High-Level Expression and Biochemical Properties of A Thermo-Alkaline Pectate Lyase From Bacillus sp. RN1 in Pichia pastoris With Potential in Ramie Degumming

Abstract

Pectate lyases play an essential role in textiles, animal feed, and oil extraction industries. Pichia pastoris can be an ideal platform for pectate lyases production, and BspPel (a thermo-alkaline pectate lyase from Bacillus sp. RN1) was overexpressed by combined strategies, reaching 1859 U/mL in a 50 L fermentator. It displayed the highest activity at 80°C, and maintained more than 60% of the activity between 30 and 70°C for 1 h. It showed an optimal pH of 10.0, and exhibited remarkable stability over a wider pH range (3.0-11.0), retaining more than 80.0% of enzyme activity for 4 h. The K _m and k _cat of BspPel on PGA (polygalacturonic acid) was 2.19 g L^-1 and 116.1 s^-1, respectively. The activity was significantly enhanced by Ca²⁺, Mn²⁺, and Cu²⁺, and a slight increase was observed with the addition of Ba²⁺ and Mg²⁺. Scanning electron microscope was used to show the degumming efficiency of BspPel on ramie fibers. The loss weight was 9.2% when treated with crude enzyme supernatant and 20.8% when treated with the enzyme-chemical method, which was higher than the 14.2% weight loss in the positive control treated with 0.5% (w/v) NaOH alone. In conclusion, BspPel could be a good candidate for the ramie degumming industry.

Keywords: Pichia pastoris; biochemical properties; high-level expression; pectate lyase; ramie degumming.

FIGURE 1

Functional expression of BspPel in P. pastoris. (A) Schematic diagrams of recombinant vectors. AOX: AOX1 promoter, AOXm: modified AOX1 promoter, α-factor: α-factor signal peptide, Native signal: native signal peptide; (B) Time course of BspPel expression in recombinant strains.

FIGURE 2

SDS-PAGE assay of the recombinant BspPel. Lane M: protein marker; lane 1: purified recombinant BspPel; lane 2: purified recombinant BspPel treated by PNGase F.

FIGURE 3

The time course of recombinant enzyme production in a 50 L fermentator and SDS-PAGE analysis. (A) Time dependence of the activity of pectate lyase and cell growth of GS115/pAmNS-4BspPel after induction with methanol, using GS115/HKA as the background (control) sample; (B) SDS-PAGE assay of the collected culture supernatant from the most effective strains in the fermentator after 168 h of induction with methanol. Lane M: protein marker; lane 1–lane 8: recombinant protein expression from 0 to 168 h.

FIGURE 4

Effect of (A) temperature, (B) pH on purified pectate lyase (PL) activity, (C) temperature stability and (D) pH stability. Values are averages of results from triplicate trials; error bars indicate the SD values.

FIGURE 5

(A) Substrate-velocity curve and (B) Lineweaver–Burk plot of purified BspPel. Values are averages of results from triplicate trials; error bars indicate the SD values.

FIGURE 6

SEM images of ramie fibers treated (a,e): with buffer only (negative control); (b,f): with BspPel; (c,g): with chemicals only (positive control); and (d,h): with the enzyme-chemical method. (a–d): External images of ramie fibers; (e–h): surface images of single fibers (1000 × magnification).