Unveiling novel insights into Bacillus velezensis 16B pectin lyase for improved fruit juice processing.

Microbial pectinolytic enzymes are important in various industries, particularly food processing. This study focuses on uncovering insights into a novel pectin lyase, BvPelB, from Bacillus velezensis 16B, with the aim of enhancing fruit juice processing. The study examines the structural and functional characteristics of pectinolytic enzyme, underscoring the critical nature of substrate specificity and enzymatic reaction mechanisms. BvPelB was successfully expressed and purified, exhibiting robust activity under alkaline conditions and thermal stability. Structural analysis revealed similarities with other pectin lyases, despite limited sequence identity. Biochemical characterization showed BvPelB's preference for highly methylated pectins and its endo-acting mode of cleavage. Treatment with BvPelB significantly increased juice yield and clarity without generating excessive methanol, making it a promising candidate for fruit juice processing. Overall, this study provides valuable insights into the enzymatic properties of BvPelB and its potential industrial applications in improving fruit juice processing efficiency and quality.

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.

Functional domains of Acinetobacter bacteriophage tail fibers.

A rapid increase in antimicrobial resistant bacterial infections around the world is causing a global health crisis. The Gram-negative bacterium Acinetobacter baumannii is categorized as a Priority 1 pathogen for research and development of new antimicrobials by the World Health Organization due to its numerous intrinsic antibiotic resistance mechanisms and ability to quickly acquire new resistance determinants. Specialized phage enzymes, called depolymerases, degrade the bacterial capsule polysaccharide layer and show therapeutic potential by sensitizing the bacterium to phages, select antibiotics, and serum killing. The functional domains responsible for the capsule degradation activity are often found in the tail fibers of select A. baumannii phages. To further explore the functional domains associated with depolymerase activity, tail-associated proteins of 71 sequenced and fully characterized phages were identified from published literature and analyzed for functional domains using InterProScan. Multisequence alignments and phylogenetic analyses were conducted on the domain groups and assessed in the context of noted halo formation or depolymerase characterization. Proteins derived from phages noted to have halo formation or a functional depolymerase, but no functional domain hits, were modeled with AlphaFold2 Multimer, and compared to other protein models using the DALI server. The domains associated with depolymerase function were pectin lyase-like (SSF51126), tailspike binding (cd20481), (Trans)glycosidases (SSF51445), and potentially SGNH hydrolases. These findings expand our knowledge on phage depolymerases, enabling researchers to better exploit these enzymes for therapeutic use in combating the antimicrobial resistance crisis.

Genetic analysis and QTL mapping of seed hardness trait in a soybean (Glycine max) recombinant inbred line (RIL) population.

Seed hardness is a critical quality trait impacting both the suitability of soybeans for consumption and their processing. The primary objective of this study was to explore the genetic foundations underlying seed hardness in soybeans. A 234 recombinant inbred line (RIL) population was evaluated for seed hardness across three years (2015 in Gansu, 2016, and 2017 in Hainan). Notably, the parent varieties, Zhonghuang35 and Jindou21, displayed significant differences in seed hardness. Also, the RIL population exhibited a wide range of genetic variation in seed hardness, with coefficients of variation between 70.53 % and 94.94 %. The frequency distribution of this trait conformed to a relatively normal distribution, making it suitable for QTL analysis. Six QTLs associated with seed hardness were identified with three located on chromosome 2 and three on chromosome 16. The major QTL, qHS-2-1, consistently exhibited the highest percentage of PVE and LOD in Gansu 2015, Hainan 2016, and Hainan 2017, suggesting its central role in determining seed hardness. Further investigation revealed four genes within the qHS-2-1 interval potentially related to seed hardness. GO enrichment analysis provided insights into their functions, including factors such as Glyma.02G307000, a pectin lyase-like superfamily protein, which could influence seed hardness through its role in pectin lyase enzyme activity. Expression analysis of these candidate genes demonstrated significant differences between the two parent varieties, further highlighting their potential role in seed coat hardness. This study offers valuable insights into the genetic mechanisms governing soybean seed coat hardness, providing a foundation for future research and crop improvement efforts.

