Enhancer Activity Research Articles

Large-scale analysis of the integration of enhancer-enhancer signals by promoters.

Genes are often regulated by multiple enhancers. It is poorly understood how the individual enhancer activities are combined to control promoter activity. Anecdotal evidence has shown that enhancers can combine sub-additively, additively, synergistically, or redundantly. However, it is not clear which of these modes are more frequent in mammalian genomes. Here, we systematically tested how pairs of enhancers activate promoters using a three-way combinatorial reporter assay in mouse embryonic stem cells. By assaying about 69,000 enhancer-enhancer-promoter combinations we found that enhancer pairs generally combine near-additively. This behaviour was conserved across seven developmental promoters tested. Surprisingly, these promoters scale the enhancer signals in a non-linear manner that depends on promoter strength. A housekeeping promoter showed an overall different response to enhancer pairs, and a smaller dynamic range. Thus, our data indicate that enhancers mostly act additively, but promoters transform their collective effect non-linearly.

Large-scale analysis of the integration of enhancer-enhancer signals by promoters

Genes are often regulated by multiple enhancers. It is poorly understood how the individual enhancer activities are combined to control promoter activity. Anecdotal evidence has shown that enhancers can combine sub-additively, additively, synergistically, or redundantly. However, it is not clear which of these modes are more frequent in mammalian genomes. Here, we systematically tested how pairs of enhancers activate promoters using a three-way combinatorial reporter assay in mouse embryonic stem cells. By assaying about 69,000 enhancer-enhancer-promoter combinations we found that enhancer pairs generally combine near-additively. This behaviour was conserved across seven developmental promoters tested. Surprisingly, these promoters scale the enhancer signals in a non-linear manner that depends on promoter strength. A housekeeping promoter showed an overall different response to enhancer pairs, and a smaller dynamic range. Thus, our data indicate that enhancers mostly act additively, but promoters transform their collective effect non-linearly.

Investigating the role of WT1 in Brugada syndrome pathophysiology

Abstract Background Brugada syndrome (BrS) is a heritable arrhythmic disorder presumably caused by conduction slowing in the right ventricular (RV) outflow tract (RVOT). A recent genome-wide association study found an association between common variants on chromosome 11 near WT1, tagged by the SNP rs72905083, and BrS susceptibility. Purpose To study whether WT1 affects cardiac conduction and could therefore represents the underlying association of the chr11 locus with BrS susceptibility. Methods We conducted in silico analyses of omics datasets to identify the likely causal gene at the chr11 BrS locus. Next, we conducted electrophysiological studies in heterozygous young adult (4 months old) Wt1-deficient (Wt1+/-) mice and wild-type littermates to explore the effect of Wt1 on cardiac conduction. To study the potential role of Wt1 in the setting of reduced conduction reserve, we compared aged Scn5a haploinsufficient mice (Scn5a+/-;Wt1+/+, 17 months) with littermates haploinsufficient for both Wt1 and Scn5a (Scn5a+/-;Wt1+/-). Optical mapping was performed in Langendorff-perfused hearts, where conduction was further challenged by acute ajmaline administration or by pacing at the effective refractory period (ERP). Transcriptomic analyses were conducted in Wt1+/- and wild-type mice by RNAseq of PCM1-sorted cardiomyocytes. Results Analysis of omics datasets uncovered WT1 as the likely causal gene since WT1 was the closest gene to, and in fact overlapped, the BrS-associated region; promoter capture Hi-C data in human tissue showed chromatin contact between the promoter of WT1 and the associated region; and H3K27ac ChIP-seq signals (i.e. enhancer activity) of putative cardiac enhancers located within the associated region were correlated with WT1 transcript abundance across multiple tissues (Z-score = 4.32, p=1.57e-05). No differences were observed in ECG parameters or optically mapped epicardial conduction between Wt1+/- mice and wild-type littermates. However, comparison of aged Scn5a+/-;Wt1+/- and Scn5a+/-; Wt1+/+ hearts, in the setting of acute ajmaline challenge or pacing at the ERP, showed faster conduction in RV/RVOT in the presence of Wt1 haploinsufficiency (genotype effect: p=0.057 and p=0.041 for RV and RVOT epicardial conduction, respectively, during ajmaline challenge; p=0.034 for RV epicardial conduction during pacing at ERP). RNAseq of RV/RVOT cardiomyocytes revealed higher Scn5a expression in Wt1+/- vs wild-type (p=0.006). Conclusion We identified WT1 as the gene that most likely underlies the effect of the chr11 BrS susceptibility locus. Functional data revealed protective effects of reduced Wt1 expression in the setting of reduced conduction reserve. Transcriptomic data suggest that this occurs through a transcriptional effect on Scn5a expression.

