Draft genome sequence of Raoultella terrigena strain Ech2A causing soft rot on sweet pepper (Capsicum annuum).

We report the draft genome sequence of Raoultella terrigena strain Ech2A causing soft rot on pepper. To verify pathogenicity, Koch's postulates were performed on sweet pepper. Genes encoding pectinolytic enzymes were found in the genome.

Proteomic analysis of Viscozyme L and its major enzyme components for pectic substrate degradation.

Enzymatic degradation of plant biomass requires the coordinated action of various enzymes. In this study, the production of reducing sugars from pectic substrates and sugar beet pulp (SBP) was investigated and compared using commercial enzyme preparations, including M2, pectinase (E1), Viscozyme L (V-L) and L-40. V-L, a cellulolytic enzyme mix produced by Aspergillus sp. was further evaluated as the most robust enzyme cocktail with the strongest SBP degradation ability in terms of the release of monosaccharides, methanol, and acetate from SBP. Mass-spectrometry-based proteomics analysis of V-L revealed 156 individual proteins. Of these, 101 proteins were annotated as containing a carbohydrate-active enzyme module. Notably, of the 50 most abundant proteins, ca. 44 % were predicted to be involved in pectin degradation. To reveal the role of individual putative key enzymes in pectic substrate decomposition, two abundant galacturonases (PglA and PglB), were heterologously expressed in Pichia pastoris and further characterized. PglA and PglB demonstrated maximum activity at 57 °C and 68 °C, respectively, and exhibited endo-type cleavage patterns towards polygalacturonic acid. Further studies along this line may lead to a better understanding of efficient SBP degradation and may help to design improved artificial enzyme mixtures with lower complexity for future application in biotechnology.

The pectinolytic activity of Burkholderia cepacia and its application in the bioscouring of cotton knit fabric.

BACKGROUND: Enzymatic catalysis in different industrial applications is often preferred over chemical methods due to various advantages, such as higher specificity, greater efficiency, and less environmental footprint. Pectinases are a group of enzymes that catalyze the degradation of pectic compounds, the key components of plant middle lamella and the primary cell wall. Pectinases have found applications in multiple industrial processes, including cotton bioscouring, fruit juice extraction and its clarification, plant fiber degumming, paper making, plant biomass liquefaction, and saccharification, among others. The purpose of this study was to taxonomically characterize a bacterial species exhibiting pectinolytic activities and assess its pectinolytic activity qualitatively and quantitatively, as well as test its bioscouring potential. RESULTS: Here, we report that Burkholderia cepacia, a previously unknown species with pectinolytic activity, exerts such activity comparable to commercially used pectinase enzymes in the textile industry, but requires less temperature for activity. CONCLUSION: Quantitative evaluation of enzyme activity indicates the potential of the bacterial species for use in the bioscouring of cotton knit fabric.

Yeasts are essential for mucilage degradation of coffee beans during wet fermentation.

During wet fermentation, mucilage layers in coffee cherries must be removed completely. To explain mucilage degradation, several controversial hypotheses have been proposed. The aim of this work was to improve our understanding of the kinetics of mucilage breakdown. Pulped coffee beans were wet fermented with seven different treatments for 36 h. Endogenous bacteria and yeasts are selectively suppressed, and pectinases or lactic acid are added. They also involve maintaining the beans at pH 7 throughout fermentation and using spontaneous fermentation without additives as a control. During spontaneous fermentation, yeast and lactic acid bacteria were detected and significantly increased to 5.5 log colony-forming units (CFU)/mL and 5.2 log CFU/mL, respectively. In the first 12 h of fermentation, there was a significant degree of endogenous pectinolytic activity, which resulted in partly destroyed beans in the absence of microorganisms. By adding pectinase and lactic acid to the fermentation mass, the breakdown process was accelerated in less than 8 h. When yeast was present throughout the fermentation, complete degradation was achieved. Bacteria played no critical role in the degradation. Klebsiella pneumoniae and Erwinia soli were found in a lower population and showed weaker pectinolytic activities compared to Hanseniaspora uvarum and Pichia kudriavzevii. During wet fermentation, mucilage degradation appears to be mediated by endogenous enzymes at the early stage, whereas microbial contributions, mainly yeasts, occur subsequently. H. uvarum and P. kudriavzevii may be promising candidates to be tested in future studies as coffee starter cultures to better control the mucilage degradation process.

