Combining binding pocket mutagenesis and substrate tunnel engineering to improve an (R)-selective transaminase for the efficient synthesis of (R)-3-aminobutanol.

(R)-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven (R)-selective transaminases were identified using homologous sequence mining. Beginning with the optimal candidate from Mycolicibacterium hippocampi, virtual mutagenesis and substrate tunnel engineering were performed to improve catalytic efficiency. The obtained variant, T282S/Q137E, exhibited 3.68-fold greater catalytic efficiency (kcat/Km) than the wild-type enzyme. Using substrate fed-batch and air sweeping processes, effective conversion of 100 mM 4-hydroxy-2-butanone was achieved with a conversion rate of 93 % and an ee value > 99.9 %. This study provides a basis for mutation of (R)-selective transaminases and offers an efficient biocatalytic process for the asymmetric synthesis of (R)-3-aminobutanol.

Study on the Characteristics of Circumferential and Longitudinal Flow of Vault Concrete during Tunnel Lining Pouring Processes.

With a large number of railroad and highway tunnels opening for operation, the diseases caused by hidden lining defects are increasing. The study of flow characteristics of freshly mixed concrete during tunnel lining casting is the key to revealing the formation mechanism of hidden defects. This paper revealed the location of blank lining formation by investigating the circumferential and longitudinal flow characteristics of concrete in the vault during tunnel pouring to provide suggestions for improving the quality of tunnel lining pouring for the various projects. This paper adopted the method of indoor testing, selected the suitable working conditions and flow parameters, validated the accuracy of the test with a numerical simulation, and simulated the secondary lining pouring process of the tunnel arch from the circumferential direction and longitudinal direction. This revealed the flow characteristics of the freshly mixed concrete in the process of pouring the arch lining. The flow of concrete in the arch lining was basically characterized by two major features which were similar to the flow in the pumping pipe and the layered flow. It also revealed the relationship between the concrete flow rate, flow distance, and the location of the formation of the blank lining risk zone with the slump of the concrete, the pumping pressure, and the radius of the tunnel.

Spacing Analysis of Casting Dolly Windows for Tunnel Sidewall Lining Based on the Flow Characteristics of Freshly Mixed Concrete.

During the actual construction of tunnel sidewall lining, construction workers often use only one or two windows per layer for pouring in order to reduce the construction sequence, which often leads to a reduction in the quality of tunnel sidewall concrete pouring. Therefore, this study analysed the necessity of the window-by-window pouring of sidewall lining through the study of concrete flow characteristics of the tunnel sidewall lining pouring process, and the reasonable spacing of pouring windows was analysed. This study firstly verified the accuracy of the simulation parameters and the feasibility of the simulation method of the lining pouring process through indoor experiments and simulation analyses, and then it numerically simulated and analysed the flow of concrete during the lining pouring process of tunnel sidewalls. The following conclusions were made: the smaller the slump of the freshly mixed concrete, the higher the pumping flow rate; additionally, the shorter the one-time pouring distance, the smaller the spacing of the trolley feeding window should be. Furthermore, this study makes suggestions for the reasonable spacing of pouring trolleys under several working conditions.

Engineering of 4-hydroxyphenylacetate 3-hydroxylase derived from Pseudomonas aeruginosa for the ortho-hydroxylation of ferulic acid.

Polyphenolic compounds have natural antioxidant properties, and their antioxidant activity is usually related to the number and position of hydroxyls. Here, we successfully applied the engineered 4-hydroxyphenylacetate 3-hydroxylases (4HPA3Hs) derived from Pseudomonas aeruginosa to catalyze ferulic acid (FA) synthesis of ortho-hydroxyferulic acid (5-hydroxyferulic acid, 5-OHFA). Through optimization of co-expression, the oxygenase component (PaHpaB) and the reductase component (PaHpaC) in E. coli, and optimization of whole-cell catalytic conditions, the engineered strain BC catalyzed ortho-hydroxylation of 2 g/L of FA with a yield of 75 % from 39 %. Through tunnel engineering of PaHpaB, the obtained mutants F301A and Q376A almost completely transformed 2 g/L of FA. Further, a multiple mutant L214A/F301A/Q376A converted 4 g/L FA into 5-OHFA within 12 h, and the yield reached 99.9 %, which was approximately 2.39-fold of the wild type. The kcat/Km value of L214A/F301A/Q376A was about 307 times greater than that of the wide type. Analysis of three-dimensional structural models showed that L214, F301, and Q376 mutated into Ala, which greatly shortened the side chain and broadened the tunnel size, thereby significantly improving the catalytic efficiency of L214A/F301A/Q376A. This biosynthesis of 5-OHFA is simple, efficient, and green, suggesting that it is useful for efficient biosynthesis of polyphenolic compounds.