Enhancing Activity Recognition After Stroke: Generative Adversarial Networks for Kinematic Data Augmentation

The generalizability of machine learning (ML) models for wearable monitoring in stroke rehabilitation is often constrained by the limited scale and heterogeneity of available data. Data augmentation addresses this challenge by adding computationally derived data to real data to enrich the variability represented in the training set. Traditional augmentation methods, such as rotation, permutation, and time-warping, have shown some benefits in improving classifier performance, but often fail to produce realistic training examples. This study employs Conditional Generative Adversarial Networks (cGANs) to create synthetic kinematic data from a publicly available dataset, closely mimicking the experimentally measured reaching movements of stroke survivors. This approach not only captures the complex temporal dynamics and common movement patterns after stroke, but also significantly enhances the training dataset. By training deep learning models on both synthetic and experimental data, we enhanced task classification accuracy: models incorporating synthetic data attained an overall accuracy of 80.0%, significantly higher than the 66.1% seen in models trained solely with real data. These improvements allow for more precise task classification, offering clinicians the potential to monitor patient progress more accurately and tailor rehabilitation interventions more effectively.

Engineering Active Sites of Metal/Metal Oxide Catalysts by Oxide Ligand Overlayers.

Engineering sites of supported metal catalysts is essential to enhancing activity and selectivity. Such enhancement is typically achieved by particle size modification, surface alloying, or attaching molecular ligands. Yet, control strategies for complex, multifunctional molecules and catalysts, where selectivity is crucial, are lacking. Here, we demonstrate that submonolayer WOx with tunable coverage preferentially decorates well-coordinated Pt terrace sites as a stable ligand. By combining experimental kinetics with probe molecules, in situ spectroscopies, and first-principles modeling, we show that the WOx coverage on Pt modifies the metal-to-acid site balance while retaining the acid strength intact and results in optimal reactivity for metal-acid catalyzed reactions at a specific metal, size, and support-dependent WOx coverage. The oxide can also alter the reactant adsorption mode, reversing selectivity and pathways from terrace- to step-dominated, as evidenced in furfural decarbonylation and hydrogenation. The insights open avenues for improving metal/metal oxide catalysts beyond the specific system.

Engineering Active Sites of Metal/Metal Oxide Catalysts by Oxide Ligand Overlayers

Engineering sites of supported metal catalysts is essential to enhancing activity and selectivity. Such enhancement is typically achieved by particle size modification, surface alloying, or attaching molecular ligands. Yet, control strategies for complex, multifunctional molecules and catalysts, where selectivity is crucial, are lacking. Here, we demonstrate that submonolayer WOx with tunable coverage preferentially decorates well‐coordinated Pt terrace sites as a stable ligand. By combining experimental kinetics with probe molecules, in situ spectroscopies, and first‐principles modeling, we show that the WOx coverage on Pt modifies the metal‐to‐acid site balance while retaining the acid strength intact and results in optimal reactivity for metal‐acid catalyzed reactions at a specific metal, size, and support‐dependent WOx coverage. The oxide can also alter the reactant adsorption mode, reversing selectivity and pathways from terrace‐ to step‐dominated, as evidenced in furfural decarbonylation and hydrogenation. The insights open avenues for improving metal/metal oxide catalysts beyond the specific system.

Analyzing super-enhancer temporal dynamics reveals potential critical enhancers and their gene regulatory networks underlying skeletal muscle development.

