Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (cel6A) of Thermobifida fusca to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of cel6A.CBC which revealed thermostability of cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass.

Streptomyces species have been exploited widely as microbial cell factories, especially for antibiotic production. However, their potential for alpha-amylase production has not been extensively studied. This study reports the isolation, molecular identification, and optimization of the physiological conditions for alpha-amylase production from Streptomyces sp., isolated from soil in Kotli Azad Kashmir. The maximum growth of Streptomyces MI-1 was observed at a neutral pH and a temperature of 35 °C. An amylase activity of 1.15 IU/mL was observed when 4% starch was added to the nutrient medium. During the submerged state fermentation, the maximum amylase activity of 2.136 IU/mL/min was observed after 144 h of incubation. The characterization of bacterial amylase revealed an optimum temperature of 40 °C, and its optimum pH was 7.0. Furthermore, during the study, examining the effect of different metal ions found that Mg2+ and Ca2+ ions had a positive effect, while Cu2+ and Fe2+ ions had an inhibitory effect compared to the control. This preliminary study provides basal line information for the discovery of novel microbes from the unexplored natural resources for amylase production, which will be used for many purposes.