Production of an endoinulinase from Aspergillus niger AUMC 9375, by solid state fermentation of agricultural wastes, with purification and characterization of the free and immobilized enzyme

Abstract

Two different substrates, sunflower (Helianthus annuus L.) tubers and lettuce (Lactuca sativa) roots, were tested. Using a mixture of both wastes resulted in higher production of endoinulinase than either waste alone. Also, ten fungal species grown on these substrates as inexpensive, carbon sources were screened for the best production of endoinulinase activities. Of these, Aspergillus niger AUMC 9375 was the most productive, when grown on the mixture using a 6:1 w/w ratio of sun flower: lettuce, and yielded the highest levels of inulinase at 50% moisture, 30°C, pH 5.0, with seven days of incubation, and with yeast extract as the best nitrogen source. Inulinase was purified to homogeneity by ion-exchange chromatography and gel-filtration giving a 51.11 fold purification. The mixture of sunflower tubers and lettuce roots has potential to be an effective and economical substrate for inulinase production. Inulinase was successfully immobilized with an immobilization yield of 71.28%. After incubation for 2 h at 60°C, the free enzyme activity decreased markedly to 10%, whereas that of the immobilized form decreased only to 87%. A reusability test demonstrated the durability of the immobilized inulinase for 10 cycles and in addition, that it could be stored for 32 days at 4°C. These results indicate that this inulinase, in the immobilized form, is a potential candidate for large-scale production of high purity fructose syrups.

Continuous generation of fructose from Taraxacum officinale tap root extract and inulin by immobilized inulinase in a packed-bed reactor

“…fumigatus on casein, alginate, chitin, DEAE-sephacel, QAE-sephadex, Dowex and Amberlite; however, no significant entrapment was achieved with calcium alginate. Inulinase sourced from A. niger AUMC 9375 was immobilized in calcium alginate and was used up to 10 cycles for generation of high fructose syrup (Housseiny, 2014). In the present study, efficient entrapment of inulinase in Ca-alginate was achieved.…”

“…dandelion, dahlia, asparagus, sunflower, lettuce root and Jerusalem artichoke (Housseiny, 2014;Mansouri et al, 2013;Rawat et al, 2015b;Singh & Singh, 2014). Kango (2008) reported A. niger NK-126, an isolate from onion peels, to produce 55 U/ml of inulinase in four days using dandelion tap root extract.…”

“…Exo-inulinae immobilized on duolite A568 after partial purification generated high fructose syrup from crude asparagus inulin and yielded 39.2 g/L fructose in four hour (Singh et al, 2007). Inulinase sourced from A. niger AUMC 9375 was immobilized in calcium alginate and was used upto 10 cycles for generation of high fructose syrup (Housseiny, 2014). Trytek et al, (2015) immobilized inulinase and also reported efficient operational stability of inulinase in packed-bed reactor for continuous hydrolysis of inulin (0.5% w/v) to generate fructose (98%) for a period of 28 days.…”

“…Microbial inulinases belong to two classes (i) exo-inulinase (EC 3.8.1.80; fructan β-fructosidases, exo-β-D-fructosidases) which splits the terminal fructose link from inulin to generate high fructose and (ii) endoinulinase (EC 3.2.1.7) that hydrolyzes internal linkage in inulin to liberate fructooligosaccharides (FOS) (Chen et al, 2009;Housseiny, 2014;Rawat, et al, 2015a). Inulin rich plant materials present a great opportunity for exploitation and generation of value-added products such as bio-ethanol, single cell protein, citric acid, FOS and high fructose syrup.…”

Exo‐inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production

“…Exo‐inulinase preparation contained 14.6 U/mg protein on account of covalent bond formation between amino group of the enzyme and the aldehyde group of glutaraldehyde (Agrawal & Kango, 2019; Suryawanshi et al., 2019). Inulinase sourced from the A. niger AUMC 9375 was immobilized in calcium alginate beads and was recycled upto 10 cycles for generation of high fructose syrup (Housseiny, 2014). Recently, A. tamarii ‐U4 thermostable exo‐inulinase was immobilized on kaolin clay by cross‐linked immobilization method using glutaraldehyde as chelating agent and 66% immobilization yield was obtained (Garuba & Onilude, 2018).…”