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J Ind Microbiol Biotechnol (2010) 37:279287 DOI 10.1007/s10295-009-0671-3
ORIGINAL PAPER
A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli
Thi Thuy Tran Gashaw Mamo Bo Mattiasson Rajni Hatti-Kaul
Received: 8 September 2009 / Accepted: 13 November 2009 / Published online: 10 December 2009 Society for Industrial Microbiology 2009
Abstract Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 2030% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed batch cultivation, glucose concentration was maintained at 23 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.
Keywords Alkaline phytase Bacillus sp.
Fed-batch cultivation Protein secretion
Introduction
Phytase is an important enzyme used as an additive in animal feed. The enzyme catalyses the sequential hydrolysis of phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate; IP6) present in plant material to less phosphorylated myo-inositol derivatives with concomitant release of inorganic phosphate [28]. The phosphate is made available to monogastric animals, such as pigs, chickens and Wsh, that do not produce phytase and also their microXora cannot degrade phytate. This eliminates the need for external addition of phosphorous to the feed, which incurs costs and also contributes to environmental pollution [3, 23, 43]. Removal of phytate from the feed also has other advantages in terms of overcoming its negative nutritional eVects such as decrease in the bioavailability of vital...