Reprogramming the Transition States to Enhance C-N Cleavage Efficiency of Rhodococcus opacusl-Amino Acid Oxidase.

l-Amino acid oxidase (LAAO) is an important biocatalyst used for synthesizing α-keto acids. LAAO from Rhodococcus opacus (RoLAAO) has a broad substrate spectrum; however, its low total turnover number limits its industrial use. To overcome this, we aimed to employ crystal structure-guided density functional theory calculations and molecular dynamic simulations to investigate the catalytic mechanism. Two key steps were identified: S → [TS1] in step 1 and Int1 → [TS2] in step 2. We reprogrammed the transition states [TS1] and [TS2] to reduce the identified energy barrier and obtain a RoLAAO variant capable of catalyzing 19 kinds of l-amino acids to the corresponding high-value α-keto acids with a high total turnover number, yield (≥95.1 g/L), conversion rate (≥95%), and space-time yields ≥142.7 g/L/d in 12-24 h, in a 5 L reactor. Our results indicated the promising potential of the developed RoLAAO variant for use in the industrial production of α-keto acids while providing a potential catalytic-mechanism-guided protein design strategy to achieve the desired physical and catalytic properties of enzymes.

A Multi-Enzyme Cascade for Efficient Production of Pyrrolidone from l-Glutamate.

Pyrrolidone is a high value-added monomer and an important active drug intermediate. However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). The carnitine-CoA ligase from Escherichia coli (EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we obtained the best EcCaiCF380M/N430D double mutant with a kcat/Km value 1.5 times higher than that of the wild type via mechanism-based protein engineering. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiCF380M/N430D, which was then cloned into the plasmid pET-EcCaiCF380M/N430D-DegoPPK2. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone. IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. The catalytic efficiency of carnitine CoA ligase from Escherichia coli (EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. Finally, we efficiently produced pyrrolidone by one pot in vivo with 95.2% conversion and 0.69 g/L/h productivity. Our study provides a new possibility for the industrial production of enzymatic synthesis of pyrrolidone.

Research on blasting cumulative dynamic damage of surrounding rock in step construction tunnel.

In the process of cyclic blasting during tunnel excavation, the reserved surrounding rock sustains irreparable damage accumulation. For safe tunnel construction, it is imperative to understand the characteristics of blasting dynamic cumulative rock damage. Sonic wave test and numerical simulation methods were applied to the research. The JH-2 model was adopted as the damage model of surrounding rock. Based on the data transfer method between solvers in ABAQUS software, the cumulative damage was calculated. The damage characteristics were obtained by combining the sonic wave test results. According to the research findings, the entire reserved surrounding rock has periodic damage characteristics. Each periodic damage area has a funnel shape along the tunnel's longitudinal direction, with a length of 160 cm, and 1.07 times the excavation footage. The latter excavation footage's blasting effect on the damaged area of the previous footage rock is 40 cm long, with three cumulative damage patterns. The three cumulative damage patterns more clearly reveal the surrounding rock's additional damage law, the degree of additional damage is greatest with the distance of 5-20 cm from the latter excavation footage. The research can provide appropriate theoretical guidance for the design of the step-blasting construction tunnel's blasting scheme and lining.

Research on structural parameter optimization of elliptical bipolar linear shaped charge based on machine learning.

Numerical simulation based on SPH method, compared with laboratory experiments, using the grey correlation theory to analyze the correlation between the parameters of the elliptical bipolar linear shaped charge and the performance of the shaped charge jet. The structure of shaped charge is optimized by machine learning to obtain the optimal structural parameters, and it is compared with the rock crack development of shaped charge blasting in practical application. The results show that the structural parameters of the shaped charge have the same influence on the jet head velocity, and there are certain differences in the impact on the jet length. The fitted curve of the support vector machine (SVM) regression model based on the genetic algorithm (GA) is high prediction accurate. By comparing the optimization results with the actual engineering application of the shaped charge structure, the rock breaking effect has been significantly improved, which has important guiding significance for the actual engineering application.