Acid Pectinase Research Articles

Pectinase Production by Rhizopus stolonifer A3 Isolated from Apple Peels

The application of pectinase in industries will continue to be developed. To broaden the applicability of enzyme manufacturing in new industries, more research to investigate pectinolytic microorganisms with high activity and stability is still required. Therefore, this study aimed to obtain pectinolytic fungi that have the potential as pectinase producers. Pectinolytic fungi were isolated from rotten apple peels and selected based on clear zone formation on pectic agar media after cetyl trimethyl ammonium bromide (CTAB) staining. Pectinolytic fungi were identified based on 18S rRNA partial gene sequences and morphological characteristics. Pectinase production used Mandels and Weber medium with citrus pectin 20 g l<sup>-1</sup> addition. Pectinase activity was determined based on the measurement of reducing sugars by a colorimetric method. A total of 5 fungal isolates were successfully isolated. All of the isolates had pectinolytic activities with clear zone diameters ranging between 0.99 to 7.32 mm. The isolate A3 showing the highest pectinolytic activity was identified as <em>Rhizopus stolonifer</em>. Microscopically, <em>R</em>. <em>stolonifer </em>A3 showed typical characteristics for <em>Rhizopus</em>, characterized by the presence of rhizoids, stolons and sporangiophores bearing a single spherical sporangium. The pectinase production of <em>R.</em> <em>stolonifer </em>A3 was optimum at initial pH 5.0, temperature 35 °C and incubation period of 3 days with pectinase activity of 14.75 U ml<sup>-1</sup>. <em>R. stolonifer </em>A3 produced acidic pectinase having optimum activity at pH 5.0 and temperature 50 °C. Thus <em>R.</em> <em>stolonifer </em>A3 has the potential to be used as a producer of acidic pectinase appropriate for use in the processing of fruit products.

Influence of Pectinase Adsorption on Scouring Efficiency of Mercerized Cotton Yarns

The low scouring efficiency of mercerized cotton yarns scoured with alkaline and acid pectinases, reported in previously published articles, raised the hypothesis of the lack of pectinase adsorption onto mercerized cotton. In an attempt to identify whether low pectinase adsorption occurs, the adsorption of alkaline and acid pectinases on raw and mercerized cotton yarns is measured using the Lowry protein assay, while laser-scanning confocal fluorescence microscopy is used to visually show the presence/absence of the enzymes on the surface of the cotton yarns. In addition, the surface zeta potential of the raw and mercerized cotton yarns is measured to identify the influence of the surface charge on the adsorption ability of the used enzymes. Data suggest that although used substrates have adsorbed more alkaline than acid pectinase, the adsorption of both enzymes is more intensive onto the mercerized cotton relative to raw cotton. The similar surface zeta potential of the raw and mercerized yarns indicates that higher enzyme adsorption on mercerized cotton is not influenced by the surface charge. Laser-scanning confocal fluorescence microscopy micrographs depict an evident increase of fluorescing signal with scouring time to raw and mercerized cotton yarn. The more intensive signals after 60 min of enzymatic scouring with both enzymes suggest enzyme presence on the yarn surface after a long treatment time. The obtained results confirm the enzyme retention on the yarn surface, thus excluding the hypothesis that the absence of enzyme adsorption is a factor for the low scouring efficiency of mercerized cotton yarns.

Thermostable and organic solvent-tolerant acid pectinase from Aspergillus terreus FP6: purification, characterization and evaluation of its phytopigment extraction potential.

The present study discusses the purification, characterization and application of pectinase from Aspergillus terreus FP6 in fruit pigment extraction. By the four-step purification involving precipitation, dialysis, ion-exchange chromatography, gel filtration chromatography, a 20.85-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the pectinase was 47kDa, as found by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme was recorded at pH 6.0 and 50°C. The enzyme retained 80.3% and 79.1% residual activity, respectively at pH 6.0 and 50°C for 90min. The pectinase was best functional in the presence of toluene and retained its activity for 30min. Cu2+ and Co2+ acted as enzyme activators, while Ca2+, β-mercaptoethanol, dimethyl sulfoxide and ethylenediaminetetraacetic acid proved to be the inhibitors. The K m and V max values of the pectinase with pectin as substrate were 0.002mM and 27.39 U/mL, respectively thus indicating the high enzyme affinity towards the substrate. After 30-min treatment of the grape skin with the partially purified enzyme, microscopic observation revealed that a short time of the enzymatic treatment resulted in substantial loss of pigment and shrinkage of the grape skin cells thereby highlighting the high efficiency of the pectinase. The current study implies that the A. terreus FP6 pectinase may be applied as a bio-agent in the food and beverage industries and has the potential to replace harmful solvents by promoting a greener approach to extract plant pigments.

