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.

Role of Volatile Organic Compounds Produced by Kosakonia cowanii Cp1 during Competitive Colonization Interaction against Pectobacterium aroidearum SM2.

The competitive colonization of bacteria on similar ecological niches has a significant impact during their establishment. The synthesis speeds of different chemical classes of molecules during early competitive colonization can reduce the number of competitors through metabolic effects. In this work, we demonstrate for the first time that Kosakonia cowanii Cp1 previously isolated from the seeds of Capsicum pubescens R. P. produced volatile organic compounds (VOCs) during competitive colonization against Pectobacterium aroidearum SM2, affecting soft rot symptoms in serrano chili (Capsicum annuum L.). The pathogen P. aroidearum SM2 was isolated from the fruits of C. annuum var. Serrano with soft rot symptoms. The genome of the SM2 strain carries a 5,037,920 bp chromosome with 51.46% G + C content and 4925 predicted protein-coding genes. It presents 12 genes encoding plant-cell-wall-degrading enzymes (PCDEWs), 139 genes involved in five types of secretion systems, and 16 genes related to invasion motility. Pathogenic essays showed soft rot symptoms in the fruits of C. annuum L., Solanum lycopersicum, and Physalis philadelphica and the tubers of Solanum tuberosum. During the growth phases of K. cowanii Cp1, a mix of VOCs was identified by means of HS-SPME-GC-MS. Of these compounds, 2,5-dimethyl-pyrazine showed bactericidal effects and synergy with acetoin during the competitive colonization of K. cowanii Cp1 to completely reduce soft rot symptoms. This work provides novel evidence grounding a better understanding of bacterial interactions during competitive colonization on plant tissue, where VOC synthesis is essential and has a high potential capacity to control pathogenic microorganisms in agricultural systems.

Fungal pectinases: an insight into production, innovations and applications.

The fungal system holds morphological plasticity and metabolic versatility which makes it unique. Fungal habitat ranges from the Arctic region to the fertile mainland, including tropical rainforests, and temperate deserts. They possess a wide range of lifestyles behaving as saprophytic, parasitic, opportunistic, and obligate symbionts. These eukaryotic microbes can survive any living condition and adapt to behave as extremophiles, mesophiles, thermophiles, or even psychrophile organisms. This behaviour has been exploited to yield microbial enzymes which can survive in extreme environments. The cost-effective production, stable catalytic behaviour and ease of genetic manipulation make them prominent sources of several industrially important enzymes. Pectinases are a class of pectin-degrading enzymes that show different mechanisms and substrate specificities to release end products. The pectinase family of enzymes is produced by microbial sources such as bacteria, fungi, actinomycetes, plants, and animals. Fungal pectinases having high specificity for natural sources and higher stabilities and catalytic activities make them promising green catalysts for industrial applications. Pectinases from different microbial sources have been investigated for their industrial applications. However, their relevance in the food and textile industries is remarkable and has been extensively studied. The focus of this review is to provide comprehensive information on the current findings on fungal pectinases targeting diverse sources of fungal strains, their production by fermentation techniques, and a summary of purification strategies. Studies on pectinases regarding innovations comprising bioreactor-based production, immobilization of pectinases, in silico and expression studies, directed evolution, and omics-driven approaches specifically by fungal microbiota have been summarized.

Insight into biodiversity of the recently rearranged genus Dickeya.

The genus Dickeya includes plant pathogenic bacteria attacking a wide range of crops and ornamentals as well as a few environmental isolates from water. Defined on the basis of six species in 2005, this genus now includes 12 recognized species. Despite the description of several new species in recent years, the diversity of the genus Dickeya is not yet fully explored. Many strains have been analyzed for species causing diseases on economically important crops, such as for the potato pathogens D. dianthicola and D. solani. In contrast, only a few strains have been characterized for species of environmental origin or isolated from plants in understudied countries. To gain insights in the Dickeya diversity, recent extensive analyzes were performed on environmental isolates and poorly characterized strains from old collections. Phylogenetic and phenotypic analyzes led to the reclassification of D. paradisiaca (containing strains from tropical or subtropical regions) in the new genus, Musicola, the identification of three water species D. aquatica, D. lacustris and D. undicola, the description of a new species D. poaceaphila including Australian strains isolated from grasses, and the characterization of the new species D. oryzae and D. parazeae, resulting from the subdivision of the species D. zeae. Traits distinguishing each new species were identified from genomic and phenotypic comparisons. The high heterogeneity observed in some species, notably for D. zeae, indicates that additional species still need to be defined. The objective of this study was to clarify the present taxonomy of the genus Dickeya and to reassign the correct species to several Dickeya strains isolated before the current classification.

