Purification and mechanistic characterisation of two polygalacturonases from Sclerotium rolfsii Enzyme and Microbial Technology, Volume 40, Issue 7, 1 June 2007, Pages 1739-1747 W. Schnitzhofer, H.-J. Weber, M. Vršanská, P. Biely, A. Cavaco-Paulo and G.M. Guebitz

Sclerotium rolfsii (strain CBS 350.80) was found to produce extraordinary high amounts of polygalacturonases (PGs). Two of these extracellular enzymes were purified by a recently introduced preparative electrophoretic device (isoelectric focusing mode of free flow electrophoresis). PG 1 (39.5 kDa, pI 6.5) and PG 2 (38 kDa, pI 5.4) exhibited quite similar properties, they were found to be both endo-acting enzymes. Both PGs cleaved penta- and trigalacturonic acid while tetragalacturonic acid was only cleaved when trigalacturonic acid was present. The latter substrate was hydrolysed much faster by PG 2. Both enzymes were active on pectins with different degrees of esterification, they were sensitive towards Ca-cations and not glycosylated. The kinetic properties were measured by viscosimetry with polygalacturonic acid as a substrate. NMR experiments on a model substrate revealed an inverting mechanism of carbohydrate hydrolysis for both enzymes.

Combined Magnetic and Chemical Covalent Immobilization of Pectinase on Composites Membranes Improves Stability and Activity Food Chemistry, In Press, Accepted Manuscript, Available online 5 May 2007,  Zhongli Lei, Shuxian Bi, Bing Hu and Hong Yang

Optimizing bioscouring condition of cotton knitted fabrics with an alkaline pectinase from Bacillus subtilis WSHB04-02 by using response surface methodology Biochemical Engineering Journal, Volume 34, Issue 2, May 2007, Pages 107-113 Qiang Wang, Xue-Rong Fan, Zhao-Zhe Hua and Jian Chen

This present study was undertaken to find optimum conditions of pH, temperature, incubation time and enzyme concentration for bioscouring of cotton knitted fabrics with an alkaline pectinase isolated from Bacillus subtilis WSHB04-02 by use of response surface methodology (RSM). A central composite design was used as an experimental design for the analysis of the allocation of treatment combination. A second-order polynomial regression model was fitted and was found adequate with a determination coefficient R2 of 0.9844 (P < 0.001). The effect of pH was the most significant factor influencing cotton bioscouring and no significant interactions between different factors were found. Estimated optimum parameters were as follows: pH 9.1, temperature 57 °C, incubation time 1.25 h and pectinase concentration 1.0 g/l. Under these conditions, a desired pectin removal percentage companied with adequate wettability was reached. In addition, a boiling water pretreatment of 30 min before enzymatic scouring was found to be useful for subsequent pectinase degradation due to the improvement of the accessibility of pectinase to the pectins in cotton fibers. Hydrolysis of grapefruit peel waste with cellulase and pectinase enzymes Bioresource Technology, Volume 98, Issue 8, May 2007, Pages 1596-1601 Mark R. Wilkins, Wilbur W. Widmer, Karel Grohmann and Randall G. Cameron Approximately 1 million metric tons of grapefruit were processed in the 2003/04 season resulting in 500,000 metric tons of peel waste. Grapefruit peel waste is usually dried, pelletized, and sold as a low-value cattle feed. This study tested different loadings of commercial cellulase and pectinase enzymes and pH levels to hydrolyze grapefruit peel waste to produce sugars. Pectinase and cellulase loadings of 0, 1, 2, 5, and 10 mg protein/g peel dry matter were tested at 45 °C. Hydrolyses were supplemented with 2.1 mg beta-glucosidase protein/g peel dry matter. Five mg pectinase/g peel dry matter and 2 mg cellulase/g peel dry matter were the lowest loadings to yield the most glucose. Optimum pH was 4.8. Cellulose, pectin, and hemicellulose in grapefruit peel waste can be hydrolyzed by pectinase and cellulase enzymes to monomer sugars, which can then be used by microorganisms to produce ethanol and other fermentation products. Loss of rutin and antioxidant activity of asparagus juice caused by a pectolytic enzyme preparation from Aspergillus niger Food Chemistry, In Press, Corrected Proof, Available online 9 April 2007,  Ting Sun, Joseph R. Powers and Juming Tang We found that a commercial