Transcriptome analysis insight into ethylene metabolism and pectinase activity of apricot (Prunus armeniaca L.) development and ripening.

Ethylene metabolism is very important for climacteric fruit, and apricots are typical climacteric fruit. The activity of pectinase is closely related to fruit firmness, which further affects fruit quality. To better understand ethylene metabolism, pectinase activity and their molecular regulation mechanisms during the development and ripening of apricot fruit, ethylene metabolism, pectinase activity and the "Luntaibaixing" apricot fruit transcriptome were analyzed at different developmental stages. Ethylene metabolic precursors, enzyme activities and ethylene release increased during fruit development and ripening, with significant differences between the ripening stage and other stages (P < 0.05). Fruit firmness decreased significantly from the S1 to S5 stages, and polygalacturonase, pectin methylesterase, and pectin lyase activities were significantly higher in the S5 stage than in other stages. RNA sequencing (RNA-seq) analysis of fruit resulted in the identification of 22,337 unigenes and 6629 differentially expressed genes (DEGs) during development and ripening, of which 20,989 unigenes are annotated in public protein databases. In functional enrichment analysis, DEGs among the three stages were found to be involved in plant hormone signal transduction. Four key genes affecting ethylene metabolism, six key ethylene signal transduction genes and seven genes related to pectinase in apricot fruit were identified by KEGG pathway analysis. By RNA-sequencing, we not only clarified the molecular mechanism of ethylene metabolism during the ripening of "Luntaibaixing" apricot fruit but also provided a theoretical basis for understanding pectin metabolism in apricot fruit.

Supporting role of lignin in immobilization of yeast on sugarcane bagasse for continuous pectinase production.

BACKGROUND: Lignocellulosic wastes are pretreated prior to their utilization in fermentation processes. Such pretreatment also alters the topological features of the substrates, and therefore the suitability of pretreated waste as immobilization matrix for microbial cells needs investigation. RESULTS: In this study, the effect of chemical pretreatment of sugarcane bagasse (SB) for its subsequent utilization as a matrix to immobilize a pectinolytic yeast, Geotrichum candidum AA15, was evaluated using cell retention, concentration of immobilized cells, immobilization efficiency, scanning electron microscopy and Fourier transform infrared spectroscopy of the substrate and pectinase titers obtained after recycling. The results revealed that untreated SB is more efficient for immobilization with higher values of cell retention and pectinase productivity (99.78%) retained for up to six production cycles. It was deduced that removal of lignin by pretreatment negatively influenced the ability of SB to support cell adhesion, as lignin acts as a sealing agent that provides strength to the substrate. CONCLUSIONS: The strategy of utilizing SB as immobilization matrix was found effective at the laboratory scale as it improved pectinase production and may be investigated further for large-scale and cost-effective production. © 2020 Society of Chemical Industry.

The nucleoid-associated protein IHF acts as a 'transcriptional domainin' protein coordinating the bacterial virulence traits with global transcription.

Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαß heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a 'transcriptional domainin' protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.

Effect of different carbon sources on the growth and enzyme production of a toxigenic and a non-toxigenic strain of Aspergillus flavus.

Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.

Effect of codon optimization and promoter choice on recombinant endo-polygalacturonase production in Pichia pastoris.

Pectinase is one of the most widely used enzymes in different fields of food industry for different purposes, such as clarification of fruit juice, extraction of vegetable oil and saccharification of agricultural substrates. The aim of this study is to produce recombinant pectinase and evaluate the effect of codon optimization and promoter selection on production. In this study, the gene encoding pectinase in Aspergillus niger was optimized according to the codon usage of Pichia pastoris. Within the scope of the study, codon-optimized and native (non-codon-optimized) pectinase genes were transferred to P. pastoris X33 strain and expressed under the regulation of methanol-inducible AOX1 and ethanol-inducible ADH2 promoter. As a result of the study, the promoter and codon combination which exhibited the highest production level was determined as the ADH2 and codon-optimized pectinase yielding an enzyme activity of 42.33 U/mL. The best producer clone was cultured in 400 mL media in 2 L shake-flask and the enzyme was purified by His-tag method. The optimum working conditions of the purified enzyme was 50 °C and pH 5.0, and after incubation at 60 °C for 1 -h, enzyme activity was maintained at 60% level. The Michelis-Menten constant (Km) and maximal velocity (Vmax) of the purified recombinant pectinase were 6.9 mg/mL and 67.57 µmol/mg/min, respectively.