The improvement of guava (Psidium guajava) juice quality using crude multi-enzymatic extracts obtained from yeasts.

Guava juice is cloudy and viscous, which hinders filtration, decreases yield, and causes the loss of quality after its processing and during storage. This study aimed to evaluate enzymatic treatment effects using crude multi-enzymatic extracts (CME) obtained from Rhodotorula mucilaginosa, Rhodotorula orizycola, and Pseudozyma sp. produced by submerse fermentation in the extraction of juice guava. Mixtures of 100 ml of guava pulp and multi-enzymatic extracts proposed by Doehlert planning were incubated under constant agitation at 150 rpm and 50°C, and a Doehlert design was applied as a multivariate optimization strategy. The optimal conditions using the multi-enzymatic extract were: 0.4% (v/v) of CME for 131 min for the multi-enzymatic treatment using Pseudozyma sp.; 3.0% (v/v) of CME for 154 min using the R. mucilaginosa CME; and 5.0% (v/v) of CME for 90 min using R. oryzicola. The maximum viscosity reduction values for the juices treated with the CME of yeasts were 10.33%, 86.38%, and 13.33% for the juices treated with the CME of Pseudozyma sp., R. mucilaginosa, and R. orizycola, respectively. The physical-chemical properties were improved after treatment with CMEs, yielding a reduction of clarity, increase of total soluble solids and reducing sugars, and decreasing the acidity (pH) for all treatments with enzymatic extracts of all strains. The yeasts studied showed a potential for CME production to be applied to juice, improving the quality of the juice, and R. mucilaginosa was the most prominent yeast due to most significant reduction of viscosity in guava juice.

Determination of bound volatiles in blueberries, raspberries, and grapes with an optimized protocol and a validated SPME-GC/MS method.

Bound volatiles are odorless aroma reservoirs that modify flavor when released during food processing, and their determination is important to understand the aroma of fruit beverages. However, the generation of oxidation/degradation artifacts during analyses of glycosidically-bound volatiles has not been compared across fruit species and their dependence on diverse acidic and enzymatic hydrolytic conditions remains unclear. This work aimed to optimize and compare different hydrolytic conditions for the analysis of glycosidically-bound volatiles in blueberries, raspberries, and grapes with a solid-phase microextraction - gas chromatography/mass spectrometry (SPME-GC/MS) methodology. Enzymatic hydrolyses using AR2000® at 100 mg.mL-1 and Pectinex Ultra SPL® at 25-100 µL.mL-1 showed profiles characterized by the expected alcohols, while using AR2000® at 200-400 mg.mL-1 and citric acid at 50-100 mM resulted in profiles defined by artifacts (hydrocarbons, norisoprenoids, and aldehydes). (Z)-3-hexen-1-ol, 3-methyl-1-butanol, linalool, citronellol, and geraniol presented Odor Activity Values (OAV) > 1 for most small fruit genotypes.

A review on pectinase properties, application in juice clarification, and membranes as immobilization support.

