Study of Different Cultivated Plants Rhizosphere Soil Fungi-Mediated Pectinase: Insights into Production, Optimization, Purification, Biocompatibility, and Application

Abstract

Microorganisms are preferred as an enzyme source due to their short lifespan, high production rate, affordability, and absence of harmful chemicals in enzymes generated from plant and animal sources. Fungi communities are biological factories for many bioactive compounds such as the important industrial enzyme pectinase. The current study dealt with production, optimization, purification, biocompatibility, and application of fungal pectinase obtained from five plant rhizospheres (banana, jarawa, lemon, tomato, and wheat) at Fayoum Governorate, Egypt. The highest pectinase degrading index (PDI) was scored for FB5, FJ2, and FW1 isolates. Pectinase production was also examined quantitively and the highest output of 1603.67, 1311.22, and 1264.83 U/ml was gained by FB5, FJ1, and FW1 fungal isolates, respectively. The most active pectinase-producing fungi were identified as Aspergillus niveus strain AUMC1624, A. niger strain AUMC16245, and A. brasiliensis strain AUMC16244, respectively. For pectinase production optimization, one factor at a time (OFAT) protocol was applied and revealed that A. niger, A. niveus, and A. brasiliensis reached maximum pectinase levels at 1% pectin after 5, 7, and 7 days, at 40, 45, and 45 °C, respectively. Obtained pectinases were partially purified using ammonium sulfate precipitation (ASP) and organic solvent precipitation (OSP) methods. The highest activity using the ASP method scored at 40-60% saturation with A. niger. The thermostability characterization of A. niger pectinase was reached with relative activities of 61.7, 69.0, 99.9, 91.3, and 90.6% at temperatures ranging between 30 and 70 °C. pH optimized at pH 5-7. The enzyme's molecular weight was approximately 30 kDa. The GC-mass analysis of pectinase end products included acetic acid ethyl ester, hexadecane carbonsaure methylase, and hexadecenoic acid. The biocompatibility was examined using a human skin cell line (HFb-4) for the first time, with a minimal half concentration (IC₅₀) of 151.86 ± 0.76 U/ml. The biocompatible pectinase was applied as a clothes bioscouring agent with different concentrations of 1893.52 U/ml achieving the highest bioscouring with 20.0%.

Keywords: Bioscouring; Human skin cell line (HFb-4); Microorganisms; Pectinase; Rhizosphere.

Fig. 1

a) Percentage distribution of fungal isolates obtained from different plants rhizosphere, b) percentage distribution of positive pectinase fungal isolates from total isolates of different plants rhizosphere

Fig. 2

a) Colony and clear zone diameter for pectinase-producing fungi, b) qualitative screening of pectinase, c) pectinase activity, and d) pectin degradation index (PDI%) of selected pectinase-producing fungi at 28 °C for 72 h on PSA (a and b) and PSF (c and d) medium. Values followed by the same letter are not significantly different, according to Duncan at a 5% level

Fig. 3

a) Morphological characterization of pectinase-producing fungal isolates cultivated at 28 °C for 72 h on PDA medium from the left A. brasiliensis, A. niger, and A. niveus, respectively. b) Phylogenetic tree of fungal isolates based on ITS sequences of 18S rRNA

Fig. 4

Effect of different concentrations of pectin on pectinase production on PMA medium by different Aspergillus spp. on PSA medium at 28 °C for 72 h. Values followed by the same letter are not significantly different, according to Duncan at a 5% level

Fig. 5

a) Time course of pectinase production by different Aspergillus spp. on PMA medium at 28 °C. b) Incubation temperature effect on pectinase production by different Aspergillus spp. on PMA medium with the suitable incubation time

Fig. 6

a) Effect of temperature. b) Effect of pH on the activity and stability of pectinase by A. niger

Fig. 7

SDS-PAGE of partially purified pectinase from A. niger compared to M protein marker of Tris–glycine 4–20% in kilo Dalton (KDa)

Fig. 8

Cell viability affected by various concentrations of pectinase from A. niger. a) Human skin cell line (HFb-4) cells viability and IC₅₀. b) Microscopic images for human skin cell line (HFb-4) before and after treatment with different concentrations of pectinase from A. niger

Fig. 9

Bioscouring of cotton fabric with different concentrations of pectinase at 50 °C, and pH 7.0 for 45 min