Maximum Pectinase Research Articles

Pectinase production from Aspergillus niger IBT-7 using solid state fermentation

Different fungal strains were isolated from the local soil, fruits and vegetables on the basis of pectin hydrolysis. All the isolated strains were identified through microscopic studies and screened for pectinase production using solid state fermentation. The fungal strain identified as Aspergillus niger IBT-7 showed the highest pectinase production. The selected strain was further subjected to optimization through different physical and nutritional parameters to enhance the production of pectinase. Amongst seven different media tested M1 containing rice bran, moistened with Czapek’s nutrient medium showed the highest pectinase production. During optimization maximum pectinase production was achieved after 72 hrs of incubation at 30 ml of moisture content, pH 5.0 and 30°C. Xylose (1.5%) and yeast extract (1%) proved to be best supplemented carbon and nitrogen sources, respectively which gave the highest pectinase production (39.1 U/ml/min).

Optimization of pectinase production by <i>Aspergillus niger</i> using central composite design

Pectinases are a group of enzymes that catalyze the breakdown of pectin. Pectinase producing Aspergillus niger was obtained from a five-day old Eba (Cassava flakes). Response surface methodology was used for optimizing the process of the pectinase produced. Four independent variables which are, temperature, pH, substrate concentration and time of Heating at 70 o C were used to optimize the significant correlation between the effects of the variables on pectinase production. A second-order polynomial was fitted to data and validated by ANOVA. The results revealed maximum pectinase production at pH 6.0, 50 o C Temperature, 0.02% substrate concentration and the enzyme lost all its activity within 7 min of heating at 70 o C. The study revealed that optimization of pectinase through RSM could improve the enzymatic characteristics and yield of the enzyme. The models used were highly significant with a correlation coefficient (R 2 ) of 0.901 Keywords: Pectinase; Aspergillus niger; Response Surface Methodology; Central Composite Design

A study on pectinases from Aspergillus tamarii: Toward greener approach for cotton bioscouring and phytopigments processing

Pectinolytic enzymes, a family of glycosyl hydrolases comprising of endo-rhamnogalacturonase, exo-polygalacturonase, endo-polygalacturonase and endo-xylogalacturonase play a significant role in food processing industries for the increase of shelf life of the food products containing pectin. This study was carried out for the production and activity of each enzyme member of the pectinase classification. Solid state fermentation process was utilized for the production of pectinase from Aspergillus tamarii using various pectin rich agro-industrial wastes like banana peel, sugarcane bagasse, lemon peel, coffee pulp and orange peel which were used for the fungal growth and wheat bran was used as the major nitrogen source. Fermentation parameters mainly pH, temperature, time, substrate concentration and moisture were optimized for maximum pectinase production. Production yield was measured using endo-polygalacturonase, exo–polygalacturonase, pectin lyase and pectin esterase assay. Results revealed that wheat bran and sugarcane bagasse at the ratio of 3:1 demonstrates appreciable Exo-pectinases production in 30 °C for 96 h at 70% moisture among the four enzymes. Characterization studies indicated the maximum activity (101.05 U/ml) and stability of Exo-PG at pH 6.0 and 55 °C. Thin layer Chromatography (TLC) was used for the identification of the hydrolysed products. Application studies were carried out for bioscouring of cotton fibers and extraction of lycopene and anthocyanin. Results suggested that use of pectinases substantially replaces the chemicals usage and will be considered as a suitable bio-agent in the current scenario on green process technology development.

Biotechnological valorization of pineapple stem for pectinase production by Bacillus subtilis BKDS1: Media formulation and statistical optimization for submerged fermentation

