Isolation and characterization of novel indigenous fungal strain and optimization of cellulase complex for hyperactivity

Abstract

The recent research is highlighted the fungal species screening for cellulase production using agricultural waste materials, rotten fruits and vegetable. The purpose of this study was to investigate hyper-production of cellulase complex from screened indigenous local strain of Aspergillus tubingensis IMMIS2, extracted from rotten tomato. Congo red test and zone of clearance method were used to confirm the cellulase complex production from this novel isolate of Aspergillus tubingensis IMMIS2. Three Aspergillus species and all Trichoderma species revealed cellulase production. Aspergillus tubingensis IMMIS2 revealed maximum beta-glucosidase production (78±0.4 µg/mL/min) as compared to exoglucanase and endoglucanase and selected for further study. Corn stover revealed maximum cellulase activity (81±1.5 µg/mL/min) after screening six substrates. Aspergillus tubingensis IMMIS2 is a filamentous fungal strain and its cellulase activity was measured on carboxy-methyl cellulose (CMC), cellulose powder, and filter paper used as substrates with Dinitrosalicylic acid (DNS) method. The optimum temperature, pH and incubation time were analyzed to be 40 ºC, 4.8, 96 hours with cellulase activity of 86.4±2.1 µg/mL/min produced from Aspergillus tubingensis-IMMIS2. Crude cellulase revealed maximum activity (112 µg/mL/min) with Response Surface Methodology (RSM) using 40mm mesh size substrate, 8g substrate, 80% moisture, 5mL inoculum, 0.5g urea, 0.1g KCl, 0.1g CaCl2 and 0.06g MgSO4 using Aspergillus tubingensis IMMIS2. Optimization of cellulase production through RSM revealed that most of the organic and inorganic parameter had significant impact on cellulase production using Aspergillus tubingensis IMMIS2. Cellulase revealed maximum activity (116 µg/mL/min) with Taguchi method using 80mm mesh size substrate, 5g substrate, 50% moisture, 3mL inoculum, 0.1g urea, 0.2g KCl, 0.3g CaCl2 and 0.01g MgSO4 using Aspergillus tubingensis-IMMIS2. Optimization of cellulase production showed that some parameters had non-significant impact on cellulase yield. Ammonium sulfate precipitation and dialysis used for partial purification of cellulase and quantity was confirmed by Biuret method. The purification factor increased from 2.12 to 5.14 with ammonium sulfate dialysis and gel filtration chromatography. Characterization of cellulase revealed that maximum activity (130.5±0.43 µg/mL/min & 133.5±0.35 µg/mL/min) was achieved at pH 4.5 and temperature 40ᵒ C, respectively. The SDS PAGE results confirmed that molecular weight of cellulase was 76 kDa. Immobilization technique was applied to enhance the stability and catalytic activity of cellulase. The immobilized and free cellulase characterization proved to enhance the thermo-stability to 82 % at 75 °C as compared to free cellulase enzyme after 26 h of incubation. Immobilization through xerogel matrix and calcium alginate incredibly increased the catalytic activity of cellulase than that of free enzyme. Cellulase activity was decreased after the 20th day of incubation of the both immobilized surfaces (calcium alginate & xerogel). Maximum cellulase activity was achieved at pH 4.5 (174 ± 0.3 µg/mL/min) and temperature 45 °C (179 ± 0.4 µg/mL/min) for xerogel matrix. Xerogel immobilization method revealed the lowest Km value as compared to free and calcium alginate immobilization. Calcium alginate and xerogel matrix immobilization increased tolerance capacity of cellulase to 75−82 % against activating agents / inhibitors like Mg+2, EDTA, SDS and Hg+. Xerogel and calcium alginate immobilization revealed good fruit scarification and hence immobilization method could be a good candidate for food industry. Cellulases are employed in food, textile, biofuel, feed and dairy industries in Pakistan.