pectolytic enzyme preparation from Aspergillus niger (pectinase AN) contained laccase activity that decreased rutin content and antioxidant activity of asparagus juice. This research investigated the effects of pH, temperature, and concentration of pectinase AN on pectinase AN’s laccase activity to decrease rutin content and antioxidant activity of asparagus juice. Asparagus juice was incubated with pectinase AN at different pHs (3.2, 4.5 and 5.8), temperatures (25, 37, and 50 °C) and enzyme concentrations (0.1%, 0.5% and 1%). Rutin content and antioxidant activity of samples was determined by HPLC and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) free radical method, respectively. The rate of loss of rutin and antioxidant activity of asparagus juice was smaller at pH 3.2 than at pH 4.5 and pH 5.8, smaller for 0.1% pectinase AN than 0.5% and 1% pectinase AN. The rate of loss of rutin of asparagus juice was greater at 25 °C than at the other two temperatures. Pectinase AN can decrease rutin content and antioxidant activity of asparagus juice at the selected conditions. But rutin content and antioxidant activity of asparagus juice produced using pectinase AN could be less decreased at pH 3.2 and 0.1% of enzyme with less than 2 h of incubation time. This information was helpful for juice industry to produce juices with high antioxidant activity using pectinase AN. Optimization of biomass, pellet size and polygalacturonase production by Aspergillus sojae ATCC 20235 using response surface methodology Enzyme and Microbial Technology, Volume 40, Issue 5, 3 April 2007, Pages 1108-1116 C. Tari, N. Gögus and F. Tokatli A two-step optimization procedure using central composite design with four factors (concentrations of maltrin and corn steep liquor (CSL), agitation speed and inoculation ratio) was used in order to investigate the effect of these parameters on the polygalacturonase (PG) enzyme activity, mycelia growth (biomass) and morphology (pellet size) of Aspergillus sojae ATCC 20235. According to the results of response surface methodology (RSM), initial concentrations of maltrin and CSL and agitation speed were significant (p < 0.05) on both PG enzyme production and biomass formation. As a result of this optimization, maximum PG activity (13.5 U/ml) was achievable at high maltrin (120 g/l), at low CSL (0 g/l), high agitation speed (350 rpm) and high inoculation ratio (2 × 107 total spore). Similarly, maximum biomass (26 g/l) could be obtained under the same conditions with only the difference for higher level of CSL requirement. The diameter of pellets in all optimization experiments ranged between 0.05 and 0.76 cm. The second optimization step improved the PG activity by 74% and the biomass by 40%. The silica-coated chitosan particle from a layer-by-layer approach for pectinase immobilization Enzyme and Microbial Technology, Volume 40, Issue 5, 3 April 2007, Pages 1442-1447 Zhongli Lei and Shuxian Bi We investigate the enzymatic activity of pectinase adsorbed on a novel type of colloidal particles. The colloidal stability is not impeded by the adsorbed proteins despite the fact that up to 247.8 mg of enzyme is adsorbed per gram of the carrier particles. Various characteristics of immobilized pectinase such as the pH stability, thermal stability, and storage stability were valuated. The immobilized pectinase revealed acceptable pH stability over a broad experimental range. The activity of immobilized pectinase adsorbed onto these particles is analyzed in terms of the Michaelis–Menten parameters. The Michaelis constant Km differs only slightly from the Km value of the native enzyme when the amount of adsorbed enzyme is raised despite the high local concentration of immobilized enzymes. Degradation kinetics of pectins by an alkaline pectinase in bioscouring of cotton fabrics Carbohydrate Polymers, Volume 67, Issue 4, 19 February 2007, Pages 572-575 Qiang Wan+g, Xuerong Fan, Zhaozhe Hua, Weidong Gao and Jian Chen   Alkaline pectinases have been proven to be effective as bioscouring agents of cotton fabrics. In order to monitor the scouring degree of cotton fabrics quantificationally, a kinetic study of the degradation of pectins in cotton by an alkaline pectinase ‘Bioprep 3000L’ was performed and the influences of initial pectinase concentration and treatment time on