A "Pro-Asp-Thr" Amino Acid Repeat from Vibrio sp. QY108 Alginate Lyase Exhibits Alginate-Binding Capacity and Enhanced Soluble Expression and Thermostability.

Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a "Pro-Asp-Thr(PDT)" fragment and two PL-7 catalytic domains (CD I and CD II). The "PDT" fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the "PDT" fragment may serve as a fusion tag in the soluble expression of recombinant proteins.

Impact of dietary Laminaria digitata with alginate lyase or carbohydrase mixture on nutrient digestibility and gut health of weaned piglets.

Laminaria digitata is a brown seaweed rich in prebiotic polysaccharides, mainly laminarin, but its alginate-rich cell wall could compromise nutrient access. Carbohydrase supplementation, such as individual alginate lyase and carbohydrases mixture (Rovabio® Excel AP), could enhance nutrient digestibility and prebiotic potential. This study aimed to evaluate the effect of these enzymes on nutrient digestibility and gut health of weaned piglets fed with 10% L. digitata. Diets did not affect growth performance (P > 0.05). The majority of the feed fractions had similar digestibility across all diets, but the supplementation of alginate lyase increased hemicellulose digestibility by 3.3% compared to the control group (P = 0.047). Additionally, we observed that algal zinc was more readily available compared to the control group, even without enzymatic supplementation (P < 0.001). However, the increased digestibility of some minerals, such as potassium, raises concerns about potential mineral imbalance. Seaweed groups had a higher abundance of beneficial bacteria in colon contents, such as Prevotella, Oscillospira and Catenisphaera. Furthermore, the addition of alginate lyase led to a lower pH in the colon (P < 0.001) and caecum (P < 0.001) of piglets, which is possibly a result of released fermentable laminarin, and is consistent with the higher proportion of butyric acid found in these intestinal compartments. L. digitata is a putative supplement to enhance piglet gut health due to its prebiotic polysaccharides. Alginate lyase supplementation further improves nutrient digestibility and prebiotic potential. These results suggest the potential use of L. digitata and these enzymatic supplements in commercial piglet-feeding practices.

The two-step strategy for enhancing the specific activity and thermostability of alginate lyase AlyG2 with mechanism for improved thermostability.

To overcome the trade-off challenge encountered in the engineering of alginate lyase AlyG2 from Seonamhaeicola algicola Gy8T and to expand its potential industrial applications, we devised a two-step strategy encompassing activity enhancement followed by thermal stability engineering. To enhance the specific activity of efficient AlyG2, we strategically substituted residues with bulky steric hindrance proximal to the active pocket with glycine or alanine. This led to the generation of three promising positive mutants, with particular emphasis on the T91S mutant, exhibiting a 1.91-fold specific activity compared to the wild type. To mitigate the poor thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were screened out. Notably, the S72Ya mutant not only displayed 17.96 % further increase in specific activity but also exhibited improved stability compared to T91S, manifesting as a remarkable 30.97 % increase in relative activity following a 1-hour incubation at 42 °C. Furthermore, enhanced kinetic stability was observed. To gain deeper insights into the mechanism underlying the enhanced thermostability of the S72Ya mutant, we conducted molecular dynamics simulations, principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. The results unveiled a reduction in the flexibility of the surface loop, a stronger correlation dynamic and a narrower motion subspace in S72Ya system, along with the formation of more stable hydrogen bonds. Collectively, our findings suggest amino acids substitutions resulting in smaller side chains proximate to the active site can positively impact enzyme activity, while reducing the flexibility of surface loops emerges as a pivotal factor in conferring thermal stability. These insights offer valuable guidance and a framework for the engineering of other enzyme types.

The structure of a pectin-active family 1 polysaccharide lyase from the marine bacterium Pseudoalteromonas fuliginea.

Pseudoalteromonas fuliginea sp. PS47 is a recently identified marine bacterium that has extensive enzymatic machinery to metabolize polysaccharides, including a locus that targets pectin-like substrates. This locus contains a gene (locus tag EU509_03255) that encodes a pectin-degrading lyase, called PfPL1, that belongs to polysaccharide lyase family 1 (PL1). The 2.2 Šresolution X-ray crystal structure of PfPL1 reveals the compact parallel ß-helix fold of the PL1 family. The back side of the core parallel ß-helix opposite to the active site is a meandering set of five α-helices joined by lengthy loops. A comparison of the active site with those of other PL1 enzymes suggests a catalytic mechanism that is independent of metal ions, such as Ca2+, but that substrate recognition may require metal ions. Overall, this work provides the first structural insight into a pectinase of marine origin and the first structure of a PL1 enzyme in subfamily 2.

