Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming

Abstract

Thermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase from Bacillus sp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca(2+). However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in the t50 (time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in the t50 value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.

FIG 1

Thermostability of single-substitution mutants. The purified wild-type and mutant enzymes were incubated at 50°C for different times, and the residual activity was then determined. The activity without incubation was taken as 100%. The measurements were performed in three independent experiments. Error bars represent standard deviations.

FIG 2

Thermostability and optimal reaction temperature of the combined mutants. (A) Thermostability curves for combined mutants. The purified wild-type and mutant enzymes were incubated at 50°C for different times. The activity without incubation was taken as 100%. (B) Optimal temperature for combined mutants. The activity was determined with 50 mM glycine-NaOH buffer (pH 10.5) containing 0.2% (wt/vol) PGA. The measurements were performed in three independent experiments. Error bars represent standard deviations.

FIG 3

Percent weight loss after degumming of ramie fibers by enzymatic or enzyme-chemical methods. The measurements were performed in six independent experiments. Error bars represent standard deviations.

FIG 4

Scanning electron microscopy imaging of degumming ramie fibers. (A and E) Ramie fibers treated with buffer only (control). (B and F) Ramie fibers treated with wild-type BspPelA. (C and G) Ramie fibers treated with the mutant EA. (D and H) Ramie fibers treated with chemicals only. Panels A to D show exterior images of ramie fibers; panels E to H show surface images of single fibers obtained by scanning electron microscopy (magnification, ×5,000).

FIG 5

Locations and structural analysis of three key mutation sites. (A) Mutations location in the crystal structure of BspPelA (PDB accession no. 3VMW). The side chains of the catalytic triad (K177, R207, and R212) and mutations (E124, T178, and S271) are represented as spheres. (B and C) Views of BspPelA with all residues in a real space with van der Waals representation, where the image in panel B is in an orientation identical to that of the image shown in panel A and panel C shows a 90°C turn of the image in panel B along the x axis. Only the respective side chains are shown in color, and the backbone is shown in gray. (D) Location and mutation of T178A. T178 and A178 are represented in green and purple, respectively. (E) Location and mutation of E124I. E124 and I124 are represented in green and purple, respectively. (F) Location and mutation of S271G. The H-bond network around Arg212 in the crystal structure of BspPelA is shown in green; S271G (last snapshot from a 10-ns MD simulation) is shown in purple.