Abstract Agro-residues are mainly comprised of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. India is one of the largest producers of pineapple and along with the expanding pineapple production, associated agro-wastes are also increasing proportionally. In this background, the present study has been endeavored to accomplish valorization of pineapple stem for the economical production of the enzyme, pectinase by Bacillus subtilis BKDS1 using pineapple stem extract (PSE) medium. The results revealed that, the effective concentration of the extract towards producing maximum pectinase enzyme was found to be 12.5% in distilled water. Enhancement of pectinase production in terms of media optimization has been attempted using Plackett-Burman design (PBD) and response surface methodology (RSM). Out of nine variables screened by PBD, only four variables; yeast extract, CaCl2, CaCO3 and inoculum have been selected to affect the pectinase production significantly. The single and interactive effects of all the selected variables have been analyzed using Central composite design (CCD) of RSM. The data revealed that, optimized medium produces a higher concentration of enzyme than unoptimized medium. Further, the efficiency of modified PSE medium for the production of pectinase by submerged fermentation (Smf) has confirmed in laboratory scale fermenter. It has also been found that, enzyme production is achieved at a faster rate in fermenter compared to shaker flasks. Thus, the study as whole highlights the fact that, pineapple stems can be successfully employed in the production of the industrially important enzyme, pectinase using B. subtilis BKDS1. This will in turn, can deliver a lot towards the proper management of agro-wastes and also ensure a better cleaner environment.

Extracellular Pectinase from a Novel Bacterium Chryseobacterium indologenes Strain SD and Its Application in Fruit Juice Clarification.

Pectinase is one of the important enzymes of industrial sectors. Presently, most of the pectinases are of plant origin but there are only a few reports on bacterial pectinases. The aim of the present study was to isolate a novel and potential pectinase producing bacterium as well as optimization of its various parameters for maximum enzyme production. A total of forty bacterial isolates were isolated from vegetable dump waste soil using standard plate count methods. Primary screening was done by hydrolysis of pectin. Pectinase activity was determined by measuring the increase in reducing sugar formed by the enzymatic hydrolysis of pectin. Among the bacterial isolates, the isolate K6 exhibited higher pectinase activity in broth medium and was selected for further studies. The selected bacterial isolate K6 was identified as Chryseobacterium indologenes strain SD. The isolate was found to produce maximum pectinase at 37°C with pH 7.5 upon incubation for 72 hours, while cultured in production medium containing citrus pectin and yeast extract as C and N sources, respectively. During enzyme-substrate reaction phase, the enzyme exhibited its best activity at pH of 8.0 and temperature of 40°C using citrus pectin as substrate. The pectinase of the isolate showed potentiality on different types of fruit juice clarification.

Characterization of Pectinase from Bacillus subtilis Strain Btk 27 and Its Potential Application in Removal of Mucilage from Coffee Beans.

The demand for enzymes in the global market is projected to rise at a fast pace in recent years. There has been a great increase in industrial applications of pectinase owing to their significant biotechnological uses. For applying enzymes at industrial scale primary it is important to know the features of the enzyme. Thus, this study was undertaken with aims of characterizing the pectinase enzyme from Bacillus subtilis strain Btk27 and proving its potential application in demucilisation of coffee. In this study, the maximum pectinase activity was achieved at pH 7.5 and 50°C. Also, the enzyme activity was found stimulated with Mg2+ and Ca2+ metal ions. Moreover, it was stable on EDTA, Trixton-100, Tween 80, and Tween 20. Since Bacillus subtilis strain Btk27 was stable in most surfactants and inhibitors it could be applicable in various industries whenever pectin degradation is needed. The enzyme Km and Vmax values were identified as 1.879 mg/ml and 149.6 U, respectively. The potential application of the enzyme for coffee processing was studied, and it is found that complete removal of mucilage from coffee beans within 24 hours of treatment indicates the potential application in coffee processing.

Algal biomass: A sustainable, economical and renewable approach for microbial production of pectinolytic enzymes using submerged and solid state fermentation techniques

