Unleashing the Power of Evolution in Xylanase Engineering: Investigating the Role of Distal Mutation Regulation.

The drive to enhance enzyme performance in industrial applications frequently clashes with the practical limitations of exhaustive experimental screening, underscoring the urgency for more refined and strategic methodologies in enzyme engineering. In this study, xylanase Xyl-1 was used as the model, coupling evolutionary insights with energy functions to obtain theoretical potential mutants, which were subsequently validated experimentally. We observed that mutations in the nonloop region primarily aimed at enhancing stability and also encountered selective pressure for activity. Notably, mutations in this region simultaneously boosted the Xyl-1 stability and activity, achieving a 65% success rate. Using a greedy strategy, mutant M4 was developed, achieving a 12 °C higher melting temperature and doubled activity. By integration of spectroscopy, crystallography, and quantum mechanics/molecular mechanics molecular dynamics, the mechanism behind the enhanced thermal stability of M4 was elucidated. It was determined that the activity differences between M4 and the wild type were primarily driven by dynamic factors influenced by distal mutations. In conclusion, the study emphasizes the pivotal role of evolution-based approaches in augmenting the stability and activity of the enzymes. It sheds light on the unique adaptive mechanisms employed by various structural regions of proteins and expands our understanding of the intricate relationship between distant mutations and enzyme dynamics.

Multi-point immobilization of GH 11 endo-ß-1,4-xylanase on magnetic MOF composites for higher yield of xylo-oligosaccharides.

GH 11 endo-ß-1,4-xylanase (Xy) was a crucial enzyme for xylooligosaccharides (XOS) production. The lower reusability and higher cost of purification has limited the industrial application of Xy. Addressing these challenges, our study utilized various immobilization techniques, different supports and forces for Xy immobilization. This study presents a new method in the development of Fe3O4@PDA@MOF-Xy which is immobilized via multi-point interaction forces, demonstrating a significant advancement in protein loading capacity (80.67 mg/g), and exhibiting remarkable tolerance to acidic and alkaline conditions. This method significantly improved Xy reusability and efficiency for industrial applications, maintaining 60 % activity over 10 cycles. Approximately 23 % XOS production was achieved by Fe3O4@PDA@MOF-Xy. Moreover, the yield of XOS from cobcorn xylan using this system was 1.15 times higher than that of the free enzyme system. These results provide a theoretical and applicative basis for enzyme immobilization and XOS industrial production.

Mechanistic Insights into the Halophilic Xylosidase Xylo-1 and Its Role in Xylose Production.

The Xylo-1 xylosidase, which belongs to the GH43 family, exhibits a high salt tolerance. The present study demonstrated that the catalytic activity of Xylo-1 increased by 195% in the presence of 5 M NaCl. Additionally, the half-life of Xylo-1 increased 25.9-fold in the presence of 1 M NaCl. Through comprehensive analysis including circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations, we elucidated that the presence of Na+ ions increased the contact frequency between the surface acidic amino acids and the surrounding water molecules. This resulted in the stabilization of the surrounding hydration layer of Xylo-1. Additionally, Na+ ions also stabilized the substrate-binding conformation and the fluctuation of water molecules within the active site, which enhanced the catalytic activity of Xylo-1 by increasing the nucleophilic attack by the water molecules. Ultimately, the optimal reaction conditions for the production of xylose by synergistic catalysis with Xylo-1 and xylanase were determined. The results demonstrated that the conversion yield of the method was high for various sources of xylan, indicating the method could have potential industrial applications. This study explored the structure-activity relationship of catalysis in Xylo-1 under high-salt conditions, provides novel insights into the mechanism of halophilic enzymes, and serves as a reference for the industrial application of Xylo-1.

pcnaDeep: a fast and robust single-cell tracking method using deep-learning mediated cell cycle profiling.

SUMMARY: Computational methods that track single cells and quantify fluorescent biosensors in time-lapse microscopy images have revolutionized our approach in studying the molecular control of cellular decisions. One barrier that limits the adoption of single-cell analysis in biomedical research is the lack of efficient methods to robustly track single cells over cell division events. Here, we developed an application that automatically tracks and assigns mother-daughter relationships of single cells. By incorporating cell cycle information from a well-established fluorescent cell cycle reporter, we associate mitosis relationships enabling high fidelity long-term single-cell tracking. This was achieved by integrating a deep-learning-based fluorescent proliferative cell nuclear antigen signal instance segmentation module with a cell tracking and cell cycle resolving pipeline. The application offers a user-friendly interface and extensible APIs for customized cell cycle analysis and manual correction for various imaging configurations. AVAILABILITY AND IMPLEMENTATION: pcnaDeep is an open-source Python application under the Apache 2.0 licence. The source code, documentation and tutorials are available at https://github.com/chan-labsite/PCNAdeep. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparison of structural and functional properties of maize starch produced with commercial or endogenous enzymes.

To explore an effective and economic method to prepare higher contents of resistant starch (RS), different enzyme treatments including single pullulanase (PUL), commercial α-amylase (AA) or/and ß-amylase (BA) with PUL, and malt endogenous amylase (MA) with PUL were used and the structural, physicochemical properties and digestibility of all modified starches (MS) were compared. All the enzyme-treated starches displayed a mixture of B and V-type diffraction patterns. The MA/PUL-MS showed higher V-type diffraction peak intensity as compared to other modified starches. Compared to the combination of commercial enzyme treatment, the combination of malt enzyme treatment led to higher apparent amylose contents (45.56%), RS content (53.93%) and thermal stability (302 °C), whereas it possessed lower solubility indices and predicted glycaemic index. The apparent viscosity and shear resistance of MA/PUL-MS were lower than that of AA/PUL-MS, whereas that of MA/PUL-MS was higher than that of BA/PUL-MS and BA/AA/PUL-MS. These findings would provide a theoretical and applicative basis to produce foods with lower GI in industrial production.