Super-enhancers (SEs) govern the expression of genes defining cell identity. However, the dynamic landscape of SEs and their critical constituent enhancers involved in skeletal muscle development remains unclear. In this study, using pig as a model, we employed CUT&Tag to profile the enhancer-associated histone modification marker H3K27ac in skeletal muscle across two prenatal and three postnatal stages and investigated how SEs influence skeletal muscle development. We identified three SE families with distinct temporal dynamics: continuous (Con, 397), transient (TS, 434), and de novo (DN, 756). These SE families are associated with different temporal gene expression trajectories, biological functions, and DNA methylation levels. Notably, several lines of evidence suggest a potential prominent role of Con SEs in regulating porcine muscle development and meat traits. To pinpoint key cis-regulatory units in Con SEs, we developed an integrative approach that leverages information from eRNA annotation, GWAS signals and high-throughput capture STARR-seq experiments. Within Con SEs, we identified 20 candidate critical enhancers with meat and carcass-associated DNA variations that affect enhancer activity and inferred their upstream TFs and downstream target genes. As a proof of concept, we experimentally validated the role of one such enhancer and its potential target gene during myogenesis. Our findings reveal the dynamic regulatory features of SEs in skeletal muscle development and provide a general integrative framework for identifying critical enhancers underlying the formation of complex traits.

A novel interpretable deep learning-based computational framework designed synthetic enhancers with broad cross-species activity.

Enhancers play a critical role in dynamically regulating spatial-temporal gene expression and establishing cell identity, underscoring the significance of designing them with specific properties for applications in biosynthetic engineering and gene therapy. Despite numerous high-throughput methods facilitating genome-wide enhancer identification, deciphering the sequence determinants of their activity remains challenging. Here, we present the DREAM (DNA cis-Regulatory Elements with controllable Activity design platforM) framework, a novel deep learning-based approach for synthetic enhancer design. Proficient in uncovering subtle and intricate patterns within extensive enhancer screening data, DREAM achieves cutting-edge sequence-based enhancer activity prediction and highlights critical sequence features implicating strong enhancer activity. Leveraging DREAM, we have engineered enhancers that surpass the potency of the strongest enhancer within the Drosophila genome by approximately 3.6-fold. Remarkably, these synthetic enhancers exhibited conserved functionality across species that have diverged more than billion years, indicating that DREAM was able to learn highly conserved enhancer regulatory grammar. Additionally, we designed silencers and cell line-specific enhancers using DREAM, demonstrating its versatility. Overall, our study not only introduces an interpretable approach for enhancer design but also lays out a general framework applicable to the design of other types of cis-regulatory elements.

Spermatogenic and Libido Enhancing Effect of Ethanol Extract of Kigelia Africana in ∆9 Tetrahydrocannabinol Induced Erectile Dysfunction in Male Wistar Rats

Erectile dysfunction has remained one of the major global health issues for the past two decades. Since the discovery of phosphodiesterase type 5 inhibitors, a significant number of patients have solved the issue of erectile dysfunction. However, the wide distribution of phosphodiesterase type 5 enzymes at various sites of the body led phosphodiesterase type 5 inhibitors to cause various unnecessary outcomes. Hence, it is vital to look for optional agents that could solve these limitations. In this present work, erectile dysfunction was induced by administering 30 mg/kgbwt THC for 3 days. Treatment given was as follows: A - Normal Control, B - Std control (THC+Viagra), C - Negative control (THC only), D - THC + 200 mg/Kgbwt ethanol leaf extract of Kigelia africana (200 mg/Kgbwt), E - THC + 400 mg/Kgbwt ethanol leaf extract of Kigelia africana for a succession of 14 days. Sexual behaviour parameters were monitored in the male rats on Days 1, 7 and 14 respectively after administration by pairing with a receptive female (1:1); thereafter, rats were sacrificed and serum was collected for some biochemical parameters using standard method. Cage side observation on the animals revealed prospective behaviours by the receptive female rats and precopulatory behaviours by the ethanol extract K.africana treated male rats.The extract at 200 and 400 mg/kg body weight significantly (P<0.05) increased the frequencies of mount and intromission.In addition, the ejaculation latency was significantly (P< 0.05) prolonged.The latencies of mount and intromission were reduced significantly (P<0.05) whereas ejaculation frequency increased. The extract also reduced the post-ejaculatory interval of the Wistar rats. Computed percentages of index of libido, mounted, intromitted, ejaculated and copulatory efficiency were significantly (p<0.05) higher in the extract-treated animals in a dosage dependent manner than the control whereas the inter copulatory interval decreased significantly. The extract also significantly (p<0.05) increased the serum testosterone, FSH and LH concentration but significantly (p<0.05) reduced serum oestrogen, prolactin and progesterone of the treated groups when compared with the control. The extract also significantly (p<0.05) increased the sperm motility, total sperm count, and sperm viability but produced a significant (p<0.05) decrease in serum immobility when compared with the THC control. It can be inferred from this work that an extract of K. africana may elicit spermatogenic, androgenic and libido enhancing activities in THC induced erectile dysfunction in a dosage dependent manner.

Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors.

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically up-regulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally acting chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

Enhancers in Plant Development, Adaptation, and Evolution.

Understanding plant responses to developmental and environmental cues is crucial for studying morphological divergence and local adaptation. Gene expression changes, governed by cis-regulatory modules (CRMs) including enhancers, are a major source of plant phenotypic variation. However, while genome-wide approaches have revealed thousands of putative enhancers in mammals, far fewer have been identified and functionally characterized in plants. This review provides an overview of how enhancers function to control gene regulation, methods to predict DNA sequences that may have enhancer activity, methods utilized to functionally validate enhancers, and the current knowledge of enhancers in plants, including how they impact plant development, response to environment, and evolutionary adaptation.

In Situ Biomineralization and Citric Acid Etching Strategy for Enhancing Activity of Immobilized Acetylcholinesterase.

Enhancing the structural stability of an enzyme and maintaining its catalytic activity are effective ways to improve enzyme utilization and reduce the cost of drug screening. However, immobilized enzyme activity tends to decrease in existing immobilization techniques due to conformational changes and microenvironmental restrictions. In this paper, we present a facile approach to prepare immobilized acetylcholinesterase (AChE) with high activity by a ZIF-8 in situ immobilization and citric acid (CA) etching strategy. CA breaks the coordination bond of ZIF-8 and produces defects, expanding the pore space, improving substrate accessibility, and fully exposing the active site of the enzyme. The enhancement of the catalytic activity of AChE@ZIF-8-CA was about 6.10-fold compared with the free enzyme. In addition, AChE@ZIF-8-CA exhibited an excellent encapsulation efficiency and good tolerance to temperature, pH, and organic solvents. The relative activity remains at the initial 83.77% even in five repeated experiments. The strategy provides a novel and efficient way to quickly construct highly active immobilized enzymes under mild conditions.

The dual-specificity kinase DYRK1A interacts with the Hepatitis B virus genome and regulates the production of viral RNA.

The genome of Hepatitis B virus (HBV) persists in infected hepatocytes as a nuclear episome (cccDNA) that is responsible for the transcription of viral genes and viral rebound, following antiviral treatment arrest in chronically infected patients. There is currently no clinically approved therapeutic strategy able to efficiently target cccDNA (Lucifora J 2016). The development of alternative strategies aiming at permanently abrogating HBV RNA production requires a thorough understanding of cccDNA transcriptional and post-transcriptional regulation. In a previous study, we discovered that 1C8, a compound that inhibits the phosphorylation of some cellular RNA-binding proteins, could decrease the level of HBV RNAs. Here, we aimed at identifying kinases responsible for this effect. Among the kinases targeted by 1C8, we focused on DYRK1A, a dual-specificity kinase that controls the transcription of cellular genes by phosphorylating transcription factors, histones, chromatin regulators as well as RNA polymerase II. The results of a combination of genetic and chemical approaches using HBV-infected hepatocytes, indicated that DYRK1A positively regulates the production of HBV RNAs. In addition, we found that DYRK1A associates with cccDNA, and stimulates the production of HBV nascent RNAs. Finally, reporter gene assays showed that DYRK1A up-regulates the activity of the HBV enhancer 1/X promoter in a sequence-dependent manner. Altogether, these results indicate that DYRK1A is a proviral factor that may participate in the HBV life cycle by stimulating the production of HBx, a viral factor absolutely required to trigger the complete cccDNA transcriptional program.

