搜索结果：Journal of bioscience and bioengineering

Strawberry cultivars have recently been diversified, and breeding objectives generally focus on production characteristics, as well as those to attract consumers. Strawberry cultivar selection and breeding rely heavily on human sensory evaluation. However, methods relying solely on sensory evaluation face challenges, such as being influenced by the subjectivity and physical condition of the panelists. Therefore, if compounds correlated with sensory profiles can be identified through instrumental analysis, combining this with sensory evaluation could enable more robust evaluation and improve the selection of new strawberry cultivars. Previous studies have investigated the relationship between strawberry metabolites and sensory profiles but have mainly focused on volatiles, certain sugars, and organic acids. However, some non-volatile compounds also influence flavor. Therefore, investigation of the correlations between non-volatile compounds and sensory profiles based on comprehensive analytical data is valuable. The objective of this study was to investigate the compounds correlated with the sensory profiles of strawberries. Sensory evaluations and gas chromatography/mass spectrometry (GC/MS)-based compound analyses were performed on five strawberry cultivars. Orthogonal partial least squares (OPLS) regression analysis was then performed using the compound data as explanatory variables and the sensory evaluation scores as response variables. Models capable of accurately predicting sensory profiles were constructed, and several non-volatile and volatile compounds showing high correlations were identified. This study confirmed the correlations between the sensory profiles of strawberries and volatile as well as non-volatile compounds, including amino and organic acids, providing useful insights into the breeding of new strawberry cultivars.

Ibuprofen, a widely used non-steroidal anti-inflammatory drug (NSAID), has been detected in the environment and is known to contaminate waterbodies. Here, we investigated the biotransformation of ibuprofen using the hyper-lignin-degrading fungus Phanerochaete sordida YK-624. This fungus exhibited excellent transformation capacity, removing 75% of ibuprofen within 5 h of incubation at a final concentration of 100 μM. Three novel metabolites of this transformation were isolated, and their structures were determined by nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry. Furthermore, experiments involving inhibition of cytochrome P450 indicated that it may play important roles in the process of ibuprofen transformation. To the best of our knowledge, this report is the first to demonstrate hydroxylation of the aromatic ring during the metabolism of ibuprofen by fungi. Additionally, a novel metabolic pathway for the transformation of ibuprofen by P. sordida YK-624 was proposed. These findings contribute to a deeper understanding of fungal-mediated pharmaceutical biotransformation and underscore the potential of white-rot fungi for environmental remediation of pharmaceutical pollutants.

Phthalate esters, widely used as plasticizers in plastic manufacturing, are known for their endocrine-disrupting effects. γ-Oryzanol derivatives, functional lipids composed of sterol and ferulic acid esters, possess diverse biological activities. In this study, we cloned and heterologously expressed a lipase-encoding gene (lipO745) from Aspergillus oryzae RIB40, deleting a 23-amino-acid N-terminal signal peptide, in Pichia pastoris using the pPICZαC vector. The resulting recombinant enzyme (rLipO745Δ23) was successfully secreted as an active extracellular protein. The purified recombinant lipase exhibited an apparent molecular mass of approximately 65 kDa, as determined using SDS-PAGE. Substrate specificity assays using p-nitrophenyl (pNP) esters (C2-C16) revealed that pNP butyrate (pNP-C4) was hydrolyzed most efficiently, with a specific activity of 369.2 ± 6.6 nmol·mL-1 mg-1. rLipO745Δ23 catalyzed the conversion of dibutyl phthalate (DBP) to monobutyl phthalate (MBP) without further degradation to phthalic acid, and exhibited an activity of 7.24 ± 0.61 nmol·mL-1 mg-1 toward DBP. The kinetic parameters (Km and kcat) were 0.66 ± 0.1 mM and 37.8 ± 6.4 s-1, respectively, for pNP-C4 substrate, and 0.40 ± 0.05 mM and 1.30 ± 0.23 s-1, respectively, for DBP. Moreover, rLipO745Δ23 showed detectable hydrolytic activity toward γ-oryzanol, including cycloartenyl ferulate, a conjugate of ferulic acid and triterpene alcohol. Notably, β-sitosterol, a major hydrolysate of phytosterol type γ-oryzanol, did not inhibit enzymatic activity. These findings highlight the potential of rLipO745Δ23 as a versatile biocatalyst for the enzymatic degradation of phthalates and γ-oryzanol derivatives in environmental and industrial applications.

