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The demand for microbial pectinase has increased due to its vast applications in different industries. The current study dealt with the synthesis of pectinase by a novel native strain Aspergillus cervinus ARS2 using agro-industrial waste. Comparative studies conducted on pectinase production by submerged fermentation (SmF) and solid-state fermentation (SSF) showed that pectinase activity was more increased in SSF (44.51 ± 1.33 IU/mL) than in SmF (40.60 ± 1.15 IU/mL) when using orange peel as a substrate. Using SSF, one-factor-at-a-time (OFAT) studies were conducted, considering different process variables such as inoculum size, initial pH, incubation time, moisture content, incubation temperature, and substrate particle size, all of which affected the pectinase activity. OFAT results showed the highest pectinase activity at an inoculum size of 106 spores/mL (43.11 ± 1.06 U/mL), an incubation time of 6 days (43.81 ± 1.21 U/mL), a moisture content of 100% (44.30 ± 1.69 U/mL), a substrate particle size of 1.7 mm (42.06 ± 1.20 U/mL), an incubation temperature of 37 °C (45.90 ± 1.33 U/mL), and an initial pH of 4 (43.31 ± 0.89 U/mL). The identified significant process variables were then optimized by response surface methodology (RSM)-central composite design (CCD). The results showed optimum pectinase activity of 107.14 ± 0.71 IU/mL for a substrate particle size of 2 mm, an incubation temperature of 31.5 °C, an initial pH of 4.9, and a moisture content of 107%, which was obtained from the Minitab optimizer. By using statistical optimization, the pectinase production from the isolated novel fungal strain A. cervinus ARS2 was increased 2.38-fold. Therefore, the A. cervinus ARS2 strain can be further explored for large-scale pectinase production which could meet the growing industrial demands. [ABSTRACT FROM AUTHOR]

Pectinolytic enzymes are related enzymes that hydrolyze pectic substances. Pectinolytic enzymes are of great interest in industrial applications for softening fruits, extracting and clarifying juices, extracting olive oil, retting textile fibers, preparing gel, and isolating protoplasts. The current work presents acidic extracellular pectinase production using low-cost agro-industrial waste with the indigenously isolated novel strain Aspergillus cervinus. Two fungal isolates, ARS2 and ARS8, with maximum pectinase activity, 41.88 ± 1.57 IU/mL and 39.27 ± 1.14 IU/mL, respectively, were screened out of 27 isolates from decayed fruit peels (orange, banana, and lemon) and soil containing decomposed vegetables. The isolate ARS2, identified as Aspergillus cervinus by molecular characterization, showed the highest pectinase activity of 43.05 ± 1.38IU/mL during screening and was further used for media component screening and optimization studies. To understand their effect on pectinase activity, one-factor-at-a-time (OFAT) studies were conducted on carbon sources, nitrogen sources, and mineral salts. The OFAT results showed the highest pectinase activity for orange peel (carbon source) at 44.51 ± 1.33 IU/mL, peptone (nitrogen source) at 45.05 ± 1.04 IU/mL, and NaH2PO4 (mineral salts) at 43.21 ± 1.12 IU/mL. The most significant media components screened by the Plackett–Burman (PB) design based on the p-value, Pareto chart, and main effect plot, were orange peel (p < 0.001), peptone (p < 0.001), NaH2PO4 (p < 0.001), and KH2PO4 (p < 0.001), which were further optimized using Response Surface Methodology (RSM) and Central Composite Design (CCD). The optimization results for the media components showed a maximum pectinase activity of 105.65 ± 0.31 IU/mL for 10.63 g orange peel, 3.96 g/L peptone, 2.07 g/L KH2PO4, and 2.10 g/L NaH2PO4. Thus, it was discovered that the indigenously isolated novel strain Aspergillus cervinus ARS2 was able to successfully produce a significant amount of pectinase using agro-industrial waste. Therefore, it can be considered for the large-scale optimized production of pectinase to meet industrial demands. [ABSTRACT FROM AUTHOR]