Flow-cytometric cell sorting coupled with UV mutagenesis for improving pectin lyase expression.

Introduction: Alkaline pectin lyase is an important enzyme with a wide range of applications in industrial production, It has been widely used in many important fields such as fruit juice processing and extraction, the dyeing and processing of cotton and linen textiles, degumming plant fibers, environmental industrial wastewater treatment, and pulp and paper production. PGLA-rep4 was previously generated as a modified alkaline pectin lyase with high specific activity at pH 11.0°C and 70°C. However, the pre-constructed high-activity pectin lyase expression strains are still difficult to apply in industrial production due to their limited enzymatic activity. We hope to solve these problems by combining modern breeding techniques with high-throughput equipment to rapidly screen alkaline pectin lyase with higher enzymatic activity and lower cost. Methods: We fused the genes encoding PGLA-rep4 and fluorescent protein egfp using a flexible linker peptide and ligated them into a temperature-sensitive plasmid, pKD46. The constructed screening plasmid pKD46-PGLA-rep4-egfp was then transformed into an expression host and screened via flow-cytometric cell sorting coupled with UV mutagenesis. Results: Following mutagenesis, primary screening, and secondary screening, the high-expression strain, named Escherichia coli BL21/1G3, was obtained. The screening plasmid pKD46-PGLA-rep4-egfp was eliminated, and the original expression plasmid pET28a-PGLA-rep4 was then retransformed into the mutant strains. After induction and fermentation, pectin lyase activity in E. coli BL21/1G3 was significantly increased (1.37-fold relative to that in the parental E. coli BL21/PGLA-rep4 strain, p < 0.001), and the highest activity was 230, 240 U/mL at 144 h. Genome sequencing revealed that genes encoding ribonuclease E (RNase E) and diadenosine tetraphosphatase (ApaH) of E. coli BL21/1G3 were mutated compared to the sequence in the original E. coli BL21 (DE3) strain, which could be associated with increased enzyme expression. Discussion: Our work provides an effective method for the construction of strains expressing pectin lyase at high levels.

Safety evaluation of a food enzyme containing endo-polygalacturonase and pectin lyase activities from the non-genetically modified Aspergillus tubingensis strain NZYM-PE.

The food enzyme with the declared activities endo-polygalacturonase ((1-4)-α-D-galacturonan glycanohydrolase; EC 3.2.1.15) and pectin lyase ((1-4)-6-O-methyl-α-D-galacturonan lyase; EC 4.2.2.10) is produced with the non-genetically modified Aspergillus tubingensis strain NZYM-PE by Novozymes A/S. It is intended to be used in four food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, refined olive oil production and wine and wine vinegar production. Since residual amounts of total organic solids (TOS) are removed during production, dietary exposure was not calculated for refined olive oil production. For the remaining three food processes, it was estimated to be up to 0.132 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 1,430 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure above 10,833. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 13 matches were found, including one food allergen (papaya). The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, in particular for individuals sensitised to papaya, but that the risk will not exceed that of consumption of papaya. In addition, oral allergy reactions cannot be excluded in pollen-sensitised individuals. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Toward gentle chokeberry juice production by ultrasound-assisted enzymatic maceration.