Optimization of Growth Condition for Acidic Pectinase at Submerged Fermentation from Fruit Waste

Optimization of Growth Condition for Acidic Pectinase at Submerged Fermentation from Fruit Waste

Environmentally benign Scouring of Cotton Knits Using Locally Produced Acid Pectinase Enzyme

The main task of the present study is to pre-screen selected fungal strain (Fusarium oxysporum M1-EGY) isolated from local agriculture soil and optimize cultivation conditions for an acid pectinase production. Experimental results demonstrated the optimum conditions for achieving the highest enzyme activity (14.80 U/ml) were found to be: incubation period (8 days) at pH (5), temperature (30 °C), and shaking rate (150 rpm), apple pectin carbon source (2%), and ammonium phosphate as nitrogen source (0.2 gN/l). The positive impacts of utilizing the produced crude acid-pectinase in eco-friendly scouring of various cotton knits as well as their subsequent bleaching and reactive dyeing have been confirmed. Additionally, the use of local resources for an acid pectinase production would encourage and promote local production of textile enzymes and their potential applications in Egyptian textile sector, taking in consideration efficiency, textile quality, environmental and economy concerns.

Green surface modification and nano-multifunctionalization of denim fabric

Eco-friendly multifunctional denim products using environmentally sound technologies are still a challenge. In current study, enzymatic pretreatments of denim fabric using acid pectinases, laccases, and acid cellulases individually and in admixtures were carried out followed by post-nanocoating of bio-treated fabric samples with ZnO- and TiO2-NPs along with citric acid/sodium hypophosphite as ester-crosslinking/binding system using the pad-dry-microwave fixation technique. The impacts of enzymatic and subsequent nano-treatments on some physico-chemical, color change, anti-microbial and UV-blocking properties were assessed. Also, surface modification and nano-metal oxides immobilization were observed and confirmed using scan electron microscope and energy dispersive X-ray spectroscopy analysis. The experimental results revealed that the degree of surface modification and subsequent multifunctionalization are governed by type of enzyme or hybrid enzyme as well as kind of nano-metal oxide and its extent of fixation onto the modified/ester-crosslinked denim fabric structure. The synergistic effect and positive impacts of enzymatic treatment followed by nanofinishing especially in case of using ZnO-NPs on imparting better and durable performance and multifunctional properties without adversely affecting the coloration properties of denim fabric samples are confirmed and discussed. Mode of interactions was also suggested.

Pectinolytic enzymes-solid state fermentation, assay methods and applications in fruit juice industries: a review.

A plethora of solid substrates, cultivation conditions and enzyme assay methods have been used for efficient production and estimation of polygalacturonase and pectin methylesterase enzymes. Recent developments in industrial biotechnology offer several opportunities for the utilization of low cost agro-industrial waste in Solid State Fermentation (SSF) for the pectinolytic enzyme production using fungi. Fruit waste mainly citrus fruit waste alone and along with other agro-industrial waste has been explored in SSF for enzyme production. Agro-industrial waste, due to the economic advantage of low procuring cost has been employed in SSF bioreactors for pectinolytic enzyme production. Acidic pectinases produced by fungi are utilized especially in food industries for clarification of fruit juices. This review focuses on the recent developments in SSF processes utilizing agro-industrial residues for polygalacturonase and pectin methylesterase production, their various assay methods and applications in fruit juice industries.

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.

Influence of Various Pre-Treatment Processes on Selective Oxidation of Cotton Yarns

Various pre-treated cotton yarns were oxidized with potassium periodate. The effects of pre-treatments on oxidation were studied by quantitatively assessing functional groups, while the extent of cotton yarn degradation was determined by investigating pre-treated yarn mechanical properties and the degree of polymerization. Pre-treatments included various scouring procedures and process sequences. The type of scouring has a significant influence on the aldehyde and carboxyl groups before oxidation, while the pre-treatment sequence did not. The number of aldehyde groups was significantly influenced by oxidation and strongly dependent on the pre-treatment sequence applied. Alkaline-scoured yarns had the least number of aldehyde groups both before and after oxidation, followed by alkaline pectinase and acid pectinase scoured, regardless of the pre-treatment sequence.

