Optimization of Aspergillus niger NRC1ami Pectinase Using Citrus Peel Pectin, Purification, and Thermodynamic Characterization of the Free and Modified Enzyme

Abstract

Enzyme cost and stability are the main problems facing industrial applications. Consequently, Aspergillus nigerNRC1ami was isolated from rotten orange and recorded a promising pectinase activity (13.8 U/ml). The enzyme was optimized using citrus peel pectin as the sole carbon source and recorded (40 U/ml). It was purified by two steps purifications and recorded 632 purification folds. The pure enzyme showed 14.7% carbohydrate content and consists of 15 amino acids. Glutamic acid was the major (22%) followed by leucine (10.67%) and threonine was the minor (2.70%). A. niger NRC1ami pectinase was conjugated by covalent coupling to sodium periodate (NaIO4) activated polysaccharides. Galactomannan showed the highest recovered activity (85%) and 94.34% reduction in viscosity. The optimum temperature for the pure enzyme shifted from 40 to 45 °C after the conjugation process. Also, the free enzyme showed its optimum activity at pH 5 compared to pH 4, 5 in the conjugated form case. The thermal stability of the free enzyme greatly improved after the modification process. The conjugated process reduced the activation energy to 36%, prolonged the enzyme half-life 5.6-fold at 60 °C, and increase the deactivation energy (Ed) by about 19% in comparison to the free form. The D value of the conjugated enzyme increased to 13.2-fold at 50 °C compared to the free form. Gibbs's free energy (ΔG) of the enzyme increased after the modification process, while the enthalpy (ΔH) and entropy (ΔS) decreased. Na+ and Zn2+ had a stimulating effect on both enzyme forms. Graphical Abstract

Improvement of fruit juice quality: novel endo-polygalacturonase II from Aspergillus tubingensis FAT 43 for enhanced liquefaction, clarification, and antioxidant potential

“…26 The thermal stability of the enzyme isolated in this research was higher than previously reported based on the D -values comparison, which is higher than other research groups reported. 19,26…”

“…28 The same relation between calculated ΔS and ΔH was reported previously for pectinase derived from A. niger isolated from orange, but the stability of this enzyme was low even after a chemical modification that resulted in a minimal increase in enzyme stability. 26 The thermal stability of the enzyme isolated in this research was higher than previously reported based on the D-values comparison, which is higher than other research groups reported. 19,26 The prevailing characterisation of PGs currently describes a predominant pattern of significantly low thermal stability.…”

“…26 The thermal stability of the enzyme isolated in this research was higher than previously reported based on the D-values comparison, which is higher than other research groups reported. 19,26 The prevailing characterisation of PGs currently describes a predominant pattern of significantly low thermal stability. A study by da Câmara Rocha et al (2020) showed that A. niger exhibits sustained PG activity over 2 h at 30 °C and 40 °C within the pH range of 4.0-6.0.…”

“…The calculated energy of denaturation PG II was 32.8 kJ mol −1 which is a much higher value compared to E d of pectinase isolated from A. niger NRC1ami. 26 Because E d is directly related to the activation enthalpy of denaturation (Δ H ), which expresses the total amount of energy required to denature the enzyme, large and positive values of E d and Δ H should be associated with high enzyme thermostability. 27 Table 2 shows the results of Δ H and other thermodynamic parameters of thermal denaturation of PG II at temperatures ranging from 40 to 60 °C.…”

Pectinases as promising green biocatalysts having broad‐spectrum applications: Recent trends, scope, and relevance

“…In paper industries, the enzymes are basically used for the manufacturing processes. In the present era of papermaking, the cationic polymers are used to improve drainage and increased retention of fine particles added to pulp in order to keep fines and filler. 153 The Alkaline peroxide bleaching of pulps solubilizes polysaccharides (pectins, or polygalacturonic acids), which are troublesome interfering substances.…”