Creatinase: Using Increased Entropy to Improve the Activity and Thermostability.

Improving protein thermostability in mutagenesis-based enzyme engineering was often achieved by enhancing interresidue interactions via mutation to increase the enthalpy penalty of unfolding. However, this approach may trade off the functional activity due to the loss of structural flexibility of the biomolecule. Here, by performing X-ray crystallography, enzymatic kinetic experiments, neutron scattering, and thermodynamical measurements, we compared the structures, catalytic behaviors, dynamics, and thermostability between a wild-type creatinase and its four-point mutant. We found that the mutant is an entropy-driven thermostable protein with higher structural flexibility, i.e., higher conformational entropy, in the folded state compared to the wild type. The increased conformational entropy of the mutant in the folded state can reduce the entropy gain during unfolding and thus renders it greater thermostability. Moreover, the increased structural flexibility, particularly around the catalytic site, can broaden the mutant's working temperature range and considerably improve its activity at ambient conditions, which is crucial for its application in diagnosing kidney diseases. Complementary all-atom molecular dynamics simulations indicated that the four mutations replaced several of the strong interresidue interactions (electrostatic interactions and hydrogen bonds) with weak hydrophobic interactions. These substitutions not only release the structural flexibility to promote the thermostability and enzymatic activity of the protein but they also preserve the protein structure from collapsing. Our findings may pave a route for the entropy-driven strategy to design proteins with high thermostability and activity.

Mechanochemical modified nitrogen-rich biochar derived from shrimp shell: Dominant mechanism in pyridinic-N for aquatic methylene blue removal.

N-doping for the preparation of functional carbon materials is a trending research topic. In this study, N-rich biochar (BC) was prepared by calcining naturally N rich shrimp shells under oxygen-limiting environment, and the calcining temperatures were controlled. BC were activated with 5% hydrochloric acid solutions and then post-modified with ball-milling to obtain a series of novel adsorbents (MBCs). All samples were characterized by SEM, BET, FT-IR, XRD, XPS, TG, and element analysis. Surface area, pore volume, and other surface functional groups were significantly improved after acidizing and ball-milling. The adsorption capacities for MB were MBC350 > MBC500 > MBC650 >BC350 > BC650 > BC500, and the equilibrium adsorption capacities were 575.01 mg/g, 506.52 mg/g, 424.59 mg/g, 113.31 mg/g, 93.53 mg/g and 86.25 mg/g, respectively. The excellent adsorption performance of MBCs for MB was ascribed to Lewis acid-base interaction, π-π interaction, electrostatic interaction and van der Waals, and the quinone group and pyridinic-N on the surface of the MBCs are identified as the major active sites. Taken together, ball-milled shrimp shell biochar is a promising material for cation dye adsorption.

[Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design].

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.