Ultrasound-Assisted Multi-Enzyme Extraction for Highly Efficient Extraction of Polysaccharides from Ulva lactuca.

Ulva polysaccharides present several physiological activities including antiviral, antitumor and anti-plasmodial effects. However, current processing usually results in low yields and high prices, thus lacking commercialization potential. The aim of this study was to develop an efficient method for the extraction of Ulva polysaccharides with high biological activity. The effect of cell wall-degrading enzymes including cellulase, hemicellulase, pectinase and protease on Ulva polysaccharide extraction was studied by statistical mixing design. Using the most effective enzyme preparations as the basic components, the optimal proportions of the enzyme mixture were determined as follows: cellulase 35.3%, pectinase 34.5%, alkaline protease 30.2%, which increased the polysaccharide yield from 6.43% in the absence of enzymes to 26.68%. Subsequently, through response surface analysis, the optimal conditions were determined: enzyme concentration of 1.5%, enzymatic time of 1.1 h, ultrasonic time of 90 min and enzymatic temperature of 60 °C. Under the optimal extraction conditions, the extraction yield of Ulva polysaccharides could be increased to 30.14%. Moreover, extracted polysaccharides exhibit strong antioxidant properties in DPPH, ABTS, hydroxyl radical, superoxide radical and H2O2-induced cellular damage models. This study laid a solid foundation for the use and development of Ulva polysaccharides.

Brazilin-7-2-butenoate inhibits amyloid ß-protein aggregation, alleviates cytotoxicity, and protects Caenorhabditis elegans.

The fibrillogenesis of amyloid ß-protein (Aß) gradually accumulates to form neurotoxic Aß aggregates in the human brain, which is the direct cause of Alzheimer's disease (AD) related symptoms. There are currently no effective therapies for AD. Brazilin, a natural polyphenol, inhibits Aß fibrillogenesis, disrupts the mature fibrils and alleviates the corresponding cytotoxicity, but it also has the high toxic. Therefore, brazilin-7-2-butenoate (B-7-2-B), a brazilin derivative, was designed and synthesized. B-7-2-B exhibited lower toxicity and stronger inhibitory effect on Aß aggregation than brazilin. B-7-2-B could prevent the formation of Aß fibrils and oligomers, and depolymerize pre-formed aggregates in a dose-dependent manner. Furthermore, B-7-2-B prominently alleviated the cytotoxicity and the oxidative stress induced by Aß aggregates in PC12 cells. The protective impacts of B-7-2-B were further demonstrated by using the Caenorhabditis elegans model, including decreasing the extent of Aß aggregation, improving the motility and sensation disorders. Eventually, B-7-2-B was proven to be no apparent damage to worms. In summarize, it can be concluded that B-7-2-B has the potential as a drug for treating AD.

Smartphone-assisted nanozyme sensor array constructed based on reaction kinetics for the discrimination and identification of phenolic compounds.

Although the research on nanozymes has attracted widespread attention in recent years, the development of highly active and multifunctional nanozymes remains a challenge. Here, a bifunctional AMP-Cu nanozyme with laccase- and catecholase-like activities was successfully prepared at room temperature with Cu2+ as the metal ion and adenosine-5'-monophosphate (AMP) as the ligand molecule. Based on the excellent catalytic performance of AMP-Cu, a three-channel colorimetric sensor array was constructed using reaction kinetics as the sensing unit to achieve high-throughput detection and identification of six common phenolic compounds at low concentrations. This strategy simplifies the construction of sensor array and demonstrates the capacity to obtain multidimensional data from a single material. Finally, with the assistance of smartphones and homemade dark boxes, a portable on-site detection method for phenolic compounds was developed. This work would contribute to the development of portable sensors and the highly efficient identification of phenolic compounds in complex samples.

Construction and Catalytic Study of Affinity Peptide Orientation and Light Crosslinking Immobilized Sucrose Isomerase.

