当前世界环境

Introduction

A phytase (myo-inositol hexakis phosphate phosphohydrolase) is any type of phosphatase enzyme that catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate) — an indigestible, organic form of phosphorus that is found in grains and oil seeds— and releases a usable form of inorganic phosphorus.¹While phytases had been found to occur in animals, plants, fungi and bacteria, phytases had been most commonly detected and characterized from fungi.²In modern age of biotechnology, enzymes have proved their market demand over other products of biotechnology with annual sales close to 2.0 billion dollars. Phytases (EC 3.1.3.8), phosphatases that catalyze the hydrolysis of phosphate moieties, have a big share in enzyme business due to its widespread application as a feed supplement.^{3- 6}The phytases enhance the bioavailibility of minerals, protein and phosphorus in monogastric animals. They reduce the phosphorus pollution in areas of intensive livestock production.^7、8The thermostability of phytase suggests potential biotechnological applications in the pulp and paper industry as a biological agent to remove plant phyticacid. The enzymatic degradation of phytic acid will not produce toxic by-products, so it is environmental friendly.¹⁰A large number of microbes including bacteria, yeast and filamentous fungi has been used for phytase production. Selection of particular microbe depends on the nature of substrate, environmental conditions and desired final product. Thermophilic fungi havecomplex or unusual nutritional requirements and well-known microbes to producephytase.^11-14针对increasingdemand植酸酶的存在ntial to produce phytase in a cost-effective manner. Phytase required for commercial feed preparation must meet following specifications i.e., thermo stability and activity over wider pH range, which is only possible withthermophilic fungi. Poultry manure is a useful nutrient source supplies phytase and high available phosphorous corn diets on the solubility and plant uptake of P, Cu, and Zn in poultry manure and soils amended with manure.Therefore, the present study was conducted with the aim to isolate apotent strain of thermophilic fungi from local habitat and optimize after screening for theproduction of phytase.

Aims and objectives

1. To isolates and characterize the fungal organism, which hydrolyses phytase enzyme in high amounts.
2. To estimate the amount of Phytase enzymeproduced from fungal micro-organisms from open natural fermenters and from soil near poultry farms.
3. Optimization of the condition for the activity of the enzyme phytase.

Material and Methods

Source of samples

Soil sample from Poultry farm waste, 2 Kali is a fermented waste water of rice and this is used by villagers to cook cereal food. These liquid feed of cattle are a good source of microbes, contributing to vitamins and enzymes in food. They contain a lot of bran – derived materials including phytase.

Isolation of organisms

Three different samples were used to isolate fungi which have the ability to produce phytase enzyme during the growth cycle. The poultry soil, kali and kudith samples were collected from Warangal and its surrounding areas. Samples were processed by serial dilution method.¹⁵Soil samples of poultry farms were collected in sterile polythene bags. One gram of sample was suspended in 100 mL of sterilized distilled water and allowed to settle over night at room temperature. The soil suspension was further diluted up to 104-106 times. One millilitre of this dilute suspension was then transferred to ndividual Petri plates containing potato dextrose agar medium. The fungal cultures were further purified from bacterial contaminants by using 10 mg/L combination of penicillin and streptomycin (1:1 ratio) in the Petri plate medium. Two other samples Kudithi and Kali were collected in sterile bottles from different cattle sheds and from different houses of suburban locatlities of Warangal town.

Morphological appearance

All the isolates of fungi were identified by microscopic examination.^16,17,18Independent colonies of each identified isolate were picked up and transferred to potato dextrose agar (PDA) slants for culture maintenance. The cultures were stored in a refrigerator at 4°C for further studies.

Inoculum

The spores from 3-5 days old slant culture were wetted by adding 10 mL ofsterilized 0.005% Monoxal O.T (diacetyl ester of Sodium sulphosuccinic acid) to each slant. The spores were scratched with sterilized inoculating needle and the tubes wereshaken gently to break the clumps of spores. sporesl suspension was used as aninoculum. Inoculum size was measured by measuring the density of spore(number of spore per unit volume) with Haemacytometer, Neubauer improved; precicdor HBG, Germany (Tiefe depth profondeur 0.10 mm and 0.0025mm2 area).