For the first time, the investigation delved into using a sustainable immobilization method using polyvinyl alcohol-alginate for pectinase, along with its application in clarifying Carica papaya juice. The pectinase produced by a newly isolated strain Aspergillus cervinus ARS2 was purified and immobilized into polyvinyl alcohol-alginate beads. Subsequently, the immobilized pectinase was subjected to characterization which revealed an optimum incubation time of 60 min, incubation pH of 5, and stability at acidic pH of 5 for 5 h storage time, and incubation temperature of 40 °C, with stability at 40 °C over a 5 h storage period. Reusability studies of immobilized pectinase showed a decrease in activity from 65.54 IU/mL (1stcycle) to 13.49 IU/mL (6thcycle) with minimum reduction in activity up to 3 cycles indicating its cost-effectiveness across a range of industrial applications. The kinetic parameters, Michaelis constant (K m), and maximum reaction rate (V max) for free and immobilized pectinase were found to be 0.78 mg/mL, 111.12 IU/mL, and 2.42 mg/mL, 71.42 IU/mL respectively. The comparison of immobilized pectinase with free pectinase showed better stability and less activity. Further based on better performance, immobilized pectinase was utilized for optimization of Carica papaya juice clarification using response surface methodology. The optimal conditions were found to be 39.49 °C incubation temperature, 62.12 min incubation time, and 3.89 mL enzyme load, resulting in juice clarity of 38.23% ± 1.12%. These findings suggest that the pectinase immobilized using polyvinyl alcohol-alginate can be a promising, economical, and sustainable industrial process in the clarification of fruit juice. [ABSTRACT FROM AUTHOR]

In this study, we compared the properties and structures of three fungal GH12 enzymes: the strict endoglucanase Bgh12A and the xyloglucanase Xgh12B from Aspergillus cervinus, and the endoglucanase Egh12 from Thielavia terrestris combining activity on linear β-glucan and branched xyloglucan. Egh12 from T. terrestris was produced in Pichia pastoris, purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides and had transglycosylase activity on (1,3)-β-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a β-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. Key points: • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides [ABSTRACT FROM AUTHOR]

The demand for microbial pectinase has increased due to its vast applications in different industries. The current study dealt with the synthesis of pectinase by a novel native strain Aspergillus cervinus ARS2 using agro-industrial waste. Comparative studies conducted on pectinase production by submerged fermentation (SmF) and solid-state fermentation (SSF) showed that pectinase activity was more increased in SSF (44.51 ± 1.33 IU/mL) than in SmF (40.60 ± 1.15 IU/mL) when using orange peel as a substrate. Using SSF, one-factor-at-a-time (OFAT) studies were conducted, considering different process variables such as inoculum size, initial pH, incubation time, moisture content, incubation temperature, and substrate particle size, all of which affected the pectinase activity. OFAT results showed the highest pectinase activity at an inoculum size of 106 spores/mL (43.11 ± 1.06 U/mL), an incubation time of 6 days (43.81 ± 1.21 U/mL), a moisture content of 100% (44.30 ± 1.69 U/mL), a substrate particle size of 1.7 mm (42.06 ± 1.20 U/mL), an incubation temperature of 37 °C (45.90 ± 1.33 U/mL), and an initial pH of 4 (43.31 ± 0.89 U/mL). The identified significant process variables were then optimized by response surface methodology (RSM)-central composite design (CCD). The results showed optimum pectinase activity of 107.14 ± 0.71 IU/mL for a substrate particle size of 2 mm, an incubation temperature of 31.5 °C, an initial pH of 4.9, and a moisture content of 107%, which was obtained from the Minitab optimizer. By using statistical optimization, the pectinase production from the isolated novel fungal strain A. cervinus ARS2 was increased 2.38-fold. Therefore, the A. cervinus ARS2 strain can be further explored for large-scale pectinase production which could meet the growing industrial demands.

