Cloning and Extracellular Expression of Thermostable Alkaline Protease Gene from Bacillus stearothermophilus AEAL2 in Pichia pastoris
Prepared by:
Prof. Dr. Essam Fadhil Alwan al-Jumaili/Institute of genetic engineering and biological technologies for postgraduate studies, University of Baghdad
Dr. Safaa’ Abdul Hadi Saleh/Department of agricultural research, Ministry of science and technology
Abstract
The thermostable, extracellular Serine alkaline protease enzyme (aprE), which can have many applications especially in detergent, which industrially an important. The aim of this study is to develop a metabolically engineered Pichia pastoris strain for production of an active extracellular thermostable alkaline protease enzyme produced from Local isolate B. stearothermophilus AEAL2 by using genetic engineering techniques. In the first part of study, Local isolate B. stearothermophilus AEAL2 was grown under the optimum conditions in production media for 48hr. at 55°C to produce alkaline protease. In the second part of the study, was designed to produce a thermostable protease in P. pastoris expression system in order to obtain higher yields of the enzyme. The highly thermostable alkaline protease gene from Bacillus stearothermophilus AEAL2 was amplified by polymerase chain reaction (PCR) using consensus primers based on the sequences of alkaline serine protease genes from related species. The DNA fragment which contain recognition enzyme site were cloned into expression vector pPICZaA (methanol inducible). This was shuttle vector, therefore the recombinant construct was transformed into Escherichia coli strain TOP 10 for propagation purposes prior to transformation into Pichia hosts. This recombinant plasmid was later introduced into AOX1 locus in P. pastoris strain X-33 genome. The selection of positive transformants was done on YPD agar containing 100 mg/ml zeocin. The recombinant P. pastoris clone carrying alkaline protease gene in its genome was named as PE1. YTPM was found to be the best medium, producing the highest expression level. In Pichia pastoris expression system, the recombinant AEAL2 protease was secreted successfully into the culture medium driven by the a-factor secretion signal. However, the expression and secretion of AEAL2 protease produced higher results to secretion by the native microorganism of AEAL2 protease (B. stearothermophilus). Recombinant protein (aprE) was secreted into the culture medium with activity reached to 7956.5 U/mg while, compared with, 1152U/mg produced from native B. stearothermophilus AEAL2. Recombinant protein purified to homogeneity in one step employing hydroxyapatite and the yield of enzyme was 17.58 With 6.796 purification fold. While apr E produced by B. stearothermophiluswas purified by three steps with yield reached to 18.63 and 10.65 for purification fold. These data show that the expression level of AEAL2 protease gene can be increased in P. pastoris system without affecting the enzyme function.