Comparative Amino Acids Studies on Phac Synthases and Proteases as Well as Establishing a New Trend in Experimental Design

Abstract

ABSTRACT: A question addressed in this study is: why similar enzymes are classified into different subclasses? As an example, PhaC synthases are classified according to four different classes (I, II, III and IV). To answer this question we proposed that besides the catalytic residues, the overall amino acids (AAs) present are responsible for the differences observed. The AAs’ composition affects the structure/function/substrate specificity (SFS) of these enzymes. The differences between the classes in various PhaC synthases and proteases were analysed to support our argument. Homology and phylogenic tree of some selected PhaC synthases of different strains (representing the four classes) were demonstrated. The properties of a specific class of enzyme could not be changed into those of another by changing the catalytic residues. Moreover, these differences could not be detected from the proteins’ 3D structures, despite clear differences at the AAs level. Another question was also addressed: could we benefit from the various existing protein databases in the field of biotechnology? To answer this, we introduced a model for an Experimental Design based on the information in the protein database (for strains available in our lab) regarding their ability to degrade castor oil. Two enzymes in the phenol degradation pathway, phenol 2-monooxygenase and catechol 1,2-dioxygenase, and a lipase enzyme were analysed. These enzymes were screened and analysed according to the BLAST-protein database and BRENDA. The comprehensive enzyme information system compared six strains against each other, including: Pseudomonas aeruginosa, Bacillus subtilis, Bacillus pumilus, Bacillus thuringiensis, Bacillus licheniformis, and Geobacillus stearothermophilus. Only P. aeruginosa proved to have the three required enzymes and was suitable for the production of lipases from castor oil (crude castor oil is usually contaminated with phenol) as indicated by the databases. In addition, in vivo castor oil degradation and in vitro lipase enzyme activity were analysed. The apparent lipase activity was 1070 Units/ml. Therefore, this new strategy is recommended to better understand the SFS as well as for using protein database in an Experimental Design.ABSTRAK: Kajian ini berkisar mengenai soalan: mengapa enzim yang sama diklassifikasikan kepada subkumpulan yang berbeza? Contohnya sintasis PhaC dikelaskan kepada empat kumpulan berbeza (I, II, III dan IV). Bagi menjawab soalan ini selain hipotesis katalitik residue diperkenalkan, faktor yang bertanggungjawab adalah asid amino (AAs) secara keseluruhannya. Komposisi AAs memberi kesan kepada struktur/fungsi/spesifikasi substrat (SFS) enzim-enzim ini. Perbezaan di antara kumpulan dalam pelbagai sintasis PhaC dan proteases telah dianalisis bagi menyokong hujah ini. Homologi dan asas phylogenic bagi sintasis PhaC tertentu yang berbeza strains (mewakili empat kumpulan) telah ditunjukkan. Sifat-sifat tidak boleh ditukarkan dengan menukarkan katalitik residue dari satu kepada yang lain. Tambahan pula, kelainannya tidak boleh dikesan dari struktur 3D protein, walaupun perbezaan yang nyata pada peringkat AAs. Soalan lain yang berkisar adalah: Adakah kita mendapat faedah dari pelbagai pengkalan data dalam bidang bioteknologi? Bagi menjawab soalan ini, model Rekabentuk Eksperimen telah diperkenalkan yang berasaskan maklumat pengkalan data protein bagi strains di makmal kami yang boleh degradasi minyak castor. Dua enzim dalam degradasi fenol telah dianalisis, fenol 2-monooxigenas dan catechol 1,2-dioxygenas serta enzim lipas. Enzim-enzim ini telah disaringkan dan dianalisis dengan merujuk kepada pengkalan data protein-BLAST dan BRENDA. Sistem maklumat enzim secara komprehensif terhadap enam strains termasuk Pseudomonas aeruginosa, Bacillus subtilis, Bacillus pumilus, Bacillus thuringiensis, Bacillus licheniformis dan Geobacillus stearothermophilus. Hanya P. Aeruginosa terbukti mempunyai tiga enzim dan sesuai bagi penghasilan lipase dari minyak kastor (minyak kastor mentah selalunya tercemar dengan fenol) seperti yang ditunjukkan dari pengkalan data. Degradasi minyak kastor in vivo dan aktiviti enzim lipase in vitro telah dilaksanakan. Aktiviti lipase jelas adalah 1070 Units/ml. Kami mencadangkan menggunakan strategi ini bagi memahami SFS serta pengkalan data protein dalam Rekabentuk Eksperimen.KEYWORDS: amino acids; model; PhaC synthase; protease; lipase; experimental design