Acoustic emission and fractal characteristics of red beds soft rock under water-force coupling.

Groundwater has a significant influence on the mechanical properties of surrounding rock. Aiming at the large deformation of surrounding rock of red layer soft rock tunnel affected by groundwater, the uniaxial graded loading tests were carried out on red beds soft rock with different water content. The failure process of the specimen was monitored by acoustic emission (AE) and the crack evolution law was analyzed, and the scanning electron microscopy (SEM) was used to compare the microstructure of the specimens before and after immersion. Combined with fractal theory, the monofractal and multifractal characteristics of AE ringing count during the loading process of red beds soft rock were analyzed. The results show that, with the gradual increase of water content, the AE ringing count before the yield stage gradually decreased, and the corresponding cumulative ringing count at the same time gradually decreased, and the decrease was large in the early stage of immersion, and decreased in the later stage. The cumulative ringing curve gradually slowed down, the internal crack appeared earlier, the cumulative ringing curve stepped significantly, the AE signal amplitude gradually weakened, and the bandwidth of each frequency band gradually decreased. The failure of red beds soft rock with different water content is dominated by shear crack, and with the gradual increase of water content, the proportion of shear crack increases gradually, and the AE b value decreases gradually. With the gradual increase of the relative peak strength, the correlation dimension D of red beds soft rock with different water content increases first and then decreases. At 80% of the relative peak strength, the correlation dimension D reaches its maximum value and then drops sharply until it is maintained at a relatively low level, and the correlation dimension D gradually decreases with the water content. The fitting correlation coefficients of different water content (lnC(r), lnr) are all above 0.9, indicating that the AE ringing count of water-bearing red beds soft rock has fractal characteristics, and the higher the correlation coefficient, the higher the self-similarity of AE ringing count sequence. As the weight q gradually increases, the generalized fractal dimension D(q) gradually decreases. When q ≠ 0, under the condition of the same q, D(q) presents a trend of first increasing and then decreasing. The multifractal characteristics of AE ringing count of red beds soft rock with different water content is inverted 'U' shape. From the natural state to immerse 1 d, the ∆α gradually increases, and from 1 to 7 d, the ∆α gradually decreases, where Δα = αmax - αmin represents the spectral width of the multifractal spectrum. When saturation is not reached, ∆f < 0 indicates that the number of cracks in the specimen is small, when saturation is reached, ∆f > 0 indicates that a large number of cracks are generated inside the specimen and macro cracks are formed, where Δf = f (αmax) - f (αmin) represents the frequency relationship between different signals of different sizes. This research can provide a reliable theoretical basis for the construction and maintenance of large deformation of water-rich soft rock tunnel excavation, and have certain engineering significance.

Structure-Guided Engineering of a Protease to Improve Its Activity under Cold Conditions.

Bacillus proteases commonly exhibit remarkably reduced activity under cold conditions. Herein, we employed a tailored combination of a loop engineering strategy and iterative saturation mutagenesis method to engineer two loops for substrate binding at the entrance of the substrate tunnel of a protease (bcPRO) from Bacillus clausii to improve its activity under cold conditions. The variant MT6 (G95P/A96D/S99W/S101T/P127S/S126T) exhibited an 18.3-fold greater catalytic efficiency than the wild-type (WT) variant at 10 °C. Molecular dynamics simulations and dynamic tunnel analysis indicated that the introduced mutations extended the substrate-binding pocket volume and facilitated extra interactions with the substrate, promoting catalysis through binding in a more favorable conformation. This study provides insights and strategies relevant to improving the activities of proteases and supplies a novel protease with enhanced activity under cold conditions for the food industry to maintain the initial flavor and color of food and reduce energy consumption.

Novel Reinforcing Techniques and Bearing Capacity Analysis for Tunnel Lining Structures with Extensive Corrosion.

