Pectinolytic lyases: a comprehensive review of sources, category, property, structure, and catalytic mechanism of pectate lyases and pectin lyases

Pectate lyases and pectin lyases have essential roles in various biotechnological applications, such as textile industry, paper making, pectic wastewater pretreatment, juice clarification and oil extraction. They can effectively cleave the α-1,4-glycosidic bond of pectin molecules back bone by β-elimination reaction to produce pectin oligosaccharides. In this way, it will not generate highly toxic methanol and has the advantages of good enzymatic selectivity, less by-products, mild reaction conditions and high efficiency. However, numerous researches have been done for several decades; there are still no comprehensive reviews to summarize the recent advances of pectate lyases and pectin lyases. This review tries to fill this gap by providing all relevant information, including the substrate, origin, biochemical properties, sequence analysis, mode of action, the three-dimensional structure and catalytic mechanism.

Genomic insights from Monoglobus pectinilyticus: a pectin-degrading specialist bacterium in the human colon

© 2019, International Society for Microbial Ecology. Pectin is abundant in modern day diets, as it comprises the middle lamellae and one-third of the dry carbohydrate weight of fruit and vegetable cell walls. Currently there is no specialized model organism for studying pectin fermentation in the human colon, as our collective understanding is informed by versatile glycan-degrading bacteria rather than by specialist pectin degraders. Here we show that the genome of Monoglobus pectinilyticus possesses a highly specialized glycobiome for pectin degradation, unique amongst Firmicutes known to be in the human gut. Its genome encodes a simple set of metabolic pathways relevant to pectin sugar utilization, and its predicted glycobiome comprises an unusual distribution of carbohydrate-active enzymes (CAZymes) with numerous extracellular methyl/acetyl esterases and pectate lyases. We predict the M. pectinilyticus degradative process is facilitated by cell-surface S-layer homology (SLH) domain-containing proteins, which proteomics analysis shows are differentially expressed in response to pectin. Some of these abundant cell surface proteins of M. pectinilyticus share unique modular organizations rarely observed in human gut bacteria, featuring pectin-specific CAZyme domains and the cell wall-anchoring SLH motifs. We observed M. pectinilyticus degrades various pectins, RG-I, and galactan to produce polysaccharide degradation products (PDPs) which are presumably shared with other inhabitants of the human gut microbiome (HGM). This strain occupies a new ecological niche for a primary degrader specialized in foraging a habitually consumed plant glycan, thereby enriching our understanding of the diverse community profile of the HGM.

Genomic insights from Monoglobus pectinilyticus: a pectin-degrading specialist bacterium in the human colon

© 2019, International Society for Microbial Ecology. Pectin is abundant in modern day diets, as it comprises the middle lamellae and one-third of the dry carbohydrate weight of fruit and vegetable cell walls. Currently there is no specialized model organism for studying pectin fermentation in the human colon, as our collective understanding is informed by versatile glycan-degrading bacteria rather than by specialist pectin degraders. Here we show that the genome of Monoglobus pectinilyticus possesses a highly specialized glycobiome for pectin degradation, unique amongst Firmicutes known to be in the human gut. Its genome encodes a simple set of metabolic pathways relevant to pectin sugar utilization, and its predicted glycobiome comprises an unusual distribution of carbohydrate-active enzymes (CAZymes) with numerous extracellular methyl/acetyl esterases and pectate lyases. We predict the M. pectinilyticus degradative process is facilitated by cell-surface S-layer homology (SLH) domain-containing proteins, which proteomics analysis shows are differentially expressed in response to pectin. Some of these abundant cell surface proteins of M. pectinilyticus share unique modular organizations rarely observed in human gut bacteria, featuring pectin-specific CAZyme domains and the cell wall-anchoring SLH motifs. We observed M. pectinilyticus degrades various pectins, RG-I, and galactan to produce polysaccharide degradation products (PDPs) which are presumably shared with other inhabitants of the human gut microbiome (HGM). This strain occupies a new ecological niche for a primary degrader specialized in foraging a habitually consumed plant glycan, thereby enriching our understanding of the diverse community profile of the HGM.