Pectic substances cause haziness and high viscosity of fruit juices. Pectinase enzymes are biological compounds that degrade pectic compounds. Nontoxicity and ecofriendly nature make pectinases excellent biocatalysts for juice clarification. However, the poor stability and nonreusability of pectinases trim down the effectiveness of the operation. The immobilization techniques have gained the attention of researchers as it augments the properties of the enzymes. Literature has reported the stability improvement of enzymes like lipase, laccase, hydrogen peroxidase, and cellulase upon immobilization on the membrane. However, only a few research articles divulge pectinase immobilization using a membrane. The catalysis-separation synergy of membrane-reactor has put indelible imprints in industrial applications. Immobilization of pectinase on the membrane can enhance its performance in juice processing. This review delineates the importance of physicochemical and kinematic properties of pectinases relating to the juice processing parameters. It also includes the influence of metal-ion cofactors on enzymes' activity. Considering the support and catalytic-separation facets of the membrane, the prediction of the membrane as support for pectinase immobilization has also been carried out.

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.

New insights in pectinase production development and industrial applications.

Pectinase, a group of pectin degrading enzymes, is one of the most influential industrial enzymes, helpful in producing a wide variety of products with good qualities. These enzymes are biocatalysts and are highly specific, non-toxic, sustainable, and eco-friendly. Consequently, both pectin and pectinase are crucially essential biomolecules with extensive applicatory perception in the biotechnological sector. The market demand and application of pectinases in new sectors are continuously increasing. However, due to the high cost of the substrate used for the growth of microbes, the production of pectinase using microorganisms is limited. Therefore, low-cost or no-cost substrates, such as various agricultural biomasses, are emphasized in producing pectinases. The importance and implications of pectinases are rising in diverse areas, including bioethanol production, extraction of DNA, and protoplast isolation from a plant. Therefore, this review briefly describes the structure of pectin, types and source of pectinases, substrates and strategies used for pectinases production, and emphasizes diverse potential applications of pectinases. The review also has included a list of pectinases producing microbes and alternative substrates for commercial production of pectinase applicable in pectinase-based industrial technology.Key points• Pectinase applications are continuously expanding.• Organic wastes can be used as low-cost sources of pectin.• Utilization of wastes helps to reduce pollution.

Pectinase hydrolysis of guava pulp: effect on the physicochemical characteristics of its juice.

The objective of this research is to assess the effect of enzymatic treatment of guava puree on the physicochemical parameters of the juice. Pectinases from Aspergillus niger were applied to the puree at 43 ± 3 °C under constant stirring. Enzyme concentrations used were: 0.033 % (w/w), 0.055% (w/w), 0.078 % (w/w) and 0.1 % (w/w). For each enzyme concentration, the treatment times were varied from 3 - 90 min. Physicochemical parameters of raw puree and enzymatically treated juice were determined. These were: viscosity, pH, electric conductivity, protein and polyphenol content, galacturonic acid content, color, TSS, and antioxidant capacity. Particle distribution, homogeneity of raw puree and juice samples dried extracts were assessed using a Field Emission Scanning Electron Microscopy (FESEM). A 91% viscosity decrease was recorded for each enzyme concentration after 3 min of enzyme reaction. That drecrase was accompanied by an increase in galacturonic acid content with increasing depectinization factors. Enzyme treatment of guava puree led to a decrease in pH, protein and polyphenol contents and an increase in conductivity and color. Analysis of FESEM images of guava samples bestowed a decrease in particle size, a scattering of particles in the medium, an increase in continuous phase proportion and an improvement of sample homogeneity with increasing values of processing parameters, due to the breaking-down of bigger particles and the solubilization during depectinization.

Systems Biology Applied to the Study of Papaya Fruit Ripening: The Influence of Ethylene on Pulp Softening.

Papaya is a fleshy fruit that undergoes fast ethylene-induced modifications. The fruit becomes edible, but the fast pulp softening is the main factor that limits the post-harvest period. Papaya fast pulp softening occurs due to cell wall disassembling coordinated by ethylene triggering that massively expresses pectinases. In this work, RNA-seq analysis of ethylene-treated and non-treated papayas enabled a wide transcriptome overview that indicated the role of ethylene during ripening at the gene expression level. Several families of transcription factors (AP2/ERF, NAC, and MADS-box) were differentially expressed. ACO, ACS, and SAM-Mtase genes were upregulated, indicating a high rate of ethylene biosynthesis after ethylene treatment. The correlation among gene expression and physiological data demonstrated ethylene treatment can indeed simulate ripening, and regulation of changes in fruit color, aroma, and flavor could be attributed to the coordinated expression of several related genes. Especially about pulp firmness, the identification of 157 expressed genes related to cell wall metabolism demonstrated that pulp softening is accomplished by a coordinated action of several different cell wall-related enzymes. The mechanism is different from other commercially important fruits, such as strawberry, tomato, kiwifruit, and apple. The observed behavior of this new transcriptomic data confirms ethylene triggering is the main event that elicits fast pulp softening in papayas.