Phytase assay

Phytase activity was assayed after some modification of¹²and¹³methods using Sodium phytate as substrateand the inorganic phosphorus released was measured spectrophotometerically by usingthe Taussky-Schoor reagent. Half milliliter of Sodium phytate (0.00682M) was added to0.1 mL of MgSO₄(0.05M) and 0.1mL of Sodium acetate buffer (0.2M). Enzyme solution(0.1 mL) was added to above mixture and the mixture was incubated at 50°C for 30minutes. After incubation, 1.0 mL of 10% tricarboxylic acid (TCA) was added along with2.0 mL of distilled water. Mixed well and 5.0 mL of Taussky-Schoor reagent was added. Taussky-Schoor reagent was prepared when 10.0 g Ammonium molybdate was mixedwith 10.0 mL H₂SO₄(10N) and further diluted with 70.0 mL of deionized water. Then 5.0g of ferrous sulphate heptahydrate (FeSO4.7H₂O) was added and made the final volumeupto 100.0 mL. Absorbance was measured at 660 nm by using spectrophotometer andliberated inorganic phosphate was estimated after comparing the absorbance with known concentration of KH₂PO₄using same assay conditions instead of enzyme. One unit ofphytase activity is defined as “the amount of enzyme that liberates one µmol of inorganicphosphate at temperature (50°C) and pH (5)”.

Statistical analysis

Duncan’s multiple range tests in the form of probability values were used to find out the significant difference among replicates. Treatment effects werecompared after Snedecor & Cochrane (1980) using computer software Costat, 3.03Berkeley, CA 94701.

Results and Discussion

Both Kudithi and Kali contained microbes which had ability of producing phytase. Fungi Seven strains of five different thermophilic fungi such as Aspergillus fumigatus,Humicola insolens, Rhizomucor miehei-I & II, Sporotrichum thermophile, Thermomyceslanuginosus-I & II were isolated from compost soil and were screened for phytaseproduction.Aspergillus fumigatus生产0.04 U /毫升/分钟的植酸酶而Humicolainsolens, Rhizomucor miehei-I & II, Sporotrichum thermophile, Thermomyces lanuginosus-I & II gave 0.22, 0.20, 0.76, 2.20, 0.20 and 0.53 U/mL/min respectively. Of the seventhermophilic fungal strains screened for phytase production, Sporotrichum thermophile wasfound to produce higher extracellular phytase when grown on solid state wheat bran (Table1). Chadha et al., (2004) isolated and screened out thermophilic fungi and found ninethermophilic strains having the potential of phytase production like Rhizomucor pusillus,Humicola grisea, Sporotrichum thermophile, Humicola insolens, Thermomyceslanuginosus-I, Thermomyces lanuginosus-II, Rhizomucor miehei-I, Rhizomucor miehei-IIand Aspergillus fumigatus. Singh & Satyanarayana (2008) also investigated thatSporotrichum thermophile has the potential for enhanced production of phytase.The main product of a fermentation process often determines the choice of carbonsource, particularly if the product results from the direct dissimilation of it. It is commonpractice to use carbohydrates as carbon source in microbial fermentation processes.

Conclusion

Phytase which is liberated during the growth cycleof microbes especially fungi and bacteria, thephytase enzyme play a vital role in improving the nourishment of plants which is a rich sourceof carbon source, the study was based on the background of Indianvillage feeds for animals which is rich source Of Proteins and carbohydrates this components are been utilized by the fungi when they grow in kali and kudhithi and release phytase enzyme. This phyatatic kali and kudhithi was been used as feed for animals but there is a complication in digestion of phyatses by animals this is liberated out as indigested produted in to environment. Execrates of animals can be used as manure in fields.