Pectinolytic enzymes are related enzymes that hydrolyze pectic substances. Pectinolytic enzymes are of great interest in industrial applications for softening fruits, extracting and clarifying juices, extracting olive oil, retting textile fibers, preparing gel, and isolating protoplasts. The current work presents acidic extracellular pectinase production using low-cost agro-industrial waste with the indigenously isolated novel strain Aspergillus cervinus. Two fungal isolates, ARS2 and ARS8, with maximum pectinase activity, 41.88 ± 1.57 IU/mL and 39.27 ± 1.14 IU/mL, respectively, were screened out of 27 isolates from decayed fruit peels (orange, banana, and lemon) and soil containing decomposed vegetables. The isolate ARS2, identified as Aspergillus cervinus by molecular characterization, showed the highest pectinase activity of 43.05 ± 1.38IU/mL during screening and was further used for media component screening and optimization studies. To understand their effect on pectinase activity, one-factor-at-a-time (OFAT) studies were conducted on carbon sources, nitrogen sources, and mineral salts. The OFAT results showed the highest pectinase activity for orange peel (carbon source) at 44.51 ± 1.33 IU/mL, peptone (nitrogen source) at 45.05 ± 1.04 IU/mL, and NaH2PO4 (mineral salts) at 43.21 ± 1.12 IU/mL. The most significant media components screened by the Plackett–Burman (PB) design based on the p-value, Pareto chart, and main effect plot, were orange peel (p < 0.001), peptone (p < 0.001), NaH2PO4 (p < 0.001), and KH2PO4 (p < 0.001), which were further optimized using Response Surface Methodology (RSM) and Central Composite Design (CCD). The optimization results for the media components showed a maximum pectinase activity of 105.65 ± 0.31 IU/mL for 10.63 g orange peel, 3.96 g/L peptone, 2.07 g/L KH2PO4, and 2.10 g/L NaH2PO4. Thus, it was discovered that the indigenously isolated novel strain Aspergillus cervinus ARS2 was able to successfully produce a significant amount of pectinase using agro-industrial waste. Therefore, it can be considered for the large-scale optimized production of pectinase to meet industrial demands.

In spite of intensive exploitation of aspergilli for the industrial production of carbohydrases, little is known about hydrolytic enzymes of fungi from the section Cervini. Novel glycoside hydrolases Bgh12A and Xgh12B from Aspergillus cervinus represent examples of divergent activities within one enzyme family and belong to the GH12 phylogenetic subgroup I (endo-(1,4)-β-glucanases) and II (endo-xyloglucanases), respectively. The bgh12A and xgh12B genes were identified in the unsequenced genome of A. cervinus using primers designed for conservative regions of the corresponding subgroups and a genome walking approach. The recombinant enzymes were heterologously produced in Pichia pastoris, purified, and characterized. Bgh12A was an endo-(1,4)-β-glucanase (EC 3.2.1.4) hydrolyzing the unbranched soluble β-(1,4)-glucans and mixed linkage β-(1,3;1,4)-D-glucans. Bgh12A exhibited maximum activity on barley β-glucan (BBG), which amounted to 614 ± 30 U/mg of protein. The final products of BBG and lichenan hydrolysis were glucose, cellobiose, cellotriose, 4-O-β-laminaribiosyl-glucose, and a range of higher mixed-linkage gluco-oligosaccharides. In contrast, the activity of endo-xyloglucanase Xgh12B (EC 3.2.1.151) was restricted to xyloglucan, with 542 ± 39 U/mg protein. The enzyme cleaved the (1,4)-β-glycosidic bonds of the xyloglucan backbone at the unsubstituted glucose residues finally generating cellotetraose-based hepta-, octa, and nona-oligosaccharides. Bgh12A and Xgh12B had maximal activity at 55 °C, pH 5.0. At these conditions, the half-time of Xgh12B inactivation was 158 min, whereas the half-life of Bgh12A was 5 min. Recombinant P. pastoris strains produced up to 106 U/L of the target enzymes with at least 75% of recombinant protein in the total extracellular proteins. The Bgh12A and Xgh12B sequences show 43% identity. Strict differences in substrate specificity of Bgh12A and Xgh12B were in congruence with the presence of subgroup-specific structural loops and substrate-binding aromatic residues in the catalytic cleft of the enzymes. Individual composition of aromatic residues in the catalytic cleft defined variability in substrate selectivity within GH12 subgroups I and II. [ABSTRACT FROM AUTHOR]