 2% corn steep liquor, 10% soluble starch and enough water to make 15 litres was sterilized in jarfermentor, adjusted to pH 7.0-7.2 and inoculated with commercial viable strain of B. Megaterium. The mixture was incubated at 30°C for 2 days with stirring where upon the broth showed an enzyme activity of 25.5 unit/ml.

The culture was centrifuged at 8000 R.P.M. for 20 minutes to remove the cells and the supernatant was mixed with enough solid (NH4)2SO4 to precipitate a crude enzyme product. It was dissolved in M/100 acetate buffer solution and the solution was dialyzed against running tap water for 3 days. Impurities still present were precipitated by adding 25% lead acetate solution drop by drop. The precipitate was removed by centrifuging.

The enzyme was again slated out with (NH4)2SO4, dissolved in M/100 acetate buffer at pH-6 and the solution was heated 15 at 60°C. It was dialyzed and enzyme from the further purified solution was absorbed on a molecular sieve chromatographic medium (SE Sephadex G-25), eluted with M/2 sodium chloride solution and get filtered to Sephadex G-100. The active fraction was freeze dried and thus product dried b- Amylase obtained activity of 100 units per mg and thus had 50% of the original broth.

Pectolytic Enzyme :

This enzyme is prepared by solid state fermentation using mold strain of commercial viable.

Preparation of culture :

Took commercial viable strain of Aspergillus Carbarierius transfer to sterile test tube containing Agar medium, then plate (Petridish) it to grow pure individual colony and then pick up well defined colony and transfer it to a test tube containing liquid nutrient media. Keep it for 24 hrs at 37°C and then transfer it to shake flask containing 250ml solid nutrient media and keep it for growth of 24 hrs. This growth is used as seed culture for the propogation.

BIOFERTILIZER

INTRODUCTION

Fertilizer a substance which helps to grow plants rapidly and produce fruits, flower, and vegetable more quantity in proper time, otherwise fertilizer be a compound, which fulfill the needed minerals or elemerts require for the growth of plants and vegetables to grow and fruits in proper time.

Fertilizer basically three types (Inorganic fertilizer (2) Organic fertilizer (3) Bio fertilizer.

Bio fertilizer are natural fertilizers which are microbial inoculants of bacteria, algae, fungi alone or in combination and they augment the availability of nutrients to the plants. Rhizobium is the best known bio fertilizer which fixes atmosphere nitrogen symbiotically with legumes. Other bio-fertilizers are Azotobacter, Azospirillum, blue green algae and Azolla.

Bio fertilizer actually counteract the toxic effects of chemical fertilizers, offers economic and ecological benefits by way of soil health and fertility to farmers. Bio fertilizers provide a dependable continued source of supply of nutrients unlike chemical fertilizers, which, besides cost, are depleting source and add upto the pollution problems.

The earliest production of Rhizobium in oculant in India started in 1934, but commercial production was undertaken in 1964 when soyabean was introduced in the country subsequently there was increase in demand for bio fertilizers for other legumes also production of other bio fertilizers such as Blue Green Algae, Azotobacter, Azospirillum and Azolla is also undertaken in the country on large scale. There is a good demand for Azospirillum from farmers of Tamil Nadu as they have obtained significant response in rice and sugar cane.

Although the bio-fertilizers cannot by themselves totally replace chemical fertilizers at least in the near future, there is need for integrated nutrient supply to crops through a judicious amalagam of biological, chemical and organic source of nutrient supply.

Among the different types of fertilizers available at present, Rhizobium is relatively more effective and more orderly used particularly the small and marginal farmers who constitute about 56% at the total farming community are reluctant to use chemical fertilizers in legume cultivation resulting in very poor yields. Considering the area cultivated under legume crop use of Rhizobium can be a promising input which can substantially increase legume crops.

Agriculture to-day Consumes high inputs of nitrogen. The present needs of nitrogen are largely met from synthetic nitrogen fertilizers their consumption has been rising every year. These fertilizers are quite expensive because of high cost of production consequently their high inputs have considerably increased agricultural production in a highly energy consuming process (13800 k cals per kg of nitrogen fixed). Therefore in the present situation of energy constraint, attention has diverted to tap alternatives to supplement nitrogen resources by directly utilizing atmosheric nitrogen through biological nitrogen through biological fixation. In the symbiotic nitrogen fixing system, photo synthetically stored energy is utilized instead of fossil fules. In this process atmospheric nitrogen is reduced to ammonia in presence of celetron donor ferredoxin and flavodoxin, ATP. Generating system and biological catalytser (nitrogenase, hydrogenase and some minerals).

