دانلود رایگان ترجمه مقاله خالص سازی و خاصیت فیتاز خارج سلولی از باسیلوس sp. KHU-10 (نشریه اسپرینگر 2001)

تخلیص و خواص فیتاز خارج سلولی از باسیلوس سوبتیلیس sp. KHU-10

Purification and Properties of Extracellular Phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl2 and pH 6.0-9.5 and 60°C with 10 mM CaCl2. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl2. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The Km value for sodium phytate was 50 μM. Its activity was inhibited by EDTA and metal ions such as Ba2+, Cd2+, Co2+, Cr3+, Cu2+, Hg2+, and Mn2+ ions.

1 Introduction

Phytase (myo-inositol hexakisphopate phosphorhydrolase, EC 3.1.3.8) catalyzes the release of phosphate from phytate (myo-inositol hexakiphosphate), which is the main form of phosphorus predominantly occurring in cereal grains, legumes, and oilseeds (Irving, 1980; Nayini and Markakis, 1986). Hydrolysis of phytic acid (phytate) to myo-inositol and phosphoric acid is considered an important metabolic process in several biosystems. Increasing awareness of agricultural pollution and demand for regulations in particular to limit the phosphorus (P) content in manure has intensified phytase research. The focus has mainly been on its production and use as a means of reducing inorganic P supplementation in feed and consequent reduction in fecal P excretion. Environmental pollution due to highphosphate manure has resulted in the accumulation of P at various locations, especially in water bodies. Phytase supplementation can reduce the amount of P in manure up to approximately 30%.

Phytase is significant for upgrading the nutritional quality of phytate-rich feed. Phytase also improves the bioavailability of phytate P in plant foods for humans (Martinez et al., 1996). Two important groups of animals, pigs and poultry, lack the enzyme needed to digest phytate efficiently in their feed (Greiner et al., 1993). As a result, they excrete large amounts of P into the environment. This results in pollution. However, for their proper skeletal growth, these animals need P at suitable concentration. This makes the situation complex. Supplementation of phytase to the feed provides an alter native to tackle both these conditions effectively.

Phytases can be derived from a number of sources including plants, animals, and microorganisms. Recent research has shown that microbial sources are more promising for the production of phytases on a commercial level. Although several strains of bacteria (Sreeramulu et al., 1996; Choi et al., 1999), yeasts (Mayer et al., 1999), and fungi (Ahmad et al., 2000; Kim et al., 1999) have been used for production under different conditions, two stratins of Aspergillus sp., A. niger (Ahmad et al., 2000), and A. ficuum (Kim et al., 1999) have most commonly been employed for its commercial production.

Although phytase shows a potential to be utilized for phytate bioconversion, the enzyme activities and yields need to be increased to make them possible for industrial application. Therefore, it is important to isolate a variety of different microorganisms and their enzymes for phytate degradation.

We reported that Bacillus sp. KHU-10 produced a high level of an extracellular phytase in a maltose, peptone, and beef extract medium. Under optimized conditions, the production of the phytase reached a highest level of 0.2 unit/ml after 4 days of fermentation (Choi et al., 1999). We report here the characterization of a novel bacterial phytase from the recently isolated heattolerant strain Bacillus sp. KHU-10.