Valorisation of wheat straw into paper with improved quality characteristics using ultrafiltered xylano-pectinolytic pulping approach.

This study has been conducted to assess the pulpability of ultrafiltered pectinase and xylanase in pulping of wheat straw. Best biopulping conditions were achieved using 107 and 250 IU of pectinase and xylanase, respectively, per gram of wheat straw, 180 min of treatment period, one gram: 10 m1 material to liquor ratio, 8.5 pH and 55 °C temperature. Ultrafiltered enzymatic treatment improved the pulp yield (6.18%), brightness (17.83%), along with reduced rejections (61.01%) and kappa number (16.95%) as compared to chemically synthesized pulp. Biopulping of wheat straw saved 14% alkali dose, with nearly same optical properties, as obtained under 100% alkali dose. Bio-chemically pulped samples resulted an increase in breaking length, tear index, burst index, viscosity, double fold and Gurley porosity by 6.05%, 18.64%, 26.42%, 7.94%, 21.6% and 15.38%, respectively, in comparison to control pulp samples. Bleached-biopulped samples showed an improvement in breaking length, tear index, burst index, viscosity, double fold number, and Gurley porosity by 7.39%, 3.55%, 28.82%, 9.1%, 53.66%, and 30.95% respectively. Thus, biopulping of wheat straw with ultrafiltered enzymes lowers alkali consumption and also improves the paper quality. This is the first study reporting, eco-friendly biopulping, for producing better quality wheat straw pulp, using ultrafiltered enzymes.

Production of eco-friendly and better-quality sugarcane bagasse paper using crude xylanase and pectinase biopulping strategy.

This research aimed to investigate the efficiency of crude xylanase-pectinase in pulping of sugarcane bagasse. Optimum biopulping was obtained, using xylanase-pectinase dose 200-60 IU/g, bagasse/liquid ratio 1:10 and 1.0% Tween 80 concentration at 55 °C temperature, pH 8.5 and period of treatment 180 min. Treatment of sugarcane bagasse samples with these enzymes generated pulp with lower rejections (58.76%), total solids (12.64%), kappa number (47.77%), higher screened pulp yield (10.66%), along with enhanced optical and physical properties, in comparison with a chemical pulp. Bagasse biopulping resulted in a 13% decrease in alkali dose to obtain the optical and physical properties similar to those achieved under the 100% alkali dose. The breaking length, burst factor, tear index, double fold, gurley porosity and viscosity were improved by 15.19, 37.64, 2.47, 37.77, 35 and 23.17%, respectively, after bleaching treatment of biopulped samples. Thus, enzymatic pulping is an eco-friendly environmentally sustainable approach, since it reduces the use of pulping chemicals and simultaneously improves the paper quality. This is the first report, showing pulping of sugarcane bagasse, with crude xylanase-pectinase, produced by an isolate.

Characterization of Novel Pectinolytic Enzymes Derived from the Efficient Lignocellulose Degradation Microbiota.

Diverse pectinolytic enzymes are widely applied in the food, papermaking, and other industries, and they account for more than 25% of the global industrial enzyme demands. Efficient lignocellulose degradation microbiota are reservoirs of pectinolytic enzymes and other lignocellulose-degrading genes. Metagenomics has been widely used to discover new pectinolytic enzymes. Here, we used a metagenomic strategy to characterize pectinolytic genes from one efficient lignocellulose-degrading microbiota derived from pulp and paper wastewater treatment microbiota. A total of 23 predicted full-length GH28 and PL1 family pectinolytic genes were selectively cloned and expressed in Escherichia coli, and 5 of the expressed proteins had pectinolytic activities. Among them, the characterization of one pectinolytic enzyme, PW-pGH28-3, which has a 58.4% identity with an exo-polygalacturonase gene of Aquipluma nitroreducens, was further investigated. The optimal pH and optimal temperature of PW-pGH28-3 were 8.0 and 40 °C, respectively, and its pectinolytic activity at the optimal condition was 13.5 ± 1.1 U/mg protein. Bioinformatics analyses and structural modeling suggest that PW-pGH28-3 is a novel secretory exo-polygalacturonase, which is confirmed by its hydrolysates of polygalacturonic acid. The detection of PW-pGH28-3 and other pectinolytic genes showed that efficient lignocellulose degradation microbiota could provide potential efficient pectinolytic enzymes for industrial application. In the future, improving metagenomic screening efficiency would discover efficient lignocellulose-degrading enzymes and lead to the sustainable and green utilization of lignocellulose.