bioscouring were evaluated quantitatively. The results showed that although the degradation products increased as pectinase concentration grew higher at same incubation time, the growth multiples of the maximum degradation rate which was used as the starting degradation rate were less than those of initial enzyme concentration. The degradation kinetics of pectins in cotton fibers with a pectinase could be described by modified Ghose–Walseth kinetic empirical equations which had been previously applied to the degradation reaction of cellulose. Effects of Neodymium on Growth, Pectinase Activity and Mycelium Permeability of Fusarium oxysporum Journal of Rare Earths, Volume 25, Issue 1, February 2007, Pages 100-105 Zhang Yufeng, Yang Lifen, Chen Kaoshan and Dong Liang The diameter of the colony of Fusarium oxysporum in solid medium, and the mycelium growth, pectinase activity, and mycelium permeability of Fusarium oxysporum in liquid medium under varying concentrations of Nd3+(0, 2, 4, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, and 400 mg·L−1) were measured. The results indicated that the growth of Fusarium oxysporum was stimulated in solid medium when the concentration of Nd3+ ranges from 2 to 180 mg·L−1, whereas it was inhibited when Nd3+ concentration was greater than 200 mg·L−1. The colonies were fewer and smaller when Nd3+ was used in the solid medium. The growth of Fusarium oxysporum was inhibited in liquid medium when Nd3+ was used. The inhibition rate showed by the dry weight of mycelium ranged from 4.83% to 52.18% and increased with Nd3+ concentration. The pectinase activity decreased compared with that of controls. When the concentration of Nd3+ was 10 and 400 mg·L−1, the pectinase activity decreased by 95% at both concentrations. Mycelium cell membrane permeability increased when Nd3+ concentrations ranged from 10 to 400 mg·L−1 but decreased when Nd3+ concentration was 2 mg·L−1. Preparation and properties of immobilized pectinase onto the amphiphilic PS-b-PAA diblock copolymers Journal of Biotechnology, Volume 128, Issue 1, 30 January 2007, Pages 112-119 Zhongli Lei and Shuxian Bi Well-defined amphiphilic block copolymers poly(styrene-b-acrylic acid) (PS-b-PAA) with controlled block length were synthesized using atom transfer radical polymerization (ATRP). Pectinase enzyme was immobilized on the well-defined amphiphilic block copolymers PS-b-PAA. The carboxyl groups on the amphiphilic PS-b-PAA diblock copolymers present a very simple, mild, and time-saving process for enzyme immobilization. Various characteristics of immobilized pectinase such as the pH and temperature stability, thermal stability, and storage stability were valuated. Among them the pH optimum and temperature optimum of free and immobilized pectinase were found to be pH 6.0 and 65 °C. Enzyme aided extraction of lycopene from tomato tissues Food Chemistry, Volume 102, Issue 1, 2007, Pages 77-81 Sheetal M. Choudhari and Laxmi Ananthanarayan Lycopene is a natural carotenoid pigment and a high value nutraceutical having wide use. The objective of the present work was to obtain a good yield of lycopene from tomato tissues, using cellulase and pectinase enzymes. Various parameters such as concentration of enzymes and time of incubation were optimised, to improve the yield of lycopene from tomatoes. Enzyme aided extraction of lycopene from whole tomatoes under optimised conditions resulted in an increase in the lycopene yield by 132 μg/g (198%) in cellulase treated sample and 108 μg/g (224%) in case of pectinase treated sample. Extraction from tomato peel under optimised conditions showed a remarkable increase in the yield of lycopene by 429 μg/g (107%) and 1104 μg/g (206%), for cellulase and pectinase treated samples, respectively. Likewise, the enzyme aided extraction of lycopene from fruit pulper waste and industrial waste of tomatoes was done to determine the potential for recovering the natural pigment from tomato waste. Chitosan-tethered the silica particle from a layer-by-layer approach for pectinase immobilization Food Chemistry, Volume 104, Issue 2, 2007, Pages 577-584 Zhongli Lei, Shuxian Bi and Hong Yang “Spherical polyelectrolyte brush” of core–shell structure were prepared by grafting poly (sodium 4-styrenesulphonate) (PSStNa) from SiO2 nanoparticle via the surface-initiated