Abstract

A novel soil isolate, MUL-14, was recognized as a hyper-producer of 1, 4-α-Dglucan glucanohydrolase (GGNH, EC 3.2.1.1), encoded by amy genes. GGNH is primarily used for starch liquefaction and has diverse applications in many industries. The study aimed to produce cost-effective GGNH from MUL-14 via solid-state fermentation. The internal transcribed spacer (ITS) sequence of MUL-14 was compared with other known sequences and the strain was identified as Aspergillus niger. The yield of gDNA was achieved 51.2-540.1 µg. Different solid substrates i.e., oat straw (Avena sativa), sugarcane bagasse (Saccharum officinarum), wheat straw (Triticum aestivum), rice straw (Oryza sativa), mustard oil cake (Brassica napus), and gram testa (Cicer arietinum) along with different moistening agents i.e., 0.05 M C2H3NaO2, 0.05 M Na3PO4, 1N HCl, 150 mM NaCl (saline solution), distilled water, and tap water were evaluated individually without starch supplementation. The optimum production of the enzyme 765±1.327 U.gds-1 (LSD~0.192) and total protein contents 19.3±0.17 mg.gds-1 (LSD~5.389) were achieved when 10 g Avena sativa at 100 % moisture level (maintained with saline solution) was inoculated with 0.6×107 spores/ml (inoculum size of 5 %) for 72 h, at 30 ?C, and pH 6.0. A high level of expressed cassettes of genes amyA (3325 bp) and amyB (3685 bp) were isolated and purified for further industrial strain development by genetic manipulation. This economically produced enzyme has great biotechnological potential in the food and textile industries.

Keywords: 1, 4-α-D-glucan glucanohydrolase ? solid state fermentation ? agro-industrial waste residues ? Aspergillus niger ? gDNA isolation ? expression cassettes