References

1. Mullaney EJ, Daly CB, Ullah AH . “Advances in phytase research”. Adv Appl Microbiol 47:157–199 (2000). doi: 10.1016/S0065-2164(00)47004-8. PMID 12876797.
2. Mullaney EJ, Ullah AH. “The term phytase comprises several different classes of enzymes”. Biochem Biophys Res Commun ., 312(1): 179-184. doi:10.1016/j.bbrc.2003.09.176. PMID 14630039
3. Becerra, M. and M.I.G. Siso. Yeast b-galactosidase in solid state fermentation. Enzyme Microb. Technol., 19: 39-44 (1996).
4. Bogar, B., G. Szakacs, J.C. Linden, A. Pandey and R.P. Tengerdy. Optimization of phytaseproduction by solid substrate fermentation. J. Ind. Microbiol. Biotechnol., 30(3): 183-189 (2003).
5. Chadha, B.S., H. Gulati, M. Minhas, H.S. Saini and N. Singh, Phytase production by thethermophilic fungus Rhizom Rhizomucor pusillus, World J. Microbiol. Biotechnol., 20: 105-109 (2004).
6. Ciofalo, V., N. Barton, K. Kretz, J. Baird, M. Cook and D. Schanahan., Safely evaluation of aphytase, expressed in Schizosaccharomyces pombe, intended for use in animal feed. Regul. Toxicol. Pharm., 37(2): 266-292 (2003).
7. Clark, W.D., K.E. Wohlt, R.L. Gilbreath and P.K. Zajar. Phytate phosphorus intake anddisappearance in the gastrointestinal tract of high producing dairy cows. J. Dair. Sci., 69: 3151-3155 (1958).
8. Cooney, D.G. and R. Emerson. Thermophilic fungi: An account of their biology, activitiesand classification. W.H. Freeman & Co., San Francisco, Calif. (1964).
9. K.H Domsch, w . gam和T.H.安德森。Compendium of soil fungi. Academic press,New York, Toronto, San Francisco. (1980).
10. Ebune, A., S. Al-Asheh and Z. Duvnjak. Production of phytase during solid statefermentation using Aspergillus ficuum NRRL 3135 in canola meal. Biores. Technol., 53: 7-12 (1995)
11. Greiner, R. and U. Konietzny. Phytase for food application. Food Technol. Biotechnol., 44(2): 125-140 (2006).
12. Harland, B.F. and J. Harland. Fermentative reduction of phytic acid in rye, wheat and wholewheat bread. Cereal Chem., 57(3): 226-229 (1980).
13. Heinonen, J.K. and R.J. Lahti. A new and convenient colorimetric determination of inorganicorthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem, 113: 313-317 (1981).
14. Hughes, M.N. and R.K. Poole. Metals and Microorganisms. Chapman and Hall, London.Hughes, M.N. and R.K. Poole. 1991. Metal speciation and microbial growth - the hard and softfacts. J. Gen. Microbiol., 137: 725-734 (1989).
15. Koneman, W., E. Allen and W.C. Washington.Diagnostic Microbiology, 3rd Ed, p. 608.Lynd, L.R., P.J. Weimer, Z.W. Van and I.S. Pretorius. 2002. Microbial cellulose utilization:fundamentals and biotechnology. Microbiol. Mol. Biol. Rev., 66: 506-577 (1991).
16. McCoy, M. Enzymes emerge as big AG feed supplements. Chem. Eng. News, 4: 29-30.Onion, A.H.S., D. Allosopp and O.W. Eggins. 1986. Smith Introduction to Industrial Mycology, 7thEd, Edward Arnold Publishers, London, 187-188 (1998).
17. Pandey, A., G. Szakacs, C.R. Soccol, J.A. Rodriguez-Leon and V.T. Soccol. Production,purification and properties of microbial phytases. Bioresour. Technol., 77(3): 203-214.Saha, C. 2003. Hemicellulose bioconversion. Ind. Microbiol. Biotechnol, 30: 279-291 (2001).
18. Singh, B. and T. Satyanarayana. Phytase production by thermophilic mold Sporotrichumthermophile in solid-state fermentation and its application in dephytinization of sesame oilcake. Appl. Biochem. Biotechnol., 133(3): 239-250 (2006).