Mediator MED23 controls oligodendrogenesis and myelination by modulating Sp1/P300-directed gene programs

Gaining the molecular understanding for myelination development and regeneration has been a long-standing goal in neurological research. Mutations in the transcription cofactor Mediator Med23 subunit are often associated with intellectual disability and white matter defects, although the precise functions and mechanisms of Mediator in myelination remain unclear. In this study, we generated a mouse model carrying an Med23Q649R mutation that has been identified in a patient with hypomyelination features. The MED23Q649R mouse model develops white matter thinning and cognitive decline, mimicking common clinical phenotypes. Further, oligodendrocyte-lineage specific Med23 knockout mice verified the important function of MED23 in regulating central nervous system myelination and postinjury remyelination. Utilizing the in vitro cellular differentiation assay, we found that the oligodendrocyte progenitor cells, either carrying the Q649R mutation or lacking Med23, exhibit significant deficits in their capacity to differentiate into mature oligodendrocytes. Gene profiling combined with reporter assays demonstrated that Mediator Med23 controls Sp1-directed gene programs related to oligodendrocyte differentiation and cholesterol metabolism. Integrative analysis demonstrated that Med23 modulates the P300 binding to Sp1-targeted genes, thus orchestrating the H3K27 acetylation and enhancer activation for the oligodendrocyte lineage progression. Collectively, our findings identified the critical role for the Mediator Med23 in oligodendrocyte fate determination and provide mechanistic insights into the myelination pathogenesis associated with MED23 mutations.

Methylation patterns at the adjacent CpG sites within enhancers are a part of cell identity

BackgroundIt is generally accepted that methylation status of CpG sites spaced up to 50 bp apart is correlated, and accumulation of locally disordered methylation at adjacent CpG sites is involved in neoplastic transformation, acting in similar way as stochastic accumulation of mutations.ResultsWe used EPIC microarray data from 596 samples, representing 12 healthy tissue and cell types, as well as 572 blood cancer specimens to analyze methylation status of adjacent CpG sites across human genome, and subsequently validated our findings with NGS and Sanger sequencing. Our analysis showed that there is a subset of the adjacent CpG sites in human genome, with cytosine at one CpG site methylated and the other devoid of methyl group. These loci map to enhancers that are targeted by families of transcription factors involved in cell differentiation. Moreover, our results suggest that the methylation at these loci differ between alleles within a cell, what allows for remarkable level of heterogeneity of methylation patterns. However, different types of specialized cells acquire only one specific and stable pattern of methylation at each of these loci and that pattern is to a large extent lost during neoplastic transformation.ConclusionsWe identified a substantial number of adjacent CpG loci in human genome that display remarkably stable and cell type specific methylation pattern. The methylation pattern at these loci appears to reflect different methylation of alleles in cells. Furthermore, we showed that changes of methylation status at those loci are likely to be involved in regulation of the activity of enhancers and contribute to neoplastic transformation.

Liquid nitrogen quenching-induced grain boundary-rich intermetallic compound/metal/carbon composites for efficient capacitive deionization

In this work, a novel liquid nitrogen quenching treatment is introduced for the first time to design and prepare a grain boundary-rich intermetallic compound/metal/carbon composite, serving as an efficient electrode material for sodium ion removal from simulated seawater. The liquid nitrogen quenching method effectively enhances the grain boundary density within the composite due to the rapid cooling-induced forces, thereby creating abundant active sites. Consequently, Co7Fe3/Co/C-700 achieves optimal performance in sodium ion embedding and de-embedding, with a capacity of 207.5 mg/g. Furthermore, sodium ion adsorption/desorption mechanisms are substantiated through ex-situ techniques, revealing dynamic atomic and electronic structure evolutions under operational conditions. Density Functional Theory (DFT) calculations validate the pivotal role of grain boundary sites in enhancing activity, elucidating the correlation between sodium ion adsorption and grain boundaries. Overall, this work elucidates the significant correlation between adsorption behavior and grain boundaries, proposing an innovative strategy for developing electrode materials for capacitive deionization.

