L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver–Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.

Polyphenol oxidase (PPO) was firstly purified from damson plum as a high antioxidant source. PPO was treated by 0–80% ammonium sulfate precipitation and dialysis. Characterization results were determined for catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05 M/pH: 7.2/25 °C; 0.2 M/pH: 4.5/10 °C; 0.01 M/pH: 6.8/5 °C, and 0.2 M/pH: 8.5/10 °C, respectively. V_max and K_M values were calculated for same substrates as 17,219.97 U/(mL*min) and 11.67 mM; 7309.72 U/(mL*min) and 5 mM; 12,580.12 U/(mL*min) and 3.74 mM; 12,100.41 U/(mL*min) and 6.25 mM, respectively. Catechol gave the highest V_max value among substrates. Affinity purification was performed by using Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose 6B-L-Tyrosine-p-aminobenzoic acid. Single bands were approximately observed at 50 kDa for each affinity sample in SDS-PAGE and Native-PAGE. 93.88 and 10.46 purification-folds were obtained for PPO by reference Sepharose-4B and original Sepharose-6B gels. Metal effects upon PPO activity were also investigated due to the importance of enzymatic browning in foods. Cu⁺² activation and Fe⁺² inhibition were observed with a final metal concentration of 1 mM at 219.66 and 43.18%, respectively. PPO purification from damson plum by affinity chromatography, its characterization, stability evaluation by statistically, and effects of metal ions on damson plum PPO have not been investigated in the literature. Graphical Abstract

A new fluorometric method was developed for the determination of α-amylase activity in human serum samples. Firstly, a saturated starch–iodine complex (SI) was prepared. The SI complex was combined with sodium fluorescein to form a starch–iodine–sodium fluorescein complex (SIF). As the SIF complex decomposes with the α-amylase enzymatic hydrolysis of starch, the intensity of its fluorescence emission increases. The α-amylase activity is determined using the increased fluorescence emission intensity following hydrolysis of the SIF complex by α-amylase. The optimum pH, optimum buffer concentration, optimum temperature, and interference effect were identified for the developed fluorometric measurement method. Under the optimum conditions, a linear calibration curve was obtained between 0.18 and 9.00 U/L for α-amylase. The α-amylase activity in the human serum sample was also determined by our prepared measurement system and compared with the result from a medical center. Both methods are in good agreement with each other. Because this newly developed fluorometric method for α-amylase activity in serum samples is inexpensive, easy to use, and carried out to detect a very low amount of human serum α-amylase with sensitivity, it can be proposed this method for alpha-amylase activity assay in all other biological samples. Graphical Abstract

Polyphenol oxidase (PPO) enzyme was purified from avocado (Persea americana) by ammonium sulfate precipitation 0-80%, dialysis and affinity chromatography. Characterization studies were performed with catechol (0.10 M, pH: 7.2, 37 °C), 4-methyl catechol (0.10 M, pH: 6.0, 37 °C), pyrogallol (0.02 M, pH: 8.5, 5 °C), chlorogenic acid (0.20 M, pH: 6.8, 10 °C) and caffeic acid (0.20 M, pH: 8.5, 10 °C), respectively. V_max and K_M values were determined for catechol (15789.96 U*mL⁻¹*min⁻¹, 10 mM), 4-methyl catechol (6768.40 U*mL⁻¹*min⁻¹, 2 mM), pyrogallol (6802.72 U*mL⁻¹*min⁻¹, 4 mM), chlorogenic acid (1377.97 U*mL⁻¹*min⁻¹, 14.29 mM) and caffeic acid (2567.24 U*mL⁻¹*min⁻¹, 5 mM). PPO was purified as 147.73-fold and 154.00-fold by Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose-6B-L-Tyrosine-p-aminobenzoic acid, respectively. 4B isolated PPO gave two bands at 35 and 50 kDa in SDS-PAGE while visible and slightly visible bands at 50–70 kDa and 100 kDa in Native-PAGE. 6B isolated PPO gave bands as distinctively at 50 kDa and unclearly at around 35 kDa in SDS-PAGE while visible and slightly visible bands at 50–70 and 100 kDa in Native-PAGE. The synthesis of original 6B-affinity gel and no any study found in literature on affinity purification of avocado PPO show importance of our study. Graphical abstract