Corn starch modification during endogenous malt amylases: The impact of synergistic hydrolysis time of α-amylase and ß-amylase and limit dextrinase.

To expand the utility of barley malts and decrease the cost of enzyme-modified starch production, the structural and physicochemical characteristics of corn starch modified with fresh barley malts at different hydrolysis time were investigated. The results indicated that compared to native starch, A chain (DP 6-12) of the enzyme-treated starches increased at hydrolysis time (≤12 h), but it decreased at hydrolysis time (>12 h). Inversely, B chains (DP > 13) decreased at hydrolysis time (≤12 h) and they generally increased at hydrolysis time (>12 h). The relative crystallinity decreased from 25.63% to 21.38% and 1047 cm-1/1022 cm-1 reduced from 1.042 to 0.942 after endogenous malt amylases at hydrolysis time from 0 to 72 h, and the thermal degradation temperatures decreased from 323.19 to 295.94 °C, whereas the gelatinization temperatures slightly increased. The gel strength decreased at hydrolysis time less than 12 h, but it increased at hydrolysis time more than 12 h. The outcomings would provide a theoretical and applicative basis about how endogenous malt amylases with lower price modify starches to obtain desirable starch derivatives and industrial production.

Structural and functional modification of kudzu starch using α-amylase and transglucosidase.

The large agglomeration of starch paste in hot water, and fast retrogradation tendency and low transparency of starch gel restrict widespread application of kudzu starch. To improve the above defects, kudzu starch was modified with sequentially α-amylase (AA) and transglucosidase (TG), the latter for varying times. The results indicated that, compared to kudzu starch, amylose content and molecular weight of AA/TG-treated starches reduced by 20.07% and 69.50%, respectively. The proportion of A chain increased by 68.68%, whereas B1, B2 and B3 chains decreased by 14.28%, 48.29% and 23.44%, respectively. The degree of branching dramatically increased by 128.3%. After AA→TG treatment, the changes of starch structure enhanced the functional properties of kudzu starch. The solubility, paste clarity and gelatinization temperature increased, whereas the relative crystallinity, viscosity, storage and loss moduli decreased. Overall, the AA→TG modification would be desirable to improve the functional properties of kudzu starch to expand more large-scale application.

Porous starches modified with double enzymes: Structure and adsorption properties.

To explore an effective enzyme combination instead of a common enzyme method, sequential α-amylase and glucoamylase, a method of sequential glycosyltransferase and branching enzyme was chosen to compare the macroscopic features, structure characteristics, porosity characteristics and adsorption quantity of potato, corn, wheat and sweet potato starches. The results indicated that after enzyme treatment, the relative crystallinity of potato, corn, wheat and sweet potato starches increased. Moreover, amylose levels decreased, while pore size and volume, and specific surface area increased after sequential glycosyltransferase and branching enzyme. In terms of pore size, sequential α-amylase and glucoamylase produced abundant mesopores (2-50 nm), whereas sequential glycosyltransferase and branching enzyme developed much more macropores (>50 nm). The adsorption quantities of the starch obtained with sequential glycosyltransferase and branching enzyme were about 2 folds higher than that of the starch obtained with sequential α-amylase and glucoamylase. Therefore, the sequential glycosyltransferase and branching enzyme may be an ideal method to create porous starch as a desirable green adsorbent for industries.

Improving waxy rice starch functionality through branching enzyme and glucoamylase: Role of amylose as a viable substrate.

The low solubility, gel strength and shear resistance of waxy rice limit utility in large-scale and widespread food applications. Herein, functional properties of waxy rice starch (WRS) have been improved through sequential branching enzyme (BE) and glucoamylase (GA) treatment with wheat amylose (WA), rice amylose (RA) and corn amylose (CA) as the substrate. The results suggest that, compared to the BE treatment, the sequential GA→BE treatment significantly improves the WRS properties. Among the three substrates, WA appears to be suited highly for the WRS through the GA→BE treatment. GABE-WRS/WA had the highest content of α-1,6-glucosidic linkages, increased number of short chains, shortest average chain length, increased crystallinity, enhanced solubility, paste clarity and swelling power, elevated gelatinization temperature and enthalpy, increased viscosity, strongest gels, and highest shear-resistance. Overall, the sequential GA→BE treatment appears to be suitable to modify the functional properties of waxy rice starch toward developing more palatable and large-scale food products.

Modification of rice starch using a combination of autoclaving and triple enzyme treatment: Structural, physicochemical and digestibility properties.

It is highly desirable to improve the physicochemical properties and nutritional functions of rice starch. In this study, the structural, physical and chemical properties and digestibility of autoclaving-modified starch (A-MS), autoclaving/pullulanase-modified starch (A/PUL-MS), autoclaving/sequential three enzymatic (ß-amylase â†’ transglucosidase â†’ pullulanase)-modified starch (A/STE-MS) and native rice starch (NRS) were compared. The results showed that compared to NRS, the structure of A/STE-MS granules became dense, and the short-chain ratio (43.17%, DP ≤ 6) was significantly increased. A/PUL-MS and A/STE-MS had significantly higher apparent amylose content (38.39% and 33.78%, respectively) and thermal stability. NRS and A/PUL-MS were A-type and B-type crystalline, respectively, while A-MS and A/STE-MS were V-type crystalline. A/STE-MS had higher swelling and solubility indices and lower apparent viscosity. In addition, A/STE-MS had a lower (62.9) glycaemic index (GI) than native rice starch and other modified starches. This study provides a new perspective for the development of the modified rice starch industry and low GI functional rice starch-based products.