Isolation, Characterization, and Insecticidal Activity of Chitinase Gene ( chi A) From Aspergillus niger (EM77)

Chitin, which is found in the structure of insect cuticles and peritrophic membranes, is degraded by the chitinase enzymes. The degradation decreases the feeding and defenses of the insect and weakens it. Therefore, chitinases are important enzymes that have potential for being used as biological control agents against harmful insects. In the present study, genes encoding exo-chitinases (e.g. chi A) have been isolated from A. niger . The partial sequence includes 1484 bp open reading frame encodes for 493 amino acid. No intron was found in the chi A of A. niger (EM77). The deduced amino acid sequence of chi A of A. niger (EM77) clearly indicated the presence of three conserved domains: ChtBD1 superfamily, chitin recognition protein, and Glyco-18 domain.


INTRODUCTION
Chitin, an insoluble homopolymer of β-(1, 4)-linked N-acetyl-D-glucosamine (GlcNAc), is an important component of exoskeletons (insects), crustacean shells (marine living) and fungal cell (Brine and Austin, 1981). After cellulose, chitin is the second most abundant component of biomass in nature (Zainol Abidin et al., 2020). Due to the presence of chitinolytic enzymes (chitinases), chitin does not accumulate in the environment and can be degraded to GlcNAc short monomers (Abo Elsoud & El Kady, 2019). Chitinases play significant physiological and ecological roles in chitin metabolism (Souza et al., 2011). Also, it has an important role in the bio-control of pathogenic fungi and insect control as a pesticide (Hamid et al., 2013). Chitinases can be found in a wide range of organisms, including bacteria, fungi, plants, insects and vertebrates (Hamid et al., 2013). Many microorganisms produce chitinase to feed on chitin as a carbon and energy source (Rathore & Gupta, 2015). Some bacterial strains (e.g. Serratia and Bacillus) are known to produce four different types of chitinases. However, most of the filamentous fungi have been recorded to produce about 20 different chitinases with higher activity levels than those of plants and bacteria (Hartl et al., 2012). Many fungi produce chitinase enzyme to degrade the substrate as a defense mechanism, as well as to gather nutrients for their survival under adverse environmental conditions (da Silva et al., 2005). Aspergillus species is a dominant and most potent species for chitinase production. Amongst, Aspergillus terreus and A. niger (LOCK62) were found to be the efficient source of chitinase production having various applications (Brzezinska & Jankiewicz, 2012). Mainly, fungi have several chitinase genes encoding enzymes with diverse chitinase activities (Ramaiah et al., 2000). Chitinase genes from several fungi species have been characterized (Danişmazoğlu et al., 2015). Genes encoding chitinase enzymes have been cloned from a wide range of yeasts and filamentous fungi (Jaques et al., 2003). All cloned chitinase enzymes belong to the glycohydrolase family18 based on the percentage of similarity to family18 (Zhong et al., 2015); two distinct classes can be identified chitinases from plants or bacteria. Chitinase genes have been cloned and characterized from many microorganisms, including Trichoderma artroviridae (Takaya et al., 1998), Aspergillus fumigatus (Xia et al., 2001), Paecilomyces sp (Dong et al., 2007), and Aspergillus nidulans (Shin et al., 2009), Spodoptera littoralis are serious pests of forest or agricultural crops throughout the world. Chemical insecticides that have a variety of side effects on the environment have been used for several years against these pests. Because of their hazardous effects, these chemicals are no longer recommended for agricultural pest management (Abdel-Aziz et al., 2013). Chitinase is able to effectively break down glycosidic bonds of chitin polymer in the content of Insects' peritrophic membranes (PM) and allow the passage of all kinds of components found in the midgut through this barrier. In this way, some bacteria and their toxic components reach the endothelial cells of the midgut and become a threat to insect life (Bahar et al., 2012). Consequently, any defect in PM reduces feeding and protection against microbial attack (Langer et al.,2000).
In the current study, an assessment of chitinase producing ability of A. niger (EM77) was performed, gene and amino acid sequences were analyzed against other reported chitinase sequences deposited into the NCBI. To the best of our knowledge, this research is the first to isolate chitinase from A. niger. Furthermore, an insecticidal assay of A. niger chitinase against S. littoralis larvae was measured.

MATERIALS AND METHODS Fungal Source and Growth Conditions:
A previously isolated and identified Aspergillus niger (EM77) (Accession no. KF774181) was maintained on potato dextrose agar (PDA) medium at 30°C for six Isolation, Characterization, and Insecticidal Activity of Chitinase Gene (chiA) 111 days in a rotary shaker (200 rpm) with a daily cycle consisting of 15 h of light and 9 h of darkness and it was preserved at -80ºC in 50% (v/v) glycerol. Preparation of Fungal Expressed Chitinase: Fungal mycelia of A. niger (EM77) were grown on chitinase inducing medium containing ground chitin (0.5 g/l) in basal salts medium following the procedure of Wang et al. (2013).