Sustainable processes accompanied by high extraction yields and minimized amounts of by-products are a major goal of current fruit juice production. Controlled degradation of cell wall polysaccharides, in particular pectin, may contribute to reduced emergence of side streams. Possible strategies for the optimization are the selection of enzyme preparations based on comprehensive studies of their activities, the adjustment of maceration temperature toward more gentle conditions, and the application of alternative technologies such as ultrasound (US) during maceration. The present study provides insights into the effects of ultrasound-assisted enzymatic maceration (UAEM) on pectin degradation, total anthocyanin content, thermal and storage stability, and juice yield during chokeberry juice production on pilot-plant scale. The two enzyme preparations applied predominantly possessed polygalacturonase or pectin lyase activity. Cell wall polysaccharide degradation was improved by US and resulted in a 3% increase in juice yield by UAEM using an enzyme preparation that shows mostly polygalacturonase activity. Thermostability of anthocyanins was improved in juices produced using pectin lyase and applying US and matched the stability of anthocyanins in juices produced using polygalacturonase. Storage stability of anthocyanins was improved in juice produced using polygalacturonase during UAEM. UAEM also resulted in lower yields of pomace making the production more resource-efficient. Overall, the use of polygalacturonase has promising potential to advance conventional chokeberry juice production by applying US at gentle conditions.

Safety evaluation of the food enzyme pectin lyase from the genetically modified Trichoderma reesei strain RF6199.

The food enzyme pectin lyase ((1-4)-6-O-methyl-α-D-galacturonan lyase; EC 4.2.2.10) is produced with the genetically modified Trichoderma reesei strain RF6199 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in six manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for fruit brandies, fruit and vegetable processing for products other than juices, wine and wine vinegar production, refined and unrefined sugar production and coffee bean demucilation. Foods obtained from fruit processing for fruit brandies and coffee bean demucilation, as well as refined sugars, were excluded from dietary exposure estimation. For the remaining four processes, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.2 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 5,000. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood of such reactions is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Modification and application of highly active alkaline pectin lyase.

Alkaline pectate lyase has developmental prospects in the textile, pulp, paper, and food industries. In this study, we selected BacPelA, the pectin lyase with the highest expression activity from Bacillus clausii, modified and expressed in Escherichia coli BL21(DE3). Through fragment replacement, the catalytic activity of the enzyme was significantly improved. The optimum pH and temperature of the modified pectin lyase (PGLA-rep4) were 11.0 and 70 °C, respectively. It also exhibited a superior ability to cleave methylated pectin. The enzyme activity of PGLA-rep4, measured at 235 nm with 0.2% apple pectin as the substrate, was 554.0 U/mL, and the specific enzyme activity after purification using a nickel column was 822.9 U/mg. After approximately 20 ns of molecular dynamics simulation, the structure of the pectin lyase PGLA-rep4 tended to be stable. The root mean square fluctuation (RMSF) values at the key catalytically active site, LYS168, were higher than those of the wildtype PGLA. In addition, PGLA-rep4 was relatively stable in the presence of metal ions. PGLA-rep4 has good enzymatic properties and activities and maintains a high pH and temperature. This study provides a successful strategy for enhancing the catalytic activity of PGLA-rep4, making it the ultimate candidate for degumming and various uses in the pulp, paper, and textile industries.

Relationship of the methanol production, pectin and pectinase activity during apple wine fermentation and aging.

Understanding pectin structure and pectinase activity was important to control methanol content in apple wine. Therefore, this study compared inoculated fermentation (I), spontaneous fermentation (S) and inoculated fermentation combined with CaCl2 treatment (I & CaCl2) to explore their differences in methanol production, pectin peak molecular weight (Mp), and the activities of pectin methyl esterase (PME), pectin lyase (PL) and polygalacturonase (PG). The results showed that the activities of PME, PL and PG were intensively inhibited during fermentation; however, they still retained 3.41-5.84% (PME), 9.46-17.71% (PL) and 9.17-10.31% (PG) of the initial activities after aging for 30 days. Therefore, the methanol content was increased in all three aged wines even aging at 4 °C. CaCl2 promoted the PME and PL activities, and thus accelerated the methanol production. Because the pectin with Mp 3.07 kDa was retained by CaCl2, the highest pectin content was found in wine I & CaCl2 (160.69 mg/L), which was 95.47 mg/L higher than that in wine I, and 107.03 mg/L higher than that in wine S. In group S, the long lag period allowed pectin to withstand the pectinases inherent in apple juice for a long time, which was conducive to the cleavage of pectin to Mp lower than 3 kDa continuously, its further degradation led to the lowest pectin content (53.65 mg/L) in wine. Hence, inhibiting the pectinases activities, or shortening the aging period would play an important role in decreasing the methanol content in apple wine.