The Influence of Non-selective Oxidation on Differently Pre-treated Cotton Yarns Properties

Differntly pre-treated cotton yarns were subjected to non-selective oxidation with perchloric acid and the effects of pre-treatment processes on oxidation were studied through functional groups quantity, and degradation through mechanical properties and degree of polymerization. Different pre-treatments include, different scouring (with alkali, alkaline and acid pectinase), mercerization-different scouring as well as different scouring-mercerization. The quantity of functional groups was studied by measuring carboxyl groups with methylene blue and calcium-acetate methods and aldehyde groups with iodine and tetrazolium (TTC) methods. Pre-treatment processes affect the degree of elimination of non-cellulosic components of cotton and have strong influence on oxidation process. The non-selective oxidation of enzymatic scoured cotton with perchloric acid has introduced carboxyl and aldehyde groups without serious degradation of cotton yarns, confirmed by only 15 % decreasing of tensile strength and 8 % decreasing of degree of polymerization. DOI: http://dx.doi.org/10.5755/j01.ms.22.3.12110

Alkaline pectinases: A review

Pectinolytic microbes have been industrially exploited for pectinases that are environment friendly enzymes and mineralize pectic substances present in the environment. This review provides structural understanding of the molecular determinants of pectin utilization, mechanisms driving catabolite selectivity and industrial applicability. The topology of pectinase with distinct catalytic machinery enables it to persist in harsh environment during infection and analysis of pectin degradation pathway has resulted in understanding the role of different pectinases in subsequent steps of substrate digestion in different cellular compartments. The pH has a definite role in altering enzyme properties. Acidic pectinases are utilized especially in food processing industries for extraction and clarification of fruit juices. Alkaline pectinases find tremendous applications in biotechnology based industries especially for cotton bioscouring and retting of plant fibers in textile processing, paper making and treatment of waste water containing pectinaceous material.

Immobilisation of acid pectinase on graphene oxide nanosheets

AbstractRecent progress in nanotechnology has prompted research interest in immobilised enzymes on graphene oxide (GO) nanosheets for their large specific surface area and abundant functional groups. In the present work, acid pectinase was immobilised on the GO via the cross-linking of amino groups on pectinase and functional groups (e.g. carboxyl group) on the GO surface. Acid pectinase was effectively immobilised on the support and high loading densities were obtained (2400 mg per g of support). In addition, the immobilised enzyme achieved a better catalytic efficiency (K cat/K m) than its free counterpart; 3.7 mg−1 min−1 mL for immobilised pectinase, 3.5 mg−1 min−1 mL for free pectinase. Under acidic conditions, pectinase immobilised on GO will be agglomerated, but the addition of surfactant PEG 6000 could solve the problem and afford higher catalytic activity and catalytic efficiency.

Identification of a fungus able to secrete enzymes that degrade regenerated cellulose films and analyses of its extracellular hydrolases

The LW03 strain was isolated from Chinese farmland soil and found to be able to secrete certain enzymes degrading regenerated cellulose films at low temperature. The LW03 strain was systematically identified as Rhizopus arrhizus var. arrhizus by morphological, physiological, and molecular methods. Incubation of regenerated cellulose films with the extracted crude enzyme of LW03 was done to measure morphological changes by using scanning electron microscopy. Microscopic observations showed that the morphology of the regenerated cellulose films changed drastically due to enzymatic hydrolysis. The extracellular hydrolases of LW03 strain incubated on bran medium were also assessed. The predominant activity in the crude enzyme was glucoamylase activity, followed by acid proteinase, phytase and pectinase activity. Interestingly, activities of β-glucosidase, endoglucanase, exoglucanase, and cellulase were also observed, but at a much lower extent. Based on initial evidence, the crude enzyme is most likely to contain some new constituents capable of degrading regenerated cellulose films.

Cold-Active Acid Pectinolytic System from PsychrotolerantBacillus: Color Extraction from Red Grape Skin

Cold-active enzymes are potentially relevant to food processing and, among them, pectinases belong to the most important enzymes in the fruit juice and wine industry. <i>Bacillus</i> sp. CH15 from grape berries was screened for the production of cold-active acidic pectinolytic enzymes. Taxonomic studies and 16S rDNA analysis revealed that the isolate was closely related to the <i>Bacillus subtilis</i> group. Maximum pectinolytic activity under acidic conditions (pH 5.0) and at low temperature (20°C) was obtained after 24 hr of incubation in medium containing citrus pectin as sole carbon source (0.305 U/mL). Polymethylgalacturonase activity was the predominant pectinase under the given assay conditions, whereas highest levels of pectate lyase activity were found at 60°C. At 5 and 10°C, the enzymatic system maintained 15 and 30% of the maximum activity, respectively. This is the first report on a pectinolytic enzyme system produced by a <i>Bacillus</i> strain and active at 20°C and pH 3.6, conditions similar to those in winemaking. According to classical and CIELab color parameters of short macerations with red grape skins at low temperature, the bacterial pectinolytic system produced a rapid color extraction and the macerates exhibited better chromatic characteristics than those obtained with commercial pectinases or after natural extraction. Total anthocyanin contents of the macerates with <i>Bacillus</i> enzyme system and after natural extraction were significantly different, whereas relative individual pigment quantities did not show any significant difference between the two treatments. In conclusion, <i>Bacillus</i> sp. CH15 could be used as a microbial source of cold-active acidic pectinases in red winemaking.