Microbial fermentation is widely used in the production of food, pharmaceuticals, and bioenergy. Proper monitoring of the fermentation process is essential to ensure consistent product quality and yield. Although the indicators of fermentation progress vary among systems, they are generally evaluated by quantifying the major metabolites in the fermentation broth. However, these measurements rely on offline analyses involving time-consuming sampling and pretreatment, which hinder the real-time detection of process deviations. In this study, volatile organic compounds (VOCs) in the fermentation gas during beer fermentation were analyzed to develop a non-invasive method for the prediction of the current state of sugar, organic acid, and ethanol concentrations in the fermentation broth at each sampling point. VOCs emitted during fermentation serve as valuable indicators reflecting the metabolic state of the yeast. Beer brewing was adopted as a model system to validate VOC monitoring, because it involves sequential sugar consumption, organic acid and ethanol formation, and abundant VOC generation. We constructed multivariate regression models using the VOC profiles obtained from gas chromatography-mass spectrometry (GC-MS) analysis using Tenax TA. Parallel sampling of the fermentation gas and broth followed by orthogonal partial least squares (OPLS) regression yielded highly accurate models for predicting key fermentation indicators at corresponding time points. Furans and aldehydes abundant at early stages showed inverse correlations with higher alcohols and esters produced later, indicating distinct metabolic transitions. This study demonstrated the feasibility of applying VOC profiles for the quantitative prediction of the current fermentation progress and highlights the potential of this approach as a novel non-invasive monitoring method linking aroma chemistry and process control.

The world's increasing demand for petrochemical, pharmaceutical, and nutraceutical products necessitates the development of new strategies for producing these high-value chemicals. The depletion of the natural fossil reserves and environmental pollution associated with their procurement further compel us to find sustainable, greener, and cost-effective alternatives. In light of this, a shift has been witnessed in deriving these products from fossil-based sources or chemical synthesis to biomanufacturing (production using living systems). However, to fully utilize the potential of biomanufacturing, novel tools and strategies that can function in bacteria, archaea, and eukaryotes, regulate multi-enzyme pathways, offer precise and conditional gene regulation, and possess versatility are highly required. This review presents a comprehensive summary of the latest gene modulation tools and strategies used by metabolic engineers, along with a mechanistic overview and their applications. In addition, we presented the tools that have the potential to be used for pathway optimization but are still less explored. Within this context, we categorized these tools based on their molecular level of gene regulation, i.e., at and beyond the central dogma. We believe that a deeper understanding of the design, development, and application of these tools would be beneficial for metabolic engineers to reprogram biosynthetic pathways by adopting system-specific approaches, as a single strategy cannot be applied to all systems. Lastly, we discussed the challenges and future prospects of developing these gene regulatory tools to further advance the biomanufacturing field.

The correction (editing) of mutated genes at the DNA level is expected to cure gene-inherited diseases and cancers. A 5'-tailed duplex (TD) with an approximately 80-base editor strand (E-strand) plus a 35-base assistant strand (A-strand) was developed for gene editing without artificial nucleases. The E-strand has the normal (or desired) sequence and the A-strand hybridizes to the 3'-region of the E-strand. In this study, the polarity-dependency of gene editing by TDs was examined. The sense and antisense E-strands for eight transcribed target genes, including the WRN (Werner syndrome) gene, were designed, and the target plasmid DNAs were co-transfected with the TDs into human U2OS cells. Most TDs with the antisense E-strand corrected the targets more efficiently than those with the sense E-strand. However, transcription had only a slight effect on gene correction efficiency. These results suggested that the TDs containing the antisense E-strand are more useful editing tools than the sense TDs.

The development of conventional antibiotics is being severely challenged by the rise of bacterial resistance and the obstacle of biofilm-associated infections. Given their enhanced permeability, nano micelles have emerged as a widely utilized platform for the delivery of hydrophobic drugs. In this study, a novel material named BS-12-PEG-OH (PB12) was synthesized, and an intelligent nanoplatform was successfully developed through thin film dispersion method by co assembling vitamin E polyethylene glycol succinate (TPGS) with PB12 into hybrid micelles via thin film dispersion method, thereby enabling the encapsulation of Honokiol (designated as HK@PB12/TPGS Ms). The hybrid micelle can effectively penetrate the extracellular polymeric substance barrier of the biofilm. Upon reaching the acidic microenvironment, it responsively releases dodecyl dimethyl betaine (BS-12) and honokiol (HK), thus eliminating the biofilm structure and killing the embedded bacteria. Under acidic conditions, HK@PB12/TPGS Ms achieved a substantial bacterial reduction of 9.60 log10 CFU/ml. Furthermore, under the acidic condition of the biofilm microenvironment, they effectively cleared 83.32 % of the biofilm and reduced the embedded S. aureus by 3.75 log10 CFU/ml. The results indicate that the HK@PB12/TPGS Ms could effectively penetrate Staphylococcus aureus biofilms, disrupt their structure, and eliminate the bacteria inside the biofilm, hence preventing new infections and providing a novel therapeutic strategy for combating stubborn biofilm-associated infections.