In the past decade, algal waste has been used as useful natural resource for production of enormous range of products that have wide economical and commercial importance. Pectinases are group of enzymes that have wide commercial applications. Hence, current study was designed to utilize algal biomass for the production of pectinases using submerged (SmF) and solid state fermentation (SSF) techniques. Different algal sources including brown (Dictyopteris polypodioides, Sargassum wightii and Dictyopteris divaricata) and green algae (Ulva lactuca and Codium tomentosum) were used and U. lactuca was found to be the most suitable substrate. Several bacterial and fungal strains were screened and among them Bacillus licheniformis KIBGE-IB4 was selected based on maximum pectinase production. SmF and SSF were studied utilizing U. lactuca as a substrate and results revealed that enzyme production was favoured by SmF (2457 ± 3.31 U mg−1) as compared to SSF (1432 ± 1.46 U mg−1). Parametric optimization of pectinase production indicated that B. licheniformis KIBGE-IB4 requires 10.0 g L–1 U. lactuca as a biomass in the medium with a pH 7.0 when incubated at 37 °C for 24 hours. Likewise, production of pectinase using algal resource was also compared with that of the conventional agricultural biomass and it was observed that when U. lactuca was used, the selected bacterial isolate produced a higher yield of enzyme than sugarcane bagasse and rice husk. Hence, it is anticipated that algal biomass can be efficiently utilized as an environmental friendly bioresource for the production of industrially important hydrolytic enzymes.

Isolation of Bacillus cereus P05 and Pseudomonas sp. X12 and their application in the ramie retting

Two bacterial strains that express high levels of pectinase and xylanase, named P05 and X12, were isolated from the soil of a ramie garden using pectinase and xylanase selective medium. A bioaugmentation experiment for ramie retting using these bacterial strains showed a significant increase in retting rate when equal ratios of P05 and X12 were mixed into the retting solution at 20% of the volume. Using bioaugmentation, the retting process was completed at 60h, reducing the retting period by 50%, and the gum removal ratio improved by 79.6%. In addition, the breaking strength of the resulting fiber was increased by 52.3%. Analysis of bacterial population structure and enzyme activity levels in the bioaugmented retting system showed that P05 and X12 quickly became the dominant bacteria within the retting solution microflora, and improved the microflora’s ability to produce key retting enzymes. The maximum pectinase and xylanase activities in the bioaugmented retting system were 80.84U/mL and 50.51U/mL, respectively, which were 68.98% and 64.21% higher respectively when compared to the control. Thus, addition of P05 and X12 into the retting solution resulted in accelerated gum degradation and fiber separation.

EXTRACELLULAR PECTINASE ACTIVITY FROM Bacillus Cereus GC Subgroup A: ISOLATION, PRODUCTION, OPTIMIZATION AND PARTIAL CHARACTERISATION

A pectin degrading bacterium was isolated from a soil and identified as Bacillus cereus GC subgroup A based on its morphological, physiochemical and FAME-GC analysis. The Solid state fermentation at 37 °C for 72h, strain produced pectinase in different substrates; among the substrates orange peel powder and wheat bran powder were good for strain but mixture of orange peel powder and wheat bran powder enhanced enzyme production upto 804 IU/ml. The maximum pectinase production was studied at 370 C and pH 7 after 96h of incubation by Bacillus cereus. Regarding carbon sources, galactose (0.02%) was the best source for strain; while malt extract (0.1%) was found as the best nitrogen source for Bacillus cereus. The enzyme was partially purified by ammonium sulphate precipitation and dialysis was carried out to remove the excess salt. A Lineweaver-Burk analysis showed a Km value of 8.3 mg/ml and Vmax of 1428.5 IU/ml. The partially purified enzyme exhibited maximal activity at a temperature of 40°C and pH 6.0. The enzyme showed stability towards salts at different concentrations 1mM, 10mM, 50mM and 100mM. In addition to that CaCl2 has increased activity of enzyme at higher concentration too. The amino acid composition of pectinase was also determined by BIOEDIT software. The molecular weight of pectinase was 38304.27 Da (348 amino acids) and was rich in glycine and valine.

Statistical Optimization of Pectinase Biosynthesis from Orange Peel by Bacillus licheniformis Using Submerged Fermentation