Affected by the erosive environment, tunnel lining concrete in the long-term service zprocess often exhibits engineering diseases such as concrete corrosion degradation and loss of strength, decreasing the stability of the tunnel lining structure and the traffic safety. Based on HTG tunnel project, the basic distribution rule of tunnel lining corrosion and macro mechanical properties of corroded concrete were explored in this paper through engineering disease site investigation. Then, on this basis, aiming at large-scale corrosion of tunnel lining structure, two reinforcement and repair schemes are proposed, corrugated steel plate reinforcement method and channel steel reinforcement method. Indoor component tests are carried out on the two reinforcement schemes. The failure characteristics and stress and deformation law of tunnel lining members after reinforcement and repair were verified. The analysis showed that the failure process of the reinforced specimens on the tensile side could be divided into the non-cracking stage and the working stage with cracks, and the cracking load and failure load of the specimens were significantly increased. The bearing capacity of the reinforced specimens was divided into the ultimate bearing capacity against cracking and the ultimate bearing capacity during failure. Finally, the calculation methods of the bearing capacity of the channel steel reinforcement method and the corrugated steel plate reinforcement method were derived. Comparative analysis shows that the results of numerical simulation, experimental testing and theoretical simplification methods are close to each other, and the maximum deviation is less than 8%. The established method for calculating the bearing capacity of corroded components after reinforcement is reliable and can be used for the design calculation of corroded lining reinforcement.

Numerical Investigation on the Mechanical Properties of Vault Void Lining and Steel Plate Strengthening.

To study the mechanism of vault lining under different void heights and verify the strengthening effect of the attached steel plate, a CDP (concrete-damaged plasticity) model and the XFEM (extended finite element method) were used to construct the local numerical model of the vault void, and an experiment was carried out for verification. The strengthened structure of the steel plate was assembled with a combination of a two-component epoxy adhesive and chemical anchor bolts. Five lining models with various void thicknesses, together with their strengthened models, were evaluated. The results of the established numerical model were compared with the experimental results in terms of failure mode, vertical displacement, and load-deformation results. The results of the two numerical models were in good agreement with the experimental results, revealing the failure mechanism of the vault lining. The rigidity of the specimen after steel plate strengthening was significantly improved. When the void height was one-fourth of the secondary lining thickness, the lining cracks were reduced from 14 to 4, and the distribution width of the cracks was also reduced from 1.047 to 0.091 m after steel plate strengthening. The level of damage caused by cracking was significantly reduced, which proves the effectiveness of the surface-sticking method for steel plate strengthening.

Research on Mechanical Behavior of the Steel-Concrete-Steel Composite Structures Subjected to High Temperature of Fire.

A new type of steel-concrete-steel composite structure was adopted and widely used in the immersed tunnel of the Shenzhen-Zhongshan access. The research on the mechanical behavior of the new composite structure under a high temperature of fire is of great engineering significance to the fire protection design of the structure. Both the model test and a numerical simulation were adopted to study the mechanical behavior and damage characteristics of the new composite structure under fire. The RABT standard temperature rise curve was used to simulate the temperature rising law under fire (it reflects the characteristics of temperature rise in case of fire in an enclosed environment: rapidly raised to 1200 °C within 5 min, maintained at 1200 °C for 120 min, then it is cooled to normal temperature after 110 min). The temperature distribution law inside the structure, the deformation development law of the roof and the crack distribution were analyzed based on the thermal-mechanical coupling analysis method. The results showed that the internal part of the composite structure close to the fire surface was directly affected by the high temperature, and the temperature presented a step distribution law, while the part far from the fire surface was affected by the lag effect of the temperature transfer, and the temperature presented a continuous growth law. The roof deformation presented a three-stage model of "rapid growth-deformation stability-deformation recovery" with time. The overall cracks of the composite structure showed a symmetrical distribution under fire. The composite structure presented a shear failure mode as a whole. The results could provide a reference for the study of fire resistance for the new composite structure and support the structural fire protection design of the immersed tunnel of the Shenzhen-Zhongshan access.

Seismic Behavior of Shear Keys Enhanced with Novel Energy Absorption Devices in Immersion Joints Based on Pseudo-Static Tests.