Safety evaluation of the food enzyme containing endo-polygalacturonase, pectinesterase, pectin lyase and non-reducing end α-l-arabinofuranosidase activities from the Aspergillus niger strain PEC.

The food enzyme has four declared activities (endo-polygalacturonase ((1→4)-α-d-galacturonan glycanohydrolase (endo-cleaving); 3.2.1.15), pectinesterase (pectin pectylhydrolase; 3.1.1.11), pectin lyase ((1→4)-6-O-methyl-α-d-galacturonan lyase; 4.2.2.10) and non-reducing end α-l-arabinofuranosidase (α-l-arabinofuranoside non-reducing end α-l-arabinofuranosidase; 3.2.1.55) and is produced with the non-genetically modified Aspergillus niger strain PEC by DSM Food Specialties B.V. The food enzyme is free from viable cells of the production organism. The food enzyme is intended to be used in the manufacture of alcoholic beverages from fruits other than grapes, fruit and vegetable processing for juice production, and wine and wine vinegar production. Dietary exposure was estimated to be up to 0.25 mg TOS/kg bodyweight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 204 mg TOS/kg bw per day, the highest dose tested which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 800. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and several matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, particularly for individuals sensitised to several pollen allergens or papaya allergens. Based on the data provided, the Panel concluded that this food enzyme did not give rise to safety concerns, under the intended conditions of use.

Differential tolerance to heat stress of young leaves compared to mature leaves of whole plants relate to differential transcriptomes involved in metabolic adaptations to stress.

Plants respond to heat shock by regulating gene expression. While transcriptomic changes in response to heat stress are well studied, it is not known whether young and old leaves reprogram transcription differently upon stress. When whole plants of Arabidopsis thaliana were subjected to heat shock, young leaves were affected significantly less than older leaves based on measurements of tissue damage. To identify quantitative changes to transcriptomes between young and old leaves upon heat stress, we used RNA sequencing on young and old leaves from plants exposed to control and heat stress at 42 °C for 1 h and 10 h. A total of 6472 differentially expressed genes between young and old leaf were identified under control condition, and 9126 and 6891 under 1 h and 10 h heat stress, respectively. Analyses of differentially expressed transcripts led to the identification of multiple functional clusters of genes that may have potential roles in the increased heat tolerance of young leaves including higher level of expression in young leaves of genes encoding chaperones, heat shock proteins and proteins known in oxidative stress resistance. Differential levels of transcripts for genes implicated in pectin metabolism, cutin and wax biosynthesis, pentose and glucuronate interconversions, cellulose degradation, indole glucosinolate metabolism and RNA splicing between young and old leaves under heat stress suggest that cell wall remodelling, cuticular wax synthesis and carbohydrate modifications impacted by alternative splicing may also have roles in the improved heat stress tolerance of young leaves.

Pectinolytic arsenal of Colletotrichum lindemuthianum and other fungi with different lifestyles.

AIM: To identify and analyse genes that encode pectinases in the genome of the fungus Colletotrichum lindemuthianum, evaluate the expression of these genes, and compare putative pectinases found in C. lindemuthianum with pectinases produced by other fungi and oomycetes with different lifestyles. METHODS AND RESULTS: Genes encoding pectinases in the genome of C. lindemuthianum were identified and analysed. The expression of these genes was analysed. Pectinases from C. lindemuthianum were compared with pectinases from other fungi that have different lifestyles, and the pectinase activity in some of these fungi was quantified. Fifty-eight genes encoding pectinases were identified in C. lindemuthianum. At least six types of enzymes involved in pectin degradation were identified, with pectate lyases and polygalacturonases being the most abundant. Twenty-seven genes encoding pectinases were differentially expressed at some point in C. lindemuthianum during their interactions with their host. For each type of pectinase, there were at least three isoenzyme groups. The number of pectinases present in fungi with different lifestyles seemed to be related more to the lifestyle than to the taxonomic relationship between them. Only phytopathogenic fungi showed pectate lyase activity. CONCLUSIONS: The collective results demonstrate the pectinolytic arsenal of C. lindemuthianum, with many and diverse genes encoding pectinases more than that found in other phytopathogens, which suggests that at least part of these pectinases must be important for the pathogenicity of the fungus C. lindemuthianum. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of these pectinases could further the understanding of the importance of this broad pectinolytic arsenal in the common bean infection and could be exploited for biotechnological purposes.