Investigating the effects of Aureobasidium pullulans on grape juice composition and fermentation.

Aureobasidium pullulans has been observed as one of the most abundant species in freshly pressed grape juice. Despite this, little is known about the consequences for the wine-making process associated with the presence and proliferation of this fungus, including its interaction with other ferment-derived microorganisms and impact on the composition of the resulting wine. In this study, the physiology of abundant A. pullulans grape juice isolates was investigated through lab scale fermentation trials, demonstrating the ability of this species to survive in grape juice while producing polysaccharides, polymers of malic acid (poly ß-malic acid) and enzymes with pectinase, ß - glucosidase and tannase activity. A possible antagonistic effect against yeast through competition for metals including Fe and Zn was also observed. Overall, the data suggests this abundant species could have important implications for wine production and quality.

Optimization of concurrent production of xylanolytic and pectinolytic enzymes by Bacillus safensis M35 and Bacillus altitudinis J208 using agro-industrial biomass through Response Surface Methodology.

Application of crude xylanolytic and pectinolytic enzymes in diverse industrial processes make these enzymes commercially valuable and demand their production process to be cost-effective. Out of four different agrowaste biomass, wheat bran (WB) and citrus peel (CP), when amended as fermentation substrates, respectively induced the highest xylanolytic enzymes and pectinolytic enzymes from both, B. safensis M35 and B. altitudinis J208. Further, the simultaneous amendment of WB and CP yielded concurrent production of these cellulase free xylanolytic and pectinolytic enzymes. Hence, the quadratic model was developed using the Central Composite Design of Response Surface Method (CCD-RSM). The model gave the concentration values for WB and CP substrates to be amended in one single production medium for obtaining two optimized predicted response values of xylanase activity and pectinase activity units, which were further practically validated for the xylanase and pectinase production responses from the optimized production medium (OPM). These practically obtained response values from OPM were found to be in accordance with a range of 95% predicted intervals (PI) values. These observations verified the validity of the predicted quadratic model from RSM and suggested that both xylanase and pectinase enzymes can be induced concurrently from both of the bacterial strains.

Banana Peels: A Promising Substrate for the Coproduction of Pectinase and Xylanase from Aspergillus fumigatus MS16.

Banana peels (BP), an under-utilized waste material, was studied for the production of xylanase and pectinase by Aspergillus fumigates MS16. The factors affecting the co-production of both the enzymes were separately studied for their influence under submerged (Smf) and solid-state fermentation (SSF) of BP. The strain was cultivated in the presence of mineral salt (MS) solution containing BP powder as a sole source of carbon and physical and nutritional factors varied to observe the change in the enzyme titers. The data revealed that the MS-based medium was appropriate for the production of both the enzymes; therefore, in subsequent experiments, the same medium was used. A temperature of 30-35°C was found better for the production of the two enzymes under Smf; however, the titers of pectinase dropped significantly at 40°C. Contrarily, xylanase production was inhibited at 40°C under SSF but not under Smf. Whereas, supplementation of xylan or pectin to BP induced the production of xylanase and pectinase, respectively. Lowering the pH value favored the production of both the enzymes under Smf; however, the production of pectinase improved significantly when a higher concentration of BP (1%) was used compared to the concentration (0.25%) required for the production of xylanase. Interestingly, the enzyme preparation obtained under SSF exhibited optimal activities of both the enzymes at higher temperatures when compared to those obtained under Smf. The data indicated that the physiology of the fungus differed greatly when the cultivation pattern varied from Smf to SSF and, hence, the enzymes produced were characteristically distinct.Banana peels (BP), an under-utilized waste material, was studied for the production of xylanase and pectinase by Aspergillus fumigates MS16. The factors affecting the co-production of both the enzymes were separately studied for their influence under submerged (Smf) and solid-state fermentation (SSF) of BP. The strain was cultivated in the presence of mineral salt (MS) solution containing BP powder as a sole source of carbon and physical and nutritional factors varied to observe the change in the enzyme titers. The data revealed that the MS-based medium was appropriate for the production of both the enzymes; therefore, in subsequent experiments, the same medium was used. A temperature of 30­35°C was found better for the production of the two enzymes under Smf; however, the titers of pectinase dropped significantly at 40°C. Contrarily, xylanase production was inhibited at 40°C under SSF but not under Smf. Whereas, supplementation of xylan or pectin to BP induced the production of xylanase and pectinase, respectively. Lowering the pH value favored the production of both the enzymes under Smf; however, the production of pectinase improved significantly when a higher concentration of BP (1%) was used compared to the concentration (0.25%) required for the production of xylanase. Interestingly, the enzyme preparation obtained under SSF exhibited optimal activities of both the enzymes at higher temperatures when compared to those obtained under Smf. The data indicated that the physiology of the fungus differed greatly when the cultivation pattern varied from Smf to SSF and, hence, the enzymes produced were characteristically distinct.

