Kinetic, Thermodynamic and Bio-applicable Studies on Aspergillus niger Mk981235 Chitinase

Chitinases have many applications in food, agricultural, medical, and pharmaceutical fields. This study succeeded in investigating Aspergillus niger MK981235 chitinase in the spot of its physiochemical, kinetic, thermodynamic, and application. The optimum temperature, pH and p-nitrophenyl-β-d-N-acetyl glucosaminide (PNP-β-GlcNAc) concentration to obtain the highest chitinase activity of 2334.79 U ml−1 were at 60 °C, 5 and 0.25%, respectively. The kinetic parameters, including Km and Vmax were determined to be 0.78 mg ml−1 and 2222.22 µmol ml−1 min−1, respectively. Furthermore, the thermodynamic parameters T1/2, D-values, ΔH, ΔG and ΔS at 40, 50 and 60 °C were determined to be (864.10, 349.45, 222.34 min), (2870.99, 1161.07, 738.74 min), (126.40, 126.36, 126.32 kJ mol−1), (101.59, 100.62, 100.86 kJ mol−1), (74.50, 76.17, 47.24 J mol−1 K−1), respectively. A. niger chitinase showed, insecticidal activity on Galleria mellonella by feeding and spraying treatments (72 and 52%, respectively), anti-lytic activity against Candida albicans, and effectiveness in improving the dye removal in the presence of crab shell powder as bio-absorbant. A. niger chitinase can be used in the pharmaceutical field for the bio-control of diseases caused by C. albicans and for the pretreatment of wastewater from the textile industry.


Introduction
Rapid progress in the industrial and agricultural fields has led to an increased demand for applicable enzymes. Chitinase is one of those enzymes that has numerous applications. Chitinases (EC 3.2.1.14), which hydrolyze chitin to release GlcNAc and N-acetyl chito-oligosaccharides (COSs), are produced by a wide range of organisms, including viruses, bacteria, fungi, insects, higher plants, and animals [1]. Chitin is a carbohydrate polymer composed of N-acetyl d-glucosamines connected by β-1,4-glycosidic linkages [2]. Chitin is present in nature in the structure of the exoskeleton and the shell of crustaceans as well as in the cell walls of fungi.
Chitinases are required in the agricultural field, for biological control of fungal pathogens and as pesticides against insects [3][4][5]. Biological control is more favorable than chemical one since the long-term use of the latter one has harmful effects on the environment and human health. In the energy industry for bioethanol production [6], In the medical and pharmaceutical fields as an antitumor agent, and in the preparation of ophthalmic solutions [7]. It is also demanded in the food industry to increase tannase release from the cell wall of fungi [8]. The products of chitinase activity, including N-acetyl-d-glucosamine [9] and N-acetyl chitooligosaccharides, are also required in the food, medicinal, and biotechnology sectors for having activity as prebiotics [10], anti-oxidants, and anti-inflammatory mediators [11].
In recent years, the accumulation of large quantities of shellfish waste from shrimp, crabs, and krill has been paid attention as a source of 20-30% chitin [7]. The conversion of these chitinous wastes into applicable products can be achieved chemically or biologically, but the former usually leads to low production efficiency and environmental pollution [12].
For dye removal, activated carbon is the most commonly used sorbent, but due to the high cost and difficulty of regeneration [13], there was a necessity for searching for alternatives. At this point, it was found that fishery wastes that contain chitin or chitosan can perform this mission successfully as a cheap and effective new choice after biological treatment [14].
Therefore, in this study, we investigated the physiochemical, kinetic, and thermodynamics of Aspergillus niger MK981235 chitinase produced utilizing molokhia stems as nutritional substrate. The anti-yeast activity of Aspergillus niger MK981235 chitinase against Candida albicans was also investigated. Also, the effectiveness of A. niger chitinase on dyes removal improvement was evaluated.

Chitinase Production
Aspergillus niger MK981235 chitinase was prepared by cultivating A. niger by the solid state fermentation (SSF) technique on molokhia stems (MS) as described previously [15].

Characterization of Aspergillus niger MK981235 Chitinase
The conditions for maximum chitinase activity were investigated by performing the reaction at different temperatures (30,

Kinetic and Thermodynamic Characterization
The kinetics, including K m (Michael ' s constant) and V max (maximum velocity), were determined from the Lineweaver-Burk plot. The E a (activation energy) and E d (energy of denaturation) were calculated from the Arrhenius plot.

Anti-yeast Activity
Candida albicans was used to investigate the yeast lytic activity of A. niger chitinase as described by Karthik et al. [18]. This was done by incubating potato dextrose agar (PDA) plates inoculated with 0.1 ml yeast cell suspension (10 7 spores ml −1 ) and pored with wells containing 200 µl chitinase (191 U) at 30 °C.

Dye Removal Enhancement by A. niger Chitinase
In this experiment, A. niger chitinase (382.86 U) in the presence of crab shell powder (1 g, untreated) were mixed with 0.1% dye (crystal violet, brilliant blue, brilliant green, methylene blue) and the reduction in color intensity was measured spectrophotometrically at 420 nm.