Shear keys are usually installed as crucial shear-resistant members of an immersion joint; thus, the mechanical behavior of the shear keys, especially under earthquake loading, deserves more attention. This paper presents a novel arc-shaped energy absorption device developed for shear keys. In order to verify the seismic performance of shear keys strengthened by the arc-shaped energy absorption devices, a series of pseudo-static tests were conducted, in which different axial pressures (300 kN, 400 kN) were also taken into consideration. The testing results indicated that failure mode of the shear key enhanced by the energy absorption devices was a synthesis of the oblique shear failure of the rubber blankets, the buckling of the energy absorption devices, and the concrete fracture of the shear key. In view of load-displacement hysteretic curves of testing specimens, loops of the reinforced shear keys were plumper than those from a traditional shear key. In addition, the load-bearing capacity (cracking load, yield load, peak load, and failing load) differences of the shear keys with and without energy absorption devices reinforcement under the same axial pressure were 33.0%, 36.7%, 26.0%, and 23.6%, respectively. The maximum equivalent viscous coefficient values of the shear keys with and without energy absorption devices reinforcement were 0.37, 0.38, and 0.32, respectively. The arc-shaped energy absorption devices can contribute to the hysteretic behavior of the shear keys. However, the axial pressure had a positive influence on the load bearing capacity, accumulated energy absorption capacity, and initial stiffness of the shear keys. In contrast to that, the axial pressure had negative influence on ductility ratio of the reinforced shear keys (equivalent viscous coefficient values of two enhanced shear keys were roughly equal). A reasonable stiffness scheme of an energy absorption device should be given attention during the anti-seismic design of an immersion joint. The study can provide scientific support for further study on the seismic responses of immersion joints and promote the application of earthquake control technology in immersed tunnels.

CsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production.

Crocetin, a high-value apocarotenoid in saffron, is widely applied to the fields of food and medicine. However, the existing method of obtaining crocetin through large-scale cultivation is far from meeting the market demand. Microbial synthesis of crocetin is a potential alternative to traditional resources, and it is found that carotenoid cleavage dioxygenase (CCD) is the critical enzyme to synthesize crocetin. So, in this study, we used "hybrid-tunnel" engineering to obtain variants of Crocus sativus-derived CsCCD2, essential for zeaxanthin conversion into crocetin, with a broader substrate specificity and higher catalytic efficiency. Variants including S323A, with a lower charge bias and a larger tunnel size than the wild-type, showed a 5-fold higher crocetin titer in yeast-based fermentations. S323A could also convert the ß-carotene substrate to crocetin dialdehyde and exhibited a 12.83-fold greater catalytic efficiency (kcat/Km) toward zeaxanthin than the wild-type in vitro. This strategy enabled the production of 107 mg/L crocetin in 5 L fed-batch fermentation, higher than that previously reported. Our findings demonstrate that engineering access tunnels to expand the substrate profile by in silico protein design represents a viable strategy to refine the catalytic properties of enzymes across a range of applications.

Shaking table test on the seismic response of the portal section in soft and hard rock junction.

Tunnel portal sections located in the soft-hard rock junction are vulnerable to the strong earthquake motions in seismically active regions. The main objective of this paper is to investigate the seismic response of tunnel portals located in the soft-hard rock junction. Taking the Baiyunding tunnel in northeast China as a background, a shaking table test with a geometric scaling ratio of 1:30 was built. Details of test setup and procedures are introduced first and then the test results are presented. The discussion of the results is based on the peak ground acceleration (PGA), the longitudinal, the contact stress, and the safety factor. The results show that the soft section of the soft-hard rock junction suffers remarkable damages under strong seismic motions, while the hard rock section is less affected by earthquakes. The increasing soft rock range causes a rise of the forced displacement of tunnel linings, which, together with the seismic inertia force, leads to the increase of the contact stress of the linings, and ultimately resulting in the deterioration of the tunnel seismic safety. To mitigate the seismic damage of tunnel portals in the soft-hard rock junction, rock grouting, bolt support, and other effective reinforced methods should be considered in the seismic design of the soft section.

Tunnel engineering for modulating the substrate preference in cytochrome P450BsßHI.

An active site is normally located inside enzymes, hence substrates should go through a tunnel to access the active site. Tunnel engineering is a powerful strategy for refining the catalytic properties of enzymes. Here, P450BsßHI (Q85H/V170I) derived from hydroxylase P450Bsß from Bacillus subtilis was chosen as the study model, which is reported as a potential decarboxylase. However, this enzyme showed low decarboxylase activity towards long-chain fatty acids. Here, a tunnel engineering campaign was performed for modulating the substrate preference and improving the decarboxylation activity of P450BsßHI. The finally obtained BsßHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; BsßHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. The study demonstrates how the substrate preference can be modulated by tunnel engineering strategy.

Effects of seepage and weak interlayer on the failure modes of surrounding rock: model tests and numerical analysis.