AQUA CULTURE

INTRODUCTION

Biotechnology is an emerging technology with great potential. The country has the necessary talent to harness the potential of this technology. Technical innovation and resulting changes is the key to the process of economic development. Determining the choice of future technologies thus becomes important economic priority for the nation. Today we are living in a society, which is witnessing results of technological development at such unprecented rate, which could not have been imagined even 50 years back.

Biotechnology the technological gap between advanced nations and to the development country. Biotechnology, defined in a broad sense, is the application of biotechnological organism and molecules to technical and industrial processes. The fermentation process which were first established after the 2^nd world war were used to produce new products such as steroid, antibiotics, and fine chemicals. And the biotechnology arises from a wave of innovation occurring within this fermentation process and results from application of different techniques. It generally implies the application of novel microbes and other living system, altered or modified through various technologies like genetic engineering, biotechnological tools enable us to manipulate the core of all living matters i.e. the DNA is a manner resulting in enhanced or even totally new properties in plants animals and micro organisms, thus providing vast scope for application in many areas like accelerated food production disease control, environment protection and improvement etc. In other words, its application is multi sectorol.

Potential application area of biotechnology are:

1. Agriculture

(a) Bio fertilizers

(b) Bioinsecticide and bio-pesticides

(c) Hybrid seeds

(d) Artificial seeds

(e) Photosynthesis improvers

(f) Stress resistant crops and plants

(g) Tissue culture

2. Animal husbandry

3. Aqua culture

4. The human health

(a) Vaccines

(b) Immunodiognistics

(c) Medicines

5. Population control

6. Fuel and fodder

7. Biomass from various sources

8. Waste

9. Chemical feedstock.

Aquaculture Techniques

With 7000 Kms of coastline and 4.5 million hectares of water area as ponds, tanks and lakes, aquaculture potential of India is yet to be harnessed fully. The potential areas of application of biotechnology in Indian fresh water aquaculture are recycling of organic matter, biological nitrogen fixation and genetic transfers. For example, average yield per hectare per annum of prawns in countries like Thailand, Philippines, Taiwan, Hong Kong etc. is 10-12 tonnes compared to 300-400 tonnes in India. Above levels of high yields is possible by systematic use of marine biology and pisciculture knowledge.

Various biotechnological tools are being utilized for use in aquaculture for improving productivity both in terms of quantity and quality or for providing value added products.

Production Techniques

It is estimated that India has a brackish water area of 11.90 lakh hectares. The major centers of brackish water prawn farming lying in the coastal regions of maritime states of India. At present the land used for brackish water aquaculture is limited to 82000 hectares from where only 62000 tonnes of prawn are produced. The rest of the area is idle or unutilized.

Site selection

Detailed engineering investigation has to be carried out to collect sufficient field data comprising of soil condition, water availability, topographical features, position of creeks canals, availability of power and road links. Marigrove land should not be used for culture and should be allowed for natural growth, which helps for soil binding and nutrient cyclings. Similarly agricultural and also should not be selected for brackish water shrimp culture. Only marginal land not used for agriculture should be used for brackish water shrimp farming. After detailed site survey topographic maps are prepared which are essential for planning lay out of the farm. Information on total height of the region and leveling of ground are very important. Subsoil investigation is necessary to obtain engineering properties of soil, which are required for designing the foundation of various farms structures. Testing of permeability of the soil is very important to measure the rate at which water sweeps vertically. After completion of engineering investigation the data used to prepare hydrographic and soil profile plan. Shrimp farm layout when properly designed should strike a balance considering economy, functionality and aesthetics. The basic principle is to minimize the number of gates, the sizes and length of dikes and canals but it should not sacrifice biological requirements of suitable environment of the culture species. The shrimp farms are designed as farmed system, which suits for semintensive culture practices.

Pond Preparation

Pond preparation is the start of the culture operation and involves planning and coordination of schedules to maximize resources without compromising basic principals of shrimp production. The pond should be prepared at least 30-45 days prior to water culture. This is important to allow physical disintegration and chemical decomposition of minerals and organic matters from soil and adjust pH to slightly alkaline (7-8).

