| Investigation of the mechanism of enzyme stability, establishing of efficientenzyme stabilization strategies, is not only a challenging hotspot in biology andprotein engineering, but also the urgent problem to be solved in industrial production.Scientists expect through the study of the enzymatic thermodynamic stability to revealthe mechanism of stabilization, while biological industry more emphasis onenhancing enzymatic kinetic stability to improve efficiency during the enzymeapplication. Many studies showed various factors affect the stability of enzyme, butthere is no general rule to follow, especially in the aspect of improving kineticstability. In recent years, with the development of bioinformatics and structuralbiology, scientists have proposed a new protein engineering strategy，which selectedhigh flexible residues (residues with a larger B factor) on molecular surface forredesign to enhance enzymatic stability. However, when this method applied onenzymes have larger molecular weight and more complex structure enzyme, theimprovement of stability, especially for kinetic stability, is not so significant.Establishing of new protein engineering strategy to improve enzyme biologicalstability, especially for kinetic stability, is an effective way to expand the applicationof enzyme. Lipase has the characteristic features of high efficiency, high selectivityand environmental friendliness, which endow them great potential for the bio-industryapplications. In this study, we selected Candida antarctica lipase B（CalB）as model, developed a novel method for enhancing the stability of enzyme that involvesmutating residues within its active site. This method increased the rigidity of theactive site to protect the enzyme against irreversible inactivation under harshconditions.(1) Effects of protein surface region and active site redesign toward enzymaticstability. We used the crystal structure of CalB from Protein Data Bank to designthermostable CalB variants. For analyzing the flexibility of the protein, the B-factorprofile of1TCA was used. The amino acids located within10of catalytic Ser105which showed the high B-factor value were chosen for saturation mutagenesis.Saturation mutagenesis libraries were created at amino acid positions F71, D223,L277, L278, A281and I285. Each saturation mutagenesis library contained200colonies. Heat treatmen（t55°C for15min）was used to identify positive mutants fromthose libraries. Some positive mutants were selected in the L278and D223libraries.In particular, sequencing revealed that the most thermostable mutants were L278Mand D223G. The half-life at48°C of D223G and L278M were13.4and24.2min,which was about3and6fold higher than wild type, respectively. To further improveenzyme stability, the gene encoding L278M was used as a template for iterativesaturation mutagenesis at the other five sites（I285, L277, A281, F71, and D223）.After screening, the best variant was identified as L278M/D223G. The half-life ofD223G/L278M was49min at48°C, which was about13-fold higher than wild type.T5015（The temperature at which50%of enzyme activity remains after incubation for15min）of D223G/L278M increased to58.5°C, which is approximately12°C higherthan that of wild type. Tmvalues of D223G/L278M was3.6°C higher than that of wildtype（56°C）, and Cmvalue（the urea concentration at the unfolding curve midpoint）of the wild type was3.44M, which shifted0.55M towards higher concentrations thenwild type. It is notable that the catalytic efficiency（kcat/Km）of all the mutants was notcompromised in lieu of enhanced stability at all temperatures.Regardless of the locations, the residues that show the highest B factor value inCalB were also chosen as the points for iterative saturation mutagenesis. Afterdeleting N terminal and C terminal amino acids, all sites were chosen on the surface of protein（R249, R309, R242, E269, L219, K13and P218）. No obvious thermostablevariants were found in all the libraries examined under the same screening conditions(2) Structural insights into the mechanism of increasing CalB kinetic stability.We used X-ray crystallography to get the3D structure of CalB wild type and mutants.Compared with the wild type CalB, the position of the main chain O atom of Pro268,Lys271, Ala274, Ala275, and Ala279moved approximately0.2-0.4, and theposition of the main chain N atom of Ala276, Leu277, and Ala279shiftedapproximately0.2–0.5. Together with the change in dihedral angle, these subtlestructural adjustments induced a new hydrogen bonding network that involves sixpairs of hydrogen bonds on segment268-281that improves its rigidity. InD223G/L278M, we found significantly lower relative B factors in the regions of211-226and257-281, indicating enhanced rigidity in these two segments. Theenhanced rigidity at257-281correlated well with a new hydrogen-bonding networkwith the268-281segments. Improvement in the stability of CalB variants was furtherelucidated by MDS at300and330K. There are two major unstable regions within thewild type CalB corresponding to5helix（142-148）and the initial portion of10helix（267-286）. In wild type CalB, fluctuation of10helix became higher at330Kthan at300K, suggesting it is a highly flexible region during heat treatment. However,the D223G/L278M mutant showed considerably greater structural rigidity comparedto wild type at both300and330K, which is consistent with relative B factor profiles.The results indicated that maintaining the original conformation of active site andincreasing the rigidity of secondary structure is an efficiency strategy to improve thekinetic stability of enzymes.(3) The co-evolution of enzymatic kinetic stability and catalytic activity byredesign of active site. Engineering of active site is effective strategy to enhance thecatalytic activity. We obtained target site which may improve enzymatic activitythrough molecular docking. After constructing and screening of saturationmutagenesis library, we got A281F and I285F which shown26and33times highercatalytic efficiency than that of wild type. The D223G/L278M/A281F mutants, which combined the thermostable and higher activity mutagenesis together, showed thethermal stability and catalytic efficiency improvement about6.5times and10timeshigher than wild type, respectively. By engineering the active site, we successfullyimplement the co-evolution between enzymatic multi-functions, which may provideeffective guidance for further protein engineering.In this study, we demonstrate the enhancement of Candida antarctica lipase Bkinetic stability by increasing the rigidity of the active site. Our specific focus on theflexible residues within the active site allowed us to understand how local rigidityaffects enzyme kinetic stability. Nevertheless, our results will help elucidate the novelthermostable mechanism of enzymes and thus provide insight into how to designmore efficient and thermostable biocatalysts and provide guidance for customizationsynthetic biology components. |
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