atom transfer radical polymerization strategy. The colloidal stability was not impeded by the adsorbed proteins despite the fact that up to 316.8 mg of enzyme was adsorbed per gram of the carrier particles. The immobilized pectinase revealed acceptable pH stability over a broad experimental range of 3.0–4.5. The activity half lives for native and bound states of enzyme were found as 13.5 d and 30 d, respectively. The activity of immobilized pectinase adsorbed onto these particles was analyzed in terms of the Michaelis–Menten parameters. Kinetic parameters were calculated as 8.28 and 9.98 g pectin ml−1 for Km and 1.165 × 10−3 g pectin s−1 g enzyme−1 1.124 × 10−3 g pectin s−1 g particle−1 for Vmax in the case of free and immobilized enzymes, respectively. Enzyme activity was found to be approximately 49.7% for immobilized enzyme after storage for 1 month. Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation Process Biochemistry, Volume 42, Issue 1, January 2007, Pages 98-101 Carolina Botella, Ana Diaz, Ignacio de Ory, Colin Webb and Ana Blandino The feasibility of using grape pomace for the production of xylanase and exo-polygalacturonase by Aspergillus awamori in solid state fermentation has been evaluated. Solid state fermentation experiments indicated that the particle size did not influence the enzyme production. The addition of extra carbon sources and the initial moisture content of the grape pomace were found to have a marked influence on the enzymes yields. Xylanase and exo-PG activities were high at 65% (w/w) initial moisture content and glucose supplementation. Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-state conditions Bioresource Technology, Volume 97, Issue 18, December 2006, Pages 2340-2344 Sarvamangala R. Patil and A. Dayanand Pectinase production studies were carried out in submerged and solid-state conditions from deseeded sunflower head employing Aspergillus niger. The two potential strains of A. niger, DMF 27 for submerged and DMF 45 for solid-state were isolated by multi-step screening technique based on coefficient of pectolysis and capability of pectinase production. Process variables such as size of inoculum, pH, temperature, particle size and moisture content were optimized with an aim to achieve the maximum production of pectinases. The increased level of pectinase production was recorded at pH 5.0 and temperature 34 °C in submerged and solid-state conditions. The optimum inoculum size was 1 × 105 ml−1 for submerged and 1 × 107 g−1 for solid-state conditions. Five hundred micrometer particle size and 65% moisture content of the substrate were optimum for the maximum production of pectinases in solid-state condition. Under optimum conditions, maximum production of exo-pectinase was 34.2 U/g in SSF and endo-pectinase was 12.6 U/ml in SmF. Production of pectinase from deseeded sunflower head by Aspergillus niger in submerged and solid-state conditions Bioresource Technology, Volume 97, Issue 16, November 2006, Pages 2054-2058 Sarvamangala R. Patil and A. Dayanand Studies were carried out on the production of pectinases using deseeded sunflower head by Aspergillus niger DMF 27 and DMF 45 in submerged fermentation (SmF) and solid-state fermentation (SSF). Higher titres of endo- and exo-pectinases were observed when medium was supplemented with carbon (4% glucose for SmF and 6% sucrose for SSF) and nitrogen (ammonium sulphate, 0.3% for both SmF and SSF) sources. Green gram husk proved to be relatively a better supplement to attain higher yield of endo-pectinase (11.7 U/g) and exo-pectinase (30.0 U/g) in solid-state conditions. Maximum production of endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by DMF 45 were recorded in SSF when compared to endo-pectinase (18.9 U/ml) and exo-pectinase (30.3 U/ml) by DMF 27 in SmF under optimum process conditions. The role of pectin in orange juice stabilization: Effect of pectin methylesterase and pectinase activity on the size of cloud particles Food Hydrocolloids, Volume 20, Issue 7, October 2006, Pages 961-965 Sarah Croak and Milena Corredig In citrus juice cloud particles impart the characteristic color and flavor, and although the chemical composition of these particles is known, the details of their stabilization are not well understood. Pectin, a major component of orange juice