Aspergillus niger production of pectinase and α-galactosidase for enzymatic soy processing.

Soybean is a most promising sustainable protein source for feed and food to help meet the protein demand of the rapidly rising global population. To enrich soy protein, the environment-friendly enzymatic processing requires multiple carbohydrases including cellulase, xylanase, pectinase, α-galactosidase and sucrase. Besides enriched protein, the processing adds value by generating monosaccharides that are ready feedstock for biofuel/bioproducts. Aspergillus could produce the required carbohydrases, but with deficient pectinase and α-galactosidase. Here we address this critical technological gap by focused evaluation of the suboptimal productivity of pectinase and α-galactosidase. A carbohydrases-productive strain A. niger (NRRL 322) was used with soybean hull as inducing substrate. Temperatures at 20 °C, 25 °C and 30 °C were found to affect cell growth on sucrose with an Arrhenius-law activation energy of 28.7 kcal/mol. The 30 °C promoted the fastest cell growth (doubling time = 2.1 h) and earliest enzyme production, but it gave lower final enzyme yield due to earlier carbon-source exhaustion. The 25 °C gave the highest enzyme yield. pH conditions also strongly affected enzyme production. Fermentations made with initial pH of 6 or 7 were most productive, e.g., giving 1.9- to 2.3-fold higher pectinase and 2.2- to 2.3-fold higher α-galactosidase after 72 h, compared to the fermentation with a constant pH 4. Further, pH must be kept above 2.6 to avoid limitation in pectinase production and, in the later substrate-limiting stage, kept below 5.5 to avoid pectinase degradation. α-Galactosidase production always followed the pectinase production with a 16-24 h lag; presumably, the former relied on pectin hydrolysis for inducers generation. Optimal enzyme production requires controlling the transient availability of inducers.

Archives of microbiology: screening of pectinase-producing bacteria from citrus peel and characterization of a recombinant pectate lyase with applied potential.

Pectinase is widely used in numerous industrial fields, including the food, wine, and paper industries. In this work, seven bacteria were isolated from orange peel and their pectinase production activity was assayed. One bacterium (OR-B2) identified as a Bacillus sp. showed the highest enzyme activity towards others. A gene encoding a pectate lyase designed as PelB-B2 in this work was amplified and heterogeneous expressed in E.coli. PelB-B2 was defined as a member of the PelB pectate lyase family after phylogenic tree analysis. 3D model of PelB-B2 was constructed by SWISS-MODEL and PelB-B2 showed conserved para-ß structure. After inducing culture and purified by Ni-affinity chromatography, the properties of the purified PelB-B2 were assayed. Optimal pH and temperature for PelB-B2 was pH 8.0 and 50 °C, respectively. PelB-B2 showed excellent pH stability and thermostability. It was stable within pH range 3.0-11.0 and retained more than 51% activity after incubation at 40 °C, 50 °C, or 60 °C for 1 h. Furthermore, we determined that PelB-B2 was a Ca2+-dependent pectinase and the pectin extracted from citrus was the benefit substrate for PelB-B2. The Km and Vmax of PelB-B2 were 1.64 g/L and 232.56 mol/(L min), respectively. The OR-B2 can be a new resource for pectinase production and the PelB-B2 has potential for industrial application. 7 bacteria were isolated from orange peel, namely OR-B1 to OR-B7 and their pectinase activities were assayed. One pectate lyase belongs to PelB family was cloned from OR-B2 and heterogeneous expressed in E. coli. Purified PelB-B2 was further studied with its properties. Effects of pH, temperature, chemicals, substrate on the enzyme activity were assayed and the enzyme kinetic was also measured.