Integrity transcriptome and proteome analyses provide new insights into the mechanisms regulating pericarp cracking in Akebia trifoliata fruit

Background: Akebia trifoliata (Thunb.) Koidz, a perennial wild woody liana, can be used as biofuel to generate bioenergy, as well as a traditional Chinese medicine plant,and new potential edible fruit crop, due to its high yields in fields, wide adaptability, high economic, medicinal and nutritive values, and tolerance tocultivation conditions. However, the pericarp of A. trifoliata cracks longitudinallyalong the ventral suture during fruit ripening, which is a serious problem that limits its usefulness and causes significant losses in yield and commercial value. Furthermore, there have been no known investigations on fruit cracking and its molecular mechanisms in A. trifoliata . Results: In this study, the dynamic structural changes in fruit pericarps were observed, revealing that the cell wall of fruit pericarp became thinner, and had reduced integrity, and that the cell walls began to degrade in the cracking fruits compared to those observed in non-cracking fruits. Moreover, analyses of the complementary RNA- sequencing-based transcriptomes and tandem mass tag-based proteomes at different development stages during fruit ripening were performed, and the expression of various genes and proteins was found to be changed after cracking, The mRNA levels of 20 differentially expressed genes and 17 differentially abundant proteins (DAPs) involved in cell wall metabolism were further analyzed; 20 DAPs were also validated through parallel reaction monitoring analysis. Among these, pectate lyases and pectinesterase involved in pentose and glucuronate interconversions, β-galactosidases 2 involved in galactose metabolism, were significantly up-regulated in cracking fruits compared to levels in non-cracking fruits, suggesting that they might play crucial roles in A. trifoliata fruit cracking. Conclusions: This study provides new insights into the molecular basis of fruit cracking in A. trifoliata fruits and important clues for further studies on the genetic improvement of A. trifoliata and the breeding of non-cracking varieties.

Exploration of Two Pectate Lyases from Caldicellulosiruptor bescii Reveals that the CBM66 Module Has a Crucial Role in Pectic Biomass Degradation

ABSTRACT Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode pectinolytic enzymes. Pectate lyases that cleave the α-1,4-galacturonosidic linkage of pectin are widely used in industries such as papermaking and fruit softening. However, there are few reports on pectate lyases with good thermostability. Here, two pectate lyases (CbPL3 and CbPL9) from a hyperthermophilic bacterium, Caldicellulosiruptor bescii, belonging to family 3 and family 9 polysaccharide lyases, respectively, were investigated. The biochemical properties of the two CbPLs were shown to be similar under optimized conditions of 80°C to 85°C and pH 8 to 9. However, the degradation products from pectin and polygalacturonic acids (pGAs) were different. A family 66 carbohydrate-binding module (CbCBM66) located in the N terminus of the two CbPLs shares 100% amino acid identity. A CbCBM66-truncated mutant of CbPL9 showed lower activities than the wild type, whereas CbPL3 with a CbCBM66 knockout portion was reported to have enhanced activities, thereby revealing the different effect of CbCBM66. Prediction by the I-TASSER server revealed that CbCBM66 is structurally close to BsCBM66 from Bacillus subtilis; however, the COFACTOR and COACH programs indicated that the substrate-binding sites between CbCBM66 and BsCBM66 are different. Furthermore, a substrate-binding assay indicated that the catalytic domains in the two CbPLs had strong affinities for pectate-related substrates, but CbCBM66 showed a weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscopy (SEM) analysis and a total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for the degradation of plant biomass. IMPORTANCE Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium, Caldicellulosiruptor bescii. The two enzymes share a high optimum temperature, a low optimum pH, and good thermostability at the evaluated temperature. A family 66 carbohydrate-binding module (CbCBM66) was identified in the two CbPLs, sharing 100% amino acid identity. The deletion of CbCBM66 dramatically decreased the activity of CbPL9 but increased the activity and thermostability of CbPL3, suggesting different roles of CbCBM66 in the two enzymes. Moreover, the degradation products of the two CbPLs were different. These results revealed that these enzymes could represent potential pectate lyases for applications in the paper and textile industries.