High-yield production of acidic pectin lyase PNLZJ5B for juice processing.

A pectin lyase gene pnlzj5b from Aspergillus niger ZJ5 was identified and overexpressed successfully in Pichia pastoris. Recombinant PNLZJ5B exhibited high activity towards citrus pectin (150 U ml-1 ). Through further codon optimization, the expression efficiency of PNLZJ5B in P. pastoris increased to 3·5-fold (532/150 U ml-1 ). PNLZJ5B was purified by ultrafiltration, anion exchange and gel chromatography. It showed optimal activity and good stability at 58°C and pH 4·5. PNLZJ5B activity improved with increasing degrees of methyl esterification of pectin. The Km and Vmax values were 0·81 mg ml-1 and 372·8 µmol min-1 mg-1 , respectively. In addition, PNLZJ5B effectively decreased the viscosity of apple juice. Compared with commercial pectin lyase, PNLZJ5B obtained a higher juice volume. These favourable enzymatic properties of PNLZJ5B show potential utility in juice-processing applications and other food-related fields.

Safety evaluation of the food enzyme pectin lyase from the genetically modified Aspergillus luchuensis strain FLOSC.

The food enzyme pectin lyase ((1→4)-6-O-methyl-α-d-galacturonan lyase; EC 4.2.2.10) is produced with the genetically modified Aspergillus luchuensis (formally Aspergillus niger) strain FLOSC by Advanced Enzyme Technologies Ltd. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in fruit and vegetable processing for juice production. Based on the maximum use level and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.268 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 794 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 2,900. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Cell-Wall-Degrading Enzymes-Related Genes Originating from Rhizoctonia solani Increase Sugar Beet Root Damage in the Presence of Leuconostoc mesenteroides.

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0-26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides.

Production and molecular characterization of tailored citrus pectin-derived compounds.

In this study, tailored-made citrus pectin-derived compounds were produced through controlled enzymatic and/or chemical modifications of commercial citrus pectin with different degrees of methylesterification (DM) and similar average molecular weight (MW). In the first treatment, degradation of the citrus pectin (CP) materials by endo-polygalacturonase (EPG) yielded pectins with average Mw's (between 2 and 60 kDa). Separation and identification of the oligosaccharide fraction present in these samples, revealed the presence of non-methylesterified galacturonic acid oligomers with degree of polymerization (DP) 1-5. In the second treatment, exploiting the combined effect of EPG and pectin lyase, compounds with MW between 2 and 21 kDa, containing methylesterified and non-methylesterified polygalacturonans (DP 1-6), were generated. Finally, CP was sequentially modified by chemical saponification and the action of EPG. A sample of DM 11% and MW 2.7 kDa, containing POS (DP 1-5), was produced. Diverse pectin-derived compounds were successfully generated for further studies exploring their functionality.

Revealing methyl-esterification patterns of pectins by enzymatic fingerprinting: Beyond the degree of blockiness.