Postharvest Treatment of Hydrogen Sulfide Delays the Softening of Chilean Strawberry Fruit by Downregulating the Expression of Key Genes Involved in Pectin Catabolism.

Hydrogen sulfide (H2S) plays several physiological roles in plants. Despite the evidence, the role of H2S on cell wall disassembly and its implications on fleshy fruit firmness remains unknown. In this work, the effect of H2S treatment on the shelf-life, cell wall polymers and cell wall modifying-related gene expression of Chilean strawberry (Fragaria chiloensis) fruit was tested during postharvest storage. The treatment with H2S prolonged the shelf-life of fruit by an effect of optimal dose. Fruit treated with 0.2 mM H2S maintained significantly higher fruit firmness than non-treated fruit, reducing its decay and tripling its shelf-life. Additionally, H2S treatment delays pectin degradation throughout the storage period and significantly downregulated the expression of genes encoding for pectinases, such as polygalacturonase, pectate lyase, and expansin. This evidence suggests that H2S as a gasotransmitter prolongs the post-harvest shelf-life of the fruit and prevents its fast softening rate by a downregulation of the expression of key pectinase genes, which leads to a decreased pectin degradation.

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

The chimeric GaaR-XlnR transcription factor induces pectinolytic activities in the presence of D-xylose in Aspergillus niger.

Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin. KEY POINTS: • Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9. • PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants. • Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.

Biopolymers and nanostructured materials to develop pectinases-based immobilized nano-biocatalytic systems for biotechnological applications.

Pectinases are the emerging enzymes of the biotechnology industry with a 25% share in the worldwide food and beverage enzyme market. These are green and eco-friendly tools of nature and hold a prominent place among the commercially produced enzymes. Pectinases exhibit applications in various industrial bioprocesses, such as clarification of fruit juices and wine, degumming, and retting of plant fibers, extraction of antioxidants and oil, fermentation of tea/coffee, wastewater remediation, modification of pectin-laden agro-industrial waste materials for high-value products biosynthesis, manufacture of cellulose fibres, scouring, bleaching, and size reduction of fabric, cellulosic biomass pretreatment for bioethanol production, etc. Nevertheless, like other enzymes, pectinases also face the challenges of low operational stability, recoverability, and recyclability. To address the above-mentioned problems, enzyme immobilization has become an eminently promising approach to improve their thermal stability and catalytic characteristics. Immobilization facilitates easy recovery and recycling of the biocatalysts multiple times, leading to enhanced performance and commercial feasibility.In this review, we illustrate recent developments on the immobilization of pectinolytic enzymes using polymers and nanostructured materials-based carrier supports to constitute novel biocatalytic systems for industrial exploitability. The first section reviewed the immobilization of pectinases on polymers-based supports (ca-alginate, chitosan, agar-agar, hybrid polymers) as a host matrix to construct robust pectinases-based biocatalytic systems. The second half covers nanostructured supports (nano-silica, magnetic nanostructures, hybrid nanoflowers, dual-responsive polymeric nanocarriers, montmorillonite clay), and cross-linked enzyme aggregates for enzyme immobilization. The biotechnological applications of the resulted immobilized robust pectinases-based biocatalytic systems are also meticulously vetted. Finally, the concluding remarks and future recommendations are also given.

Phylogenetic Relationships and Potential Functional Attributes of the Genus Parapedobacter: A Member of Family Sphingobacteriaceae.

