Design, Cloning And Expression And Characterization Of Thermostable Hybrid Enzymes

In the past years, the application of enzyme in industrial production has becomemore and more important. However, some natural enzymes couldn’t meet the demandsof industrial applications, which arouses the necessity of transforming natural enzymevia technique of protein molecular design. According to the numbers of transformedpart in protein, protein molecular design could be defined as minor transformation,moderate transformation and major transformation. Among the three transformations,minor transformation could be realized through site mutation or chemicalmodification, moderate transformation means the jointing or assembling of structuraldomains from different proteins to produce hybrid enzyme, and major transformationrefers to designing entirely new proteins.In this paper, two hybrid enzymes wereconstructed via mutual exchange of structural domains. Studies have been performedin our group on the molecular evolutionary mechanism of hyperthermophilicacylpeptide releasing enzyme APE1547from the thermophilic archaeon Aeropyrumpernix K1and thermostable acylpeptide releasing enzyme ST0779from Sulfolobustokodaii strain7T(JCM10545). Sequence similarity between APE1547and ST0779was found37.6%through sequence alignment and structural analysis, and thesequence similarity of their catalytic domains up to41.7%. But significant differenceswere found between the propeller domains of APE1547and ST0779, thus theirsubstrate specificity was extraordinarily different. Therefore, an assumption oftransforming propeller domain from ST0779into catalytic domain from APE1547toobtain hybrid enzyme SA was emerged. The first α-helix at N-end played animportant role in maintaining the stability of APE1547. To prove whether this α-helixinfluences the stability of hybrid enzyme SA or not, hybrid enzyme NO-SA withoutthe first α-helix at N-end was constructed. Stability of hybrid enzymes was confirmed via computer software assistance. The reasonable region of jointing points wasconfirmed and optimum jointing scheme was ascertained through moleculardynamics.The results showed that enzyme activity, substrate specificity and thermal stabilitybetween hybrid enzyme SA and NO-SA were enormously different. Enzymaticproperties of hybrid enzyme NO-SA with removing the first α-helix at N-end andhybrid enzyme SA also caused enormous difference. The optimum substrate of thetwo hybrid enzymes (SA and NO-SA) was different to original enzymes, and highercatalytic activity of two hybrid enzymes was found towards substrates with longerchain (including more than8carbons). The optimum temperature of two hybridenzymes was found between that of parent enzymes. Under this condition, specificactivity of hybrid enzyme SA was1501.59U/mg to the optimum substrate. Theoptimum pH of APE1547and ST0779was8.0. The optimum pH of two hybridenzymes was also8.0. As for thermal stability, APE1547was found to show higheststability, stability of hybrid enzyme SA was higher than that of ST0779, but thestability of hybrid enzyme NO-SA was lower than both APE1547and ST0779. Mostmetal ions exerted little influence on enzyme activity, but EDTA had differentinhibitory effects on all four enzymes. The kinetic parameter perspective of twohybrid enzymes also showed difference with two parent enzymes. The stability of SAand NO-SA in organic solvents was relatively low, activity were tested in DMSO after24h. There were significant differences between NO-SA and SA in catalytic activityand thermal stability, indicating thisα-helix could help hydrolysis of substrate andstabilize the whole enzyme.Hybrid enzyme technology not only plays an important role in changing theproperties of enzymes and studying structure-function relationship, but also expandsthe application range of nature enzymes. New catalysts that catalyze reactions thatnature enzymes can’t catalyze can be builded with enzyme or enzyme fragmentsthrough hybrid enzyme technology. Hybrid enzyme technology is a coalition thattraditional enzymatic activity screening methods and recombinant DNA technology.This will enable molecular biologists easier to design and tailor enzyme(or protein) molecular, but also make protein engineering more powerful and attractive.