Characterization of A. niger Chitinase
As shown in Fig. 1a A. niger chitinase was active over a wide temperature range of 30-70 °C, emphasizing its usefulness in a variety of industrial fields. Its maximal activity was recorded at 60 °C (956.70 U ml −1 ), which was similar to Cohnella sp. A01 chitinase [19]. According to Vincy et al. [20] for Vibrio alginolyticus at 45 °C, Abdel Wahab et al. [17] for Trichoderma longibrachiatum KT693225 at 40 °C, and Subramanian et al. [21] for Achromobacter xylosoxidans at 45 °C, most chitinases have their maximal activity around 40 °C.
A. niger chitinase was almost unaffected by heat pretreatment at 60 °C for 60 min, retaining 95.41% of its initial activity, as shown in Fig. 1b demonstrating its high thermal stability. Thermal pretreatment of A. niger chitinase at higher temperatures (65, and 70 °C) for various periods of time caused gradual decrease in enzyme activity due to protein denaturation. The activity of A. niger chitinase peaked at pH 5 (956.70 U ml −1 ) and then declined drastically below and above, as reported by Aliabadi et al. [19] and Dai et al. [22]. Vincy et al. [20] and Subramanian et al. [21] found that pH 9 and 8 were the best for chitinase from Vibrio alginolyticus and Achromobacter xylosoxidans, respectively. With 0.25% PNP-GlcNAc, the maximal A. niger chitinase activity of 2334.79 U ml −1 was obtained, after which any substrate increase had no effect on enzyme activity (data not shown) and this may be due to the full saturation of enzyme active sites with the substrate. The activity of A. niger chitinase, as shown in Fig. 1c, was unaffected by any of the metal ions tested. In contrast, they had a variable inhibitory effect on chitinase activity, with Hg 2+ causing a 72% drop in activity. In addition, Cu 2+ and Co 2+ reduced activity by 52.6 and 35.6 per%, respectively, as reported by Dai et al. [22]. Aliabadi et al. [19] found that Cu 2+ had a favorable effect on Cohnella sp. A01 chitinase.

Kinetics and Thermodynamics Characterization of A. niger Chitinase
It's crucial to understand the kinetics and thermodynamics of every enzyme before deciding whether it's suitable for industrial use. The K m and V max values are important factors that determine the enzyme's sensitivity to the substrate. K m and V max were found from the Lineweaver Burk plot (Fig. 2a) to be 0.78 mg ml −1 and 2222.22 mol ml −1 min −1 , respectively. K m for chitinases from Cohnella sp. A01 and T. longibrachiatum were determined to be 5.6 mg ml −1 [19] and 8 mg ml −1 [17], respectively, due to the strong affinity of chitinase for the PNP-GlcNAc. E a , E d (Fig. 2b, c), T 1/2 , K d , D-value, ΔH, ΔG, and ΔS are some thermodynamic characteristics that characterize the stability of the enzyme (Table 1). At 60 °C, the half-lives of A. griseoaurantiacus KX010988 [23] and T. longibrachiatum KT693225 [17] were 205.63 and 220.64 min, respectively, compared to 864.10 min for A. niger chitinase. The A. niger chitinase stability is highlighted by the low E a (3.87 kJ) and high E d (129.11 kJ mol −1 ) values (Fig. 2b, c). The lower the E a value, the less energy is required to produce the active complex (enzyme-substrate), and the higher the E d value, the more energy is required to denaturate the enzyme [17]. E d value for A. niger chitinase (129.11 kJ mol −1 ) reflected the higher thermostability than those for A. griseoaurantiacus KX010988 (50.72 kJ mol −1 ) [23] and T. longibrachiatum KT693225 (28.87 kJ mol −1 ) [17] meaning that A. niger chitinase required more energy for denaturation.

Aspergillus niger Chitinase Anti-yeast Activity
The majority of the identified chitinases have antifungal activity against Fusarium oxysporum, Trichoderma viride, Aspergillus oryzae, Penicillium oxysporium, Rhizocotonia solani, Fusarium solani, and Colletotrichum sp. [4,18,[24][25][26][27]. Candida albicans causes superficial mucosal candidosis and a variety of severe infections [28], and its hyphal development is critical for virulence [29,30]. As a result, the most effective treatment should target hyphal morphogenesis rather than pathogen survival. A. niger chitinase showed antimicrobial activity against Candida albicans (3 cm). Due to the presence of chitinase activity, Farag et al. [26] and Allonsius et al. [31] found antimicrobial action for A. terreus and Lactobacillus rhamnosus GG, respectively, against C. albicans. Streptomyces sp. chitinase, on the other hand, had no effect on C. albicans growth [18]. Figure 3 revealed some observations, First, there was a variance in dye color reduction (10-70%) depending on the dye, and second, the presence of chitinase improved dye reduction. Brilliant blue had the biggest drop in dye intensity (70%) in the presence of chitinase and crab shell powder, compared to just 35% in the presence of crab shell powder. For dye removal, Liang et al. [14] used squid pen powder fermented with chitinolytic bacteria and found that color reduction was more noticeable in the presence of fermented squid pen than in the presence of unfermented squid pen. According to Laing et al [14], color adsorption into chitinous waste occurs by physical adsorption in fermented waste and chemical adsorption in unfermented waste.

Dyes Removal
The presence of functional groups such as amino and hydroxyl groups serving as dye-binding material could explain the dye removal action.

Insecticidal Activity
Insect control can be achieved by changing their peritrophic membrane, which protects the midgut epithelium  and hence reduces their feeding [32]. Table 2 shows that mixing Galleria's food with A. niger chitinase or spraying it with A. niger chitinase resulted in mortality rates of 72 and 52%, respectively. Galleria's chitin polymer was rapidly depolymerized by the chitinase enzymes, resulting in chitin breakdown and the pest's death. Bahar et al. [32] discovered a substantial link between bacterial isolates' insecticidal and chitinase activity. Insecticidal actions of chitinases on Galleria mellonella were reported by Awad et al. [33] and Abulikemu et al. [34].