Pectinases Secretion by Saccharomyces cerevisiae: Optimization in Solid-State Fermentation and Identification by a Shotgun Proteomics Approach.

A sequential design strategy was applied to optimize the secretion of pectinases by a Saccharomyces cerevisiae strain, from Brazilian sugarcane liquor vat, on passion fruit residue flour (PFRF), through solid-state fermentation (SSF). A factorial design was performed to determine the influence variables and two rotational central composite designs were executed. The validated experimental result was of 7.1 U mL-1 using 50% PFRF (w/w), pH 5, 30 °C for 24 h, under static SSF. Polygalacturonase, pectin methyl esterase, pectin-lyase and pectate-lyase activities were 3.5; 0.08; 3.1 and 0.8 U mL-1, respectively. Shotgun proteomics analysis of the crude extract enabled the identification of two pectin-lyases, one pectate-lyase and a glucosidase. The crude enzymatic extract maintained at least 80% of its original activity at pH values and temperatures ranging from 2 to 8 and 30 to 80 °C, respectively, over 60 min incubation. Results revealed that PFRF might be a cost-effective and eco-friendly substrate to produce pectinases. Statistical optimization led to fermentation conditions wherein pectin active proteins predominated. To the extent of our knowledge, this is the first study reporting the synthesis of pectate lyase by S. cerevisiae.

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.

An eco-friendly biocatalytic process for producing better quality paper from sugarcane bagasse using ultrafiltered enzymes concoction.

In the current study, pretreatment of sugarcane bagasse has been carried out with ultrafiltered xylano-pectinolytic enzymes, before conventional chemical bleaching process. Optimized enzymatic dose (4 IU xylanase and 1.2 IU pectinase per g of oven dried pulp) and retention time (180 min) were determined on the basis of maximum decrement in kappa number (from 20.93 to 15.32), release of maximum sugars (7.4 mg/g) as well as attainment of maximum brightness (25.1% ISO), whiteness (from - 57.3 to - 41.9) and minimum yellowness (from 48.7 to 35.3) of the pulp samples. Enzymatically treated samples also showed release of phenolic, lignin and hydrophobic compounds in their filtrates. Nearly 30% decrement in the exhaustion of bleaching chemical dose was detected as compared to control samples. The physical properties such as tear index, burst index, double fold number, breaking length, gurley porosity and viscosity of enzymo-chemically treated bagasse pulp samples were improved by 6.68%, 33.86%, 22.92%, 13.43%, 17.5% and 9.64%, respectively. Additionally, a decrement of 36.75% and 28.29% in the values of BOD and COD of the effluents was also noted, which demonstrated the fact that, inclusion of enzymes in chemical based protocols of paper and pulp industries could be a highly beneficial and eco-friendly approach in upcoming decades. This is the first report mentioning the effect of ultrafiltered xylano-pectinolytic enzymes concoction on sugarcane bagasse pulp.

Machine learning prediction of novel pectinolytic enzymes in Aspergillus niger through integrating heterogeneous (post-) genomics data.

Pectinolytic enzymes are a variety of enzymes involved in breaking down pectin, a complex and abundant plant cell-wall polysaccharide. In nature, pectinolytic enzymes play an essential role in allowing bacteria and fungi to depolymerize and utilize pectin. In addition, pectinases have been widely applied in various industries, such as the food, wine, textile, paper and pulp industries. Due to their important biological function and increasing industrial potential, discovery of novel pectinolytic enzymes has received global interest. However, traditional enzyme characterization relies heavily on biochemical experiments, which are time consuming, laborious and expensive. To accelerate identification of novel pectinolytic enzymes, an automatic approach is needed. We developed a machine learning (ML) approach for predicting pectinases in the industrial workhorse fungus, Aspergillus niger. The prediction integrated a diverse range of features, including evolutionary profile, gene expression, transcriptional regulation and biochemical characteristics. Results on both the training and the independent testing dataset showed that our method achieved over 90 % accuracy, and recalled over 60 % of pectinolytic genes. Application of the ML model on the A. niger genome led to the identification of 83 pectinases, covering both previously described pectinases and novel pectinases that do not belong to any known pectinolytic enzyme family. Our study demonstrated the tremendous potential of ML in discovery of new industrial enzymes through integrating heterogeneous (post-) genomimcs data.