RNA Isolation:
Grown mycelia were harvested flashfrozen in liquid nitrogen then ground to a fine powder in a porcelain mortar. Total RNA was isolated from homogenized fungal mycelia using the RNeasy® Mini Kit (Qiagen, Netherlands). Concentrations of RNA were determined using the Nanodrop ND-1000 (Nanodrop). First-strand cDNA was prepared using 1 μg of RNA, and following the manufacturer's instructions of SuperScript® II system (Invitrogen) including the reverse trancriptase and the oligo-dT primer. Specific primers chiF, 5'-ATGTCAGTCATTTCGGCATTTC-3', and chiR, 5'-TCATGGTACCATCATCCAGA-3' were designed to amplify the open reading frame of the 5'termini of chitinase gene. The primers were designed according to the data derived by the automatic computational analysis of the annotated genomic sequence of A. niger chitinase that was accessioned as XM-001396587.
PCR was conducted using the Go Taq DNA polymerase (Promega) in a thermocycler machine following the stepwise condition for; 30 cycles, as denaturation step for 60 sec. at 94 °C, annealing step for 90 sec. at 55 °C, extension step for 60 sec. at 72 °C, and a final extension step for 7 min of 70 °C. The amplified fragment was separated by electrophoresis on 1.0% agarose gel stained with ethidium bromide (0.5 μg/ml) and was visualized on a UV gel documentation system (BioRad, USA). The amplicon was excised from the agarose gel then purified from unincorporated PCR primers and dNTPs using the QIAquick PCR purification kit following the manufacturer's instructions (Qiagen, Netherlands). The sequence of A. niger chitinase fragment was sent for sequencing to LGC Genomics GmbH on a 3730xl DNA Analyzer (Applied BiosystemsTM/Thermo Fisher Scientific). Then it was BLAST searched on the National Center for Biotechnology Information (NCBI) database and compared with the obtained sequence on the Sequence® Software. Sequencing runs were performed at the Chitinase gene (chiA) of A. niger (EM77) was submitted to the NCBI database (accession No. MH878942).

Sequence Alignments and Phylogenetic Analysis:
Sequence of chitinase gene (chiA) which obtained for our isolate A. niger (EM77) was compared to other sequences of other Aspergillus species deposited to the NCBI GenBank using the "BLASTN" and "BLASTX" tools at the National Center for Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment of nucleotide and deduced amino acid sequences with selected Chitinase genes from other Aspergillus species were done using T-Coffee Multiple Sequence Alignment Tools (http://www.tcoffee.org/Projects/tcoffee/inde x.html#DOWNLOAD). Box shading of conserved regions was applied using the sequence manipulation suite (http://www.bioinformatics.org/sms2/color_a lign_cons.html). The phylogenetic tree was constructed based on the amino acid sequences using the neighbor-joining method of MEGA X software Kumar et al. (2016); www.megasoftware.net). The conserved domains within the amino acid or the coding nucleotide sequence were detected using the website of https://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi.

Exochitinase Crude Enzyme Production:
A culture of A. niger (EM77) was grown on a basal medium broth (BM) supplemented with chitin as the following (g/l): chitin 0.5; K2HPO4, 3.0; yeast extract, 3.5; peptone, 2.0; KCL, 3.0; NaNO3, 3.0, MgSO4, 0.5; FeSO4 0.01 (pH 6.2) at 30°C for six days with a daily cycle consisting of 15 h of light and 9 h of darkness. Then 50 ml of the BM broth were inoculated with one ml of spore suspension approximately equal (1×10 6 CFU/ml). The inoculated flasks were incubated at 30 °C for six days in a rotary shaker (200 rpm). At the end of the incubation period, the cultures were centrifuged for 15 min. using a cooling centrifuge. The culture filtrate was used as the crude enzyme. Exochitinase Partial Purification: Exochitinase of A.niger (EM77) was fractionated using ethanol concentrations (25-75%). Each fraction has produced a precipitate that was obtained by centrifugation at 10000 xg, for 15 min. at 4°C then assayed for enzymatic activity. Exochitinase Activity Assay: Exochitinase activity was determined following the method of Matsumoto et al (2004) using the chromogenic substrate p-nitrophenyl-β-D-Nacetylglucosaminide (PNP-β-GlcNAc) as a substrate. One unit of the enzyme activity was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per minute under the specified assay conditions. Polyacrylamide Gel Electrophoresis: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique (Laemmli 1970) was performed at a 12% concentration to determine the molecular mass of the exochitinase enzyme that was fractionated with the highest purification fold. The standard protein marker contained a mixture of 11 known proteins that were resolved specific bands with known molecular weight ranged between 10 and 175 kDa. Insecticidal Activity: The insecticidal effect using surface contamination bioassay of partially-purified chitinase was applied to the first-instar larvae of the Egyptian cotton leafworm (Spodoptera littoralis). Briefly, castor bean leaves (Ricinus communis) were soaked with partially purified exo-chitinase as 0.5 and 1.0 ml of partially -purified enzyme (42.39 Units/ml). The assay was performed in cups of artificial diet. For the control treatment, water has substituted the exo-chitinase doses in the artificial diet. Three replicates were done for each treatment, where each replicate has contained 30 larvae. The cups were incubated at 26 ± 1 °C, 60 % humidity, and a 12 h light/dark photoperiod (El-Defrawi et al., 1964 andEl-barky et al., 2008). Mortality was monitored daily for seven days after treatment by counting the number of dead larvae.

