Soil Samplse In Khartoum State Biology Essay

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Two hundred and seventy Bacillus species were isolated from soil samplse in Khartoum State and tested for amylase production. 20 potential isolates of amylase producer were obtained during primary screening. Characterization of the extra-cellular crude amylase was further evaluated for its biochemical properties as an enzyme for industrial use. The production of amylase following growth of the microorganism was found to be at optimum temperature and pH of 70°C and 9.0, respectively.

Introduction: Amylase is an enzyme that catalyses the breakdown of starch into sugars. Amylase is present in human saliva, where it begins the chemical process of digestion. Foods that contain much starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed because amylase turns some of their starch into sugar in the mouth. The pancreas also makes amylase (alpha amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylase. As diastase, amylase was the first enzyme to be discovered and isolated (by Anselme Payen in 1833). Specific amylase proteins are designated by different Greek letters. All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds.

Amylases have been reported to occur in microorganisms, although they are also found in plants and animals. Two major classes of amylases have been identified in microorganisms, namely a-amylase and glucoamylase. a-Amylases are extracellular enzymes that randomly cleave the 1,4-a-D-glucosidic linkages between adjacent glucose units in the linear amylose chain. Glucoamylase hydrolyzes single glucose units from the nonreducing ends of amylose and amylopectin in a stepwise manner. Among various extracellular enzymes, a-amylase ranks first in terms of commercial exploitation. Spectrum of applications of a-amylase has widened in many sectors such as clinical, medicinal and analytical chemistry. Besides their use in starch saccharification, they also find applications in baking, brewing, detergent, textile, paper and distilling industry. The cost of enzyme production in submerged fermentation (SmF) is high, which necessitates reduction in production cost by alternative methods. The contents of synthetic media are very expensive and these contents might be replaced with more economically available agricultural by-products for the reduction of cost of the medium. The use of agricultural wastes makes solid-state fermentation (SSF) an attractive alternative method have reported a-amylase production in solid-state fermentation with wheat bran and rice husk as substrates have checked the potential of coconut oil cake as substrate for the production of a-amylase using Aspergillus oryzae, a GRAS strain have described the selection of a suitable low cost fermentation medium for the production of a-amylase by using agricultural by-products. Glucoamylase production with an Aspergillus sp. has been reported using cheap rice flake manufacturing wastes as substrate.

The most effective amylases are those that are thermostable. They are generally preferred as their application minimizes contamination risk and reduces reaction time, thus enabling considerable energy saving. Thermostable a-amylases are used for the liquefaction of starch at high temperature and thermolabile a-amylases are used for the saccharification of starch in baking. Babu and Satyanarayana have reported production of a-amylase by a thermophilic Bacillus sp. and optimization of culture conditions for maximum enzyme production. Suitability of thermophilic Bacillus coagulans for a-amylase production by solid-state fermentation in flasks, reactor and trays has been reported. In the present study a-amylase production from Bacillus cereus MTCC 1305 using solid-state fermentation has been investigated and the enzyme is reported to show activity at high temperature.

Bacillus is a genus of Gram-positive, rod-shaped bacteria and a member of the phylum Firmicutes. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species. Under stressful environmental conditions, the cells produce oval endospores that can stay dormant for extended periods. These characteristics originally defined the genus, but not all such species are closely related, and many have been moved to other genera.

Many Bacillus species are able to secrete large quantities of enzymes. Bacillus amyloliquefaciens is the source of a natural antibiotic protein barnase (a ribonuclease), alpha amylase used in starch hydrolysis, the protease subtilisin used with detergents, and the BamH1 restriction enzyme used in DNA research.

Amylases find use in breadmaking and to break down complex sugars, such as starch (found in flour), into simple sugars. Yeast then feeds on these simple sugars and converts it into the waste products of alcohol and CO2. This imparts flavour and causes the bread to rise. While amylases are found naturally in yeast cells, it takes time for the yeast to produce enough of these enzymes to break down significant quantities of starch in the bread. This is the reason for long fermented doughs such as sour dough. Modern breadmaking techniques have included amylases (often in the form of malted barley) into bread improver, thereby making the process faster and more practical for commercial use.

Alpha and beta amylases are important in brewing beer and liquor made from sugars derived from starch. In fermentation, yeast ingest sugars and excrete alcohol. In beer and some liquors, the sugars present at the beginning of fermentation have been produced by "mashing" grains or other starch sources (such as potatoes). In traditional beer brewing, malted barley is mixed with hot water to create a "mash," which is held at a given temperature to allow the amylases in the malted grain to convert the barley's starch into sugars. Different temperatures optimize the activity of alpha or beta amylase, resulting in different mixtures of fermentable and unfermentable sugars. In selecting mash temperature and grain-to-water ratio, a brewer can change the alcohol content, mouthfeel, aroma, and flavor of the finished beer.

In some historic methods of producing alcoholic beverages, the conversion of starch to sugar starts with the brewer chewing grain to mix it with saliva. This practice is no longer in general use. Factory workers who work with amylase for any of the above uses are at increased risk of occupational asthma. Five to 9% of bakers have a positive skin test, and a fourth to a third of bakers with breathing problems are hypersensitive to amylase. An inhibitor of alpha-amylase, called phaseolamin, has been tested as a potential diet aid.

