Pectinase plays a crucial role in ramie bio-degumming. A pectate lyase gene ( pel4J4) from the high-efficiency degumming bacteria Dickeya dadantii DCE-01 of bast fibers was cloned and connected to pET28a, and then the recombinant plasmid was successfully transformed into Escherichia coli BL21(DE3). The pectate lyase (Pel4J4) induced was purified by ultrafiltration and Sephadex G-100 gel chromatography. The enzymatic properties of Pel4J4 were studied in detail. pel4J4 (GenBank accession number: KC900167) had a sequence length of 1179 bp, encoding 392 amino acids. The extracellular pectate lyase activity of pET28a- pel-BL was up to 204.4 IU/mL. The optimal temperature and pH of the purified Pel4J4 were 55℃ and 8.5, respectively. The stable temperature and pH of Pel4J4 activity were 45℃ and 8.5–10.0, respectively. The catalytic activity is Ca²⁺ dependent and promoted by 1 mmol/L Zn²⁺, Fe³⁺, Ca²⁺, and NH₄⁺, but seriously inhibited by Cu²⁺ and Pb²⁺. The optimal substrate is citrus pectin with more than 85% esterification. The heat-resistant alkaline Pel4J4 could strongly degrade natural ramie pectin, indicating a promising application prospect in ramie bio-degumming.

To explore high-quality microbial resources with the capability of ramie degumming, we collected soil samples from rotten ramie and straw heaps. After enrichment culture by ramie raw materials, bacterial strains with the potential ramie-degumming function were screened using a pectin-hydrolysis plate. Dominant bacteria were identified by combining colonial morphological characteristics with the molecular biology method, and their ramie-degumming effects were verified through comprehensive biological degumming indices. Results demonstrated that Bacillus aryabhattai, Bacillus thuringiensis, Lysinibacillus fusiformis, and Acidovorax temperans were successfully obtained. The highest pectinase activity, 98.2 U/mg, was found by A. temperans. B. thuringiensis showed the best ramie-degumming effect. The residual gum content, single-fiber linear density, and bundle-breaking strength of the degummed ramie fiber treated with B. thuringiensis were 8.32%, 6.80 dtex, and 7.84 cN/dtex, respectively. The residual gum content of the ramie fiber treated with B. thuringiensis met the textile requirement (<10%), and the values of all other indicators were also satisfactory. Therefore, B. thuringiensis was an excellent strain for ramie degumming, indicating potential industrial applications.

Ramie (Boehmeria nivea and Boehmeria tenacissima) is a widely used fiber crop. Traditional water retting or chemical boiling method performed in order to extract ramie fiber seriously pollute the environment and severely damage the fiber, so biological method is the general trend of the fiber-extracting industry. Some strains (687), involving 26 genera and 43 species, were collected from the three samples, which produce hydrolyzed circles in the selective culture medium in order to detect the degumming effect and to compare the enzyme activity. Among these strains, 13 of them did not produce cellulase and had a ramie decreasing weight rate above 25 %, which were regarded as efficient ramie-degumming strains named from R1 to R13. R1 to R13 belonged to Amycolata autotrobutylicun, Bacillus subtilis, Clostridium acetobutylicum, Bacillus subtilis, Rhizobium leguminosarum, Bacteroides finegoldii, Streptomyces lividans, Bacillus amyloliquefaciens, Clostridium acetobutylicum, Pseudomonas brassicacearum, Bacillus pumilus, Bacillus licheniformis, Pectobacterium wasabiae respectively. Bacteroides sp., Rhizobium sp. and Pseudomonas sp. were firstly reported to be used in ramie-degumming. At the same time, the pectinase was the key enzyme in the ramie-degumming process

To improve the thermal stability of pectate lyase for ramie degumming, we modified the novel pectate lyase gene (pelG403) derived from the Dickeya dadantii DCE-01 high-efficiency ramie degumming strain by site-directed mutagenesis. Twelve mutants were acquired, wherein a prospective mutant (A129V) showed better enzyme activity and thermal stability. Compared with the wild type (PelG403), the specific enzyme activity and the optimal reaction temperature of A129V in the fermentation broth increased by 20.1%, and 5 °C, respectively. Under the conditions of 55 °C and pH 9.0, the weightlessness rate of ramie raw materials of A129V increased by 6.26%. Therefore, this study successfully improved the enzyme activity and heat resistance of PelG403 in an alkaline environment, which may contribute to the development of enzyme preparations and the elucidation of the mechanism for ramie bio-degumming.

In order to obtain a thermostable pectate lyase for ramie degumming, a rational design based on structural analysis was carried out on a novel pectate lyase (Pel419) derived from the Dickeya Dadantii DCE-01 for high-efficiency ramie degumming. A total of five potential amino acid sites were chosen to replace residues. Then, the mutant enzymes were subjected to the heterologous expressions in Escherichia coli and their enzymatic characteristics were determined. The optimal reaction temperature for the five mutants kept consistent with that for the wild type. The enzyme activity and thermal stability of mutant V52A were significantly improved. Meanwhile, the weight loss rate obtained by V52A with the best enzymatic characteristics in the ramie degumming process at 50 °C is comparable with that obtained by commercial cotton-ramie processing pectinases, indicating that V52A was a potential industrial enzyme that could be applied to large-scale ramie degumming. In this study, the biological functions of conservative residues of Pel419 were preliminarily explored. The mutant V52A with both enzymatic activity and improved heat resistance was acquired, providing a superior material for developing enzyme preparations of ramie degumming, and rendering an effective method for the rational design aiming to improve the thermostability of pectate lyase.