Explore the vast range of titles available.

Alkaline pectate lyases (Pels) have potential application in bioscouring of the textile industry. In this study, a thermo-alkaline Pel (BacPelA) gene from an alkaliphilic Bacillus clausii strain was cloned and overexpressed in Escherichia coli . The mature BacPelA exhibited maximum activity at pH 10.5 and 70 °C and showed high cleavage capability on methylated pectins. BacPelA showed the highest specific activity of 936.2 U mg −1 on ≥85% methylated pectin and 675.5 U mg −1 on standard substrate polygalacturonic acid (PGA) upon evaluation of the absorbance at 235 nm (A 235 ). The K m and k cat values for PGA were 0.54 g l −1 and 346.5 s −1 , respectively. Moreover, the 3,5-dinitrosalicylic acid (DNS) assay, which detects the released reducing oligogalacturonic acids, was confirmed to be inaccurate and unsuitable for endo-acting pectinase activity assay because of the difference in the reducibility by DNS reagent between the standard galacturonic acid and the catalytic oligomer products. Significant ramie fiber weight loss was observed following treatment with BacPelA (24.8%) and combined enzyme-chemical method (30.9%), which indicated that the degumming efficiency of BacPelA was the highest of all alkaline and thermostable Pels reported to date. The total activity of the recombinant mature BacPelA reached 8378.2 U ml −1 (A 235 ) by high-cell-density cultivation in fed-batch fermentation with productivity of 239.4 U ml −1  h −1 using E. coli as host, which represents the highest Pel yield reported to date. Therefore, BacPelA, with promising properties for bioscouring, shows potential applications for ramie degumming in the textile industry.

Pectin is a major component and structural polysaccharide of the primary cell walls and middle lamella of higher plants. Pectate lyase (PEL, EC 4.2.2.2), a cell wall modification enzyme, degrades de-esterified pectin for cell wall loosening, remodeling and rearrangement. Nevertheless, there have been few studies on PEL genes and no comprehensive analysis of the PEL gene family in cotton. We identified 53, 42 and 83 putative PEL genes in Gossypium raimondii (D5), Gossypium arboreum (A2), and Gossypium hirsutum (AD1), respectively. These PEL genes were classified into five subfamilies (I-V). Members from the same subfamilies showed relatively conserved gene structures, motifs and protein domains. An analysis of gene chromosomal locations and gene duplication revealed that segmental duplication likely contributed to the expansion of the GhPELs. The 2000 bp upstream sequences of all the GhPELs contained auxin response elements. A transcriptomic data analysis showed that 62 GhPELs were expressed in various tissues. Notably, most (29/32) GhPELs of subfamily IV were preferentially expressed in the stamen, and five GhPELs of subfamily V were prominently expressed at the fiber elongation stage. In addition, qRT-PCR analysis revealed the expression characteristics of 24 GhPELs in four pollen developmental stages and significantly different expression of some GhPELs between long- and short-fiber cultivars. Moreover, some members were responsive to IAA treatment. The results indicate that GhPELs play significant and functionally diverse roles in the development of different tissues. In this study, we comprehensively analyzed PELs in G. hirsutum, providing a foundation to better understand the functions of GhPELs in different tissues and pathways, especially in pollen, fiber and the auxin signaling pathway.

Background Ramie degumming is often carried out at high temperatures; therefore, thermostable alkaline pectate lyase (PL) is beneficial for ramie degumming for industrial applications. Thermostable PLs are usually obtained by exploring new enzymes or reconstructing existing enzyme by rational design. Here, we improved the thermostability of an alkaline pectate lyase (PelN) from Paenibacillus sp. 0602 with rational design and structure-based engineering. Results From 26 mutants, two mutants of G241A and G241V showed a higher thermostability compared with the wild-type PL. The mutant K93I showed increasing specific activity at 45 °C. Subsequently, we obtained combinational mutations (K93I/G241A) and found that their thermostability and specific activity improved simultaneously. The K93I/G241A mutant showed a half-life time of 15.9 min longer at 60 °C and a melting temperature of 1.6 °C higher than those of the wild PL. The optimum temperature decreased remarkably from 67.5 °C to 60 °C, accompanied by a 57% decrease in Km compared with the Km value of the wild-type strain. Finally, we found that the intramolecular interaction in PelN was the source in the improvements of molecular properties by comparing the model structures. Rational design of PelN was performed by stabilizing the α-helices with high conservation and increasing the stability of the overall structure of the protein. Two engineering strategies were applied by decreasing the mutation energy calculated by Discovery Studio and predicting the free energy in the process of protein folding by the PoPMuSiC algorithm. Conclusions The results demonstrated that the K93I/G241A mutant was more suitable for industrial production than the wild-type enzyme. Furthermore, the two forementioned strategies could be extended to reveal engineering of other kinds of industrial enzymes.

