Saccharomyces cerevisiae OS303 expression of an alkaline protease from a newly isolated Bacillus subtilis D9

Abstract

The aim of this study was to produce high yield of a local bacterial alkaline protease in the yeast system because the scientific involvement of microorganisms in enzyme production is still not given enough attention in Saudi Arabia. Soil samples were collected from the rhizosphere of some desert plants in Saudi Arabia. Ninety-three alkaline protease producing bacterial isolates were recovered on skimmed-milk agar at pH 9.4 and 45°C for 48 hr. Isolate D9 obtained from the rhizosphere of Heliotropium digynum at Dhahran City was the most potent isolate in respect to enzyme productivity (184.6 U/ml). The full gene of alkaline protease was amplified and showed the expected size (1300 bp). Restriction enzymes analysis also verified the integrity of the PCR product. The sequence of the protease gene revealed an open reading frame of 1329 nt correspond to the full length of the protease gene of isolate D9 encoding a 443 aa protein. After ligation of the amplified gene by the TA cloning method, digestion with appropriate restriction enzymes confirmed the integrity of the cloned gene. The insert was prepared by two PCRs that were conducted with a pair of primers specifically designed for this purpose. The digested and purified cloning vector pRS426/GAL1p-207-Glu-MS was ligated with the insert then transformed into various strains of Saccharomyces cerevisiae via the electroporation method. Maximum protease expression was done by recombinant OS303 in galactose containing media (145.5 U/ml) with an approximately 2-fold increase when compared with the wild OS303 strain., this may be due to ability to activate gal operon.

Isolation, Screening, and Identification of Alkaline Protease-Producing Bacteria and Application of the Most Potent Enzyme from Bacillus sp. Mar64

“…Mar64P was homogenously purified, and the final specific activity was 8.5-fold with a recovery of 12.4%. SDS-PAGE indicated a prominent band at 28 kDa with similarity with the alkaline protease of B. horikoshii [21] while it was different from the alkaline proteases of Priestia megaterium gasm32 (30 kDa, [10]), B. alveayuensis CAS 5 (33 kDa, [22]), Salmonella typhimurium UcB5 (35 kDa, [23]), B. cereus (35 kDa, [16]), B. subtilis D10 (35 kDa, [24]), and B. subtilis D9 (48 kDa, [25,26]).…”