Identification of Phytase producing Bacteria C43 isolated from cattle shed soil samples of Hyderabad, A.P

Author Name: *Sreedevi S, Reddy B.N.
Author Email: sreedevi163@yahoo.com

Abstract

Phytases are the enzymes which hydrolyze phytic acid, a major storage form of phosphorus in cereals and legumes, to less phosphorylated myo-inositol derivatives releasing inorganic phosphate. The supplementation of animal feed with phytases reduces the cost of diets by removing or reducing the need for supplemental inorganic phosphate and increases the bioavailability of phosphorous in monogastric animals such as pig, poultry and fish which lack gastrointestinal phytases. These feed enzymes can also have a positive impact on the environment by allowing better use of natural resources and reducing pollution. Different soil samples – Rhizosphere soil, Cattle shed soil and poultry farm soil were collected from various regions of Hyderabad, Andhra Pradesh and were used as a source material for isolation and screening of phytase producing bacteria. Isolate C43 produced significantly higher phytase yield than other isolates and was chosen for species identification. Preliminary identification by microscopic and biochemical tests identified the isolate C43 as Bacillus sp. FAME analysis of the isolate by Gas Chromatography identified the isolate C43 as Bacillus licheniformis. Further, the identification of the strain was confirmed by subjecting it to 16S rRNA gene sequence analysis. The isolate C43 showed high similarity to Bacillus subtilis subsp. spizizenii strain 3EC5A1 and was thus designated as Bacillus subtilis C43.

Keywords

Phytases, Phytic acid, FAME analysis, 16S rRNA, Gene sequence analysis.

Introduction

Phytic acid (myo-inositol hexakisphosphate) is an anhydrous storage form of phosphate accounting for more than 80% of the total phosphorus in cereals and legumes [1, 2, 3]. Under normal physiological conditions phytic acid chelates essential minerals such as calcium, iron, zinc, magnesium, manganese, copper and molybdenum and preventing their absorption [4, 5, 6, 7, 8, 9]. The organically bound phosphate of phytic acid is not metabolized by monogastric animals such as pig, poultry and fish due to lack of phytase and consequently contributes to the phosphorus pollution problems in areas of intensive livestock production [10, 11, 12, 13, 14]. The anti-nutritive properties and its value as possible phosphorus Source; have stimulated researchers to develop phytate hydrolysis methods. Phytases are the enzymes (myo-inositol hexakisphosphate phosphohydrolases) which hydrolyze phytic acid to less phosphorylated myo-inositol derivatives (in some cases to free myo-inositol), releasing inorganic phosphate [15]. The supplementation of animal feed with phytases reduces the cost of diets by removing or reducing the need for supplemental inorganic phosphate and increases the bioavailability of phosphorous in monogastric animals. Apart from contributing to improving nutritive value, these feed enzymes can also have a positive impact on the environment by allowing better use of natural resources and reducing pollution. These Phytases are widespread in nature and can be derived from a host of sources including plants, animals and microorganisms. Microbial sources are more promising for the production of phytases on a commercial scale [16, 17]. Some of the phytase producing microorganisms include bacteria such as Bacillus subtilis[18], Escherichia coli[19], Yeasts such as Saccharomyces cerevisiae[20], Schwannoiomyces castellii [21] and fungi such as Aspergillus niger [22], A. oryzae [23], A. flavus [24] and Penicillium sp.[25]. Due to several biological characteristics, such as substrate specificity, resistance to proteolysis and catalytic efficiency, bacterial phytases have considerable potential in commercial applications. The increasing potential of phytase application prompts screening for newer phytase producing microorganisms, which can meet the conditions favourable to the industrial production. Bacteria are though ubiquitous in their occurrence, the most common sources for their isolation are soils [26, 27].
The historical method for identification of unknown bacteria is dependent on the comparison of an accurate morphologic and phenotypic description of type strains with the accurate morphologic and phenotypic description of the isolate to be identified using standard references such as Bergey’s Manual of Systematic Bacteriology. Frequently, there would be no perfect match and a judgment would have to be made about the most probable identification which could vary among laboratories [28]. A new standard for identifying bacteria was by comparing a stable part of the genetic code i.e. 16S rRNA gene [29, 30, 31, 32, 33, 34, 35]. Thus, if the goal is to identify an unknown organism on the basis of no prior knowledge, the 16S rRNA gene sequence is an excellent and extensively used choice. Therefore, the present research study was taken up with the objective to isolate a potential phytase producing bacteria from various soil samples and identify the strain by studying morphological and biochemical characteristics and 16S rRNA gene sequence analysis.

Conclusion

Thus phytase producing bacteria were isolated from various soil sources and the high yielding bacterial isolate C43 was selected for characterization. Microscopic, Biochemical and 16S rRNA gene sequence studies revealed the newly isolated phytase producing bacterial isolate as Bacillus subtilis. In addition to phytase production, the isolate B.subtilis has other characters of commercial importance. The target protein is secreted to the culture medium which makes its purification simpler and more economical. Moreover, B.subtilis has a GRAS (Generally Regarded As Safe) status, allowing its use as feed additive. Further optimization studies will be made to develop cost effective phytase with improved properties to be used for the animal feed industries.

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