RESULTS Characterization of Exo-Chitinase cDNA and Sequence Analysis:
A fragment at the size of 1484 bp was amplified from the cDNA synthesized from the RNA of the A. niger (EM77) (Fig.  1). The obtained fragment was sequenced and identified as a partial sequence of the chitinase gene using the BLASTx and BLASTn tools of the NCBI GenBank database. The exo-chitinase fragment could not be produced when chitin was not supplemented in the growing medium of A. niger (EM77).
Isolation, Characterization, and Insecticidal Activity of Chitinase Gene (chiA) 113 The nucleotide sequence of the obtained fragment has deduced an ORF of 1484 bp and 493 of amino acid as shown in Fig. (2). No introns were detected in the nucleotide sequence of chiA of A. niger (EM77). Further analysis of the deduced amino acid sequence of chiA of A. niger (EM77) has clearly indicated three conserved domains. The first domain was allocated between the amino acids numbered 53 and 94 (42aa), which corresponds to ChtBD1 super-family with e-value of 6.67e-09. The second domain was allocated between the amino acids numbered 112 and 134 (20 aa), which corresponds to the chitin recognition protein with e-value 1.17e-09. The third domain was allocated between the amino acids numbered 150 and 491 (440-aa), which corresponds to the Glyco-18 domain (Fig. 2). The third domain is proof that the chiA of A. niger (EM77) belongs glycosyl hydrolase family18 of chitinases.

Fig. 3:
Phylogenetic analysis of A. niger (EM77) chitinase (chi) partial protein sequence with chi genes from selected Aspergillus species. Tree was constructed with Neighbor-joining method using MEGA X software. The analysis involved 493 amino acid sequences. All positions containing gaps and missing data were eliminated. Alignment of the sequences was done with CLUSTALW, bootstrap values (in percent) are calculated from 1000 resampling.

Exo-Chitinase Activity and Molecular Weight:
The chitinase activity was evaluated based on the ethanol fractions methods of Matsumoto et al. (2004). Fractions obtained at 50 and 75% of ethanol have scored the heights purification folds 2.9 and 3.4-fold respectively, and were collected together. Also, the maximum activity was observed by using the basal media supplemented with 0.5 g/l chitin then incubates at 30°C for six days. The protein concentration was 42.39 units/ml.

Protein Electrophoresis Analysis:
From the SDS-PAGE analysis, the purified protein of chitinase enzyme was observed at the molecular weight of ~ 30 kD in comparison to the with crud and purified protein (Fig. 5).

Bioassay of Insecticidal Activity:
Different concentrations of partiallypurified A. niger EM77 chitinase were tested for its efficiency on first instar Spodoptera littoralis larvae. Death of S. littoralis larvae has begun after 48 h. using only 1.0 ml of (42.39 Units/ml) partially -purified chitinase.