Expression and Characterization of an Efficient Alginate Lyase from Psychromonas sp. SP041 through Metagenomics Analysis of Rotten Kelp.

Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn2+, Co2+, Ca2+, Mg2+, Ni2+, and Ba2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na+, and upon treatment with Mn2+, its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed.

High-Level Extracellular Production of a Trisaccharide-Producing Alginate Lyase AlyC7 in Escherichia coli and Its Agricultural Application.

Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 µg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7.

Construction and characterization of a novel fusion alginate lyase with endolytic and exolytic cleavage activity for industrial preparation of alginate oligosaccharides.

Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2-4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

Efficacious bioconversion of alginate/cellulose to value-added oligosaccharides by alginate-degrading GH5 endoglucanase from Trichoderma asperellum.

Enzymatic degradation of lignocellulosic biomass provides an eco-friendly approach to produce value-added macromolecules, e.g., bioactive polysaccharides. A novel acidophilic GH5 ß-1,4-endoglucanase (termed TaCel5) from Trichoderma asperellum ND-1 was efficiently expressed in Komagataella phaffii (∼1.5-fold increase, 38.42 U/mL). TaCel5 displayed both endoglucanase (486.3 U/mg) and alginate lyase (359.5 U/mg) enzyme activities. It had optimal pH 3.0 and strong pH stability (exceed 86 % activity retained over pH range 3.0-5.0). 80 % activity (both endoglucanase and alginate lyase) was retained in the presence of 15 % ethanol or 3.42 M NaCl. Analysis of action mode revealed that hydrolytic activity of TaCel5 required at least three glucose (cellotriose) residues, yielding mainly cellobiose. Glu241 and Glu352 are essential catalytic residues, while Asp106, Asp277 and Asp317 play auxiliary roles in cellulose degradation. TaCel5 displayed high hydrolysis efficiency for glucan and alginate substrates. ESI-MS analysis indicated that the enzymatic hydrolysates of alginate mainly contained disaccharides and heptasaccharides. This is the first detailed report of a bifunctional GH5 endoglucanase/alginate lyase enzyme from T. asperellum. Thus TaCel5 has strong potential in food and feed industries as a catalyst for bioconversion of cellulose- and alginate-containing waste materials into value-added products oligosaccharides, which was of great benefit both for the economy and environment.

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I.

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP's richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Biochemical Characterization of a Novel Thermostable Ulvan Lyase from Tamlana fucoidanivorans CW2-9.

Ulvan is a complex sulfated polysaccharide extracted from Ulva, and ulvan lyases can degrade ulvan through a ß-elimination mechanism to obtain oligosaccharides. In this study, a new ulvan lyase, EPL15085, which belongs to the polysaccharide lyase (PL) 28 family from Tamlana fucoidanivorans CW2-9, was characterized in detail. The optimal pH and salinity are 9.0 and 0.4 M NaCl, respectively. The Km and Vmax of recombinant EPL15085 toward ulvan are 0.80 mg·mL-1 and 11.22 µmol·min -1 mg-1·mL-1, respectively. Unexpectedly, it is very resistant to high temperatures. After treatment at 100 °C, EPL15085 maintained its ability to degrade ulvan. Molecular dynamics simulation analysis and site-directed mutagenesis analysis indicated that the strong rigidity of the disulfide bond between Cys74-Cys102 in the N-terminus is related to its thermostability. In addition, oligosaccharides with disaccharides and tetrasaccharides were the end products of EPL15085. Based on molecular docking and site-directed mutagenesis analysis, Tyr177 and Leu134 are considered to be the crucial residues for enzyme activity. In conclusion, our study identified a new PL28 family of ulvan lyases, EPL15085, with excellent heat resistance that can expand the database of ulvan lyases and provide the possibility to make full use of ulvan.

Rational design of PspAlgL to improve its thermostability and anti-biofilm activity against Pseudomonas aeruginosa.

