production and characterization of extracellular phytase from

PRODUCTION AND CHARACTERIZATION OF
EXTRACELLULAR PHYTASE FROM PSEUDOMONAS SP.
WEERACHAT SRI-AKKHARIN
A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR
THE DEGREE OF MASTER OF SCIENCE (BIOTECHNOLOGY)
FACULTY OF GRADUATE STUDIES
MAHIDOL UNIVERSITY
2004
ISBN 974-04-5062-8
COPYRIGHT OF MAHIDOL UNIVERSITY
Fac. of Grad. Studies, Mahidol Univ.
Thesis / iv
PRODUCTION AND CHARACTERIZATION OF EXTRACELLULAR PHYTASE
FROM PSEUDOMONAS SP.
WEERACHAT SRI-AKKHARIN 4536525 SCBT/M
M.Sc.(BIOTECHNOLOGY)
THESIS ADVISORS : SAOVANEE DHARMSTHITI, Ph.D. (GENETICS),
SITTIWAT
LERTSIRI, Ph.D. (AGRICULTURAL SCIENCE), SUCHAT
UDOMSOPAGIT, Ph.D. ( MICROBIOLOGY)
Abstact
A bacterial strain capable of producing an extracellular phytase was isolated
from soil. According to its colony morphology, Gram stain, biochemical
characteristics, and ability to produce greenish pigment and growth on a selective
medium, it was identified as Pseudomonas aeruginosa strain K2. Maximum phytase
activity was obtained when it was cultivated in a 250-ml Erlenmeyer flask containing
75 ml of LB (Luria Bertani ) medium with initial pH of 5 under 200 rpm agitation at
400C for 24 hours. General properties of crude K2 phytase produced in LB medium
were determined. Crude K2 phytase exhibited maximum activity at pH 5.0 and 450C.
The enzyme was stable at acidic pH range of 3-5 and at temperatures lower than 400C.
A study on the effect of EDTA (a chelating agent) and metal ions showed that the
enzyme was stable with EDTA, Ca2+, Fe2+ and Fe3+ but it was moderately sensitive to
Cu2+ and was strongly inhibited by Zn2+. Addition of Fe2+ to the crude phytase helped
increase its temperature stability. This enzyme was sensitive to trypsin and bile acids,
i.e. taurocholic acid and deoxycholic acid, whereas it was highly stable with lactic and
propionic acid at either 0.1%(v/v) or 1.0%(v/v) concentration. Adding lactic and
propionic acid could also promote the enzyme activity. K2 phytase could hydrolyse a
range of non-phytate-based phosphorylated substrates, but only at lower activities than
phytate. The crude enzyme also showed protease, alkaline and acid phosphatase
activities at level of 4.10, 0.43 and 1.29 U/ml, respectively, and some carbohydratehydrolyzing enzymes at low levels of activity. Insensitivity to 2-mercaptoethanol
indicated the absence of essential disulfide bond(s) in this phytase molecule. A
complex medium, WTY, suitable for K2 phytase production has been developed. It
was composed of compositions of PSM (Kerovuo et al., 1998) with reduction of
glucose concentration to 1.5% (w/v) and omission of phytate in the formulae, and
supplemented with 2.0% (w/v) yeast extract, 0.1% (v/v) Tween-80 and 10% (v/v) of
wheat bran extract. Digestion of mixed chicken feed, rice bran, wheat bran and
soybean meal by crude K2 phytase at 5 U per 1 g of such materials could markedly
increase phosphate content and also increase reducing sugar and protein content. This
digestion efficiency suggested that this enzyme was a potential candidate for use in
animal feed applications.
KEY WORDS : EXTRACELLULAR PHYTASE / Pseudomonas / PRODUCTION /
CHARACTERIZATION
127 pp. ISBN 974-04-5062-8
Fac. of Grad. Studies, Mahidol Univ.
Thesis / v
Pseudomonas sp. (PRODUCTION
AND CHARACTERIZATION OF EXTRACELLULAR PHYTASE FROM
PSEUDOMONAS SP.)
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ISBN 974-04-5062-8
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