Modification and application of highly active alkaline pectin lyase

Abstract

Alkaline pectate lyase has developmental prospects in the textile, pulp, paper, and food industries. In this study, we selected BacPelA, the pectin lyase with the highest expression activity from Bacillus clausii, modified and expressed in Escherichia coli BL21(DE3). Through fragment replacement, the catalytic activity of the enzyme was significantly improved. The optimum pH and temperature of the modified pectin lyase (PGLA-rep4) were 11.0 and 70 °C, respectively. It also exhibited a superior ability to cleave methylated pectin. The enzyme activity of PGLA-rep4, measured at 235 nm with 0.2% apple pectin as the substrate, was 554.0 U/mL, and the specific enzyme activity after purification using a nickel column was 822.9 U/mg. After approximately 20 ns of molecular dynamics simulation, the structure of the pectin lyase PGLA-rep4 tended to be stable. The root mean square fluctuation (RMSF) values at the key catalytically active site, LYS168, were higher than those of the wildtype PGLA. In addition, PGLA-rep4 was relatively stable in the presence of metal ions. PGLA-rep4 has good enzymatic properties and activities and maintains a high pH and temperature. This study provides a successful strategy for enhancing the catalytic activity of PGLA-rep4, making it the ultimate candidate for degumming and various uses in the pulp, paper, and textile industries.

Improvement of optimum pH and specific activity of pectate lyase from Bacillus RN.1 using loop replacement

“…Pectate lyase activity was determined by measuring the increase in unsaturated bonds generated at 235 nm. Enzyme activity assays were performed as described by Li et al (2022) with some modifications. The reaction mixture consisted of 1900 μL 50 mM Gly-NaOH (pH 11) containing 0.2% substrate and 100 μL appropriately diluted enzyme solution.…”

“…The box shape was a cube, and the size of the boundary atoms in all directions extended outward by 1.5 nm. After the box was filled with water molecules, ions were added to make the entire system electrically neutral, and then the energy was minimized ( Li, et al, 2022 ). The system was simulated for 40 ns The atomic displacement parameter (B-factor) of amino acid residues was simulated and calculated using B-FITTER.…”

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification

“…6 Therefore, these enzymes are extensively used in various alkaline conditions (pH 9.0− 11.0) such as pulp processing, wastewater treatment, and textile material preparation. 11 In addition, acidic PNLs are mainly used in juice processing to minimize the requirement for additional enzymes to attain synergistic effects. 12 Moreover, they exhibit a remarkable ability to degrade pectin in highly esterified fruits without altering the ester content, thereby imparting distinctive flavors to fruit juices.…”

“…Alkaline PNLs play a crucial role in the retting and degumming of natural plant fibers by removing heavily coated, noncellulosic gummy material from the cellulose part . Therefore, these enzymes are extensively used in various alkaline conditions (pH 9.0–11.0) such as pulp processing, wastewater treatment, and textile material preparation . In addition, acidic PNLs are mainly used in juice processing to minimize the requirement for additional enzymes to attain synergistic effects .…”

Rational Design of Disulfide Bonds Increases Thermostability of a β-1,3-1,4-Glucanase from Paenibacillus

“…With developments in rational and semi-rational design, many methods have been developed to improve the properties of enzymes (Sun et al 2020), and these include fragment substitution (Li et al 2022), disul de bond addition or deletion (Niu et al 2016), site-directed mutation (Niu et al 2015), surface charge engineering (Zhang et al 2023), and random mutation and replacement of linked peptides (Lu et al 2006). Of these, increasing or decreasing disul de bonds has been well applied to improve the heat tolerance of many enzymes, and this requires an analysis of the protein structure and the selection of disul de bond sites.…”