Pseudomonas aeruginosa biofilm enhances tolerance to antimicrobials and immune system defenses. Alginate is an important component of biofilm and a virulence factor of P. aeruginosa. The degradation of alginate by alginate lyases has come to serve as an adjunctive therapeutic strategy against P. aeruginosa biofilm, but poor stability of the enzyme limited this application. Thus, PspAlgL, an alginate lyase, can degrade acetylated alginate but has poor thermostability. The 3D structure of PspAlgL was predicted, and the thermostability of PspAlgL was rationally designed by GRAPE strategy, resulting in two variants with better stability. These variants, PspAlgLS270F/E311P and PspAlgLG291S/E311P, effectively degraded the alginate in biofilm. In addition, compared with PspAlgL, these variants were more efficient in inhibiting biofilm formation and degrading the established biofilm of P. aeruginosa PAO1, and they were also able to destroy the biofilm attached to catheters and to increase the sensitivity of P. aeruginosa to the antibiotic amikacin. This study provides one potential anti-biofilm agent for P. aeruginosa infection.

Pectate lyase genes from Radopholus similis and their application in pathotype identification.

Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguishable pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes and as a target gene for the molecular identification of these two pathotypes. KEY POINTS: • Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed. • The expression of Rs-pels is different in two pathotypes of Radopholus similis. • A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target gene was established.

Genome Analysis of Multiple Polysaccharide-Degrading Bacterium Microbulbifer thermotolerans HB226069: Determination of Alginate Lyase Activity.

Polysaccharide-degrading bacteria are key participants in the global carbon cycle and algal biomass recycling. Herein, a polysaccharide lyase-producing strain HB226069 was isolated from Sargassum sp. from Qingge Port, Hainan, China. Results of the phylogenetic of the 16S rRNA gene and genotypic analysis indicated that the isolate should be classified as Microbulbifer thermotolerans. The whole genome is a 4,021,337 bp circular chromosome with a G+C content of 56.5%. Analysis of the predicted genes indicated that strain HB226069 encoded 161 carbohydrate-active enzymes (CAZymes), and abundant putative enzymes involved in polysaccharide degradation were predicted, including alginate lyase, fucosidase, agarase, xylanase, cellulase, pectate lyase, amylase, and chitinase. Three of the putative polysaccharide lyases from PL7 and PL17 families were involved in alginate degradation. The alginate lyases of strain HB226069 showed the maximum activity of 117.4 U/mL at 50 °C, pH 7.0, and 0.05 M FeCl3, while exhibiting the best stability at 30 °C and pH 7.0. The Thin Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analyses indicated that the alginate oligosaccharides (AOSs) degraded by the partially purified alginate lyases contained oligosaccharides of DP2-DP5 and monosaccharide while reacting for 36 h. The complete genome of M. thermotolerans HB226069 enriches our understanding of the mechanism of polysaccharide lyase production and supports its potential application in polysaccharide degradation.

Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

Litchi aspartic protease LcAP1 enhances plant resistance via suppressing cell death triggered by the pectate lyase PlPeL8 from Peronophythora litchii.

Plant cell death is regulated in plant-pathogen interactions. While some aspartic proteases (APs) participate in regulating programmed cell death or defense responses, the defense functions of most APs remain largely unknown. Here, we report on a virulence factor, PlPeL8, which is a pectate lyase found in the hemibiotrophic pathogen Peronophythora litchii. Through in vivo and in vitro assays, we confirmed the interaction between PlPeL8 and LcAP1 from litchi, and identified LcAP1 as a positive regulator of plant immunity. PlPeL8 induced cell death associated with NbSOBIR1 and NbMEK2. The 11 conserved residues of PlPeL8 were essential for inducing cell death and enhancing plant susceptibility. Twenty-three LcAPs suppressed cell death induced by PlPeL8 in Nicotiana benthamiana depending on their interaction with PlPeL8. The N-terminus of LcAP1 was required for inhibiting PlPeL8-triggered cell death and susceptibility. Furthermore, PlPeL8 led to higher susceptibility in NbAPs-silenced N. benthamiana than the GUS-control. Our results indicate the crucial roles of LcAP1 and its homologs in enhancing plant resistance via suppression of cell death triggered by PlPeL8, and LcAP1 represents a promising target for engineering disease resistance. Our study provides new insights into the role of plant cell death in the arms race between plants and hemibiotrophic pathogens.