Two pectate lyases from Caldicellulosiruptor bescii with the same CALG domain had distinct properties on plant biomass degradation

Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that carry pectinolytic enzymes. Pectate lyases that cleave α-1,4-galacturonosidic linkage of pectin are widely used in industries, such as paper making and fruit softening. However, reports on pectate lyases with high thermostability are few. Two pectate lyases (CbPL3 and CbPL9) from a thermophilic bacterium Caldicellulosiruptor bescii were investigated. Although these two enzymes belonged to different families of polysaccharide lyase, both were Ca2+-dependent. Similar biochemical properties were shown under optimized conditions 80 °C–85 °C and pH 8–9. However, the degradation products on pectin and polygalacturonic acids (pGA) were different, revealing the distinct mode of action. A concanavalin A-like lectin/glucanase (CALG) domain, located in the N-terminus of two CbPLs, shares 100% amino acid identity. CALG-truncated mutant of CbPL9 showed lower activities than the wild-type, whereas the CbPL3 with CALG knock-out portion was reported with enhanced activities, thereby revealing the different roles of CALG in two CbPLs. I-TASSER predicted that the CALG in two CbPLs is structurally close to the family 66 carbohydrate binding module (CBM66). Furthermore, substrate-binding assay indicated that the catalytic domains in two CbPLs had strong affinities on pectate-related substrates, but CALG showed weak interaction with a number of lignocellulosic carbohydrates, except sodium carboxymethyl cellulose and sodium alginate. Finally, scanning electron microscope analysis and total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for degradation of plant biomass.ImportanceThermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium Caldicellulosiruptor bescii. The two enzymes had high optimum temperature, low optimum pH, and good thermostability at evaluated temperature. A family-66 carbohydrate binding module (CBM66) was identified in two CbPLs with sharing 100% amino acid identity. Deletion of CBM66 obviously decreased the activity of CbPL9, but increase the activity and thermostability of CbPL3, suggesting the different roles of CBM66 in two enzymes. Moreover, the degradation products by two CbPLs were different. These results revealed these enzymes could represent a potential pectate lyase for applications in paper and textile industries.

On a Cold Night: Transcriptomics of Grapevine Flower Unveils Signal Transduction and Impacted Metabolism

Low temperature is a critical environmental factor limiting plant productivity, especially in northern vineyards. To clarify the impact of this stress on grapevine flower, we used the Vitis array based on Roche-NimbleGen technology to investigate the gene expression of flowers submitted to a cold night. Our objectives were to identify modifications in the transcript levels after stress and during recovery. Consequently, our results confirmed some mechanisms known in grapes or other plants in response to cold stress, notably, (1) the pivotal role of calcium/calmodulin-mediated signaling; (2) the over-expression of sugar transporters and some genes involved in plant defense (especially in carbon metabolism), and (3) the down-regulation of genes encoding galactinol synthase (GOLS), pectate lyases, or polygalacturonases. We also identified some mechanisms not yet known to be involved in the response to cold stress, i.e., (1) the up-regulation of genes encoding G-type lectin S-receptor-like serine threonine-protein kinase, pathogen recognition receptor (PRR5), or heat-shock factors among others; (2) the down-regulation of Myeloblastosis (MYB)-related transcription factors and the Constans-like zinc finger family; and (3) the down-regulation of some genes encoding Pathogen-Related (PR)-proteins. Taken together, our results revealed interesting features and potentially valuable traits associated with stress responses in the grapevine flower. From a long-term perspective, our study provides useful starting points for future investigation.