meso-Diaminopimelate dehydrogenase (DAPDH) holds considerable promise for D-amino acid synthesis but is limited by its low catalytic efficiency toward non-natural substrates. To address this, we present a Rosetta-driven evolution framework that enables the rapid tuning of DAPDH substrate specificity. This approach leverages Rosetta design to guide the construction of a smart-small mutant library, facilitating evolution in just one or two rounds with minimal screening (100–1,000 transformants), thus offering superior efficiency and cost-effectiveness. Using this strategy, the activities of two DAPDHs toward model substrates─benzoylformic acid (1b) and 3-indolepyruvic acid (10b)─were enhanced from undetectable levels to 29.5 and 5.1 U/mg, respectively. The activity toward 1b was nearly ten times higher than the best-reported mutant. The evolved DAPDHs displayed an expanded substrate spectrum and high catalytic performance, enabling the efficient synthesis of structurally diverse bulky D-amino acids at both analytical and preparative scales. The highest concentration of 1b achieved was 0.5 M (100 mL scale), with a space-time yield of 128.46 g/L/day. This study underscores the power of Rosetta-based evolution in accelerating enzyme engineering, offering a versatile and efficient platform for optimizing DAPDHs and other industrially significant enzymes.
(Pyridine-2,5-diylbis(azaneylylidene)) bis-(methaneylylidene)) bis(benzene-5,1,3-triyl) tetrakis (hydrogen phosphonate) (PZMB) was successfully synthesized for Th(IV) and Ce(IV) adsorption, the adsorption parameters evaluated and found to be pH 3.5, time 60 min, dose 0.15 g. Adsorption kinetics, isotherm, and thermodynamics studies. Adsorption kinetics studies show that adsorbent follow pseudo-second-order model while isotherm studies conclude that adsorption processes fitted Langmuir and D-R isotherm model. The maximum adsorption capacity of BZMB of Th(IV) and Ce(IV) are 366.4 and 244.53 mg/g respectively, Thermodynamic studies indicate both adsorption processes exhibited by the PZMB were endothermic, spontaneous and sorption occurs in a random manner.
Qian Zhang,Shuai Fan,Mengjia Tang,Chenyu Wang,Xiaoxiao Li,Yuanyuan Jin,Zhaoyong Yang
The engineered human cystathionine-γ-lyase (hCGL) resulting in enhanced activity toward both cysteine and cystine unveils a potential robust antitumor activity. However, the presence of cysteine residues has the potential to induce oligomerization or incorrect disulfide bonding, which may decrease the bioavailability of biopharmaceuticals. Through a meticulous design process targeting the cysteine residues within engineered hCGL, a set of potential beneficial mutants were obtained by virtual screening employing Rosetta and ABACUS. Experimental measurements have revealed that most of the mutants showed increased activity toward both substrates l-Cys and CSSC. Furthermore, mutants C109V and C229D demonstrated Tm value increases of 8.2 and 1.8 °C, respectively. After an 80 min incubation at 60 °C, mutant C229D still maintained high residual activity. Unexpectedly, mutant C109V, displaying activity approximately 2-fold higher than the activity of wild type (WT) for both substrates, showed disappointing instability in plasma, which suggests that computational design still requires further consideration. Analysis of their structure and molecular dynamics (MD) simulation revealed the impact of hydrophobic interaction, hydrogen bonds, and near-attack conformation (NAC) stability on activity and stability. This study acquired information about mutants that exhibit enhanced activity or thermal resistance and serve as valuable guidance for subsequent specific cysteine modifications.
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