A novel affinity peptide orientation and light-controlled covalent immobilized method was developed. Sucrose isomerase (SI) was selected as the model enzyme. Molecular simulation was first performed to select the targeted immobilization region. Subsequently, a short peptide (H2N-VNIGGX-COOH, VG) with high affinity to this region was rationally designed. Thereafter, 4-benzoyl-l-phenylalanine with the photosensitive group of benzophenone was introduced. Then, the affinity between the ligand and the SI was validated using molecular dynamics simulation. Thereafter, the SI was directionally immobilized onto the surface of the epoxy resin (EP) guided by VG via photo-crosslinking, and thus the oriented photo-crosslinking enzymes were obtained. The enzymatic activity, thermostability, and reusability of the affinity directional photo-crosslinked immobilized sucrose isomerase (hv-EP-VG-SI) were systematically studied. The oriented immobilization enzymes were significantly improved in recycling and heat resistance. Moreover, hv-EP-VG-SI retained more than 90% of the original activity and 50% of the activity after 11 cycles.

Engineering of a fungal steroid 11α-hydroxylase and construction of recombinant yeast for improved production of 11α-hydroxyprogesterone.

Cytochrome P450 enzyme CYP68J5 from filamentous fungus Aspergillus ochraceus is industrially used for selective C11α-hydroxylation of canrenone and progesterone. To improve its selectivity of C11α-hydroxylation for relevant steroid substrates, a sequence-based targeted mutagenesis combined with saturation mutagenesis was conducted to search for variants with improved hydroxylation reaction specificity toward progesterone and D-ethylgonendione. Recombinant yeast expressing triple mutant V64F/E65G/N66T showed significantly increased C11α-hydroxylation selectivity (85 % VS WT 69.7 %). Saturation mutagenesis of V64, E65 and N66 resulted in the identification of single mutant V64K with greatly enhanced 11α-hydroxylation specificity toward progesterone (90.6 % VS WT 69.7 %). Furthermore, mutant N66D showed significant enhanced selectivity of C11α-hydroxylation toward D-ethylgonendione (70.8 % VS WT 58 %). Evaluation of recombinant yeast over-expressing V64K for progesterone transformation in 50 mL scale resulted in product 11α-OH progesterone concentrations of 432.5 mg/L, a 30.2 % increase compared with the CYP68J5 control. Our results also reveal that V64, E65 and N66 are key residues of CYP68J5 influencing its selectivity of C11α-hydroxylation, thus offering opportunities for further engineering of CYP68J5 for expanded industrial applications.

Targeted profiling of rodent unconjugated bile acids using GC-MS to determine the influence of high-fat diet.

Unconjugated bile acids (BAs) have gained more attention than conjugated BAs in the association studies among diet, gut microbiota, and diseases. GC-MS is probably a good choice for specialized analysis of unconjugated BAs due to its high separation capacity and identification convenience. However, few reports have focused on the rodent unconjugated BAs using GC-MS, and the main library for identification has not included rodent-specific BAs. We developed a GC-MS method for targeted profiling of eight main unconjugated BAs in rodent models, which showed excellent performance on sensitivity, reproducibility, and accuracy. Quantitative reproducibility in terms of relative standard deviation (RSD) was in the range of 2.05-2.91%, with detection limits of 3-55 ng/mL, quantitation limits of 9-182 ng/mL, and the recovery rate of 72-115%. All the calibration curves displayed good linearity with correlation coefficient values (R2 ) more than 0.99. Using the established method, the influence of high-fat diet on the metabolism of unconjugated BAs was revealed. Significant increase in fecal unconjugated BAs induced by high-fat diet would be a potential risk to gut inflammation and cancer. The study provides a convenient and targeted GC-MS method for specialized profiling of rodent unconjugated BAs in physiological and pathological studies.

Novel Detection Method for Evaluating the Activity of an Alkaline Serine Protease from Bacillus clausii.

Until now, the detection methods for serine proteases have been quite time-consuming or cannot indicate the "real" protease activity. Here, a rapid and simple method for determining the "real" activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 standard amino acids) was developed. This AAPX method has high reliability, sensitivity, and repeatability and can be used for detecting the serine protease activity spectrophotometrically. Additionally, the site-directed saturation mutagenesis library of alkaline serine protease PRO (BcPRO) from Bacillus clausii was screened with this AAPX method. Three beneficial mutants S99R, S99H, and S99W were identified, and S99W displayed the highest activity. In comparison to wild-type BcPRO, S99W exhibited enhanced catalytic performance toward eight AAPX monomers, and the molecular dynamics simulation revealed the mechanism responsible for its improved activity toward AAPM. Consequently, this work provides an efficient method for detecting, characterizing, mining, and high-throughput screening of serine proteases.

