Submerged production, partial purification and characterization of extracellular chitinase from local endophytic fungus for Culex pipiens biocontrol: Strategy for protein stabilization

Abstract

The major barrier which restricts the enzymatic industrial application is their insufficient stability during processing. In this study, a native strain Aspergillus niger M-8, identified and deposited in GenBank under accession number MW940924, showed high chitinase production via submerged fermentation. The optimized operational conditions increased the enzyme production about 13 fold (up to 572.0 U/mg) using medium containing 0.1% untreated chitin, 0.1% starch and 0.3% tryptone at 35°C and pH 5. To increase the enzyme purification efficiency and stability, two co-solvent protein stabilizers (tryptone and yeast extract) were applied in the production medium and fractionation buffers. The results revealed that medium constituents, stabilizer type and pH greatly affected the yield and stability. Tryptone-optimized medium potentiated the enzyme specific activity (8885.0 U/mg), purification fold (16.3) and recovery (10.2%) more than the same medium supplemented with yeast extract (0.4%), under the same fractionation conditions. Furthermore, the fractionation buffer (acetate buffer, pH 5) containing 2 mM tryptone as stabilizer resulted in double the recovery (18.8%) in comparison to the yeast-containing buffer. The purified chitinase fraction using 40% (v/v) ethanol (F40) reached its optimum catalytic activity at a temperature of 30°C and pH 5. Yet, it was also stable and retained most of its initial activity at a wide range of temperature (25-70°C) and pH values (4-7). Interestingly, the more stabilized F40 chitinase had more virulence effect against mosquito larvae (LC95= 222.9 ppm) after 48 h, suggesting that it can be applied for biological control of Culex pipiens.

Gene Cloning, Heterologous Expression, and In Silico Analysis of Chitinase B from Serratia marcescens for Biocontrol of Spodoptera frugiperda Larvae Infesting Maize Crops

“…The resulting cell lysate was then centrifuged for 10 min. Chitinase protein was precipitated according to previously described methods [61,62]. Briefly, a volume of approximately 250 mL of lysate was subjected to fractional precipitation at 4 • C using cooled ethanol at varying concentrations of 0-20%, 20-40%, 40-60%, 60-70%, and 70-90%.…”

“…SAVES v6.0 comprises five programs designed to assess the general coherence of a protein's structural arrangement. Among these programs, PROCHECK [60], VERIFY-3D [61], and ERRAT [62] scores were employed to evaluate the 3D protein models, while PROCHECK including Ramachandran plots and Ramachandran statistics was employed to evaluate the model's quality by examining the allowed and disallowed regions on the plot [44]. The ProSA web server generated a Z-score value, providing an indication of the overall quality of the model and its similarity to native nuclear magnetic resonance/X-ray crystal structures [43].…”