Imran Ali

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.

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.

The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5′-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5′-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type (cel6A.wt) and mutant (cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A.mut) with ∆G = −2.7 kcal mol⁻¹was observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A.wt) having ∆G = −7.8 kcal mol⁻¹ and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg⁻¹ on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg⁻¹), alkali treated bagass (1.7 Umg⁻¹), filter paper (1.2 Umg⁻¹) and BMCC (0.3 Umg⁻¹). Metal ions affect endoglucanase activity in different ways. Only Fe²⁺ exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2⁺ (91.8 %).