Use of Agro-Industrial Wastes for the Production of a Wild Yeast Enzyme with Disintegration Activity on Plant Tissues

Abstract The objective of the present study was to develop a cost-effective medium, using agro-industrial wastes for the production of a polygalacturonase by Wickerhanomyces anomalus of interest in cassava starch industries. The effect of several raw agro-industrial wastes and others nutrients on polygalacturonase production by W. anomalus, were evaluated, in a reference fermentation medium, using statistical designs, by batch culture. The ability of the cell-free supernatant to extract cassava starch was evaluated. Lemon peel was the best inducer for the production of PGase. Statistical analysis of the data showed that lemon peel, Mg+2 and PO4HK2 had significant effect on PGase production, and the others variables (yeast extract, Ca+2, Fe+2, amino acid and trace element solution) were no significant. PGase synthesis reached ~31 EUmL-1, in the OFM (glucose, lemon peel, urea, vitamins, KH2PO4 and MgSO4), after 12 h of culture, at a lab scale bioreactor. PGase of W. anomalus, was able to disintegrate cassava tuber tissue, and the starch granules contained within the cells were released into the reaction medium. Lemon peel can be used as inducer for PGase production by W. anomalus, in a low cost culture medium, appropriate for the production of the enzyme at large scale.

Transcriptome Profiling Unravels a Vital Role of Pectin and Pectinase in Anther Dehiscence in Chrysanthemum.

Chrysanthemum (Chrysanthemum morifolium (Ramat.) Kitamura) plants have great ornamental value, but their flowers can also be a source of pollen contamination. Previously, morphological and cytological studies have shown that anthers of some chrysanthemum cultivars such as 'Qx-115' fail to dehisce, although the underlying mechanism is largely unknown. In this study, we investigated the molecular basis of anther indehiscence in chrysanthemum via transcriptome analysis of a dehiscent cultivar ('Qx-097') and an indehiscent cultivar ('Qx-115'). We also measured related physiological indicators during and preceding the period of anther dehiscence. Our results showed a difference in pectinase accumulation and activity between the two cultivars during dehiscence. Detection of de-esterified pectin and highly esterified pectin in anthers during the period preceding anther dehiscence using LM19 and LM20 monoclonal antibodies showed that both forms of pectin were absent in the stomium region of 'Qx-097' anthers but were abundant in that of 'Qx-115' anthers. Analysis of transcriptome data revealed a significant difference in the expression levels of two transcription factor-encoding genes, CmLOB27 and CmERF72, between 'Qx-097' and 'Qx-115' during anther development. Transient overexpression of CmLOB27 and CmERF72 separately in tobacco leaves promoted pectinase biosynthesis. We conclude that CmLOB27 and CmERF72 are involved in the synthesis of pectinase, which promotes the degradation of pectin. Our results lay a foundation for further investigation of the role of CmLOB27 and CmERF72 transcription factors in the process of anther dehiscence in chrysanthemum.

Kinetic and thermodynamic investigations of cell-wall degrading enzymes produced by Aureobasidium pullulans via induction with orange peels: application in lycopene extraction.

The production of cell-wall degrading enzymes (CWDE) such as cellulase and pectinase by Aureobasidium pullulans NAC8 through induction using orange peels was investigated for the potential application of these enzymes in the extraction of lycopene from tomato skin, waste, and paste (SWP). The CWDE was then immobilized via entrapment in alginate beads for lycopene extraction and the kinetic/thermodynamic properties of the free and immobilized CWDE investigated. The optimum production of CWDE occurred at pH, temperature, and orange peel concentration of 6.0, 50 °C, and 2.0% (w/v), respectively. The values obtained for some kinetic and thermodynamic parameters such as Ed*, t1/2, ΔGd*, and ΔHd* indicate that both free and immobilized cellulase and pectinase were thermostable between 40 and 50 °C. Maximum lycopene extracted from the tomato SWP was 80 ± 2.4 mg/kg, 42 ± 1.3 mg/kg and 60 ± 1.2 mg/kg, respectively, using the immobilized CWDE. The entrapped CWDE was able to extract lycopene with yields of 58 ± 4.2, 51 ± 1.2 and 57 ± 4.2% for tomato SWP respectively after the fifth cycle. Using orange peels for the induction of CWDE by A. pullulans offers a unique and cheaper approach to obtaining thermostable multi-enzyme complexes employable for easy lycopene extraction from tomato SWP.

[Heterogenous expression and enzymatic property analysis of microbial-derived pectinases in pig PK15 cells].