Sub-Saharan Africa (SSA) is experiencing an epidemiological transition where non-communicable diseases are becoming the leading cause of disability and mortality alongside infectious diseases such as HIV/AIDS. Multimorbidity, the coexistence of two or more long-term conditions, is increasing in SSA. However, the cost of managing multimorbidity is largely unknown. This study aimed to estimate the economic cost of public outpatient primary care for adults with multimorbidity (HIV, hypertension, and/or diabetes, and their associated conditions: cardiovascular disease, and TB) in rural South Africa. This study used a cross-sectional, retrospective cost-of-illness approach to estimate the direct and indirect costs of multimorbidity management in Bushbuckridge, Mpumalanga, in 2022. Data were synthesized from patient-level data from eight public primary healthcare facilities within the Agincourt study site-a rapidly transitioning rural South African setting. Additionally, government reports and an existing study on transport costs and productivity losses conducted within the Agincourt study site were used to estimate the costs of managing patients in the primary care facilities. Results showed that patients with multimorbidity had higher average economic costs per patient compared to those with single conditions. Overall, patients with multimorbidity increase costs above the baseline of a patient with a single condition (R4 900/annum) by between 42% and 83%. Patients with multimorbidity also incur slightly higher costs associated with accessing primary care services compared to those with a single condition. However, our model shows that the additive cost of managing multiple conditions in separate consultations is higher than managing all conditions in one visit. This shows that managing patients within an integrated care model seems to have a cost-limiting effect. However, treatment guidelines for managing multimorbidity in South Africa should be developed to ensure standardized care.

Methylotrophic yeasts are promising hosts for bioproduction from methanol, but their growth is significantly reduced in the presence of methanol. In this study, we investigated metabolic bottlenecks in Komagataella phaffii, Ogataea polymorpha, and Candida boidinii during methanol assimilation through comparative metabolome analysis. We found a common decrease in α-ketoglutarate-derived amino acids and tricarboxylic acid (TCA) cycle intermediates in all three species during methanol assimilation, suggesting a metabolic bottleneck around the TCA cycle entry. Additionally, K. phaffii and O. polymorpha accumulate trehalose in the presence of methanol. ΔΔG analysis suggested reduced activity of pyruvate kinase and TCA cycle entry enzymes as potential rate-limiting steps. Biosynthesis of TCA cycle-derived amino acids, including Glu, Arg, and Pro, is a limiting factor because supplementation with these amino acids significantly enhanced growth rates and final cell density for all three species. Furthermore, disruption of the trehalose biosynthesis gene (TPS2) in K. phaffii further improved growth, indicating that trehalose accumulation negatively affects methanol-based growth. Amino acid supplementation with TPS2 disruption in K. phaffii resulted in a 1.2-fold increase in the specific growth rate and a 2.73-fold increase in final cell density. These findings provide insights into the metabolic limitations of methylotrophic yeast growth in the presence of methanol and offer strategies for improving bioproduction efficiency.

Substrate specificity of amino acid racemases is a key determinant of their biological function, yet the structural principles governing this specificity, particularly with respect to substrate side-chain structure, remain poorly understood. In this study, we investigated whether substrate specificity in the pyridoxal 5'-phosphate (PLP)-independent 2,4-diaminobutyrate racemase PddB can be progressively redirected through minimal substitutions, guided by structural and phylogenetic variation within a conserved enzyme scaffold. Comparative analyses identified residues in the distal region of the substrate-binding pocket as candidate determinants for substrate accommodation. Substitutions at three positions (92, 164, and 206) progressively shifted substrate preference from C4 to C5, and ultimately to C6 diamino acids. These stepwise transitions involved the acquisition of activity toward previously unrecognized substrates, accompanied by loss of the original specificity in the triple mutant. Catalytic efficiencies of active variants toward their preferred substrates were maintained within the same order of magnitude, indicating that catalytic competence was preserved despite substantial changes in substrate preference. Structural modeling indicates that these shifts arise from remodeling of the distal binding pocket, thereby altering its capacity to accommodate substrates of different chain lengths without perturbing the catalytic machinery. These findings demonstrate that substrate specificity in this enzyme family can be redirected in a stepwise manner through minimal substitutions, and that such changes define a plausible evolutionary trajectory toward alternative substrate specificities within a conserved scaffold. This study provides insights into the structural basis of substrate specificity in diamino acid racemases and highlights the evolutionary accessibility of alternative substrate specificities within this scaffold.