The genus Parapedobacter was established to describe a novel genus within the family Sphingobacteriaceae and derives its name from Pedobacter, with which it is shown to be evolutionarily related. Despite this, Parapedobacter and Pedobacter do not share very high 16S rRNA gene sequence similarities. Therefore, we hypothesized whether these substantial differences at the 16S rRNA gene level depict the true phylogeny or that these genomes have actually diverged. Thus, we performed genomic analysis of the four available genomes of Parapedobacter to better understand their phylogenomic position within family Sphingobacteriaceae. Our results demonstrated that Parapedobacter is more closely related to species of Olivibacter, as opposed to the genus Pedobacter. Further, we identified a significant class of enzymes called pectinases with potential industrial applications within the genomes of Parapedobacter luteus DSM 22899T and Parapedobacter composti DSM 22900T. These enzymes, specifically pectinesterases and pectate lyases, are presumed to have largely different catalytic activities based on very low sequence similarities to already known enzymes and thus may be exploited for industrial applications. We also determined the complete Bacteroides aerotolerance (Bat) operon (batA, batB, batC, batD, batE, hypothetical protein, moxR, and pa3071) within the genome of Parapedobacter indicus RK1T. This expands the definition of genus Parapedobacter to containing members that are able to tolerate oxygen stress using encoded oxidative stress responsive systems. By conducting a signal propagation network analysis, we determined that BatD, BatE, and hypothetical proteins are the major controlling hubs that drive the expression of Bat operon. As a key metabolic difference, we also annotated the complete iol operon within the P. indicus RK1T genome for utilization of all three stereoisomers of inositol, namely myo-inositol, scyllo-inositol, and 1D-chiro-inositol, which are abundant sources of organic phosphate found in soils. The results suggest that the genus Parapedobacter holds promising applications owing to its environmentally relevant genomic adaptations, which may be exploited in the future.

Effect of the Fiber Treatment on the Stiffness of Date Palm Fiber Reinforced PP Composites: Macro and Micromechanical Evaluation of the Young's Modulus.

The present work aims at determining the potential of date palm wastes to be applied as reinforcement in polypropylene. For this, fibers were separated from the raw biomass via mechanical defibration in Sprout Waldron equipment. Then, three different treatment strategies were adopted on the fibers, being (i) mechanical, (ii) chemical with NaOH, and (iii) enzymatical with xylanases and pectinases. Fibers were characterized in terms of chemical composition, morphology and SEM. Additionally, PP was reinforced with date palm fibers and the composites' stiffness was evaluated. The analysis was performed from a macro and micro mechanical viewpoint. The incorporation of 40 and 60 wt.% of DPF-E enhanced the Young's modulus of PP by 205 and 308%, respectively. The potential of enzymatically treated fibers to replace glass fibers in composites was studied, exhibiting similar stiffening abilities at 60 wt.% of date palm fiber (6.48 GPa) and 40% of glass fibers (6.85 GPa). The intrinsic Young's modulus of the fibers was set at values around 16, 20 and 24 GPa for mechanical, chemical and enzymatic fibers. From the micromechanical analysis, the efficiency of the reinforcement as well as the contribution of the length and orientation to the Young's modulus of the composite was evaluated.

The Homogalacturonan Deconstruction System of Paenibacillus amylolyticus 27C64 Requires No Extracellular Pectin Methylesterase and Has Significant Industrial Potential.