Background pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli – B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence ( ori ) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. Results The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN , an alkaline protease spro1 and a pullulanase gene pulA11 , respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. Conclusion The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis . Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

Background Aspergillus niger is widely used in industrial enzyme production due to its strong secretion capacity and the status of generally recognized as safe (GRAS). However, heterologous protein expression in A. niger is frequently constrained by high levels of background endogenous protein secretion, limited access to native high transcription loci, and limitations in the efficiency of the secretory machinery. To address these limitations, this study genetically engineered a chassis strain based on an industrial glucoamylase-producing A. niger strain AnN1 for constructing the improved heterologous protein expression. Results In this study, by using CRISPR/Cas9-assisted marker recycling, we deleted 13 of the 20 copies of the heterologous glucoamylase TeGlaA gene and disrupted the major extracellular protease gene PepA , resulting in the low-background strain AnN2. Compared to the parental strain AnN1, AnN2 exhibited 61% less extracellular protein and significantly reduced glucoamylase activity, while retaining multiple transcriptionally active integration loci. Four diverse proteins were integrated into the high-expression loci originally occupied by the TeGlaA gene in the chassis AnN2. These recombinant protein included a homologous glucose oxidase (AnGoxM), a thermostable pectate lyase A (MtPlyA), a bacterial triose phosphate isomerase (TPI), and a medical protein Lingzhi-8 (LZ8). All target proteins were successfully expressed and secreted within 48–72 h, with yields ranging from 110.8 to 416.8 mg/L in 50 mL shake-flasks cultivation. The enzyme activities of AnGoxM, MtPlyA and TPI reached ~ 1276 − 1328 U/mL, ~ 1627. 43 − 2105.69 U/mL, and ~ 1751.02 to 1906.81 U/mg after 48 h, respectively. Additionally, Overexpression of Cvc2, a COPI vesicle trafficking component, further enhanced MtPlyA production by 18%, highlighting the benefit of combining transcriptional and secretory pathway engineering. Conclusions Our results demonstrated that the chassis AnN2 served as a robust, modular, and time-efficient platform for heterologous protein expression in A. niger . Through site-specific integration of target genes into native high-expression loci and strategic modulation of the secretory pathway, we successfully enabled the rapid production of functional enzymes and bioactive proteins from diverse origins. This dual-level optimization strategy, which integrates rational genomic engineering with targeted enhancement of the secretory pathway, enabled high-yield expression while minimizing background interference. Together, these findings offer a practical framework for constructing versatile fungal expression systems and highlight the potential of combining genetic and cellular engineering to improve recombinant protein production in filamentous fungi.

In this paper, we report cloning of a pectate lyase gene from Bacillus amyloliquefaciens S6 (pelS6), and biochemical characterization of the recombinant pectate lyase. PelS6 was found to be identical with B. subtilis 168 pel enzyme with 100% amino acid sequence homology. Although these two are genetically very close, they are distinctly different in physiology. pelS6 gene encodes a 421-aa protein with a molecular mass of 65,75 kDa. Enzyme activity increased from 12.8 ± 0.3 to 49.6 ± 0.4 units/mg after cloning. The relative enzyme activity of the recPel S6 ranged from 80% to 100% at pH between 4 and 14. It was quite stable at different temperature values ranging from 15 to 90 °C. The recPEL S6 showed a maximal activity at pH 10 and at 60 °C. 0.5 mM of CaCl2 is the most effective metal ion on the recPEL S6 as demonstrated by its increased relative activity with 473%. recPEL S6 remained stable at − 20 °C for 18 months. In addition recPEL S6 increased juice clarity. This study introduces a novel bacterial pectate lyase enzyme with its characteristic capability of being highly thermostable, thermotolerant, and active over a wide range of pH, meaning that it can work at both acidic and alkaline environments, which are the most preferred properties in the industry.

Gravity is known as an important environmental factor involved in the regulation of plant architecture. To identify genes related to the gravitropism of Tartary buckwheat, a creeping line was obtained and designated as lazy1 from the mutant bank by 60 Co-γ ray radiation. Genetic analysis indicated that the creeping phenotype of lazy1 was attributed to a single recessive locus. As revealed by the horizontal and inverted suspension tests, lazy1 was completely lacking in shoot negative gravitropism. The creeping growth of lazy1 occurred at the early seedling stage, which could not be recovered by exogenous heteroauxin, hormodin, α-rhodofix, or gibberellin. Different from the well-organized and equivalent cell elongation of wild type (WT), lazy1 exhibited dilated, distorted, and abnormally arranged cells in the bending stem. However, no statistical difference of indole-3-acetic acid (IAA) levels was found between the far- and near-ground bending sides in lazy1 , which suggests that the asymmetric cell elongation of lazy1 was not induced by auxin gradient. Whereas, lazy1 showed up-expressed gibberellin-regulated genes by quantitative real-time PCR (qRT-PCR) as well as significantly higher levels of gibberellin, suggesting that gibberellin might be partly involved in the regulation of creeping growth in lazy1 . RNA sequencing (RNA-seq) identified a number of differentially expressed genes (DEGs) related to gravitropism at stages I (before bending), II (bending), and III (after bending) between WT and lazy1 . Venn diagram indicated that only Pectate lyase 5 was down-expressed at stages I [Log 2 fold change (Log 2 FC): −3.20], II (Log 2 FC: −4.97), and III (Log 2 FC: −1.23) in lazy1 , compared with WT. Gene sequencing revealed that a fragment deletion occurred in the coding region of Pectate lyase 5 , which induced the destruction of a pbH domain in Pectate lyase 5 of lazy1 . qRT-PCR indicated that Pectate lyase 5 was extremely down-expressed in lazy1 at stage II (0.02-fold of WT). Meanwhile, lazy1 showed the affected expression of lignin- and cellulose-related genes and cumulatively abnormal levels of pectin, lignin, and cellulose. These results demonstrate the possibility that Pectate lyase 5 functions as the key gene that could mediate primary cell wall metabolism and get involved in the asymmetric cell elongation regulation of lazy1 .