Developmental and housekeeping transcriptional programs display distinct modes of enhancer-enhancer cooperativity in Drosophila

Genomic enhancers are key transcriptional regulators which, upon the binding of sequence-specific transcription factors, activate their cognate target promoters. Although enhancers have been extensively studied in isolation, a substantial number of genes have more than one simultaneously active enhancer, and it remains unclear how these cooperate to regulate transcription. Using Drosophila melanogaster S2 cells as a model, we assay the activities of more than a thousand individual enhancers and about a million enhancer pairs toward housekeeping and developmental core promoters with STARR-seq. We report that housekeeping and developmental enhancers show distinct modes of enhancer-enhancer cooperativity: while housekeeping enhancers are additive such that their combined activity mirrors the sum of their individual activities, developmental enhancers are super-additive and combine multiplicatively. Super-additivity between developmental enhancers is promiscuous and neither depends on the enhancers’ endogenous genomic contexts nor on specific transcription factor motif signatures. However, it can be further boosted by Twist and Trl motifs and saturates for the highest levels of enhancer activity. These results have important implications for our understanding of gene regulation in complex multi-enhancer developmental loci and genomically clustered housekeeping genes, providing a rationale to interpret the transcriptional impact of non-coding mutations at different loci.

Context transcription factors establish cooperative environments and mediate enhancer communication.

Many enhancers control gene expression by assembling regulatory factor clusters, also referred to as condensates. This process is vital for facilitating enhancer communication and establishing cellular identity. However, how DNA sequence and transcription factor (TF) binding instruct the formation of high regulatory factor environments remains poorly understood. Here we developed a new approach leveraging enhancer-centric chromatin accessibility quantitative trait loci (caQTLs) to nominate regulatory factor clusters genome-wide. By analyzing TF-binding signatures within the context of caQTLs and comparing episomal versus endogenous enhancer activities, we discovered a class of regulators, 'context-only' TFs, that amplify the activity of cell type-specific caQTL-binding TFs, that is, 'context-initiator' TFs. Similar to super-enhancers, enhancers enriched for context-only TF-binding sites display high coactivator binding and sensitivity to bromodomain-inhibiting molecules. We further show that binding sites for context-only and context-initiator TFs underlie enhancer coordination, providing a mechanistic rationale for how a loose TF syntax confers regulatory specificity.

ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency

The primed epiblast acts as a transitional stage between the relatively homogeneous naïve epiblast and the gastrulating embryo. Its formation entails coordinated changes in regulatory circuits driven by transcription factors and epigenetic modifications. Using a multi-omic approach in human embryonic stem cell models across the spectrum of peri-implantation development, we demonstrate that the transcription factors ZIC2 and ZIC3 have overlapping but essential roles in opening primed-specific enhancers. Together, they are essential to facilitate progression to and maintain primed pluripotency. ZIC2/3 accomplish this by recruiting SWI/SNF to chromatin and loss of ZIC2/3 or degradation of SWI/SNF both prevent enhancer activation. Loss of ZIC2/3 also results in transcriptome changes consistent with perturbed Polycomb activity and a shift towards the expression of genes linked to differentiation towards the mesendoderm. Additionally, we find an intriguing dependency on the transcriptional machinery for sustained recruitment of ZIC2/3 over a subset of primed-hESC specific enhancers. Taken together, ZIC2 and ZIC3 regulate highly dynamic lineage-specific enhancers and collectively act as key regulators of human primed pluripotency.

Surfactant-tolerance evolution of Bacillus clausii protease for enhancing activity and stability by reshaping the substrate access tunnel

Alkali proteases are crucial in numerous industries, especially in the laundry industry, but their inactivation by surfactants limits their effectiveness. This study employed substrate access tunnel engineering to improve the performance of WT bcPRO in surfactants. By modifying the key residues in the substrate pocket, the best variant N212S showed higher stability and activity in both AES and LAS. Molecular dynamics (MD) simulations provided insights into the enhanced stability and activity. The Asn212Ser mutation weakened the anti-correlation motion, increased the number of hydrogen bonds between amino acid residues, and made the protein structure more compact, contributing to its stability. Additionally, the mutation extended the substrate access tunnel and enabled additional interactions with the substrate, enhancing its catalytic activity in surfactants. This study demonstrates a strategy for reshaping the substrate access tunnel to improve protease stability and activity in surfactant environments, offering a promising protease candidate for the laundry industry.