Volatile benzenoids such as veratrole and methyleugenol, together with terpenoids, constitute the characteristic floral scent of anise magnolia (Magnolia salicifolia), and the composition of these volatiles varies among natural populations in Japan. However, the enzymes responsible for biosynthesis of these volatile benzenoids remain poorly understood. In this study, we identified and biochemically characterized an S-adenosyl-l-methionine-dependent O-methyltransferase (MsOMT) from M. salicifolia. Recombinant MsOMT catalyzed methylation of guaiacol and eugenol to produce veratrole and methyleugenol, respectively. Kinetic analysis revealed apparent Km values of 18 μM for guaiacol and 16 μM for eugenol. In contrast, the enzyme showed only weak activity toward bulkier phenolic substrates such as isorhapontigenin and isorhamnetin, despite their structural similarity to guaiacol-derived compounds. Homology modeling of MsOMT supported these biochemical findings and suggested that a compact substrate-binding pocket underlies the enzyme's preference for small phenolic substrates. These results provide the first biochemical characterization of a guaiacol O-methyltransferase from M. salicifolia and provide new insights into the structural basis of substrate recognition in plant O-methyltransferases.

This study aimed to investigate whether that microwave pretreatment of glycogen-containing Synechococcus elongatus UTEX 2973 could enhance subsequent enzymatic saccharification and ethanol fermentation. The first experiment found that pretreatment with microwave was more effective than either pretreatment with ultrasound or lysozyme under the specific operational conditions examined, when a glycogen-containing cyanobacterial suspension (100 g/L) was pretreated and subjected to the saccharification assay at low biomass loading (10 g/L). Next, to investigate the appropriate microwave pretreatment time for enzymatic saccharification of glycogen in S. elongatus UTEX 2973, a cyanobacterial suspension (100 g/L) was pretreated by microwave (200 W) for 0-200 s and subjected to the saccharification assay at low biomass loading (10 g/L). The glucose yield was only 18% in the case of pretreatment for 0 s (negative control). On the contrary, the value increased to almost 100% in the case of microwave pretreatment for 100 s and more. Finally, to perform saccharification and ethanol fermentation of microwave-pretreated glycogen-containing S. elongatus UTEX 2973, a cyanobacterial suspension (100 g/L) was pretreated by microwave for 0-150 s and subjected to high biomass loading (100 g/L) enzymatic saccharification followed by ethanol fermentation. When the pretreatment time was 150 s, the glucose concentration at the end of saccharification was 44 g/L (glucose yield of 94%). The ethanol concentration was 21 g/L during the ethanol fermentation, which is 88% of the theoretical value. These results suggest that microwave pretreatment of glycogen-containing S. elongatus UTEX 2973 could facilitate the subsequent enzymatic saccharification and ethanol fermentation.

A robust heterologous expression system was developed in Pseudomonas putida KT2440 for the production of recombinant xanthine oxidase (XOD) from Cellulosimicrobium sp. TH20. To alleviate metabolic burden and enable inducer-free expression, a constitutive expression plasmid, pPegP119, was constructed by replacement of the pUCP18 origin with the low-copy-number pSC101 origin. Evaluation of the expression products from two gene clusters indicated that the complete xodCBA cluster yielded significantly higher activity (CsXodCBA, 5.6 U/mL) compared to the truncated xodBA cluster (CsXodBA, 2.1 U/mL), directly supporting the role of XodC in enzyme maturation. This role was further substantiated biochemically: purified CsXodCBA was found to contain approximately 2.6 times more molybdenum than CsXodBA and exhibited a substantially higher specific activity (28.1 U/mg versus 15.3 U/mg). Despite the genetic differences, the final assembled forms of CsXodCBA and CsXodBA were identical, consisting solely of the XODA (106 kDa) and XODB (30 kDa) subunits. Biochemical characterization of purified CsXodCBA demonstrated optimal activity at pH 7.0 and 50 °C, with kinetic parameters of Km = 85 ± 4 μM and Vmax = 20.6 ± 1.3 μM min-1. Enzyme activity was potently inhibited by Cu2+, Hg2+, and Zn2+. Thus, an efficient and cost-effective platform for the production of active XOD has been established through this work.

