Extracellular expression of a thermostable phytase (phyA) in Kluyveromyces lactis

• Phytase from A. niger NII 08121 was expressed in Kluyveromyces lactis in secretory form.
• Purified protein was heavily glycosylated.
• Recombinant enzyme was thermostable at 100 °C for 1 h.
• Role of asparagine 345 in thermostability was enlightened.

Functional expression of a thermostable phytase from A. niger was achieved in Kluyveromyces lactis GG799 cells. Effective secretion of recombinant enzyme (198 U ml−1) in the fermentation broth at 72 h incubation at 22 °C was obtained. Purified enzyme showed a specific activity of 72 U mg−1) and was detected on SDS-PAGE as a heavily glycosylated protein with a molecular weight of ≥140 kDa. Optimum temperature of the enzyme was at 55 °C and it showed a characteristic bi-hump pH profile with two pH optima (at pH 2.5 and 5.5). Enzyme showed considerable pepsin resistance with 60% activity retention after incubation with pepsin at the ratio of 1:1000. Enzyme was thermostable retaining 69 and 37% activity at 90 and 100 °C for 10 min respectively and remained active at these temperatures till 1 h. Deglycosylation studies demonstrated negligible effect of N-linked glycans on thermal properties. Multiple sequence alignment data revealed a conserved Asn at position 345 of this phytase which might contribute to its thermal properties. This thermostable phytase coupled with its noticeable protease resistance could be a better alternative to current commercial phytases.