Chitin
oligosaccharide exists naturally in many organisms including exteriors of insects and fungi. Upon degradation of chitin by a number of organisms, severe damage and even death may occur in pathogens and pests whose external surfaces contain this polymer. Currently, chemical fungicides and insecticides are the major means of controlling these disease-causing agents (Veliz et al., 2017). However, due to the potential harm that these chemicals cause to the environment and to human and animal health, new strategies are being developed to replace or reduce the use of fungal-and pestkilling compounds in agriculture. Thus, microbes producing chitinases have attained great attention for the last few decades. Microorganisms producing chitinolytic enzymes were considered as bio-control agents (Farag et al., 2016), amongst were the Aspergillus species (Ni et al., 2015 andStoykov et al., 2015), that more specifically have included the A. niger that produces the chitinase with the highest activity (Awad et al., 2017).
In the current study, an ORF (1484 bp) of chiA gene was isolated and encodes as a chitinase enzyme of fungal class A. niger (EM77). The ORF was submitted to the NCBI database (accession No. MH878942) and analyzed using bioinformatics tools, which showed no previous record with a sequence of chiA gene from A. niger. All published sequences of chitinase genes from some Aspergillus species that occur in NCBI database were derived by automatic computational analysis using the gene prediction method for genomic sequences. This suggests that the ORF sequence is the first record of chiA gene from A. niger.
Based on the similarity percentages of amino acid sequences, most chitinase genes that have been characterized to date are classified into two families of glycosyl hydrolases (family18 and 19) (Meng et al., 2015). Thus, the deduced amino acid Isolation, Characterization, and Insecticidal Activity of Chitinase Gene (chiA) 119 sequence of chitinase (chiA) of the isolate EM77 has included the characteristic substrate-binding site and the catalytic motifs cited by Vocadlo and Davies (2008). Therefore, it can be denoted as a member of the family18 of glycosyl hydrolases. The characterized chitin-binding domain (ChtBD1) was found important to specifically bind chitinases to insoluble chitin (Limón, et al., 2004). Despite the fact that some fungal chitinases may lack the presence of ChtBD domain (Duo-Chuan, 2006), however, the chitinase in the current study has included the ChtBD.
Chitinase enzyme produced by A. niger (EM77) was partially purified using ethanol fraction due to the insolubility of precipitated proteins. Coagulation of chitinase proteins with ethanol fractions agreed with Tandjaoui et al. (2015) who reported that precipitation of enzymes by ethanol and acetone was not stable in most enzymes and insoluble in water and buffer solutions that in turn would hinder the purification processes. On the other side, other workers preferred precipitation of chitinase with ammonium sulphate, for instance; Bacillus cereus, Streptomyces griseorubens and A. tereus (Liang et al., 2014;Karthik et al., 2015;and Rashad et al., 2017). The chitinase was exhibited maximum activity (42.39 Units/ml). These results were in agreement with those other chitinase preparation obtained from B. thuringiensis (25.11 U/ml) (Gomaa and El-Mahdy, 2018).
Chitinases enzyme is glycosyl hydrolases with sizes ranging from 20 kDa to about 90 kDa (Bhattachrya et al., 2007). Great varieties of chitinase can be found among fungal species, and each species can produce different chitinase isomers, which have different catalytic properties (Homthong et al., 2016). In the present study, a single band at ~ 30 KDa was determined as the molecular weight of the partially purified, which is approximately close to that of the chitinase produced from Aspergillus flavus (30 kDa); Monacrosporium thaumasium (30KDa),  (Beltagy et al., 2018, Karthik et al., 2015Nguyen et al., 2015;Soares et al.,2015;Hammami et al., 2013;Waghmare and Ghosh, 2010;Yamazaki et al., 2007). Our finding does not in agreement with the results of identified purified 45 kDa chitinase from A. fumigatus (Jaques et al., 2003).
The potential use of chitinase for controlling insect pests has been demonstrated in several studies. In our study, partially-purified exo-chitinase of A. niger EM77 had insecticidal activity against S. littoralis larvae after 96 hours (38.9%). That was because of the ability of chitinase to disrupting peritrophic membrane of insect cells (Zhong et al., 2015). As well as chitinase of family18 and 19 were known for its catalytic activity similar to lysozyme and chitosanase in their activities (Sahai and Manocha, 1993), which could be serving as an effective biopesticide to control harmful pests which destroy importantly economic crops instead of hazardous chemical pesticide.

Conclusion
Several reports have extensively highlighted the importance of chitinases for various biotechnological and environmental applications. So, the increasing number of databases describing and chitinase genes and protein structure and function can help to streamline the engineering of chitinases in a more rational manner to yield chitinases with improved activity and stability specifically for the various biotechnological applications. In our study, gene encoding exo-chitinases (chiA) were characterized by the isolate EM77 as the first to be published in Aspergillus niger. Characterization of the new chitinase has included molecular, biochemical, and bioassay techniques. Results indicated that the A. niger EM77 is a fungal strain that can produce amounts of chitinase (42.39 Units/ml) within six days. In addition, the partial-purified chitinases enzyme has shown a promising insecticidal activity (~39 % mortality in four days) against S. littoralis larvae.