Background: Alkaline pectate lyase plays an important role in papermaking, biological refining and wastewater treatment, but its industrial applications are largely limited owing to its low activity and poor alkali resistance. Methods: The alkaline pectate lyase BspPel from Bacillus RN.1 was heterologously expressed in Escherichia coli BL21 (DE3) and its activity and alkali resistance were improved by loop replacement. Simultaneously, the effect of R260 on enzyme alkaline tolerance was also explored. Results: Recombinant pectate lyase (BspPel-th) showed the highest activity at 60°C and pH 11.0, and showed significant stability over a wide pH range (3.0–11.0). The specific enzyme activity after purification was 139.4 U/mg, which was 4.4 times higher than that of the wild-type enzyme. BspPel-th has good affinity for apple pectin, since the V max and K m were 29 μmol/min. mL and 0.46 mol/L, respectively. Molecular dynamics simulation results showed that the flexibility of the loop region of BspPel-th was improved. Conclusion: The modified BspPel-th has considerable potential for industrial applications with high pH processes.

In this study, we first established a PEG-mediated protoplast transformation system of C. camelliae and used it to investigate the biological functions of seven pectate lyase genes ( CcPEL s) which were abundantly expressed during infection. The results provided insights into the contributions of pectate lyase to mycelial growth, conidial production, appressorium formation, and the pathogenicity of C. camelliae. We also confirmed the secretory function of CcPEL proteins and their role in suppressing Bax-induced cell death. Colletotrichum camelliae is the dominant species causing foliar diseases of tea plants ( Camellia sinensis ) in China. Transcriptome data and reverse transcription-quantitative PCR (qRT-PCR) analysis have demonstrated that the pectate lyase genes in C. camelliae ( CcPEL s) were significantly upregulated during infectious development on tea plants (cv. Longjing43 ). To further evaluate the biological functions of CcPEL s, we established a polyethylene glycol (PEG)-mediated protoplast transformation system of C. camelliae and generated targeted deletion mutants of seven CcPEL s. Phenotypic assays showed that the genes contribute to mycelial growth, conidiation, and appressorium development. The polypeptides encoded by each CcPEL gene contained a predicted N-terminal signal peptide, and a yeast invertase secretion assay suggested that each CcPEL protein could be secreted. Cell death-suppressive activity assays confirmed that all seven CcPELs did not suppress Bax-induced cell death in tobacco leaf cells. However, deletion of CcPEL16 significantly reduced necrotic lesions on tea leaves. Taken together, these results indicated that CcPEL s play essential roles in regulating morphological development, and CcPEL16 is required for full virulence in C. camelliae . IMPORTANCE In this study, we first established a PEG-mediated protoplast transformation system of C. camelliae and used it to investigate the biological functions of seven pectate lyase genes ( CcPEL s) which were abundantly expressed during infection. The results provided insights into the contributions of pectate lyase to mycelial growth, conidial production, appressorium formation, and the pathogenicity of C. camelliae. We also confirmed the secretory function of CcPEL proteins and their role in suppressing Bax-induced cell death. Overall, this study provides an effective method for generating gene-deletion transformants in C. camelliae and broadens our understanding of pectate lyase in regulating morphological development and pathogenicity.

Pectin is a structural polysaccharide and a major component of plant cell walls. Pectate lyases are a class of enzymes that degrade demethylated pectin by cleaving the α-1,4-glycosidic bond, and they play an important role in plant growth and development. Currently, little is known about the gene family members and their involvement in salt stress in potato. In this study, we utilized bioinformatics to identify members of the potato pectate lyase gene family and analyzed their gene and amino acid sequence characteristics. The results showed that a total of 27 members of the pectate lyase gene family were identified in potato. Phylogenetic tree analysis revealed that these genes were divided into eight groups. Analysis of their promoters indicated that several members' promoter regions contained a significant number of hormone and stress response elements. Further, we found that several members responded positively to salt treatment under single salt and mixed salt stress. Since exhibited a consistent expression pattern under both single and mixed salt stress conditions, its subcellular localization was determined. The results indicated that StPL18 is localized in the endoplasmic reticulum membrane. The results will establish a foundation for analyzing the functions of potato pectate lyase family members and their expression under salt stress.