Pectinase from Microorganisms and Its Industrial Applications.

The utilization of microbial pectinase in different industries has been increased in its world demand. The major sources of pectinase are microorganisms mainly bacteria, fungi and yeast. The utilization of low-cost agro-industrial wastes as substrates has been preferable in pectinase production. Pectinase production faced various parameters optimization constraints such as temperature, pH and production times which are the main factors in pectinase production. The pectinase enzyme is getting attention due to its several advantages; hence, it needs to be explored further to take its maximum advantage in different industries. This review discusses the pectin substance structure, substrate for pectinase production, factors influencing pectinase production, the industrial application of microbial pectinase and also discusses challenges and future opportunities of applying microbial pectinase in industry.

Ultrasound-assisted enzymatic extraction of Corni Fructus alpha-glucosidase inhibitors improves insulin resistance in HepG2 cells.

Corni Fructus (CF) is a traditional medicine and beneficial food with multifaceted protective effects against diabetes and its complications. Since alpha-glucosidase inhibitors (GIs) are promising first-choice oral antihyperglycemic drugs for diabetes, we examined whether GIs from CF (GICF) are useful for diabetes treatment. Therefore, GICF was extracted by ultrasound-assisted enzymatic extraction (UAEE) that is optimized by a three-level, four-factor Box-Behnken design and determined by ultra-performance liquid chromatography. Compared to 36.31 mg g-1 without enzyme treatment, the GICF yield increased to 70.44 mg g-1via UAEE under optimum conditions (0.5% compound enzyme extracted in 23 min at 46 °C and pH 4.8). The activity (91.99%) of GICF was as predicted (93.28%). When GICF was used in an insulin-resistant HepG2 cell model, it significantly ameliorated the glucose metabolism in a dose-dependent manner. Our findings indicate that UAEE may be an innovative method for functional food extraction and a potential strategy for high-quality food ingredient (such as GI) production with high efficiency and productivity.

From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18.

BACKGROUND: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. OBJECTIVES: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. METHODS: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. RESULTS: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. CONCLUSION: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

Enterocin M-Producing Enterococcus faecium CCM 8558 Demonstrating Probiotic Properties in Horses.

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

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.

Biotechnological potential of an exo-polygalacturonase of the new strain Penicillium janthinellum VI2R3M: biochemical characterization and clarification of fruit juices.

AIMS: The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. METHODS AND RESULTS: The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent Km , Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56 mg ml-1 , 163·1 U mg-1 and 277 s-1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. CONCLUSIONS: The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.

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.

Optimization of Process Parameters for Production of Pectinase using Bacillus Subtilis MF447840.1.

BACKGROUND: Pectinase enzyme has immense industrial prospects in the food and beverage industries. OBJECTIVE: In our investigation, we find out the optimum process parameters suitable for better pectinase generation by Bacillus subtilis MF447840.1 using submerged fermentation. METHOD: 2% (OD600 nm = 0.2) of pure Bacillus subtilis MF447840.1 bacterial culture was inoculated in sterile product production media. The production media components used for this study were 1 g/l of pectin, 2 g/l of (NH4)2SO4, 1 g/l of NaCl, 0.25 g/l of K2HPO4, 0.25 g/l of KH2PO4 and 1 g/l of MgSO4 for pectinase generation. We reviewed all recent patents on pectinase production and utilization. The various process parameters were observed by changing one variable time method. RESULTS: The optimum fermentation condition of different parameters was noticed to be 5% inoculums, 25% volume ratio, temperature (37°C), pH (7.4) and agitation rate (120 rpm) following 4 days incubation. CONCLUSION: Maximum pectinase generation was noticed as 345 ± 12.35 U following 4 days incubation.

Immobilization of an alkaline endopolygalacturonase purified from Bacillus paralicheniformis exhibits bioscouring of cotton fabrics.