Large language models (LLMs) are being applied across diverse fields due to their capability to derive various insights from complex data. In biotechnology, where complex multimodal data including images is rapidly expanding, LLMs offer powerful capabilities for data analysis. However, unlocking the full potential of these models depends critically on prompt engineering, which is often a labor-intensive process that requires specialized expertise and lacks reproducibility. To address these challenges, we developed novel automatic prompt engineering (APE) approaches tailored for multimodal tasks in the bioscience and bioengineering domains. This study introduces two types of approaches: the Batch APE method, an efficient method for optimizing prompts for powerful black-box models, and a fine-tuning method with supervised fine-tuning (SFT) and direct preference optimization (DPO) on a local vision-language model (VLM). These methods were systematically evaluated across four diverse scientific datasets of microscopic images of protein crystals, human cell images, molecular structure images, and medical radiography images of Chest X-ray. Experimental results demonstrated that Gemini with Batch APE and SFT-based local LLM alignment generally outperformed baseline APE techniques, though DPO alone showed inconsistent results across datasets. Furthermore, qualitative analysis of the generated prompts revealed key characteristics of prompts that enhance image classification performance in multimodal LLMs. These findings highlight the potential of advanced APE to improve the utility of both local and black-box multimodal models for specialized scientific applications, particularly in domains where fine-tuning was restricted or infeasible.

Sake yeast Kyokai no. 11 (K11) is an ethanol-tolerant mutant of Kyokai no. 7 (K7) and produces a higher ethanol concentration in the sake mash than K7. A previous study revealed that stress-induced genes under the control of STRE elements were upregulated in K11. To elucidate the causal mutation responsible for ethanol tolerance, we compared the genome sequences of the ethanol-tolerant mutants (K11 and K7AT2, a newly isolated ethanol-tolerant mutant of K7) with that of their parental strain, K7. We identified a shared loss of heterozygosity region in the left arm of chromosome X in both mutants. We focused on CYR1 in this region, as it encodes adenylate cyclase, which negatively regulates expression of STRE-regulated genes through the upregulation of protein kinase A. Nucleotide 2066 of CYR1 was changed from G/A (amino acids Arg/His) in K7 to A/A (amino acids His/His) in K11 and K7AT2. When the plasmid containing CYR12066G was introduced into K11 or K7AT2, the stress tolerance of the transformants decreased to the level of K7, whereas the introduction of CYR12066A had a minimal effect. Consistently, disruption of the CYR12066G allele in K7 increased stress tolerance, whereas disruption of CYR12066A decreased stress tolerance. Furthermore, when CYR1 in a laboratory haploid strain was disrupted and either the CYR12042A or CYR12042G allele of S288C (corresponding to K7CYR12066) was introduced into the disruptant, the transformants with CYR12042A showed higher stress tolerance than those with CYR12042G. We concluded that the CYR1G2066A mutation was responsible for ethanol tolerance in K11.

Harnessing solar energy more efficiently and sustainably remains a key challenge in advancing renewable, bio-based production systems. Artificial photosynthesis tends to mimic natural photosynthesis by using catalytic systems and semiconductor assemblies to capture light and convert H2O and CO2 into energy-rich fuels such as H2 or hydrocarbons, whereas biophotovoltaics utilize living organisms or biological components (such as photosystems, chloroplasts, microalgae, or bacteria) integrated with electrodes for solar-to-electrical conversion. The review paper provides novel insights into exploring the integration of artificial photosynthesis and biophotovoltaics, discussing how their amalgamation can enhance and solidify solar-to-chemical and solar-to-electrical energy conversion. It emphasizes the crucial role of biocatalysts, such as microalgae, cyanobacteria, and bacteria, which can operate within these biohybrid systems. It also discusses the advanced strategies for enhancing biocatalyst efficiency, including genetic engineering, boosting carotenoid biosynthesis for better photoprotection and energy transfer, and integrating machine learning and Internet of Things to optimize the performance of microorganisms. In addition, the potential applications of artificial photosynthesis systems and biophotovoltaics are outlined, including biorefineries, biohydrogen production, chemical synthesis, and sustainable biofuel and food production.