The presence of weak interlayers and groundwater are common adverse geological conditions in tunnels. To investigate the modes of failure of rock masses surrounding tunnels owing to weak interlayers and groundwater, model tests and numerical simulations were conducted in this study based on two cases, and a model that considers only the weak interlayer was conducted for comparison. Based on the tests, differences between two models in terms of rock pressure, displacement, cracks and strain were analysed. The results reveal that the presence of groundwater has a significant effect on the space-time distribution of stress, displacement and cracks in the surrounding rock. Furthermore, based on the numerical model, the seepage field was analysed in terms of pore water pressure, permeability and the seepage process to understand the joint action of groundwater and weak interlayer on the failure mechanism of tunnels. The results show that the groundwater and interlayer complement each other to induce the failure mode of the surrounding rock. The water accelerates slip in the interlayer and the development of cracks. Conversely, low strength, muddy weak interlayers serve as the channels of water flow, resulting in deformations and cracks at different locations and different failure modes.

Entropy-Based Risk Control of Geological Disasters in Mountain Tunnels under Uncertain Environments.

Uncertainty is one of the main sources of risk of geological hazards in tunnel engineering. Uncertainty information not only affects the accuracy of evaluation results, but also affects the reliability of decision-making schemes. Therefore, it is necessary to evaluate and control the impact of uncertainty on risk. In this study, the problems in the existing entropy-hazard model such as inefficient decision-making and failure of decision-making are analysed, and an improved uncertainty evaluation and control process are proposed. Then the tolerance cost, the key factor in the decision-making model, is also discussed. It is considered that the amount of change in risk value (R1) can better reflect the psychological behaviour of decision-makers. Thirdly, common multi-attribute decision-making models, such as the expected utility-entropy model, are analysed, and then the viewpoint of different types of decision-making issues that require different decision methods is proposed. The well-known Allais paradox is explained by the proposed methods. Finally, the engineering application results show that the uncertainty control idea proposed here is accurate and effective. This research indicates a direction for further research into uncertainty, and risk control, issues affecting underground engineering works.

Building information modelling review with potential applications in tunnel engineering of China.

Building information modelling (BIM) can be applied to tunnel engineering to address a number of problems, including complex structure, extensive design, long construction cycle and increased security risks. To promote the development of tunnel engineering in China, this paper combines actual cases, including the Xingu mountain tunnel and the Shigu Mountain tunnel, to systematically analyse BIM applications in tunnel engineering in China. The results indicate that BIM technology in tunnel engineering is currently mainly applied during the design stage rather than during construction and operation stages. The application of BIM technology in tunnel engineering covers many problems, such as a lack of standards, incompatibility of different software, disorganized management, complex combination with GIS (Geographic Information System), low utilization rate and poor awareness. In this study, through summary of related research results and engineering cases, suggestions are introduced and an outlook for the BIM application in tunnel engineering in China is presented, which provides guidance for design optimization, construction standards and later operation maintenance.

Study on the Fire Damage Characteristics of the New Qidaoliang Highway Tunnel: Field Investigation with Computational Fluid Dynamics (CFD) Back Analysis.

In the New Qidaoliang Tunnel (China), a rear-end collision of two tanker trunks caused a fire. To understand the damage characteristics of the tunnel lining structure, in situ investigation was performed. The results show that the fire in the tunnel induced spallation of tunnel lining concrete covering 856 m³; the length of road surface damage reached 650 m; the sectional area had a maximum 4% increase, and the mechanical and electrical facilities were severely damaged. The maximum area loss happened at the fire spot with maximum observed concrete spallation up to a thickness of 35.4 cm. The strength of vault and side wall concrete near the fire source was significantly reduced. The loss of concrete strength of the side wall near the inner surface of tunnel was larger than that near the surrounding rock. In order to perform back analysis of the effect of thermal load on lining structure, simplified numerical simulation using computational fluid dynamics (CFD) was also performed, repeating the fire scenario. The simulated results showed that from the fire breaking out to the point of becoming steady, the tunnel experienced processes of small-scale warming, swirl around fire, backflow, and longitudinal turbulent flow. The influence range of the tunnel internal temperature on the longitudinal downstream was far greater than on the upstream, while the high temperature upstream and downstream of the transverse fire source mainly centered on the vault or the higher vault waist. The temperature of each part of the tunnel near the fire source had no obvious stratification phenomenon. The temperature of the vault lining upstream and downstream near the fire source was the highest. The numerical simulation is found to be in good agreement with the field observations.