Pectin degrading enzyme has been increasing interest in an industrial application as biocatalysts, such as juice, textile, and wine industry. Bacillus paralicheniformis CBS3, isolated from popular traditional Korean food (kimchi), produced a novel extracellular thermostable alkaline endopolygalacturonase (BPN3). In this study, BPN3 was purified to 22.04-fold with a recovery yield of 18.93% and specific activity of 2216.41 U/mg by gel filtration and anion exchange column chromatography. The molecular mass of BPN3 was approximately 53 kDa as analyzed by SDS-PAGE and pectic zymography. The N-terminal sequence of BPN3 was AIPVILAX. BPN3 was stable over a broad pH range (8.14-11.47), was thermally stable at 50-60 °C, and functioned optimally in pH 9.1 at 60 °C. BPN3 had Km and Vmax values of 0.039 mg/mL and 747.9 ± 1.2 U mg- 1, respectively, whereas pectin from apple as substrate. BPN3 activity was remarkably affected by metal ions, modulators, and detergents. Digalacturonic acid (GA2) was the major oligosaccharide produced by hydrolysis of BPN3. Immobilized BPN3 was active over a pH range (8.1-11.5), temperature (50-60)°C, and remained stable with 63.34 and 43.41% of its relative activity during second and third cycle, respectively. Desized cotton exhibited highest reducing sugar liberation through optimized conditions of bioscouring. Bioscouring effectiveness of BPN3 was characterized by the comparison of weight loss for purified BPN3 with commercial pectinase and comparison of BPN3 with grey fabric. BPN3 was simple to purify, had high thermal stability, and was stable over a broad pH range that suggests its suitability for bioscouring application as an industrial catalyst.

Physicochemical characterization of pectinase activity from Bacillus spp. and their accessory role in synergism with crude xylanase and commercial cellulase in enzyme cocktail mediated saccharification of agrowaste biomass.

AIM: The aim of this study was to evaluate the physicochemical properties of the crude pectinase activity from three Bacillus isolates of ruminant dung origin and study their synergism with crude xylanases from the same Bacillus spp. and a commercial cellulase to evaluate their accessory role in improved biomass saccharification. METHODS AND RESULTS: Pectinolytic crude culture filtrate obtained from three ruminant dung isolates, Bacillus safensis M35, Bacillus altitudinis R31 and Bacillus altitudinis J208, on crude pectin containing medium possessed polygalacturonate hydrolase, pectate lyase and pectin lyase activities. Studies regarding their stability under various temperature and pH conditions revealed their mild acidic to alkaline and mesophilic nature with enzyme activity falling within the pH range 6·0-9·0 and temperature range 30-60°C. The pectinase activity was categorized as endolytic as it brought about ~50% reduction in relative viscosity of pectic polymer within initial 10 min of incubation. Synergism of pectinase activity with crude xylanase activities and/or commercial cellulase was clearly demonstrated as ~1·6 to ~1·9-fold increase in agrowaste biomass saccharification was obtained confirming the role of pectinases as accessory enzymes. CONCLUSION: Synergism of the broad-spectrum endopectinase activity obtained from three Bacillus isolates with accessory crude xylanases from the same isolates and commercial cellulase enhanced the agrowaste saccharification and confirmed the accessory role of crude pectinase as they formed an efficient enzyme cocktail functioning in a contributive manner for improvement of agrowaste biomass saccharification. SIGNIFICANCE AND IMPACT OF THE STUDY: Mesophilic crude endopectinases obtained from Bacillus spp. isolated from ruminant dung possessed activity in broad pH and temperature ranges as well as broad substrate specificity. Moreover, their synergism with crude xylanase and Primfast® 200 cellulase demonstrated the potential to form efficient enzyme cocktail for application in plant biomass saccharification process.

Production optimization, purification and characterization of a heat-tolerant acidic pectinase from Bacillus sp. ZJ1407.

Medium compositions for a heat-tolerant acidic pectinase production from Bacillus sp. ZJ1407 were optimized via response surface methodology (RSM) and its enzymatic properties were investigated. A 2-level factorial design was used to estimate the main effect of factors, and to screen the significant factors. A central composite design was used to find out the optimal concentrations of screened key factors. Lactose, tryptone and (NH4)2SO4 were found to have a significant influence on the pectinase activity (p <0.05). The optimal medium compositions were as follows: lactose 44.8g/l, tyrptone 30.9g/l, (NH4)2SO4 1.35g/l, MnSO4·H2O 0.2g/l, MgSO4 0.4g/l and NaCl 3.5g/l. Pectinase was purified to homogeneity by ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephadex G-100 size-exclusion chromatography. The molecular weight of the purified pectinase determined by SDS-PAGE was about 23kDa, and its final specific activity was 110.47U/mg. Its optimal temperature and pH were 37°C and 5.0, respectively. Pectinase was very stable within a pH range of 3.0-5.0, and showed a high thermo-stability at 80 and 90°C. Ba2+ could significantly promote the activity of pectinase, and Mn2+ heavily inhibited its activity. This study provides new insight into the future development and use of pectinase from Bacillus sp. ZJ1407.

