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The study addresses the investigation of a novel alkaline protease produced by a fungus involved in the fermentation of cassava and its potential application as a detergent additive. This study aims to investigate the potential of a newly discovered alkaline protease, Rhizopus oligosporus alkaline protease (ROAP), produced during cassava fermentation, as a detergent additive. The objective is to understand its characteristics, stability, and stain-removing capabilities. ROAP was partially purified using acetone precipitation. The study extensively assessed its optimal conditions, including temperature and pH, and explored the impact of inorganic salts, chemical agents, and surfactants on enzyme activity. Kinetic parameters (Vmax, Km, Kcat) were determined, along with thermal stability indicators. Cloth washing experiments using stained white cotton fabrics were conducted to evaluate stain removal efficiency. After purification, ROAP exhibited an impressive increase in specific activity. Optimal conditions for its activity were identified at 40 °C and pH 10.0. The enzyme showed varying responses to different salts, some of which did not affect its activity while others influenced it. Most notably, ROAP proved highly effective in removing stains, achieving a 94% removal rate for grease stains when used alone and an 86% efficiency when combined with Rin. This study underscores the potential of ROAP as a valuable detergent additive, especially for tackling challenging protein-based stains like blood and grease. The enzyme's stability, efficiency, and versatility in various conditions hold promise for its application in industries requiring effective stain removal, contributing to improved cleaning processes. • Specific activity of ROAP increased from 1972.6 U/mg proteins in the crude extract to 5956.1 U/mg. • ROAP exhibited more than 70% relative activity when tested on casein substrate at temperatures ranging from 30 to 60 °C. • The purified ROAP has a broad pH tolerance, with maximum activity observed at pH 10.0. • ROAP activity was positively affected by low concentrations of oleic acid, as well as high concentrations of glycerin and DMSO (dimethyl sulfoxide). [ABSTRACT FROM AUTHOR]

α-Amylase is a foremost hydrolytic enzyme used for various industrial processes. In the present study, Rhizopus oligosporus α-amylase (ROAA) produced during solid state fermentation of cassava (Manihot esculenta) tubers by the fungus was partially purified and biochemically characterized. The kinetic and thermal inactivation thermodynamics properties of the partially purified enzyme were also evaluated as well as the potential application in cloth washing. The ROAA was purified by 4.11-fold and had a molecular weight of 60 kDa. ROAA was optimally active at 60 °C, pH 5.0 and had a salt tolerance of 1–100 mM NaCl for 120 min. The activity of the α-amylase was significantly (p < 0.05) increased in the presence of various inorganic salts especially at 10 mM. Ethylenediaminetetraacetic acid (EDTA), urea and thiourea enhanced ROAA activity. Triton X-100 enhanced ROAA activity by 141.97%, whereas sodium dodecyl sulphate (SDS) decreased it by 28.58%. Tween 20 and Tween 80 did not significantly affect ROAA activity at 1 mM. V max , K m and K cat of ROAA were 37.59 U/mg protein, 16.39 mg/mL and 0.016 s−1, respectively. Thermal stability indicators such as half-life, D-value at 70 °C and Z-value were 101.91 min, 338.70 min, and 48.78 °C, respectively. Cloth washing efficiency of a mixture of the isolated enzyme and commercial detergents was high relative to detergent-only trial. The enzyme had high thermal stability, optimized activity in the presence of some inorganic salts, and resistance to several surfactants and a chelating agent. The fungal α-amylase reported in this study would find relevance not only in starch-based industries but also in laundry. [Display omitted] • R. oligosporus secreted α-amylase during the SSF of cassava tubers. • The α-amylase was optimally active at 60 °C and pH 5, activated by EDTA. • Thermal inactivation thermodynamics followed first order kinetics. • The α-amylase (ROAA) produced was moderately thermally stable (D-value, Z-value). • ROAA showed resistance to the inhibitory action of some surfactants. [ABSTRACT FROM AUTHOR]

In this study, α-amylases from fresh and dried Cyperus esculentus seeds were isolated and characterized for biochemical and thermodynamic properties. The α-amylases (FTNE-α-amylase and DTNE-α-amylase) were optimally active at 60–70 °C, pH 7.0 (FTNE-α-amylase) and pH 6.0 (DTNE-α-amylase) with activation energies (Ea) of 3.56 and 6.81 kJ mol-1, respectively. Both enzymes retained above 70% of their hydrolytic activities after 1 h at pH 6.0–8.0. The enzyme activities were more enhanced in the presence of divalent metal ions (Ca2+, Mg2+, Zn2+, Cu2+, Ba2+, Co2+, Pb2+, and Hg2+) than monovalent ions. SDS, cyclohexanol, ether and diethylamine inhibited them except EDTA. Using soluble starch as substrate, DTNE-α-amylase had higher V max , K cat and lower K m than FTNE-α-amylase. Both enzymes expressed high substrate preferences for yam flour amidst other substrates with catalytic efficiencies (K cat /K m) of 2.71 and 4.44 mL. mg−1sec−1, respectively. The higher catalytic efficiency of DTNE-α-amylase was due to its transition state stabilization (ΔG E-S = -0.90 kJ mol-1 and ΔG E-T = -2.22 kJ mol-1). Fourier Transformed-Infrared (FTIR) spectroscopy revealed changes in C–O stretching and -N-H bending in the substrate-bound enzymes. Thermal inactivation of the amylases followed first-order rate kinetics and was non-spontaneous (ΔG#>0). FTNE-α-amylase half-lives and D-values at 70 and 80 °C, and Z-value (256.41 °C), were however higher than in DTNE-α-amylase (Z-value = 63.29 °C; t 1/2 = 22.43 min at 70 °C) indicating that FTNE-α-amylase is more thermally stable than DTNE-α-amylase and would be more industrially applicable as a thermostable starch-hydrolyzing enzyme. Image 1 • Enzymes exhibited pH/thermal stabilities; enhanced catalysis with divalent ions. • Both enzymes expressed high catalytic efficiencies (K cat /K m) for yam flour. • Infrared spectroscopy revealed changes in enzymes' structures during interactions. • Enzymes thermal inactivation obeyed first order kinetics; ΔH#<0, ΔS#<0 and ΔG#>0. • α-Amylase from fresh seeds of Cyperus esculentus is unique and thermostable. [ABSTRACT FROM AUTHOR]