As one of plant cell wall components, pectin is the main anti-nutritional factor in livestock and poultry feeds and has an adverse effect on utilization efficiency of feed energy and nitrogen. Pectinases, which are widely found in microorganisms such as bacteria, yeast and filamentous fungi in nature,can improve feed efficiency by relieving the anti-nutritional effect of pectin through promoting the hydrolysis reaction of feed pectin. To explore the feasibility of expressing microbial-derived pectinase genes in pig cells, we introduced microbial-derived pectinase genes pg5a, pgI, pga3A, and pgaA into porcine PK 15 cells by lipofection for heterogenous expression. Enzymatic activities of the pectinases encoded by these genes were analyzed using the 3,5 dinitrosalicylic acid (DNS) method. Results showed that all four pectinase genes were able to be transcribed into mRNAs in porcine PK 15 cells, but only pg5a and pgI were adapted to the porcine cell expression system. Among them, the maximum activity of pectinase PG5A was 0.95 U/mL, the optimum pH was pH 4.0, and the enzymatic activity was maintained above 46% in the range of pH 4.6 to 6.0. Pectinase PGI obtained the highest enzymatic activity at pH 5.0, which was 0.30 U/mL, and maintained more than 35% of the activity in the range of pH 4.0 to 6.0. The results of digestive protease tolerance test showed that PG5A and PGI were highly resistant to pepsin and trypsin. After treatment with 1 mg/mL pig pepsin for two hours, the residual enzymatic activities of PG5A and PGI were 76% and 71%, respectively. And after two hours treatment with 1 mg/mL of pig trypsin, the remaining enzymatic activities of PG5A and PGI were 44% and 93%, respectively. In summary, pectinase PG5A and PGI can be effectively expressed in pig cells, and have strong tolerance to pig intestinal pH environment and digestive proteases. Therefore, both pg5a and pgI can be used as candidate genes for production of transgenic pigs.

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.

High-level secretive expression of a novel achieved Talaromyces cellulolyticus endo-polygalacturonase in Pichia pastoris by improving gene dosage for hydrolysis of natural pectin.

Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides.

Simultaneous production of industrially important alkaline xylanase-pectinase enzymes by a bacterium at low cost under solid-state fermentation conditions.

Simultaneous production of alkaline xylanase and all seven types of pectinases by a bacterial isolate, under solid-state fermentation was checked in this study. Under optimized conditions, high concurrent production of xylanase (22,800 ± 578 IU/g substrate) and pectinase (4,832 ± 189 IU/g substrate) was achieved. The different types of pectinases produced were exo-polymethylgalacturonase (782 IU/g), endo-polymethylgalacturonase (6.42 U/g), exo-polygalacturonase (2,250 IU/g), endo-polygalacturonase (11.57 U/g), polymethylgalacturonate lyase (53.99 IU/g), polygalacturonate lyase (59.78 IU/g), and pectin esterase (5.78 IU/g). Wheat bran resulted in the highest titer of both enzymes. The maximum xylanase-pectinase yield was detected after 7 days of incubation with 2 mM MgSO4 and 1.5 g/L K2 HPO4 at wheat bran to moisture ratio 1:1.5 (w/v), media to flask volume ratio 1:25, pH 7.0, temperature 37 °C, and inoculum size 15%. Xylanase was most stable at pH 8.0, retained more than 75% activity up to 24 H, whereas pectinase was most stable at pH 9.0, having full activity even after 24 H. At 45 °C, the xylanase showed 82% residual activity after 6 H of incubation. The pectinase was 97% and 61% stable up to 3 H at 50 and 55 °C, respectively. This is the first report showing the production of xylanase-pectinases by bacterium along with high titer of seven types of pectinases, suitable for industries.

Optimizing Culture Conditions by Statistical Approach to Enhance Production of Pectinase from Bacillus sp. Y1.