Toward Enhancing the Enzymatic Activity of a Novel Fungal Polygalacturonase for Food Industry: Optimization and Biochemical Analysis.

BACKGROUND: The review of literature and patents shows that enhancing the polygalacturonase (PG) production and activity are still required to fulfill the increasing demands. METHODS: A dual optimization process, which involved Plackett-Burman design (PBD), with seven factors, and response surface methodology, was applied to optimize the production of extracellular PG enzyme produced by a novel strain of Aspergillus flavus isolated from rotten orange fruit. The fungal PG was purified and biochemically characterized. RESULTS: Three variables (harvesting time, pH and orange pomace concentration), that were verified to be significant by the PBD analysis, were comprehensively optimized via Box-Behnken design. According to this optimization, the highest PG activity (4073 U/mL) was obtained under pH 7 after 48 h using 40 g/L orange pomace as a substrate, with enhancement in PG activity by 51% compared to the first PBD optimization step. The specific activity of the purified PG was 1608 U/mg with polygalacturonic acid and its molecular weight was 55 kDa. The optimum pH was 5 with relative thermal stability (80%) at 50˚C after 30 min. The PG activity improved in the presence of Cu2+ and Ca2+, while Ba2+, Fe2+ and Zn2+ greatly inhibited the enzyme activity. The obvious Km and Vmax values were 0.8 mg/mL and 2000 µmol/min, respectively. CONCLUSION: This study is a starting point for initial research in the field of optimization and characterization of A. flavus PG. The statistical optimization of A. flavus PG and its biochemical characterization clearly revealed that this fungal strain can be a potential producer of PG which has a wide range of industrial applications.

Biochemical characterization and low-resolution SAXS structure of an exo-polygalacturonase from Bacillus licheniformis.

Among the structural polymers present in the plant cell wall, pectin is the main component of the middle lamella. This heterogeneous polysaccharide has an α-1,4 galacturonic acid backbone, which can be broken by the enzymatic action of pectinases, such as exo-polygalacturonases, that sequentially cleave pectin from the non-reducing ends, releasing mono or di-galacturonic acid residues. Constant demand for pectinases that better suit industrial requirements has motivated identification and characterization of novel enzymes from diverse sources. Bacillus licheniformis has been used as an important source for bioprospection of several industrial biomolecules, such as surfactants and enzymes, including pectate lyases. Here we cloned, expressed, purified, and biochemically and structurally characterized an exo-polygalacturonase from B. licheniformis (BlExoPG). Its low-resolution molecular envelope was derived from experimental small-angle scattering data (SAXS). Our experimental data revealed that BlExoPG is a monomeric enzyme with optimum pH at 6.5 and optimal temperature of approximately 60°C, at which it has considerable stability over the broad pH range from 5 to 10. After incubation of the enzyme for 30min at pH ranging from 5 to 10, no significant loss of the original enzyme activity was observed. Furthermore, the enzyme maintained residual activity of greater than 80% at 50°C after 15h of incubation. BlExoPG is more active against polygalacturonic acid as compared to methylated pectin, liberating mono galacturonic acid as a unique product. Its enzymatic parameters are Vmax=4.18µM.s-1,Km=3.25mgmL-1 and kcat=2.58s-1.

Purification and Biochemical and Kinetic Properties of an Endo-Polygalacturonase from the Industrial Fungus Aspergillus sojae.

An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.

Production optimization of a heat-tolerant alkaline pectinase from Bacillus subtilis ZGL14 and its purification and characterization.