Paenibacillus amylolyticus 27C64, a Gram-positive bacterium with diverse plant cell wall polysaccharide deconstruction capabilities, was isolated previously from an insect hindgut. Previous work suggested that this organism's pectin deconstruction system differs from known systems in that its sole pectin methylesterase is cytoplasmic, not extracellular. In this work, we have characterized the specific roles of key extracellular pectinases involved in homogalacturonan deconstruction, including four pectate lyases and one pectin lyase. We show that one newly characterized pectate lyase, PelC, has a novel substrate specificity, with a lower Km for highly methylated pectins than for polygalacturonic acid. PelC works synergistically with PelB, a high-turnover exo-pectate lyase that releases Δ4,5-unsaturated trigalacturonate as its major product. It is likely that PelC frees internal stretches of demethylated homogalacturonan which PelB can degrade. We also show that the sole pectin lyase has a high kcat value and rapidly depolymerizes methylated substrates. Three cytoplasmic GH105 hydrolases were screened for the ability to remove terminal unsaturated galacturonic acid residues from oligogalacturonide products produced by the action of extracellular lyases, and we found that two are active on demethylated oligogalacturonides. This work confirms that efficient homogalacturonan deconstruction in P. amylolyticus 27C65 does not require extracellular pectin methylesterase activity. Three of the extracellular lyases studied in this work are also thermostable, function well over a broad pH range, and have significant industrial potential.IMPORTANCE Pectin is an important structural polysaccharide found in most plant cell walls. In the environment, pectin degradation is part of the decomposition process that turns over dead plant material and is important to organisms that feed on plants. Industrially, pectinases are used to improve the quality of fruit juices and can also be used to process coffee cherries or tea leaves. These enzymes may also prove useful in reducing the environmental impact of paper and cotton manufacturing. This work is significant because it focuses on a Gram-positive bacterium that is evolutionarily distinct from other well-studied pectin-degrading organisms and differs from known systems in key ways. Most importantly, a simplified extracellular deconstruction process in this organism is able to break down pectins without first removing the methyl groups that inhibit other systems. Moreover, some of the enzymes described here have the potential to improve industrial processes that rely on pectin deconstruction.

Extraction of sunflower head pectin with superfine grinding pretreatment.

Effect of superfine grinding on pectin extraction was investigated. Sunflower heads were grinded into microparticles of ~50 µm, thus pectin chains were fully exposed due to cell wall breakdown. A good pectin yield of 14.5 ± 0.36% (w/w) was subsequently achieved at mild conditions (pH 5.0, 25 °C, 2 h) associated with 0.8% (w/v) sodium citrate (SC). However, the molecule weight of pectin was greatly reduced (Mw = 7.87 ± 0.21 kDa) due to the action of endo-pectinases. With heating (pH 5.0, 85 °C for 20 min, 25 °C for 1.5 h) the endo-pectinases were effectively inhibited, thus Mw was increased to 338.07 ± 12.37 kDa. With superfine grinding, pectin extracted with different conditions presented various properties. Pectin extracted at pH 2.5 had higher esterification degree (DE, ~40%) and gelled at 3% (w/v) concentration. In contrast, pectin extracted under mild condition with low DE (21%) cannot gel at the same concentration. Conclusively, superfine grinding has potential application in pectin extraction.

Effect of Bacillus tequilensis SALBT crude extract with pectinase activity on demucilation of coffee beans and juice clarification.

Pectinases are a group of enzymes, which catalyze the breakdown of pectin with numerous applications in various industries. Microbes are the predominant pectinase producers. In the present study, bacterial species were isolated from the soil of a vegetable and fruit dump yard area in a market. The species screened and isolated were identified as Bacillus tequilensis SALBT, and the media and culture conditions were optimized for enhanced production of total pectinases. Maximum pectinolytic activity was observed with 1.5% (w/v) pectin concentration with a combination of yeast extract as nitrogen source and MgSO4 as a metal ion source. Carbon/nitrogen in 2:1 ratio (w/v) yielded the maximum pectinase production with pH and temperature of the medium of 7.5°C and 40°C, respectively. Pectinase activity was determined by the dinitrosalicylic acid method. The pectinase production was relatively stable in the presence of various surfactants like Tween (20, 40, 60, and 80) and sodium dodecyl sulfate (SDS), whereas Triton X-100 showed an inhibitory effect. Mass production of the enzyme in optimized media and partial purification was performed by ammonium sulfate precipitation followed by dialysis. The approximate molecular weight of the partially purified pectinase was found to be 35 kDa by SDS-polyacrylamide gel electrophoresis. Application studies such as demucilaging coffee beans and juice clarification were also performed. The findings revealed that B. tequilensis SALBT with pectinase activity has the ability to remove the mucilage layer of pulped coffee seeds, and the partially purified pectinases found to be effective in clarifying juice.