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 Ca 2+ , Mn 2+ , and Cu 2+ , and a slight increase was observed with the addition of Ba 2+ and Mg 2+. 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.

Characterization of a Novel Thermophilic Mannanase and Synergistic Hydrolysis of Galactomannan Combined with Swollenin

“…However, it was observed that Cu 2+ ions could significantly enhance the mannanase activity of AfMan5A. A similar phenomenon was also observed in other studies regarding enzymes, such as amylase [28], pectate lyase [29] and laccase [30]. We speculate that this is likely caused by the structure changes of AfMan5A in the presence of Cu 2+ ions.…”

Green Process: Improved Semi-Continuous Fermentation of Pichia pastoris Based on the Principle of Vitality Cell Separation

“…pastoris GS115 strain containing the pPICHKAPel vector was used for the heterologous expression of pectate lyase (Pel from Bacillus sp. RN1) under control of the AOX1 promoter G/Pel ( Zheng et al, 2020 ). The P .…”

“…Pectate lyase activity was determined according to the method described by Zheng et al (2020) , with one modification: measuring the absorbance change at 235 nm with 2 mg PGA ml-1 as the substrate in 50 mM glycine–NaOH (pH 10.0) buffer containing 1 mM CaCl 2 for 40°C, 30 min. One unit (U) of pectin lyase activity was defined as the amount of enzyme that is required to produce unsaturated oligogalacturonide equivalent to 1 μmol of unsaturated diglucuronide min–1 using a molecular extinction coefficient of 4,600 M −1 cm −1 at 235 nm ( Zheng et al, 2020 ).…”

“…The P. pastoris GS115 strain containing the pPICHKAPel vector was used for the heterologous expression of pectate lyase (Pel from Bacillus sp. RN1) under control of the AOX1 promoter G/Pel (Zheng et al, 2020). The P. pastoris GS115 strain containing the pPICZαAphy vector was used for heterologous expression of phytase (Phy from C. amalonaticus CGMCC 1696) under the control of the AOX1 promoter G/Phy (Weidner et al, 2010).…”

Modification and application of highly active alkaline pectin lyase

“…Nevertheless, its industrial applications are limited. Under some of the required processing conditions, its stability and enzyme activity is decreased, and its efficiency in degumming ramie, cotton textiles, and pulp processing is not ideal (Xu et al 2021 ; Zheng et al 2020 ; Cheng et al 2019 ; Chiliveri and Linga 2014 ). Therefore, an alkaline pectin lyase with increased stability, heat resistance, and enzymatic activity is imperative to industrial processing.…”