cloud, is thought to play an important role in juice destabilization: in the presence of the active enzyme pectin methylesterase (PME), pectin forms calcium pectate complexes and causes the precipitation of cloud particles. This research summarizes a study on the changes occurring to orange juice cloud particles after addition of pectinase and PME. The particle size of juice cloud did not change with addition of pectinase. On the other hand, at the natural pH (3.8) of the juice, the addition of PME caused aggregation of the cloud particles within a few minutes, and the amount of enzyme added affected the kinetics of the aggregation. A higher amount of enzyme was necessary to cause the same effects at pH 6.0, while at low pH (2.5) cloud particles showed faster aggregation and a higher particle size, possibly because of a decreased surface charge at this pH. The results obtained by dynamic light scattering provided evidence of a direct effect of the PME on cloud and question our current understanding of the mechanism of cloud loss. The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour Journal of Biotechnology, Volume 125, Issue 4, 1 October 2006, Pages 447-461 C. Louw, D. La Grange, I.S. Pretorius and P. van Rensburg Commercial polysaccharase preparations are applied to winemaking to improve wine processing and quality. Expression of polysaccharase-encoding genes in Saccharomyces cerevisiae allows for the recombinant strains to degrade polysaccharides that traditional commercial yeast strains cannot. In this study, we constructed recombinant wine yeast strains that were able to degrade the problem-causing grape polysaccharides, glucan and xylan, by separately integrating the Trichoderma reesei XYN2 xylanase gene construct and the Butyrivibrio fibrisolvens END1 glucanase gene cassette into the genome of the commercial wine yeast strain S. cerevisiae VIN13. These genes were also combined in S. cerevisiae VIN13 under the control of different promoters. The strains that were constructed were compared under winemaking conditions with each other and with a recombinant wine yeast strain expressing the endo-β-1,4-glucanase gene cassette (END1) from B. fibrisolvens and the endo-β-1,4-xylanase gene cassette (XYN4) from Aspergillus niger, a recombinant strain expressing the pectate lyase gene cassette (PEL5) from Erwinia chrysanthemi and the polygalacturonase-encoding gene cassette (PEH1) from Erwinia carotovora. Wine was made with the recombinant strains using different grape cultivars. Fermentations with the recombinant VIN13 strains resulted in significant increases in free-flow wine when Ruby Cabernet must was fermented. After 6 months of bottle ageing significant differences in colour intensity and colour stability could be detected in Pinot Noir and Ruby Cabernet wines fermented with different recombinant strains. After this period the volatile composition of Muscat d’Alexandria, Ruby Cabernet and Pinot Noir wines fermented with different recombinant strains also showed significant differences. The Pinot Noir wines were also sensorial evaluated and the tasting panel preferred the wines fermented with the recombinant strains. Pectinase production by solid fermentation from Aspergillus niger by a new prescription experiment Ecotoxicology and Environmental Safety, Volume 64, Issue 2, June 2006, Pages 244-250 Jing Debing, Li Peijun, Frank Stagnitti, Xiong Xianzhe and Ling Li The Ordos Plateau in China is covered with up to 300,000 ha of peashrub (Caragana) which is the dominant natural vegetation and ideal for fodder production. To exploit peashrub fodder, it is crucially important to optimize the culture conditions, especially culture substrate to produce pectinase complex. In this study, a new prescription process was developed. The process, based on a uniform experimental design, first optimizes the solid substrate and second, after incubation, applies two different temperature treatments (30 C for the first 30 h and 23 C for the second 42 h) in the fermentation process. A multivariate regression analysis is applied to a number of independent variables (water, wheat bran, rice dextrose, ammonium sulfate, and Tween 80) to develop a predictive model of pectinase activity. A second-degree polynomial model is developed which accounts for an excellent proportion of the explained