Response surface methodology-artificial neural network based optimization and strain improvement of cellulase production by Streptomyces sp.

ABSTRACT Thirty seven different colonies were isolated from decomposing logs of textile industries. From among these, a thermotolerant, grampositive, filamentous soil bacteria Streptomyces durhamensis vs15 was selected and screened for cellulase production. The strain showed clear zone formation on CMC agar plate after Gram’s iodine staining.  Streptomyces durhamensis vs15 was further confirmed for cellulase production by estimating the reducing sugars through dinitrosalicylic acid (DNS) method. The activity was enhanced by sequential mutagenesis using three mutagens of ultraviolet irradiation (UV), N methyl-N’-nitro-N-nitrosoguanidine (NTG) and Ethyl methane sulphonate (EMS). After mutagenesis, the cellulase activity of GC23 (mutant) was improved to 1.86 fold compared to the wild strain (vs15). Optimal conditions for the production of cellulase by the GC 23 strain were evaluated using Response Surface Methodology (RSM) and Artificial Neural Network (ANN). Effect of pH, temperature, duration of incubation, , and substrate concentration on cellulase production were evaluated. Optimal conditions for the production of cellulase enzyme using Carboxy Methyl Cellulase as a substrate are 55 oC of temperature, pH of 5.0 and incubation for 40 h. The cellulase activity of the mutant Streptomyces durhamensis GC23 was further optimised to 2 fold of the activity of the wild type by RSM and ANN

Comparative analysis of structural variations due to genome shuffling of Bacillus subtilis VS15 for improved cellulase production

Cellulose is one of the most abundant and renewable biomass products used for the production of bioethanol. Cellulose can be efficiently hydrolyzed by Bacillus subtilis VS15, a strain isolate obtained from decomposing logs. A genome shuffling approach was implemented to improve the cellulase activity of Bacillus subtilis VS15. Mutant strains were created using ethyl methyl sulfonate (EMS), N-Methyl-N′ nitro-N-nitrosoguanidine (NTG), and ultraviolet light (UV) followed by recursive protoplast fusion. After two rounds of shuffling, the mutants Gb2, Gc8, and Gd7 were produced that had an increase in cellulase activity of 128%, 148%, and 167%, respectively, in comparison to the wild type VS15. The genetic diversity of the shuffled strain Gd7 and wild type VS15 was compared at whole genome level. Genomic-level comparisons identified a set of eight genes, consisting of cellulase and regulatory genes, of interest for further analyses. Various genes were identified with insertions and deletions that may be involved in improved celluase production in Gd7.. Strain Gd7 maintained the capability of hydrolyzing wheatbran to glucose and converting glucose to ethanol by fermentation with Saccharomyces cerevisiae of the wild type VS17. This ability was further confirmed by the acidified potassium dichromate (K2Cr2O7) method