Agricultural, fruit and vegetable wastes were investigated as cost effective source for pectinase production. Orange peel proved as the best inducer of pectinase in a very simple culture medium used. Optimization of various cultural parameters was carried out by statistical method using response surface methodology and central composite design. The basic aim was to find out the best among local agri-wastes for maximum production of pectinase keeping in mind the agro-based economy of the country. Also the aim was to find the best pectinase of alkaline and thermophilic nature having maximum activity in minimum time, to be used in various industrial processes mainly textile industry which is the key industry in a country like Pakistan. The Bacillus licheniformis used in the present study was of special worth as it gave maximum pectinase activity of 219 U/ml in submerged fermentation, in a very simple medium with very few components (NaNO3, KH2PO4, KCl, MgSO4, Tryptone and Orange peel) under optimum conditions of pH 9.5, when incubated at 37 °C for 120 h in an alkaline culture medium of pH 9.5 supplementing with 0.3 % inoculum, 2.5 % orange peel and 0.5 % tryptone. The enzyme exhibited thermophilic nature by showing maximum activity when incubated with substrate (0.5 % citrus pectin solution pH 8.0) at 70 °C for 10 min. The study proved that orange peel has nutrients that enabled the microorganism understudy to produce high quantity (219 U/ml) of pectinase in very simple medium and can be better option for pectinase production on large scale for commercial use in industries.

Conversion of orange peel waste biomass to bioelectricity using a mediator-less microbial fuel cell

Microorganisms have the potential to become a game-changer in sustainable energy production in the coming generations. Microbial fuel cells (MFCs) as an alternative renewable technology can capture bioenergy (electricity) from carbon-based sources by utilizing microorganisms as biocatalysts. This study demonstrated that MFC technology can be explored for bioelectricity production from orange peel waste (OPW), an agricultural byproduct and an organic substrate, without any chemical pretreatment or the addition of extra mediators. A maximum voltage generation of 0.59±0.02V (at 500Ω) was achieved in a dual chamber MFC during stable voltage generation stages. The maximum power density and current density obtained were 358.8±15.6mW/m2 and 847±18.4mA/m2, respectively. Key components of OPW, namely pectin and cellulose, were also tested in their pure form, with pectin giving a stable current, while no significant current generation was achieved using cellulose alone as the substrate, thus demonstrating the absence of cellulose-degrading bacteria. Maximum pectinase and polygalacturonase enzyme activities of 18.55U/g and 9.04U/g (per gram of substrate), respectively were achieved during orange peel degradation in MFCs. Bacterial identification using 16S rRNA analysis of the initial inoculum fed to the MFC, the biofilm attached to the anode, and the anode suspension, showed significant diversity in community composition. A well-known exoelectrogen, Pseudomonas, was present among the predominant genera in the anode biofilm.

Morphological and molecular based identification of pectinase producing Bacillus licheniformis from rotten vegetable.

Pectinase catalyzed the degradation of pectin substances and has been used in various biotechnological industries. In the current study, 23 bacterial strains were isolated from rotten vegetables, soil and air. The isolated bacterial strains were qualitatively screened for pectinase production on pectin agar medium and only three strains HR 4, HR 21 and HR 23 were observed to produce extracellular pectinase. These strains were further screened quantitatively for pectinase production through submerged fermentation technology in pectin containing fermentation medium. Strain HR 4 from rotten brinjal (Solanum melongena) was found to produce higher pectinase as compared to others. The maximum pectinase producing bacterial strain was identified as Bacillus licheniformis on the basis of morphological, physiological and biochemical characteristics. For further confirmation of identification, 16S rDNA sequence analysis was performed. The 16S rDNA sequences were aligned and the phylogenetic tree was constructed. The phylogenetic tree confirmed that the strain was belonging to B. licheniformis. The 16S rDNA sequences of this new strain were submitted to GenBank and designated as B. licheniformis KIBGE-IB21 with the GenBank accession number JQ 411812. The newly isolated pectinase producing B. licheniformis used apple pectin as carbon and yeast extract as nitrogen source for maximum pectinase production.

Optimization of Cultural Conditions for the Production of Pectinase from Selected Fungal Strain