Ultrafiltered biopulping strategy for the production of good quality pulp and paper from sugarcane bagasse.

This research was carried out with an objective to examine the efficacy of ultrafiltered xylano-pectinolytic enzymes in pulping of sugarcane bagasse. Maximum biopulping was achieved with enzyme dose of xylanase (175 IU / g bagasse) and pectinase (75 IU / g bagasse) at treatment period of 180 min. The temperature, pH, and bagasse to liquid ratio for biopulping experiments were kept constant at 55o C, 8.5, and 1:10 (g/ml), respectively. The ultrafiltered biopulping improved chemical pulping, resulted in 25.11%, 9.17% increase in brightness, unscreened pulp production and 11.81, 59.50, and 49.14% decrease in total solids, rejections. and kappa number, respectively. The bagasse biopulping also resulted in 15% decrease of alkali load to attain similar kappa number and optical properties as obtained under 100% alkali dosage. Ultrafiltered biopulped-unbleached samples showed significant increase in breaking length (13.55%), burst index (40.21%), tear index (19.04%), double fold (42.5%), Gurley porosity (28.21%) and viscosity (13.37%) in comparison with non-enzymatically treated control pulp samples. In comparison with non biotreated-bleached pulp samples, ultrafiltered biopulped-bleached samples also resulted in higher burst index (56.80%), breaking length (17.38%), double fold (39.58%), tear index (3.38%), viscosity (30.68%), and Gurley porosity (52.50%). This environmentally sustainable ultrafiltered biopulping approach for sugarcane bagasse has the potential to decrease the demand of chemicals, ultimately pollution along with enhance the quality of paper.

Eco-friendly pulping of wheat straw using crude xylano-pectinolytic concoction for manufacturing good quality paper.

In this study, suitability of xylano-pectinolytic enzymes in pulping of wheat straw has been explored. The suitable biopulping conditions were optimized, with xylanase dose of 400 and pectinase dose of 120 IU/g wheat straw, 1:10 (g/ml) material to liquid ratio, 55 °C temperature, 3 h treatment time, 0.75% Tween 80 and pH 8.5. Enzymatic pretreatment efficiently increased the pulpability of wheat straw, generated pulp with higher yield, lower kappa number (15.67%) and rejections (59.65%) in comparison with chemical pulp. The brightness of pretreated wheat straw pulp with enzyme was 16.04% higher than that of the non-enzyme treated wheat straw pulp. The biopulping resulted in 12% reduction of pulping chemicals along with more residual alkali content, in order to achieve similar optical and chemical properties as obtained by 100% chemically treated pulp. Physical properties of pulp also improved after enzymatic pretreatment, increasing burst index (26.50%), tear index (18.22%) and breaking length (5.56%). The enzyme plus chemical (88% pulping chemicals) treated pulp showed improvement in brightness and whiteness, with reduction in yellowness at all bleaching stages. In comparison with chemically bleached pulp, biopulp with reduced alkali dose (88%) had higher breaking length (6.63%), double fold number (51.28%), tear index (2.83%), burst index (24.31%), along with increased viscosity (6.12%) and Gurley porosity (27.50%). These results clearly suggest that biopulping of wheat straw with xylano-pectinolytic enzymes can reduce chemical loading during soda-anthraquinone pulping and also improve the quality of paper. This is the first report demonstrating the biopulping of wheat straw using crude xylano-pectinolytic enzymes.

Lon Protease Is Important for Growth Under Stressful Conditions and Pathogenicity of the Phytopathogen, Bacterium Dickeya solani.

