ABSTRACT 

The objectives of this research were to screen, produce, assay, perform physio-chemical and molecular characterization and sequence analysis of the phytase gene from A.niger.  We isolated different fungal species from cereal grains and agricultural soil.  Five isolates designated as SB1, SB2, BS, WF and STD was confirmed positive for phytase production. The isolates were obtained from Soybean and sorghum flour and were identified morphologically as A.niger. Phytase enzyme was in produced in Cornstarch media (CSM) and Potatoes Dextrose Broth (PDB). Physiochemical, molecular Characterization of phytase producing A, niger and amplified phytase gene were performed. The activities of SB1, SB2, BS, WF  and STD  phytase when CSM was used were 2.97±0.005UmL-1, 2.87±0.007 UmL-1, 3.18 ±0.009 UmL-1, 4.37±0.04 UmL-1 and 3.11UmL-1 respectively higher than the  activity  of  extracellular phytase produced PDB i.e 2.07±0.06 UmL-1, 2.17±0.05 UmL-1, 2.22±0.01 UmL-1, 2.78±0.03 UmL-1 and 2.05±.0.1UmL-1. The optimal temperature of isolate SB1 and WF phytase was 40 °C, and SB and BS phytase were 50 °C and 60 °C respectively. The optimal pH of phytase was 5.0 for isolate SB1 and WF while SB2 and BS phytase was 6.0, and 4.0 respectively.  0.1 M and 0.01 M Ca2+, Mg2+, Cu2+were observed to slightly inhibit the activity of SB2, BS and WF and STD while 0.1M of Fe3+ enhanced the activity of SB1 phytase.  Sequence analysis of 18S rRNA sequences assured that isolates  SB1 was 99% identical to A. niger ANTS (KY825168), SB2, BS, and WF were 99% identical to A. niger isolate Moriga leaf (MG889596.1). Phylogenetic analysis confirmed that all isolates were closely related to A.niger. Moreover, an ITS1, 5.8S and ITS2 sequences analysis revealed that SB1 and SB2 were 97% and 99% respectively similar with A.niger voucher MRC200804 (MF078659.1). In addition, isolates BS was 98% similar with A.niger strain IHB F 2919 (KM817216) and isolates WF was 99% identical to A.niger isolate SS5 (KJ432863). BLAST and phylogenetic evolutionary analysis of amplified phytase gene confirmed that the amplified phytase genes are PhyA, which are closely related to Aspergillus spp phytase A. The phyA_SB1 was 98% identical to phytase A of A. ficuum AAB26466 and, 97% similar with A. niger ADP05107, while phyA_STD showed 96% homology with phyA of A.japonicus ACE79228, 98% homology with A.niger ADP05105 and 99% homology with A.ochraceoroseus PLB29348. Multiple sequence alignments showed the presence of conserved signature motif RHGARYPTD at N – terminal of protein sequences while HD  motif was not amplified. The amino acid sequence of phyA from isolating SB1 and A.niger STD  had 4 glycosylation sites while SB2 phytase had three N-glycosylation sites it was confirmed that the SB1, SB2 and STD phytase are the members of histidine acid phosphatase family. These findings are potential for further phytase research in Indonesia A.niger as a potential source of phytase enzymes for use in feeds or food Nutrition. 

Keywords: Phytase, Phytate, A.niger, Physio-Chemical, Molecular Characterization