Pectinase are the group of enzymes that catalyze the degradation of pectin substances through de-polymerisation and de-esterification. This study is concerned on the isolation, screening and selection of suitable strain of pectinolytic organism and optimization of cultural conditions for the biosynthesis of pectinase. From the soil samples collected from Lalitpur, Kathmandu, Gulmi, Manang and Dang, 18 fungal colonies were isolated on the basis of halozone formation on Potato dextrose agar and identified. Enzyme production was carried out by submerged state fermentation. The partially purified enzymes showing higher pectinolytic activity were characterized on the basis of temperature of incubation, substrate concentration and pH of the substrate by Dinitro salicylic acid assay (DNSA) method. The fungal isolate showing highest enzyme activity was subjected to optimization of culture medium for the production of enzymes. On optimization, it was found that MG1 (Aspergillus niger) was the most potent strain at 1.5% substrate (pectin) concentration, pH 4.5 and temperature of 30°C. On the enzyme application, the yield of the orange juice, Total Soluble Solid and absorbance increased as the concentration of the enzyme increased and hence increasing the possibility of being used commercially for maximum pectinase production. DOI: http://dx.doi.org/10.3126/jfstn.v8i0.11752 J. Food Sci. Technol. Nepal, Vol. 8 (65-70), 2013

Immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 on chitosan beads for continuous degradation of pectin polymers

Abstract Pectinase catalyzes the degradation of pectin substances and has been used in various food and textile industries. In this study, pectinase from Bacillus licheniformis KIBGE-IB21 was immobilized on chitosan beads using formaldehyde as a crosslinking agent. Various concentration of formaldehyde was tried during immobilization procedure and 10% was found to immobilize maximum pectinase. The optimum crosslinking time and temperature for the activation of chitosan beads were observed to be 3.0 h and 30 °C, respectively. The immobilization did not influence the optimum pH and temperature of pectinase for maximum activity and both the native and immobilized pectinase showed maximum activity at same pH and temperature. However, the stability of pectinase against different pH and temperatures was increased after immobilization. The chitosan immobilized pectinase showed excellent recycling efficiency and retained more than 90% of its initial activity during 4th cycle.

Assessment of pectinase production by Aspergillus oryzae in growth-limiting liquid medium under limited and non-limited oxygen supply

The cultivation of Aspergillii in liquid media is often hindered by the intense consumption of oxygen by the fungi. In this work, pectinase production by Aspergillus oryzae IPT-301 was assessed in cultures grown with limiting and non-limiting oxygen supplies to evaluate the effects of pH and the concentration of dissolved oxygen on the process. Different strategies – such as the use of a growth-limiting medium and the delayed addition of inducer (citrus pectin) to the bioreactor – were employed to provide an effective oxygen supply to the microorganisms and to avoid excessively high impeller speeds that could damage the fungal hyphae. In a limited oxygen environment, it was observed that the maintenance of a constant pH value of 4.0 throughout the cultivation resulted in high cell mass concentration but no enzyme activity. However, when the pH was kept at 4.0 only during the period of intense cell growth followed by sustained control at pH 2.7, significant enzyme activity was detected (43UmL−1). It was observed that this low pH favors both the production of enzyme and their stability. Under a non-limiting dissolved oxygen concentration, over 30% of saturation, the delayed pH control approach resulted in the maximum pectinase activity of 120UmL−1.

Enzymatic properties of oyster mushroom (Pleurotus florida) exudate

Pleurotus florida Mont. exudate, appearing as reddish- brown droplets of fluid was characterized for enzymatic properties. Oyster mushroom exudate grown on mango peel residue and wheat straw in 10:1 ratio at 25±1°C possessed high antioxidant value (920 mM/mL) and exhibited pectinase, cellulase, b-glucosidase and endoglucanase activities. Predominant enzyme activity was identified as pectinase with maximum activity of 0.35 U/ ml. The temperature of 35 ºC and pH 5.0 were found optimum for maximum pectinase specific activity. Kinetic analyses of pectinase in terms of Michaelis-Menten constant (Km), velocity maximum (Vmax) were 0.54 mg/ml, 0.25 mole/ml/min, respectively. This is first report of mushroom exudate enzyme characterization.