The Lon protein is a protease implicated in the virulence of many pathogenic bacteria, including some plant pathogens. However, little is known about the role of Lon in bacteria from genus Dickeya. This group of bacteria includes important potato pathogens, with the most aggressive species, D. solani. To determine the importance of Lon for pathogenicity and response to stress conditions of bacteria, we constructed a D. solani Δlon strain. The mutant bacteria showed increased sensitivity to certain stress conditions, in particular osmotic and high-temperature stresses. Furthermore, qPCR analysis showed an increased expression of the lon gene in D. solani under these conditions. The deletion of the lon gene resulted in decreased motility, lower activity of secreted pectinolytic enzymes and finally delayed onset of blackleg symptoms in the potato plants. In the Δlon cells, the altered levels of several proteins, including virulence factors and proteins associated with virulence, were detected by means of Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) analysis. These included components of the type III secretion system and proteins involved in bacterial motility. Our results indicate that Lon protease is important for D. solani to withstand stressful conditions and effectively invade the potato plant.

Identification of a gene encoding the last step of the L-rhamnose catabolic pathway in Aspergillus niger revealed the inducer of the pathway regulator.

In fungi, L-rhamnose (Rha) is converted via four enzymatic steps into pyruvate and L-lactaldehyde, which enter central carbon metabolism. In Aspergillus niger, only the genes involved in the first three steps of the Rha catabolic pathway have been identified and characterized, and the inducer of the pathway regulator RhaR remained unknown. In this study, we identified the gene (lkaA) involved in the conversion of L-2-keto-3-deoxyrhamnonate (L-KDR) into pyruvate and L-lactaldehyde, which is the last step of the Rha pathway. Deletion of lkaA resulted in impaired growth on L-rhamnose, and potentially in accumulation of L-KDR. Contrary to ΔlraA, ΔlrlA and ΔlrdA, the expression of the Rha-responsive genes that are under control of RhaR, were at the same levels in ΔlkaA and the reference strain, indicating the role of L-KDR as the inducer of the Rha pathway regulator.

Microbial synergy between Pichia kudriazevii YS201 and Bacillus subtilis BS38 improves pulp degradation and aroma production in cocoa pulp simulation medium.

Interactions between two major microorganisms from Ivorian cocoa fermentation, namely Bacillus subtilis BS38 and Pichia kudriazevii YS201, were investigated during fermentation in cocoa pulp simulation medium. The strains were mutually inhibitory, with Bacillus being more susceptible to this antagonistic effect than Pichia. However, both strains yielded different pulp-degrading enzymes, namely polygalacturonase (PG) from Pichia and pectate lyase (Pel) from Bacillus, that cooperate to efficiently breakdown pectin and vegetable pulp. The quantification of aromas from microbial cultures using Gas Chromatography-Mass Spectroscopy (GC-MS) coupled with headspace microextration (SPME) method, showed that P. kudriazevii produce mainly alcohols such as ethanol (63.165 g/L), phenylethanol (1.005 g/L), methylbutanol (0.138 g/L) and esters, notably ethyl acetate (0.037 g/L) and isoamyl acetate (0.032 g/L). The volatile fraction produced by Bacillus was dominated by butanediol (5.707 g/L), acetoin (1.933 g/L), phenylethanol (0.035 g/L) and acetic acid (0.034 g/L). In co-culture, Bacillus produced low levels of aroma compounds whereas a moderate decrease in the production of these compounds was observed in the yeasts strain. Thus, the dominant aromas present in the co-culture were mainly those from the yeasts strain; however, a 1.37 fold increase of ethanol production was observed in co-culture indicating a synergy between the strains. This study showed that cooperation between B. subtilis BS38 and P. kudriazevii YS201 leads principally to increasing pulp degradation and ethanol production, known as desirable properties for a well processing of cocoa fermentation.

Development of novel economical methodologies for zymographic analysis of purified xylano-pectinolytic enzymes using agrowaste-based substrates.