Alkaline pectinase has important applications in the pretreatment of waste water from food processing and in both the fabric and paper industries. In this study, a 2-level factorial design was used to screen significant factors that affect the activity of alkaline pectinase, and the response surface methodology (RSM) with a Box-Behnken design (BBD) was used to optimize their concentrations. Starch, peptone, KH2PO4 and K2HPO4·3H2O were found to significantly affect the activity of alkaline pectinase. Their optimal concentrations were as follows: 4.68% starch, 1.6% peptone, 0.26% KH2PO4 and 0.68% K2HPO4·3H2O. Under the above conditions, the activity of alkaline pectinase was significantly improved to 734.11 U/mL. Alkaline pectinase was purified to homogeneity with a recovery rate of 9.6% and a specific activity of 52372.52 U/mg. Its optimal temperature and pH were 50°C and 8.6, respectively. The purified enzyme showed strong thermo-stability and good alkali resistance. In addition, the activity of alkaline pectinase was improved in the presence of Mg2+. Cu2+, Mn2+, and Co2+ significantly inhibited its activity. This study provides an important basis for the future development and use of a heat-tolerant alkaline pectinase from B. subtilis ZGL14.

Purification, Kinetic, and Thermodynamic Characteristics of an Exo-polygalacturonase from Penicillium notatum with Industrial Perspective.

An extracellular exo-polygalacturonase (exo-PG) produced by Penicillium notatum was purified (3.07-folds) by ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Two distinct isoforms of the enzyme, namely exo-PGI and exo-PGII, were identified during column purification with molecular weights of 85 and 20 kDa, respectively, on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme displayed its optimum activity at pH 6.0 and 50 °C and was found to be stable in the slightly acidic pH (ranging from 4.5 to 6.0). Michaelis-Menten parameters, i.e., K m (app) and V max for pectin hydrolysis, were calculated to be 16.6 mg/mL and 20 µmol/mL/min, respectively. The enzyme followed biphasic deactivation kinetics. Phase I of the exo-PGI showed half-lives of 6.83 and 2.39 min at 55 and 80 °C, respectively, whereas phase II of the enzyme exhibited a half-life of 63.57 and 22.72 min at 55 and 80 °C, respectively. The activation energy for denaturation was 51.66 and 44.06 kJ/mol for phase I and phase II of the exo-PGI, respectively. The enzyme activity was considerably enhanced by Mn2+, whereas exposure to a hydrophobic environment (urea and sodium azide solution) drastically suppressed the enzyme activity. Results suggest that exo-PGI might be considered as a potential candidate for various applications, particularly in the food and textile industries.

A polygalacturonase localized in the Golgi apparatus in Pisum sativum.

Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 µM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed.

Identification of an acidic endo-polygalacturonase from Penicillium oxalicum CZ1028 and its broad use in major tropical and subtropical fruit juices production.

Endo-polygalacturonases play an important role on depectinization in fruit juices industry. A putative endo-polygalacturonase gene PoxaEnPG28A was cloned from Penicillium oxalicum CZ1028. PoxaEnPG28A consisted of a putative signal peptide and a catalytic domain belonging to glycoside hydrolase family 28, and it shared 72% identity with that of a functionally characterized endo-polygalacturonase from Trichoderma harzianum. Gene PoxaEnPG28A was successfully expressed in Pichia pastoris with a high yield of 1828.7 U/mL. The purified recombinant enzyme PoxaEnPG28A hydrolyzed polygalacturonic acid in endo-manner releasing oligo-galacturonates. PoxaEnPG28A showed maximal activity at pH 5.5 and 55°C, and was stable between pH 3.0 to 10.0 and below 45°C. The kinetic constants Km and Vmax of PoxaEnPG28A were calculated as 1.57 g/L and 14,641.29 U/mg, respectively. PoxaEnPG28A significantly improved the yields of fruit juices from banana, plantain, papaya, pitaya and mango. The high production level of the recombinant enzyme PoxaEnPG28A by P. pastoris and remarkable catalytic activity of PoxaEnPG28A toward five kinds of fruit juices made the enzyme a potential application in agriculture and food industries.

Scaling-up and ionic liquid-based extraction of pectinases from Aspergillus flavipes cultures.

The viability of the scaling-up of pectinases production by Aspergillus flavipes at 5L-bioreactor scale has been demonstrated by keeping constant the power input, and a drastic increase in the endo- and exopectinolytic enzyme production was recorded (7- and 40-fold, respectively). The main process variables were modelled by means of logistic and Gompertz equations. In order to overcome the limitations of the conventional downstream strategies, a novel extraction strategy was proposed on the basis of the adequate salting-out potential of two biocompatible cholinium-based ionic liquids (N1112OHCl and N1112OHH2PO4) in aqueous solutions of Tergitol, reaching more than 90% of extraction.

Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation.

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. Ea(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG(d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.