Utilization of Soybean Oil Waste for a High-Level Production of Ceramide by a Novel Phospholipase C as an Environmentally Friendly Process.

Ceramide is a natural functional ingredient as food additive and medicine that has attracted extensive attention in the food, medical, and cosmetic industries. Here, we developed a biotechnological strategy based on a recombinant whole-cell biocatalyst for efficiently producing ceramide from crude soybean oil sediment (CSOS) waste. A novel phospholipase C (PLCac) from Acinetobacter calcoaceticus isolated from soil samples was identified and characterized. Furthermore, recombinant Komagataella phaffii displaying PLCac (dPLCac) on the cell surface was constructed as a whole-cell biocatalyst with better thermostability (30-60 °C) and pH stability (8.0-10.0) to successfully produce ceramide. After synergistical optimization of reaction time and dPLCac dose, the ceramide yield of hydrolyzing from CSOS using dPLCac was 51% (the theoretical maximum yield of converting sphingomyelin, ∼70%) and the relative yield was over 50% after seven consecutive 4 h batches under the optimized conditions. Our study provides a potentially promising strategy for the commercial production of ceramide.

An Enzymatic Biosensor for the Detection of D-2-Hydroxyglutaric Acid in Serum and Urine.

D-2-hydroxyglutaric acid (D2HG) is overproduced as a result of the D-2-hydroxyglutaric aciduria and relevant cancers, caused by gene mutation. Accurate analysis of D2HG could help rapid diagnosis of these diseases and allow for timely treatment. In this work, a D-2-hydroxyglutarate dehydrogenase from Ralstonia solanacearum (RsD2HGDH) is cloned and recombinantly expressed. This enzyme features the direct electron transfer to chemical electron mediators (such as methylene blue (MB)) in the absence of additional coenzymes. Therefore, NAD+, a natural electron acceptor for the commercial D2HGDH and usually known for being unstable and difficult for immobilization can be avoided in the preparation of biosensors. The RsD2HGDH and MB are co-immobilized on a two-dimensional material, Ti3C2 MXene, followed by drop-coating on the gold screen-printed electrode (AuSPE) to construct a compact and portable biosensor. The D2HG in samples can be catalyzed by RsD2HGDH, where the current change is measured by chronoamperometry at -0.23 V. The biosensor shows a D2HG detection range of 0.5 to 120 µM (R2 = 0.9974) with a sensitivity of 22.26 µA mM-1 cm-2 and a detection limit of 0.1 µM (S/N = 3). The biosensor retains 72.52% performance of its incipient state after 30 days of storage. The samples of D2HG-containing fetal bovine serum and artificial urine were analyzed with the recovery of 99.56% to 106.83% and 97.30% to 102.47% further indicating the great application potential of our portable D2HG biosensor.

Design of carboxylated single-walled carbon nanotubes as highly efficient inhibitors against Aß40 fibrillation based on the HyBER mechanism.

Misfolding and the subsequent self-assembly of amyloid-ß protein (Aß) is very important in the occurrence of Alzheimer's disease (AD). Thus, inhibition of Aß aggregation is currently an effective method to alleviate and treat AD. Herein, a carboxylated single-walled carbon nanotube (SWCNT-COOH) was rationally designed based on the hydrophobic binding-electrostatic repulsion (HyBER) mechanism. The inhibitory effect of SWCNT-COOH on Aß fibrillogenesis was first studied. Based on the results of thioflavin T fluorescence and atomic force microscopy imaging assays, it was shown that SWCNT-COOH can not only effectively inhibit Aß aggregation, but also depolymerize the mature fibrils of Aß. In addition, its inhibitory action will be affected by the content of carboxyl groups. Moreover, the influence of SWCNT-COOH on cytotoxicity induced by Aß was investigated by the MTT method. It was found that SWCNT-COOH can produce an anti-Aß neuroprotective effect in vitro. Molecular dynamics simulations showed that SWCNT-COOH significantly destroyed the overall and internal structural stability of an Aß40 trimer. Moreover, SWCNT-COOH interacted strongly with the N-terminal region, turn region and C-terminal region of the Aß40 trimer via hydrogen bonds, salt bridges and π-π interactions, which triggered a large structural disturbance of the Aß40 trimer, reduced the ß-sheet content of the Aß40 trimer and led to more disorder in these regions. All the above data not only reveal the suppressive effect of SWCNT-COOH on Aß aggregation, but also reveal its inhibitory mechanism, which provides a useful clue to exploit anti-Aß drugs in the future.