In this study, zymographic analysis for xylanase and pectinase enzymes has been carried out using agrowaste residues, wheat bran and citrus peel as well as their extracts. Isozymic forms of xylanase as well as pectinase enzyme displayed comparable zymographic bands onto agar petriplates containing either commercial substrates (xylan and pectin), agrowaste-based substrates (wheat bran and citrus peel), or polysaccharides extracted from these agrowastes (crude xylan and pectin extracted from wheat bran and citrus peel, respectively), indicating the fact that agro residues and their extracts can be utilized as a substitute of cost-intensive commercial substrates, xylan and pectin for zymographic analysis. This is the first report revealing the zymographic analysis of xylano-pectinolytic enzymes using agro-based solid residues particles or polysaccharides extracted from agro-based residues.

Antioxidant and Potentially Anti-Inflammatory Activity of Anthocyanin Fractions from Pomace Obtained from Enzymatically Treated Raspberries.

Raspberry pomace was obtained from raspberries subjected to enzymatic maceration using three commercial pectinolytic preparations (Pectinex Ultra SP-L, Pectinex Yield Mash, and Ultrazym AFP-L). Phenolic compounds were extracted and anthocyanin fractions were isolated using the SPE solid phase extraction technique. In the separated anthocyanin fractions, the content of individual compounds was determined by the HPLC technique and the antioxidant activity was assessed with four complementary methods (DPPH and ABTS radical scavenging activity, chelating Fe(II) power, and ferric reducing power). Potential anti-inflammatory properties were also identified as the ability to inhibit the activity of lipoxygenase and cyclooxygenase 2. For these enzymes, the type of inhibition was determined based on the Lineweaver-Burke plot.

A novel and cost-effective methodology for enhanced production of industrially valuable alkaline xylano-pectinolytic enzymes cocktail in short solid-state fermentation cycle.

The aim of this study was to enhance the production of xylano-pectinolytic enzymes concurrently and also to reduce the fermentation period. In this study, the effect of agro-residues extract-based inoculum on yield and fermentation time of xylano-pectinolytic enzymes was studied. Microbial inoculum and fermentation media were supplemented with xylan and pectin polysaccharides derived from agro-based residues. Enzymes production parameters were optimized through two-stage statistical design approach. Under optimized conditions (temperature 37°C, pH 7.2, K2 HPO4 0.22%, MgSO4 0.1%, gram flour 5.6%, substrate: moisture ratio 1:2, inoculum size 20%, agro-based crude xylan in production media 0.45%, and agro-based crude xylan-pectin in inoculum 0.13%), nearly 28,255 ± 565 and 9,202 ± 193 IU of xylanase and pectinase, respectively, were obtained per gram of substrate in a time interval of 6 days only. The yield of both xylano-pectinolytic enzymes was enhanced along with a reduction of nearly 24 h in fermentation time in comparison with control, using polysaccharides extracted from agro-residues. The activity of different types of pectinase enzymes such as exo-polymethylgalacturonase (exo-PMG), endo-PMG, exo-polygalacturonase (exo-PG), endo-PG, pectin lyase, pectate lyase, and pectin esterase was obtained as 1,601, 12.13, 5637, 24.86, 118.62, 124.32, and 12.56 IU/g, respectively, and was nearly twofold higher than obtained for all seven types in control samples. This is the first report mentioning the methodology for enhanced production of xylano-pectinolytic enzymes in short solid-state fermentation cycle using agro-residues extract-based inoculum and production media.

Stability/activity features of the main enzyme components of rohapect 10L.

Rohapect 10L is an enzyme cocktail commercialized for juice clarification. Here, we characterized the activity and stability of five enzymatic activities present in this cocktail: total pectinase (PE), polygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME), and total cellulase (CE) activities. All these enzyme activities have the maximum activity and stability at pH 4, conditions near those found in most fruit juices. However, if the enzymes need to be handled under different conditions (e.g., to immobilize them), their stability becomes extremely low in some cases, just at pH values slightly higher than the optimal one. For example, at pH 10 only CE was reasonably stable at 25°C, while many other enzyme activities were rapidly almost inactivated, even at 4°C. For these cases, different additives were evaluated, and we found that polyethylene glycol was positive or very positive for all enzyme stabilities, allowing keeping reasonable activities after several hours at pH 10 and 25°C. Another additive, that is, dextran, has a small positive effect for PE, PG, and CE, and a very positive effect for PL, albeit significantly destabilizing PME. Thus, the handling and use of this extract requires some care when is performed out of optimal conditions.