Design Of Thermophilic Liquid Lipases

Liquid enzyme is a novel solvent-free liquid nanohybrid biocatalyst with extremely low water contents?0.3–0.6 wt%?and homogeneous components?solvent-free liquid enzyme?.It has been reported to show high thermostability,and to be able to function at extremely high temperatures?¹50 ^oC?and directly solvate and convert substrates,providing a new direction in industrial catalysis.Currently,the research on liquid enzymes is located in the exploration stage,as the product of protein surface charge modification,liquid enzymes still exhibit several unfavorable features in terms of severe drop in activity.In the present study,we combined site-directed mutagenesis and chemical modification in order to investigate the effects of number and distribution of enzyme surface charges upon preparation,structure,activity and stability of either native or liquid enzymes,using Bacillus subtilis lipase A?BsLA?as a model.Here,it is demonstrated that improved BsLA variants can be engineered via site-mutagenesis by a rational design,either with enhanced activity in aqueous solution in native form,or with enhanced biophysical property and increased activity in solvent-free liquid system,and all solvent-free liquid BsLA lipases displayed hydrolytic activities at 100 ^oC.Furthermore,we designed a new modification strategy that was proved to significantly enhance liquid lipase activity.Liquid lipase is produced via lyophilization and subsequent thermal annealing of aqueous lipase-polymer conjugates,which comprise a compact globular protein core and a soft polymer surfactant corona.It's the corona that extends the spatial range of the interprotein attractive interactions,thereby achieving liquid phase of protein without solvent.The electrostatically attractive interactions between enzyme surface and polymers play a key role in synthesis of a liquid lipase,and cationization is a key step that equips the protein with a highly positively charged surface,to which a certain amount of anion polymer surfactants?abbreviated as S polymer?can be conjugated electrostatically,followed by ultimate formation of cation-type liquid enzymes.The charges in a protein are generated via ionization of carboxyl or amine groups under certain pH conditions.Most of the naturally occurring proteins cannot maintain their structures and functions without the existence of charges,such as protein folding and stability,enzyme functionality in binding substrates and catalytic reactions,protein aggregation and precipitation etc.In enzyme engineering,enzyme surface charge modification has been proved as a powerful tool in achieving improved enzymes with higher stability,activity and solubility via chemical modification or site-directed mutagenesis strategies,among which anionization and mutations that introduced acidic residues usually equipped enzyme with improved activity and stability.BsLA is a typical model enzyme in several reported surface charge modification studies,which focused on improving enzyme activity and stability by introducing surface carboxylates with site-directed mutagenesis.Accordingly,we rationally designed BsLA surface charges with various distributions to investigate the effects of the number and distribution of BsLA surface charges on the properties of cation-type liquid lipases.In order to increase surface carboxylates near the active site,we performed two groups of four mutations,which were originated from two reported studies targeted towards enhanced ionic liquid-tolerance and thermostability,respectively.Two quadruple mutants?4M1 and4M2?were constructed,followed by incorporation of the eight mutations to generate a novel octuple mutant?8M?.Characterization of lipase activity and stability exhibited significantly enhanced resistance of mutant 8M against ionic liquid?[BMIM][Cl]?,with the half-life time increased from 474 min?4M1?to 965 min;meanwhile,8M displayed extremely high thermostability with T₅₀ value elevated from 63.2 ^oC?4M2?to 97 ^oC.The above findings indicated an additive effect of the mutations in altering enzyme ionic liquid-tolerance and thermostability.Alteration of the number and distribution of BsLA surface charges via site-directed mutagenesis allows different chemical modifications of protein surface charges in cation-type liquid enzyme synthesis.The wild-type BsLA?WT?,along with the mutants 4M1,4M2 and 8M,was used as the precursor for liquid enzyme synthesis,accompanied with activity assays and structural characterizations with several techniques including synchrotron radiation circular dichroism?SRCD?and small angle X-ray scattering?SAXS?.The results suggested that the number and distribution of binding sites towards S polymers played a key role in the physical state,structure and activity of synthesized liquid lipases.Therefore,production of solvent-free liquid enzymes could be ultimately affected by alteration of the number and distribution of enzyme surface charges via site-directed mutagenesis.Inspired by the scientific findings that anionization could better enhance enzyme activity and thermostability,we designed a new synthetic strategy via anionization of a BsLA mutant 8M,followed by electrostatic conjugation with cationic polymer surfactants?abbreviated as cS polymer?and ultimate synthesis of an anion-type solvent-free liquid lipase.The findings displayed that anionization caused much less change in enzyme structure and activity than cationization,and the synthesized anion-type liquid 8M lipase showed a 2.5-fold improvement in activity over its cation-type counterpart.In addition,we further studied on myoglobin?Mb?as the model in order to evaluate the applicability of this new anionization strategy in producing solvent-free liquid proteins.Although anionization resulted in complete unfolding of Mb,its structure was partially recovered in the form of anion-type liquid protein,showing higher?-helical level than its cation-type counterpart.In this study,we obtained a more active liquid lipase via anionization,and we got better understanding of mechanism of the influences of different modifications on different proteins by comparing them in synthesis of different liquid proteins,laying the foundation for further research.The present study indicated that protein surface charge modification is a powerful tool in improving enzyme activity and stability in its own right,and play an even more significant role in solvent-free liquid enzyme research.In this study,we took advantage of the combination of site-directed mutagenesis and chemical modification,which not only improved enzyme activity and stability in native form and in aqueous solution,but also improve enzyme activity and biophysical properties in the form of a liquid protein.Significantly,a new anionization strategy was designed for production of better liquid enzymes,providing a new direction for further development and application of solvent-free liquid enzymes.