Efficient preparation of alginate oligosaccharides by using alginate lyases and evaluation of the development promoting effects on Brassica napus L. in saline-alkali environment.

Enzymatic degradation of alginate for the preparation of alginate oligosaccharides (AOS) is currently receiving significant attention in the field. AOS has been shown to promote crop growth and improve plant resistance to abiotic stresses. In this study, two PL6 family alginate lyases, AlyRmA and AlyRmB, were expressed and characterized. These enzymes demonstrate exceptional activity and stable thermophilicity compared to other known alginate lyases. AlyRmA (8855.34 U/mg) and AlyRmB (7879.44 U/mg) exhibited excellent degradation activity towards sodium alginate even at high temperatures (70 °C). The AlyRmA and AlyRmB were characterized and utilized to efficiently produce AOS. The study investigated the promotional effect of AOS on the growth of Brassica napus L. seedlings in a saline-alkaline environment. The results of this study demonstrate the high activity and thermal stability of AlyRmA and AlyRmB, highlighting their potential in the preparation of AOS. Moreover, the application of AOS prepared by AlyRmB could enhance the resistance of Brassica napus L. to saline-alkali environments, thereby broadening the potential applications of AOS.

Glycosaminoglycan lyase: A new competition between bacteria and the pacific white shrimp Litopenaeus vannamei.

Horizontal gene transfer (HGT) is an important evolutionary force in the formation of prokaryotic and eukaryotic genomes. In recent years, many HGT genes horizontally transferred from prokaryotes to eukaryotes have been reported, and most of them are present in arthropods. The Pacific white shrimp Litopenaeus vannamei, an important economic species of arthropod, has close relationships with bacteria, providing a platform for horizontal gene transfer (HGT). In this study, we analyzed bacteria-derived HGT based on a high-quality genome of L. vannamei via a homology search and phylogenetic analysis, and six HGT genes were identified. Among these six horizontally transferred genes, we found one gene (LOC113799989) that contains a bacterial chondroitinase AC structural domain and encodes an unknown glycosaminoglycan (GAG) lyase in L. vannamei. The real-time quantitative PCR results showed that the mRNA expression level of LOC113799989 was highest in the hepatopancreas and heart, and after stimulation by Vibrio parahaemolyticus, its mRNA expression level was rapidly up-regulated within 12 h. Furthermore, after injecting si-RNA and stimulation by V. parahaemolyticus, we found that the experimental group had a higher cumulative mortality rate in 48 h than the control group, indicating that the bacteria-derived GAG lyase can reduce the mortality of shrimp with respect to infection by V. parahaemolyticus and might be related to the resistance of shrimp to bacterial diseases. Our findings contribute to the study of the function of GAGs and provide new insights into GAG-related microbial pathogenesis and host defense mechanisms in arthropods.

Identification and characterization of a PL35 GAGs lyase with 4-O-sulfated N-acetylgalactosamine (A-type)-rich structures producing property.

Glycosaminoglycan (GAG) lyases are important tools for investigating the structure of GAGs and preparing low-molecular-weight GAGs. The PL35 family, a recently established polysaccharide lyase family, should be further investigated. In this study, we discovered a new GAG lyase, CHa1, which belongs to the PL35 family. When expressed heterologously in Escherichia coli (BL21), CHa1 exhibited high expression levels and solubility. The optimal activity was observed in Tris-HCl buffer (pH 7.0) or sodium phosphate buffer (pH 8.0) at 30 °C. The specific activities towards HA, CSA, CSC, CSD, CSE, and HS were 3.81, 13.03, 36.47, 18.46, 6.46, and 0.50 U/mg protein, respectively. CHa1 digests substrate chains randomly that acting as an endolytic lyase and shows a significant preference for GlcA-containing structures, prefers larger oligosaccharides (≥UDP8) and can generate a series of oligosaccharides composed mainly of the A unit when digesting CSA. These oligosaccharides include ΔC-A, ΔC-A-A, ΔC-A-A-A, ΔC-A-A-A-A, and ΔC-A-A-A-A-A. The residues Tyr257 and His421 play crucial roles in the catalytic process, and Ser211, Asn212, Asn213, Trp214, Gln216, Lys360, Arg460 and Gln462 may participate in the binding process of CHa1. This study on CHa1 contributes to our understanding of the PL35 family and provides valuable tools for investigating the structure of GAGs.