Non Acidic Sulphate Soil

The non acidic sulphate soil has pH value of more than 6. The soil is recognized by a dark to light brown colour and a clay to sandy clay texture.

Acidic Sulphate Soil

Acidic sulphate soil has very less pH (3-4). The soil is recognizable by the reddish colour (of iron oxide) that may form on the pond bottom after flooding. In the process concentration of iron and aluminium, which one harmful to the shrimp are released from soil.

Pond preparation for reclaiming acid sulphate soil to pH above 5 includes steps such as (a) drying, cultivating and submerging and (b) refilling, flushing and liming. In case where a thin layer of non acid top soil, overlay soil materials with high potential acidity, it may be advisable to cut the pond bottom to a desired depth and fill back with non acid top soil.

Components of Farm

The various farm components are reservoir system of ponds comprising nursery rearing ponds, bio ponds, supply canal dikes. Water control structures, pumps and supporting facilities like buildings, pump house, storage sheds and roads.

Pond

Before pond layout is planned it is necessary to keep in mind the requirement of size and shape of ponds such as pre growing pond, rearing pond and bio-pond. While designing the farm, a minimum space of 500m (buffer zone) between adjacent farms and drinking water source should be maintained.

Pre-Growing Pond

Pre-growing pond are nursery pond used for rearing fry up PL 20 stage after which they are transferred to rearing ponds. Pre growing pond will be in 10% of the total production area, shape will be square or rectangular. Pre growing ponds can be detected if PL 20 seeds are obtained from hatchey.

Rearing Ponds

It is used for growing from PL 20 to marketable size at the harvest stage. It’s size will be 70% of the total production area, it’s shape will be rectangular of the length and breadth ratio 1.5:1. Sometimes the shape of the ponds depends on the shape of site. The size of the grow out pond range from 1 ha. But for management convenience ponds beyond 5 ha is not preferred. Bottom should be free from projected rocks, tree stumps etc. The pond bottom must have a gradual slope (3/1000) from the inlet gate towards drainage gate.

Bio Ponds

It is for satisfaction of pollution control authorities and to prevent environment degradation, drainage water from the farm should be acceptable to flow to the area or river system or sea. Bio ponds are necessary as part of the culture pond system. Bio ponds are large setting where the rich nutritional affluent are treated naturally. Bio ponds are also used for culture of fish, mussel etc. Bio ponds constitute about 7-10% of the total farm area.

Dike

The entire farm is provided with dikes or bands there are three types of dike :-

1. Main dike

2. Secondary dike

3. Tertiary dikes

1. Main Dike

Is to protect the prawn farm from destruction due to flood, storm and tide etc.

2. Secondary Dike

Smaller in size to the main peripheral dike, they are usually provided on both sides of the main supply or drainage canal of and secondary supply or drainage canal. The total height is obtained by providing suitable free board of 0.3 m. The top secondary dike is usually 1.5 m and side slopes 1:1.5

3. Tertiary Dike

Tertiary dikes are constructed between ponds. They should be able to contain the desired depth of water in the pond. The total slope of the top will be fixed in the same manner as the secondary dike.

Water Control Structure

The water flow in the pond system are controlled by various control structures, which are (1). Main supply gate of farm, (2). Secondary supply canal control gate (3). Supply canal drain gate (4). Pond inlet gate (5). Pond outlet gate and (5). Farm main drainage gate.

Pumping station

Calculation of pump capacity requirement will be based on parameters such as pond area, average water depth in ponds, duration of pumping, water exchanges and efficiency of the pump. Pumping requirement of farm is of high capacity and low head type.

Outlet Gate Screen

The most size of the screen is related to average body weight (ABW) of the prawn. For prawn weight ranging from 0.5 gms to 15 gms, mesh size varies from 0.5 mm to 10 mm at the entrance gate. Screen is necessary to prevent loss of culture prawn.

Flushing

Flush is performed immediate after harvest of the pond. For preparing the pond, the bottom soil of the pond has to be ploughed. It overturn the wet soil and oxidize thoroughly.

Liming

When the pH of the water is above than 6.5, there is no requirement of liming. When the pH is below 6, CaCO3 or Ca (OH)2 may be used according to requirements.

^ Top