Characterization on Surface of Mercerized and Enzymatic Scoured Cotton after Different Temperature of Drying

The influence of different temperature of drying after mercerizing on enzymatic scouring through changes in the surface properties and scouring efficiency were investigated. Mercerized cotton yarns dried at room temperature (20 o C) and 80 o C were scoured with alkaline and acid pectinases. Alkaline scouring was also done for comparing. Surface properties and scouring efficiency were followed through the CIE whiteness degree, CIELAB coordinates, X-ray photoelectron spectroscopy, staining the non-cellulosic components with appropriate dyes, wettability and surface fee energy. Mercerization changes the cotton surface, and removes part of the cuticle components. Drying at 80 o C melted down the residual waxes covering the surface of the fiber, enhancing the alkaline and alkaline pectinase scouring and enabling acid pectinase scouring. While way of scouring has significant influence on the most measured properties, temperature of drying has significant influence on CIE whiteness, CIELAB coordinates a*, b*, L*BB and on x-ray photoelectron spectroscopy value O/C.

Wax removal for accelerated cotton scouring with alkaline pectinase

A rational approach has been applied to design a new environmentally acceptable and industrially viable enzymatic scouring process. Owing to the substrate specificity, the selection of enzymes depends on the structure and composition of the substrate, i.e. cotton fibre. The structure and composition of the outer layers of cotton fibre has been established on the basis of thorough literature study, which identifies wax and pectin removal to be the key steps for successful scouring process. Three main issues are discussed here, i.e. benchmarking of the existing alkaline scouring process, an evaluation of several selected acidic and alkaline pectinases for scouring, and the effect of wax removal treatment on pectinase performance. It has been found that the pectinolytic capability of alkaline pectinases on cotton pectin is nearly 75% higher than that of acidic pectinases. It is concluded that an efficient wax removal prior to pectinase treatment indeed results in improved performance in terms of hydrophilicity and pectin removal. To evaluate the hydrophilicity, the structural contact angle (theta) was measured using an auto-porosimeter.

Combined Bioscouring and Bleaching of Cotton Fibres

SUMMARY Cotton fibres were scoured with acid and alkaline pectinases and bleached with peracetic acid. Because of similar treatment conditions of both processes, they were combined in one bath as one-step and two-step processes. At the beginning of the treatments, pH 5 was set for acid pectinases and pH 8 for alkaline pectinases. To activate the peracetic acid, pH was also adjusted during the process. Tetrasodium pyrophosphate was added to improve the effects of bleaching and scouring. The highest CIE whiteness values and water absorbencies were obtained in a one-step process with alkaline pectinases and peracetic acid.

Effect of some process parameters in the enzymatic scouring of cotton using an acid pectinase

An analysis of variance has been applied to study the individual effects and the interaction between the process parameters, temperature, pH, and surfactant concentration, in the bioscouring of cotton fibers with an acid pectinase. This study indicates that increasing the temperature does not increase the total percentage of bioscouring but accelerates the rate of the process. The pH and surfactant seem to be determinant for the optimal enzyme performance under the studied conditions. The enzyme–substrate ratio has been analyzed and a kinetic study at different enzyme concentrations has been carried out.

Viscometric method for assaying of total endodepolymerase activity of pectinases.

An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, polymethylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5 min at 40 degrees C and the appropriate pH. Depending on the pH-optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH 5.0, and 2) for neutral (mildly alkaline) pectinases at pH 8.0. The modifications differed in the control and in the calculation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was linear in the range of at least 10-fold change in the enzyme concentration). The relative error of the method did not exceed 10%.

Effect of Sonication on Cotton Preparation with Alkaline Pectinase

Pectinase bioscouring of greige cotton fabric is a very promising new approach for preparing cotton textiles for subsequent wet processing treatments such as mercerizing, bleaching, dyeing, printing, or finishing. Such biopreparation of greige cotton fabric is an energy efficient and environmentally benign process. Our research has shown that at the laboratory scale, introducing ultrasonic energy into the reaction chamber during enzymatic scouring of the greige cotton fabric significantly improves pectinase efficiency, but does not decrease the tensile strength of cotton fabric. In line with our previous data, current research also confirms that sonication of pectinase processing solutions does not impair the complex structures of the enzyme molecules, but significantly improves their performance. Data show that alkaline pectinase appears to be a more efficient agent for biopreparation of greige cotton than acidic pectinase, resulting in better wettability and whiteness. We also establish that the combination of pectinase bioscouring with desizing and after-washing insures sufficient fabric wettability with adequate uniformity. The results are comparable to or better than those for fabric after traditional alkaline scouring. Introducing ultrasonic energy into the reaction chamber during enzymatic treatment of cotton fabric could help overcome the major disadvantage of pectinase scouring—a longer processing time compared to conventional alkaline scouring.