It was found that Bacillus sp. Y1 could secrete alkaline pectinase with suitable enzyme system for powerful and fast degumming of ramie fiber. In this study, the medium components and fermentation conditions were optimized by some statistical methods including mixture design, fractional factorial design, central composite design and response surface methodology, and single factor method for enhancing the alkaline pectinase production. The optimized conditions for pectinase production were that the culture was shaken at 34°C for 60 h in 50 mL of medium containing 10.5% (w/v) carbon source (consisting of 3.8% starch, 4.2% wheat bran, and 2.5% sucrose), 0.37% (NH4)2SO4, 0.3% MgSO4, and 0.1% Tween-80, with initial pH 8.2 and inoculation amount of 1.3 mL (with the OD600 of the seed medium about 5.77). Using the optimizing conditions, the activities of polygalacturonate lyase (PGL) and polygalacturonase (PG) in fermentation liquor were increased to 2.00-fold and 3.44-fold, respectively, and the fermentation time shortened 12 hours (from 72 h to 60 h), which showed good application potential in degumming of ramie.

Pectinase secreted by psychrotolerant fungi: identification, molecular characterization and heterologous expression of a cold-active polygalacturonase from Tetracladium sp.

BACKGROUND: Pectinolytic enzymes, which are used in several industries, especially in the clarification process during wine and fruit juice production, represent approximately 10% of the global enzyme market. To prevent the proliferation of undesired microorganisms, to retain labile and volatile flavor compounds, and to save energy, the current trend is to perform this process at low temperatures. However, the commercially available pectinases are highly active at temperatures approximately 50 °C and poorly active at temperatures below 35 °C, which is the reason why there is a constant search for cold-active pectinases. In preliminary studies, pectinolytic activity was detected in cold-adapted yeasts and yeast-like microorganisms isolated from Antarctica. The aim of the present work was to characterize pectinases secreted by these microorganisms and to express the best candidate in Pichia pastoris. RESULTS: Degradation of pectin by extracellular protein extracellular extracts obtained from 12 yeast cultures were assayed in plates at 4 °C to 37 °C and pH from 5.4 to 7.0, obtaining positive results in samples obtained from Dioszegia sp., Phenoliferia glacialis and Tetracladium sp. An enzyme was purified from Tetracladium sp., analyzed by peptide mass fingerprinting and compared to genome and transcriptome data from the same microorganism. Thus, the encoding gene was identified corresponding to a polygalacturonase-encoding gene. The enzyme was expressed in Pichia pastoris, and the recombinant polygalacturonase displayed higher activity at 15 °C than a mesophilic counterpart. CONCLUSIONS: Extracellular pectinase activity was found in three yeast and yeast-like microorganisms from which the highest activity was displayed by Tetracladium sp., and the enzyme was identified as a polygalacturonase. The recombinant polygalacturonase produced in P. pastoris showed high activity at 15 °C, representing an attractive candidate to be applied in clarification processes in the production of fermented beverages and fruit juices.

Development of strategy for simultaneous enhanced production of alkaline xylanase-pectinase enzymes by a bacterial isolate in short submerged fermentation cycle.

The aim of this study is to enhance the production of industrially valuable xylanase and pectinase enzymes in short duration, using agrowaste extracted substrates. Conventional cum statistical multifactor analysis approaches were used in order to evaluate the effect of crude extracted substrates, supplemented for the production of xylanase-pectinase enzymes. Incorporation of crude extracted xylan (1.2 mg/ml of inoculum) and pectin (4.8 mg/ml of inoculum) substrates in inoculum resulted in maximal xylanase (320 ± 15) and pectinase titre (90 ± 8) after 48 h, using 2% wheat bran and 2% citrus peel in production medium with 48 h of fermentation time, with one variable factor at a time approach. The best condition obtained after performing statistical multifactor interaction analysis includes 5.50 mg/ml of pectin in inoculum,1.50 mg/ml of xylan in inoculum, wheat bran 3%, temperature 37.5 °C, time 48 h, 7 mg/ml of pectin in production medium, peptone 1.05%, inoculum size 2% and inoculum age of 20 h, with alkaline xylanase activity of 415.22 ± 18.50 IU/ml and alkaline pectinase activity of 109.10 ± 8.80 IU/ml. Activity of different pectinolytic enzymes per ml was also calculated, with 18.98 IU of exo-polymethylgalacturonase, 0.14 IU of endo-polymethylgalacturonase, 80 IU of exo-polygalacturonase, 0.28 IU of endo-polygalacturonase, 1.42 IU of polymethylgalacturonate lyase, 1.47 IU of polygalacturonate lyase, 0.15 IU of pectin esterase. This is the first report mentioning the utilization of crude extracted xylan and extracted pectin in inoculum to get the increment in the activity of both alkaline xylanase-pectinase enzymes simultaneously under short submerged fermentation cycle.