The food additive fast green FCF inhibits α-synuclein aggregation, disassembles mature fibrils and protects against amyloid-induced neurotoxicity.

α-Synuclein (α-syn) aggregates into cytotoxic amyloid fibrils, which are recognized as the defining neuropathological feature of Parkinson's disease (PD). Therefore, inhibiting α-syn fibrillogenesis and disrupting the preformed fibrils are both considered attractive strategies to cure PD. We discovered that a safe food additive, fast green FCF, is capable of inhibiting α-synuclein fibrillogenesis and reducing the related cytotoxicity. Thioflavin T fluorescence assays demonstrated that fast green FCF could inhibit the fibrillogenesis α-synuclein. In the presence of 100 µM fast green FCF, amorphous aggregates were formed and observed by atomic force microscopy. Toxicity assays in cell cultures revealed that fast green FCF significantly reduced the cytotoxicity of α-syn. Molecular dynamics simulations revealed the potential mechanism of the interactions between fast green FCF and α-synuclein. Fast green FCF greatly disrupted the α-synuclein pentamer and reduced the ß-sheet content by reducing both nonpolar and polar interactions. Furthermore, two binding sites were identified, named region I (Y39-K45) and region II (H50-Q62). Our data reveal that electrostatic interactions, hydrogen bonds, and π-π interactions synergistically contribute to the binding of fast green FCF to the α-synuclein pentamer. These results indicate that fast green FCF is a candidate prototype for the development of drugs against the aggregation of amyloid fibrils in PD.

Friend or foe? The roles of inulin-type fructans.

Inulin-type fructans (ITFs) as functional fructans and soluble dietary fiber are a mixture of inulin, oligofructose and fructooligosaccharide with ß configuration. They are modified by gut microbiota at the end of ileum, subsequently, improve digestive system, metabolic syndrome, immune system and inflammatory diseases, and prevent against infection and cancer. However, it has been reported that inadequate consumption of ITFs aggravates the development of non-alcoholic fatty liver disease, results in gastrointestinal symptoms, liver cancer and intestinal inflammation. Therefore, this review summarizes the health benefits, pharmaceutical applications and safety evaluation of ITFs, which would direct their rational applications.

Co-fermentation of lentils using lactic acid bacteria and Bacillus subtilis natto increases functional and antioxidant components.

We identified lentil products with both nutritional value and antioxidant capacity by studying the changes of probiotics and functional substances during single fermentation with lactic acid bacteria (LAB) or co-fermentation using LAB and Bacillus subtilis natto. After fermentation, the best growth of LAB was observed in anaerobic solid-state co-fermentation, whereby the viable counts of Lactobacillus plantarum TK9 and Lactobacillus paracasei TK1501 reached 2.77 × 109 and 2.78 × 109 CFU/g, respectively. Furthermore, the total phenol and genistin content produced by the two mixed groups, respectively, increased by 0.52- and 0.66-fold, as well as 0.63- and 0.64-fold, compared with unfermented samples. Similarly, the free amino acid content increased by 0.53- and 0.49-fold, respectively. The 50% inhibitory concentrations for the radical-scavenging against 1,1-diphenyl-2-picrylhydrazyl and α-glucosidase inhibitory activity were lower following anaerobic co-fermentation. Consistently, products of anaerobic mixed solid-state fermentation had higher oxygen radical absorbance capacity. Therefore, anaerobic solid-state co-fermentation of lentils using B. subtilis natto may promote the multiplication of LAB and enhance the antioxidant activity of fermented lentil products. PRACTICAL APPLICATION: Simple and efficient food handling is more suitable for industrial production. Co-fermentation is a good method to optimize the fermentation process. Co-culture technology has high potential in terms of functionality and antioxidant capacity.

Dual Effect of the Acidic Polysaccharose Ulvan on the Inhibition of Amyloid-ß Protein Fibrillation and Disintegration of Mature Fibrils.