variation (R2=97.7%). Using unconstrained mathematical programming, an optimized substrate prescription for pectinase production is subsequently developed. The mathematical analysis revealed that the optimal formula for pectinase production from Aspergillus niger by solid fermentation under the conditions of natural aeration, natural substrate pH (about 6.5), and environmental humidity of 60% is rice dextrose 8%, wheat bran 24%, ammonium sulfate ((NH4)2SO4) 6%, and water 61%. Tween 80 was found to have a negative effect on the production of pectinase in solid substrate. With this substrate prescription, pectinase produced by solid fermentation of A. niger reached 36.3 IU/(g DM). Goats fed on the pectinase complex obtain an incremental increase of during the initial 25 days of feeding, which is a very promising new feeding prospect for the local peashrub. It is concluded that the new formula may be very useful for the sustainable development of arid and semiarid pastures such as those of the Ordos Plateau. Purification and characterization of a novel pectinase from Acrophialophora nainiana with emphasis on its physicochemical properties Journal of Biotechnology, Volume 123, Issue 1, 3 May 2006, Pages 33-42 S. Maria C. Celestino, S. Maria de Freitas, F. Javier Medrano, M. Valle de Sousa and E. Ximenes Ferreira Filho An extracellular pectinase (PECI) was purified to apparent homogeneity from liquid state cultures of the thermophilic fungus Acrophialophora nainiana by ultrafiltration and a combination of gel filtration and ion-exchange chromatographic procedures. The molecular masses of PECI were 35,500 and 30,749 Da, as determined by SDS-PAGE and mass spectrometry, respectively. It was more active at 60 °C and pH 8.0 and showed high stability at 50 °C with half-life of 7 days. However at 60 and 70 °C, PECI was much less stable with half lives of approximately 20 and 3 min, respectively. The thermostability of purified PECI was also investigated by fluorescence and circular dichroism spectroscopy. Fluorescence revealed that the unfolding transition region was observed between 45 and 70 °C. A major decrease in the stability was found at 70 °C. Circular dichroism measurements at pH between 5.0 and 9.0 showed a transition temperature (Tm) range of 50–55°. The thermodynamic analysis of these results showed that EPGI is thermal stable protein exhibiting maximum stability (ΔG25) of 22.65 and 19.19 kcal/mol at pH 8.0 and 9.0, respectively. The apparent Km value on pectin from citrus fruits was 4.22 mg ml−1. PECI exhibited no detectable activity of pectin methylesterase, endo-polygalacturonase, mannanase, xylanase and cellulase. However, it showed exo-polygalacturonase and pectin lyase activities. The presence of carbohydrate was detected in the pure PECI. It was activated by l-tryptophan, DEPC, DTT, DTNB, DTP, l-cystein and β-mercaptoethanol and inhibited by NBS, Fe2+, Cu2+, Zn2+, Mn2+, Al3+ and Ca2+. The enzyme showed homology with a pectin lyases from Xanthomonas campestris and Bacillus licheniformis. A marked enhancement in the production of a highly alkaline and thermostable pectinase by Bacillus pumilus dcsr1 in submerged fermentation by using statistical methods Bioresource Technology, Volume 97, Issue 5, March 2006, Pages 727-733 D.C. Sharma and T. Satyanarayana The production of a highly alkaline and thermostable pectinase of Bacillus pumilus was optimized in submerged fermentation using Plackett–Burman design and response surface methodology. Three fermentation variables (C:N ratio, K2HPO4, and pH), which were identified to significantly affect pectinase production by Plackett–Burman design were further optimized using response surface methodology of central composite design (CCD). An over all 34- and 41-fold increase in enzyme production was achieved in shake flasks and lab fermenter by the optimization of variables using statistical approaches, respectively. The enzyme was optimally active at pH 10.5 and 50 °C, and selectively degraded only the noncellulosic gummy material of ramie (Boehmeria nivea) fibres causing 10.96% fibre weight loss, and therefore, the enzyme could find application in fibre processing industry. The use of the enzyme in fibre processing reduces the use of alkali, and the associated alkalinization of water bodies.

Last update: 05-July-2007