In molecular biology, the presence of amylase can serve as an additional method of selecting for successful integration of a reporter construct in addition to antibiotic resistance. As reporter genes are flanked by homologous regions of the structural gene for amylase, successful integration will disrupt the amylase gene and prevent starch degradation, which is easily detectable through iodine staining.

Materials required

Students will work in pairs

§ Cultures : all the Bacilli spp. in the ITT Dublin culture collection

§ Nutrient Agar (NA) + 1% starch plates

§ Iodine to flood the starch plates

§ B. cereus/B subtilis cultures prepared as described in Experimental procedure (2)

§ DNS reagent

§ 10 mMolar glucose solution for DNS standard curve

§ Starch solution (1 % prepared in 0.1M phosphate buffer, pH 7.0

§ Water bath at 40 o C

§ Boiling water baths

§ Testtubes X 12 (for DNS assay)

§ Cuvettes

§ Range of pipettes (15ml volume)

§ UVVIS spectrophotometer

Result and Discussion:

The first step was to isolate the desired microorganism that produces α- amylase. Among the seven selected Bacillus strains that showed the highest amylase synthesis, Strain 7 was selected because it gave a larger diameter zone of clearance and the highest relative amylolytic activity compared to the other species. These culture were further screened for amylase production by submerge fermentation using the hydrolysis method.

Results for screening of Bacillus species for amylase production

18th October 2012 Group 2 Results

Bacillus Species

Group Number

Group1

Group2

Group3

Group4

Group5

Group6

Group7

Group8

B. sphaerius

-

-

-

-

-

-

-

N/A

B. pumilus

-

-

-

-

-

-

-

N/A

B. subtilis

++

++

++

++

+

N/A

++

++

B. megaterium

-

-

+

+

-

+

N/A

+

B.stearothermophilus

+

+

+

N/A

N/A

+

+

++

B. cereus

++

N/A

++

++

++

++

++

+++

B. mycoides

N/A

+

N/A

+

-

+

+

+

Legend: - No effect

+ Small effect

++ Medium effect

+++ Large effect

Bacillus species are considered to be the most important sources of a-amylase and have been used for enzyme production using SSF. Production of pullulanase and amylase using Bacillus cereus has been studied.

Although many microorganisms produce this enzyme, the ones most commonly used for their industrial production are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and Aspergillus niger.

Production of amylase by Bacillus has been studied by growing the isolate on various natural solid substrates. The results in the present study indicated that amylase production pattern varied with type of agro-residue. Among Bacillus sp. are considered to be the most important source of amylase and have been used for which wheat bran was found to be most suitable, while minimum amylase production was noticed with gram bran. The suitability of wheat bran may be due to the fact that it contains sufficient nutrients and is able to remain loose even in moist conditions, thereby producing a large surface area. The mixtures of substrates containing WB gave significant increase in enzyme production, but the highest enzyme activity was found with WB+GIOC.

The production of enzyme was determined at different temperatures ranging from 35 to 75 °C and optimum enzyme production was obtained at 55 °C. Similar patterns were reported for B. licheniformis (Saito and Yamamoto, 1975), B. stearothermophilus (Wind et al, 1994) and Bacillus sp. (Mamo and Gessesse, 1999), where as Bajpai and Bajpai (1989) reported that the enzyme synthesis and growth temperature of B. licheniformis TCRDC-B13 strain was 25-50 °C and maximum enzyme production was obtained at 35 °C.

pH of the growth medium plays an important role by inducing morphological changes in microbes and in enzyme secretion. The pH change observed during growth of microorganism also effects the product stability in the medium. Maximum amylase production was achieved at pH 7.0 by B. cereus MK, although pH 4.0 to 12.0 supported amylase production.

The incubation time for achieving the maximum enzyme level is governed by the characteristics of the culture and is based on the growth rate and enzyme production. Maximum amylase production was observed at 24h of incubation time. Further increase in incubation time showed decreased enzyme yields. The decrease in enzyme yield may be because of the decomposition or degeneration of amylase due to interaction with other components in the.

Among the several factors that are important for microbial growth and enzyme production under solid state fermentation using a particular substrate, moisture level / water activity is one of the most critical factors. Because, solid state fermentation processes are different from submerged fermentation culturing since microbial growth and product formation occurs at or near the surface of the solid substrate particle having low moisture contents.

Inoculum level selected for this study ranged from 5 to 25% of 24h. Enzyme production is varied with inoculum level and showed parabolic nature in the present study. Maximum amylase synthesis was noticed with 15% inoculum size.

Supplementation of carbon sources in the form of mono saccharides, di saccharides and poly saccharides to solid medium at 1.0% level showed different impact on enzyme production with different compounds

Conclusions:

Amylases have been obtained as a result of screening of Bacillus sp. strains, isolated from different natural substrates. They have shown 30-100% of their activities after 60 min of incubation at 100 degrees C. Seven thermostable a-amylases demonstrated 100% of their activities after 60 min of incubation at 100 degrees C. These enzymes showed maximal activities at pH 9.0 and exhibited 10-30% of activities at pH 11.0 and 12.0. These properties make them promising for future research and possible practical use.

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