Pectinase production by Aspergillus niger isolated from decomposed apple skin

Pectinase activity among twelve different fungal strains, Aspergillus niger IM09 was identified as a potential one to produce maximal level 831 U/g at pH 4.0. Media composition, incubation temperature, incubation time, substrate concentration, aeration, inoculum size, assay temperature and nitrogen sources were found to effect pectinase activity. Moisture content did not affect the activity significantly. Media composition was varied to optimize the enzyme production in solid state fermentation. It was observed that the highest pectinase activity of 831.0 U/g was found to produce in presence of yeast extract as a nitrogen source in combination with ammonium sulfate in assay media. Aeration showed positive significant effects on pectinase production 755 U/g at 1000 ml flasks. The highest pectinase production was found at 2 g pectin (521 U/g) used as a substrate. Pectinolytic activity was found to have undergone catabolite repression with higher pectin concentration (205 U/g at 5 g pectin). The incubation period to achieve maximum pectinase activity by the isolated strain Aspergillus niger IM09 was 3 days, which is suitable from the commercial point of view. DOI: http://dx.doi.org/10.3329/bjsir.v48i1.15410 Bangladesh J. Sci. Ind. Res. 48(1), 25-32, 2013

Pectinase production from lemon peel pomace as support and carbon source in solid-state fermentation column-tray bioreactor

Pectinase is an important enzyme that finds application in many food processing industries and solid-state fermentation (SSF) is an attractive technology for enzyme production. In this work, a SSF process is described for the production of pectinase by Aspergillus niger Aa-20 and lemon peel pomace (LPP) as support and carbon source in a solid-state bioreactor. The process consists of three steps. (1) Selection of microorganism for SSF. Eight different fungal strains from the genus Aspergillus and Penicillium were screened for invasion ability on LPP; (2) Selection of particle size. Invasion ability of selected fungal strain was analyzed on four particle sizes of LPP; (3) SSF process was operated in a column-tray bioreactor at 30°C and 70% moisture content, 194mL/min of air flow rate and substrate particle size (2–0.7mm) of LPP for 96h. Results showed, that high levels of pectinase activities were obtained. The maximum pectinase activity obtained was 2181U/L. Maximum biomass and maximum specific growth rate of A. niger Aa-20 were Xmax=8mgglucosamine/g of LPP and μmax=0.1271/h. The LPP and the use of A. niger Aa-20 in SSF suggest as a very promising process for pectinase production.

Screening and identification of Acinetobacter junii for Apocynum vernetum L. fiber enzymatic retting

There are many kinds of ketones with antisepsis functions in the Apocynum vernetum L. fiber (ALF), but its enzymatic retting is difficult. In order to select the appropriate ALF enzymatic retting strain, wastewater from ALF retting was analyzed to screen a strain with high pectinase activity. Of the analyzed strains, the maximum pectinase activity measured was of the #2 strain: 103.2 IU/ml at a zymogenic time of 12 hrs at 37°C. The retting experiments verified that this strain was well suited for ALF; the residual gum rate of ALF was 15.26% and its fiber physicochemical properties were similar to those fibers which were chemically retted. The strain was determined to be a new retting strain, Acinetobacter junii, according to a 16S rDNA sequence analysis in combination with morphology, and biophysical and biochemical tests using fermentation, catalase, methyl red and gelatin liquefaction. As a new pectinase‐producing strain, Acinetobacter junii could be utilized in industrial enzymatic retting productions after further process optimization and can replace the conventional chemical retting process due to its improved fiber quality and reduced environmental pollution.

Utilization of agro-industrial residues for pectinase production by the novel strain Pseudozyma sp. SPJ under solid state cultivation

Production of high titers of an alkaline, extracellular and thermo-tolerant pectinase by a newly isolated yeast Pseudozyma sp. SPJ was carried out in solid state fermentation (SSF). Among all the agro-industrial residues used as substrate, citrus peel was found to be the best. Maximum pectinase production was observed after 72 h of incubation at 32°C. The moistening agent containing MgSO4⋅7H2O, KH2PO4 and (NH4)2SO4, at pH 7.0 with a substrate-to-moisture ratio of 1:3, was found to be most effective. No additive was required to supplement the substrate for production of the enzyme. Use of an economical substrate (citrus peel) and only a little mineral salt solution without any other supplement, made pectinase production cost-effective. A commendable enzyme titer (279,600 ± 897 U g−1 solid substrate) by Pseudozyma sp. SPJ under such an economic solid state cultivation is reported for the first time. This enzyme could be applied in various industries (works within a good temperature and pH range) and in biodegradation of citrus waste.