Lignin is a complex aromatic polymer and a major component of lignocellulosic biomass, whose sustainable utilization is critical for the development of a low-carbon society. The white-rot fungus Phanerochaete sordida YK-624 exhibits high lignin-degrading activity. However, the pathways underlying the metabolism of lignin-derived aromatic compounds by this fungus remain unclear. Here, we combined genomic, transcriptomic, and metabolomic analyses to elucidate the catabolism of lignin-derived aromatic compounds in P. sordida YK-624. Multi-omics data suggest that this strain primarily metabolizes lignin-derived aromatic compounds using 1,2,4-trihydroxybenzene (THB) as a central intermediate, subsequently converting it first into 3-hydroxy-cis,cis-muconic acid (HMA) and then into 3-hydroxyhex-2-enedioic acid. The genes Psthbd and Pshmar 1 or 2, encoding putative THB dioxygenase and HMA reductase in P. sordida YK-624, were heterologously expressed in Escherichia coli. Functional assays confirmed that recombinant PsTHBD catalyzes the dioxygenation of THB and catechol, whereas PsHMAR2 exhibits HMA reductase activity. In contrast, PsHMAR1 showed no detectable activity, suggesting differences in cofactor binding or catalytic function. Our results demonstrate the involvement of PsTHBD and PsHMAR2 in THB degradation and identify for the first time an HMA reductase gene in white-rot fungi. This study advances our understanding of the catabolism of lignin-derived aromatic compounds and highlights the potential of P. sordida YK-624 as a platform for lignin valorization in biotechnological applications.

The prolonged use of current anti-inflammatory therapies has significant limitations for chronic inflammation management, requiring safer alternatives. In this study, we investigated the anti-inflammatory effect of methylxanthine derivative, pentoxifylline. MTT assay was used for the determination of the cytotoxicity of pentoxifylline. Lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells were used for the determination of the anti-inflammatory effect of pentoxifylline using Griess assay, qRT-PCR, Western blot, gene reporter assay and cell migration assay. Pentoxifylline exhibited a high level of safe therapeutic window with no cytotoxicity up to 20 &#x3bc;M in the MTT assay on RAW 264.7 cells. Pentoxifylline demonstrated concentration and time-dependent anti-inflammatory effects with significantly (p < 0.05) reduced NO production at 10 and 20 &#x3bc;M concentrations. Gene expression revealed significant downregulation of inflammatory mediator genes: Nos2, Tnf&#x3b1;, and Il1&#x3b2; (p < 0.05). Protein analysis confirmed these effects with reductions in iNOS, TNF&#x3b1;, IL1&#x3b2;, and IL6 (p < 0.05). Pentoxifylline caused a significant reduction (p < 0.05) of transcriptional activity of NF-&#x3ba;B and AP-1. Further, a significant reduction of nuclear translocation of NF-&#x3ba;B by pentoxifylline confirmed the transcriptional inhibition. Additionally, in the scratch assay, pentoxifylline exhibited a significant reduction (p < 0.05) in macrophage migration, supporting its role in inhibiting inflammation progression. These comprehensive multi-target anti-inflammatory effects of pentoxifylline support its promising potential for use as a therapeutic candidate for inflammatory diseases.

Selective quantification of individual d-amino acids is crucial for functional studies and biomarker development. Enzymatic assays enable convenient and rapid detection of d-amino acids. d-Amino acid oxidases (DAAOs) and d-aspartate oxidases (DDOs) are widely used for such assays; however, their broad substrate specificity limits selective detection in mixed samples. Here, we identified determinants of substrate recognition in DDO from the yeast Vanrija humicola (ChDDO) and reprogrammed its substrate specificity by structure-guided engineering. Crystal structure analysis highlighted Arg243 at the active-site entrance, and mutational analysis confirmed that substitution of Arg243 abolished detectable activity toward d-Asp and uncovered activity toward d-His and d-Phe. Molecular dynamics simulations of ChDDO indicated that Arg243 preferentially samples an inward-facing conformation toward His56 relative to the crystal-like outward orientation, accompanied by local rearrangements, suggesting a dynamic d-Asp uptake mechanism involving Arg243. Using R243A as a scaffold, we introduced second-site mutations to refine substrate specificity: L58N/R243A and F60Q/R243A increased d-His selectivity primarily by decreasing the apparent kcat for d-Phe, whereas H56E/R243A increased d-Phe selectivity primarily by decreasing the apparent kcat for d-His and increasing the apparent kcat for d-Phe. The engineered variants enabled quantification of d-His or d-Phe in various media, even in the presence of a mixture of 17 non-target d-amino acids. This study suggests a potentially distinct gating mechanism in ChDDO and provides a structure-guided route to convert strict DDOs into tailored biocatalysts for analytical and biotechnological applications.