Citrus pectins were studied by enzymatic fingerprinting using a simultaneous enzyme treatment with endo-polygalacturonase (endo-PG) from Kluyveromyces fragilis and pectin lyase (PL) from Aspergillus niger to reveal the methyl-ester distribution patterns over the pectin backbone. Using HILIC-MS combined with HPAEC enabled the separation and identification of the diagnostic oligomers released. Structural information on the pectins was provided by using novel descriptive parameters such as degree of blockiness of methyl-esterified oligomers by PG (DBPGme) and degree of blockiness of methyl-esterified oligomers by PL (DBPLme). This approach enabled us to clearly differentiate citrus pectins with various methyl-esterification patterns. The simultaneous use of PG and PL showed additional information, which is not revealed in digests using PG or PL alone. This approach can be valuable to differentiate pectins having the same DM and to get specific structural information on pectins and therefore to be able to better predict their physical and biochemical functionalities.

Pectin Modification in Seed Coat Mucilage by In Vivo Expression of Rhamnogalacturonan-I- and Homogalacturonan-Degrading Enzymes.

The cell wall is essential for plant survival. Determining the relationship between cell wall structure and function using mutant analysis or overexpressing cell wall-modifying enzymes has been challenging due to the complexity of the cell wall and the appearance of secondary, compensatory effects when individual polymers are modified. In addition, viability of the plants can be severely impacted by wall modification. A useful model system for studying structure-function relationships among extracellular matrix components is the seed coat epidermal cells of Arabidopsis thaliana. These cells synthesize relatively simple, easily accessible, pectin-rich mucilage that is not essential for plant viability. In this study, we expressed enzymes predicted to modify polysaccharide components of mucilage in the apoplast of seed coat epidermal cells and explored their impacts on mucilage. The seed coat epidermal-specific promoter TESTA ABUNDANT2 (TBA2) was used to drive expression of these enzymes to avoid adverse effects in other parts of the plant. Mature transgenic seeds expressing Rhamnogalacturonate lyase A (RglA) or Rhamnogalacturonate lyase B (RglB) that degrade the pectin rhamnogalacturonan-I (RG-I), a major component of mucilage, had greatly reduced mucilage capsules surrounding the seeds and concomitant decreases in the monosaccharides that comprise the RG-I backbone. Degradation of the minor mucilage component homogalacturonan (HG) using the HG-degrading enzymes Pectin lyase A (PLA) or ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2) resulted in developing seed coat epidermal cells with disrupted cell-cell adhesion and signs of early cell death. These results demonstrate the feasibility of manipulating the seed coat epidermal cell extracellular matrix using a targeted genetic engineering approach.

Effect of solid loading on the behaviour of pectin-degrading enzymes.

BACKGROUND: Pectin plays a role in the recalcitrance of plant biomass by affecting the accessibility of other cell wall components to enzymatic degradation. Elimination of pectin consequently has a positive impact on the saccharification of pectin-rich biomass. This work thus focused on the behaviour of different pectin-degrading enzymes in the presence of low (5%) to high (35%) solid loading of lemon peel. RESULTS: High solid loading of lemon peel affected pectin solubilisation differently depending on the pectinase used. Pectin lyase was less sensitive to a reduction of water content than was a mixture of endopolygalacturonase and pectin methylesterase, regardless of whether or not the latter's mode of action is processive or not. Marked changes in water mobility were observed along with enzymatic degradation depending on the enzyme used. However, the pectin lyase resulted in less pronounced shifts in water distribution than polygalacturonase-pectin methylesterase mixtures. At similar pectin concentration, pectin solutions hindered the diffusion of hydrolases more than the solid substrate. This can be attributed to the high viscosity of the highly concentrated pectin solutions while the solid substrate may provide continuous diffusion paths through pores. CONCLUSIONS: The increase in solid substrate loading reduced the efficiency of pectin-degrading enzymes catalysing hydrolysis more significantly than those catalysing ß-elimination. LF-NMR experiments highlighted the impact of solid loading on water mobility. Compared to other enzymes and whatever the solid loading, pectin lyase led to longer relaxation times linked with the most destructuration of the solid substrate. This new information could benefit the biorefinery processing of pectin-rich plant material when enzymes are used in the treatment.