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.

Keywords: Alkaline pectin lyase; Enzymatic activity; Fragment replacement; Molecular dynamics simulation.

Fig. 1

The three-dimensional structure and multiple sequence alignment of the modified alkaline pectin lyase a PGLA-rep1, b PGLA-rep2, c PGLA-rep3, d PGLA-rep4, e Constructed 5 sequence alignment maps, The part drawn with a green horizontal line is the part of the replacement fragment—the purple part in the 3D structure depicts replacement fragment

Fig. 2

pH and temperature profiles of alkaline pectin lyase, a Optimum pH, b Optimum temperature, c Stability of PGLA and PGLA-rep4 at pH range, d Thermal stability of PGLA and PGLA-rep4 in glycine-NaOH buffer (pH 11.0) at 60 °C—the activities of all enzymes were determined under standard enzyme assay conditions using 0.2% apple pectin as substrate and all data are presented as the mean of three experiments; error bars represent standard deviation

Fig. 3

Effects of metal ions on the activity of PGLA and PGLA-rep4

Fig. 4

Substrate specificity of PGLA and PGLA-rep4

Fig. 5

Nonlinear regression curves for the enzymatic reaction of pectin lyase. a PGLA reaction, b PGLA-rep4 reaction

Fig. 6

RMSF values of PGLA and PGLA-rep4 at three key catalytic sites