The abnormal folding and aggregation of amyloid-ß protein (Aß) is the main reason for the occurrence and development of Alzheimer's disease (AD). The discovery of novel inhibitors against Aß aggregation is still the current research focus. Herein, we report the inhibitory effect of ulvan, an acidic polysaccharide from green algae of the genus Ulva, against Aß fibrillation using thioflavin T (ThT) fluorescence and atomic force microscopy (AFM) assays. It is shown that ulvan effectively inhibits Aß fibrillogenesis in a concentration-dependent manner and actively inhibits the formation of A11-reactive Aß oligomers, the most toxic Aß species. The circular dichroism spectrum reveals that ulvan blocks the conformational transition of Aß40 from the initial random coil to a ß-sheet structure, but it only delays the conformational transition of Aß42. It is also found that ulvan greatly reduces Aß-induced cytotoxicity by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, ulvan effectively downregulates intracellular reactive oxygen species production and protects PC12 cells from the damage caused by Aß fibrillation. Moreover, ulvan disaggregates preformed mature fibrils into off-pathway oligomers and greatly decreases their associated cytotoxicity, as revealed using ThT fluorescence, AFM, MTT, and dot-blotting assays. The above results not only fully describe the inhibitory effect of ulvan on Aß fibrillation and its related cytotoxicity but also provide novel ideas for the development of functional food ingredients from seaweed to treat AD.

Screening of the candidate inhibitory peptides of subtilisin by in vitro RNA display technique.

The application of inhibitors facilitates the stable preservation of enzyme in liquid detergent by mitigating the proteolytic activity of subtilisin. The conventionally used subtilisin inhibitors such as boric acid pose a threat to the environment and human health. Thus, the formulation of novel subtilisin inhibitors demands immediate attention. In the current study, we have screened the peptide inhibitors for subtilisin by employing the in vitro mRNA display technique. It is a sensitive screening technique with a high library capacity. The affinity screening was performed between the biotin-modified subtilisin immobilized on the streptavidin magnetic beads and the cDNA-mRNA-peptide fusion molecular library acquired from the in vitro translation and reverse transcription. The candidate peptides with high affinity were obtained after multiple rounds of screening. Furthermore, the inhibitory effect was evaluated, showing that some candidate peptides had inhibitory effects, but the isothermal titration calorimetry and time dependent experiments ultimately proved that these candidate peptides were not stable inhibitors. However, the in vitro mRNA display method explored in this study can be used as a preliminary screening method to provide candidate peptides for the screening of subtilisin inhibitors.

Cyanidin-3-O-glucoside inhibits Aß40 fibrillogenesis, disintegrates preformed fibrils, and reduces amyloid cytotoxicity.

Alzheimer's disease (AD) is mainly caused by the fibrillogenesis of amyloid-ß protein (Aß). Therefore, the development of effective inhibitors against Aß fibrillogenesis offers great hope for the treatment of AD. Cyanidin-3-O-glucoside (Cy-3G) is a commonly found anthocyanin that is mainly present in fruits, with established neuroprotective effects in situ. However, it remains unknown if Cy-3G can prevent Aß fibrillogenesis and alleviate the corresponding cytotoxicity. In this study, extensive biochemical, biophysical, biological and computational experiments were combined to address this issue. It was found that Cy-3G significantly inhibits Aß40 fibrillogenesis and disintegrates mature Aß fibrils, and its inhibitory capacity is dependent on the Cy-3G concentration. The circular dichroism results showed that Cy-3G and Aß40 at a molar ratio of 3 : 1 slightly prevents the structural transformation of Aß40 from its initial random coil to the ß-sheet-rich structure. Co-incubation of Aß40 with Cy-3G significantly reduced the production of intracellular reactive oxygen species induced by Aß40 fibrillogenesis and thus reduced Aß40-induced cytotoxicity. Molecular dynamics simulations revealed that Cy-3G disrupted the ß-sheet structure of the Aß40 trimer. Cy-3G was found to mainly interact with the N-terminal region, the central hydrophobic cluster and the ß-sheet region II via hydrophobic and electrostatic interactions. The ten hot spot residues D7, Y10, E11, F19, F20, E22, I31, I32, M35 and V40 were also identified. These findings not only enable a comprehensive understanding of the inhibitory effect of Cy-3G on Aß40 fibrillogenesis, but also allow the identification of a valuable dietary ingredient that possesses great potential to be developed into functional foods to alleviate AD.

Brazilin Inhibits α-Synuclein Fibrillogenesis, Disrupts Mature Fibrils, and Protects against Amyloid-Induced Cytotoxicity.

The inhibitory effect of brazilin against α-synuclein (α-syn) fibrillogenesis, disruption effect against mature fibrils, and the following cytotoxicity were examined by systematical biochemical, biophysical, cellular biological, and molecular simulation experiments. It is found that brazilin inhibited α-syn fibrillogenesis and disrupted the performed fibrils with a concentration-dependent manner. Moreover, cellular experimental data showed that brazilin effectively reduced the cytotoxicity induced by α-syn aggregates. Finally, molecular dynamics simulations were performed to explore the interactions between brazilin and α-syn pentamer. It is found that brazilin directly interacts with α-syn pentamer, and the hydrophobic interactions are favorable for brazilin binding with the α-syn pentamer, while the electrostatic part provides adverse effects. Three binding regions were identified to inhibit α-syn fibrillogenesis or disrupt the preformed aggregates. Furthermore, six important residues (i.e., G51, V52, A53, E61, V66, and K80) of α-syn were also identified. We expected that brazilin is an effective agent against α-syn fibrillogenesis and associated cytotoxicity.

Inhibitory Effect of a Flavonoid Dihydromyricetin against Aß40 Amyloidogenesis and Its Associated Cytotoxicity.

Misfolding and fibrillogenesis of amyloid-ß protein (Aß) play a key role in the onset and progression of Alzheimer's disease (AD). Screening for inhibitors against Aß amyloidogenesis is helpful for rational designing and developing new anti-AD drugs and therapeutic strategies. Dihydromyricetin, a natural flavonoid extracted from a Chinese herb, Ampelopsis grossedentata, has been proven with antioxidative, anti-inflammatory, and neuroprotective effects against neurodegenerative disease. Herein, we found that dihydromyricetin could inhibit Aß40 aggregation, impede the protofibril formation, disassemble preformed Aß40 fibrils, and protect PC12 cells from the Aß40-induced cytotoxicity using a series of biochemical and biophysical assays, including thioflavin T fluorescence, atomic force microscopy, and cell toxicity assays. Circular dichroism spectroscopy data proved that dihydromyricetin delayed the Aß40 conformational conversion. In addition, the results of molecular dynamics simulations indicated that the interaction between dihydromyricetin and Aß40 trimer is mainly nonpolar interactions. Key residues (i.e., V18, A21, and D23) of the Aß40 interacting with dihydromyricetin were also identified. This study suggested that dihydromyricetin shows great potential to be developed as a novel Aß40 inhibitor.

Facile synthesis of (-)-vibo-quercitol from maltodextrin via an in vitro synthetic enzymatic biosystem.

(-)-vibo-Quercitol (VQ: 1L-1,2,4/3,5-cyclohexanepentol), a form of deoxyinositol, is an alternative chiral building block in the synthesis of bioactive compounds to control diabetes. In this study, an adenosine triphosphate-free in vitro synthetic enzymatic biosystem composed of five enzymes (including one enzyme for NADH regeneration) was constructed to produce VQ from maltodextrin in one-pot. After optimization of reaction conditions, 7.6 g/L VQ was produced from 10 g/L maltodextrin with a product yield (mol/mol) of 77%, and 25.3 g/L VQ with a purity of 87% was produced from 50 g/L maltodextrin through simple scaling up of this nonfermentative enzymatic biosystem. Therefore, this study provides an economical and environmentally friendly method for the envisioned quercitol biosynthesis.

Improvement of the alkali stability of Penicillium cyclopium lipase by error-prone PCR

BACKGROUND: Lipases are extensively exploited in lots of industrial fields; cold-adapted lipases with alkali-resistance are especially desired in detergent industry. Penicillium cyclopium lipase I (PCL) might be suitable for applications of detergent industry due to its high catalytic efficiency at low temperature and relatively good alkali stability. In this study, to better meet the requirements, the alkali stability of PCL was further improved via directed evolution with error-prone PCR. RESULTS: The mutant PCL (N157F) with an improved alkali stability was selected based on a high-throughput activity assay. After incubating at pH 11.0 for 120 min, N157F retained 70% of its initial activity, which was 23% higher than that of wild type PCL. Combined with the three-dimensional structure analysis, N157F exhibited an improved alkali stability under the high pH condition due to the interactions of hydrophilicity and ß-strand propensity. Conclusions: This work provided the theoretical foundation and preliminary data for improving alkali stability of PCL to meet the industrial requirements, which is also beneficial to improving alkali-tolerance ability of other industrial enzymes via molecular modification.