Aspergillus fumigatus was grown on chopped wheat straw in a solid state fermentation (SSF) process carried out in constant presence of isolated free water inside the fermentation chamber. The system allowed maintaining a constant vapor pressure inside the fermentor throughout the fermentation process. Crude endoglucanase produced by A. fumigatus under such conditions was more thermostable than previously reported enzymes of the same fungal strain which were produced under different conditions and was also more thermostable than a number of other previously reported endoglucanases as well. Various thermostability parameters were calculated for the crude endoglucanase. Half lives (T1/2) of the enzyme were 6930, 866, and 36&#x2009;min at 60&#xb0;C, 70&#xb0;C, and 80&#xb0;C, respectively. Enthalpies of activation of denaturation (<inline-formula><mml:math id="M1" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi mathvariant="normal">&#x394;</mml:mi><mml:msubsup><mml:mi>H</mml:mi><mml:mi>D</mml:mi><mml:mo>&#x2a;</mml:mo></mml:msubsup></mml:mrow></mml:math></inline-formula>) were 254.04, 253.96, and 253.88&#x2009;K&#x2009;J&#x2009;mole&#x2212;1, at 60&#xb0;C, 70&#xb0;C and 80&#xb0;C, respectively, whereas entropies of activation of denaturation (<inline-formula><mml:math id="M2" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi mathvariant="normal">&#x394;</mml:mi><mml:msubsup><mml:mi>S</mml:mi><mml:mi>D</mml:mi><mml:mo>&#x2a;</mml:mo></mml:msubsup></mml:mrow></mml:math></inline-formula>) and free energy changes of activation of denaturation (<inline-formula><mml:math id="M3" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi mathvariant="normal">&#x394;</mml:mi><mml:msubsup><mml:mi>G</mml:mi><mml:mi>D</mml:mi><mml:mo>&#x2a;</mml:mo></mml:msubsup></mml:mrow></mml:math></inline-formula>) were 406.45, 401.01, and 406.07&#x2009;J&#x2009;mole&#x2212;1&#x2009;K&#x2212;1 and 118.69, 116.41, and 110.53&#x2009;K&#x2009;J&#x2009;mole&#x2212;1 at 60&#xb0;C, 70&#xb0;C and 80&#xb0;C, respectively.

Abdul A. N. Saqib

Ansa Farooq

Maryam Iqbal

Jalees Ul Hassan

Umar Hayat

Shahjahan Baig

A Thermostable Crude Endoglucanase Produced by Aspergillus fumigatus in a Novel Solid State Fermentation Process Using Isolated Free Water

Apoptosis is a major feature in neural development and important in traumatic diseases. The presence of active caspases is a widely accepted marker of apoptosis. We report here the development of a method to study neuronal apoptotic death in whole-mounted brain preparations using fluorochrome-labeled inhibitors of caspases (FLICA). As a model we used axotomy-induced retrograde neuronal death in the CNS of larval sea lampreys. Once inside the cell, the FLICA reagents bind covalently to active caspases causing apoptotic cells to fluoresce, whereas nonapoptotic cells remain unstained. The fluorescent probe, the poly caspase inhibitor FAM-VAD-FMK, was applied to whole-mounted brain preparations of larval sea lampreys 2 weeks after a complete spinal cord (SC) transection. Specific labeling occurred only in identifiable spinal-projecting neurons of the brainstem previously shown to undergo apoptotic neuronal death at later times after SC transection. These neurons also exhibited intense labeling 2 weeks after a complete SC transection when a specific caspase-8 inhibitor (FAM-LETD-FMK) served as the probe. In this study we show that FLICA reagents can be used to detect specific activated caspases in identified neurons of the whole-mounted lamprey brain. Our results suggest that axotomy may cause neuronal apoptosis by activation of the extrinsic apoptotic pathway.

Ant&#xf3;n Barreiro-Iglesias

Michael I. Shifman

Use of Fluorochrome-Labeled Inhibitors of Caspases to Detect Neuronal Apoptosis in the Whole-Mounted Lamprey Brain after Spinal Cord Injury

A highly conserved arginine residue is close to the catalytic center of PPM/PP2C-type protein phosphatases. Different crystal structures of PPM/PP2C homologues revealed that the guanidinium side chain of this arginine residue can adopt variable conformations and may bind ligands, suggesting an important role of this residue during catalysis. In this paper, we randomly mutated Arginine 13 of tPphA, a PPM/PP2C-type phosphatase from Thermosynechococcus elongatus, and obtained 18 different amino acid variants. The generated variants were tested towards p-nitrophenyl phosphate and various phosphopeptides. Towards p-nitrophenyl phosphate as substrate, twelve variants showed 3–7 times higher Km values than wild-type tPphA and four variants (R13D, R13F, R13L, and R13W) completely lost activity. Strikingly, these variants were still able to dephosphorylate phosphopeptides, although with strongly reduced activity. The specific inability of some Arg-13 variants to hydrolyze p-nitrophenyl phosphate highlights the importance of additional substrate interactions apart from the substrate phosphate for catalysis. The properties of the R13 variants indicate that this residue assists in substrate binding.

Jiyong Su

Karl Forchhammer

The Role of Arg13 in Protein Phosphatase M tPphA from Thermosynechococcus elongatus

The various studies on tyrosinase have recently gained the attention of researchers due to their potential application values and the biological functions. In this study, we predicted the 3D structure of human tyrosinase and simulated the protein-protein interactions between tyrosinase and three binding partners, four and half LIM domains 2 (FHL2), cytochrome b-245 alpha polypeptide (CYBA), and RNA-binding motif protein 9 (RBM9). Our interaction simulations showed significant binding energy scores of −595.3 kcal/mol for FHL2, −859.1 kcal/mol for CYBA, and −821.3 kcal/mol for RBM9. We also investigated the residues of each protein facing toward the predicted site of interaction with tyrosinase. Our computational predictions will be useful for elucidating the protein-protein interactions of tyrosinase and studying its binding mechanisms.

Su-Fang Wang

Sangho Oh

Yue-Xiu Si

Zhi-Jiang Wang

Hong-Yan Han

Jinhyuk Lee

Guo-Ying Qian

Computational Prediction of Protein-Protein Interactions of Human Tyrosinase

Land snails, Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation.

Ryan A. V. Bell

Neal J. Dawson

Kenneth B. Storey

Insights into the In Vivo Regulation of Glutamate Dehydrogenase from the Foot Muscle of an Estivating Land Snail

Rhus laccase was isolated and purified from acetone powder obtained from the exudates of Chinese lacquer trees (Rhus vernicifera) from the Jianshi region, Hubei province of China. There are two blue bands appearing on CM-sephadex C-50 chromatography column, and each band corresponding to Rhus laccase 1 and 2, the former being the major constituent, and each had an average molecular weight of approximately 110 kDa. The purified and crude Rhus laccases were immobilized on zirconium chloride in ammonium chloride solution, and the kinetic properties of free and immobilized Rhus laccase, such as activity, molecular weight, optimum pH, and thermostability, were examined. In addition, the behaviors on catalytic oxidation of phenols also were conducted.

Rong Lu

Tetsuo Miyakoshi

Studies on Acetone Powder and Purified Rhus Laccase Immobilized on Zirconium Chloride for Oxidation of Phenols

Alane Beatriz Vermelho

Claudiu T. Supuran

Jose M. Guisan

Microbial Enzyme: Applications in  Industry and in Bioremediation

This study aimed to develop an optimal continuous process for lipase immobilization in a bed reactor in order to investigate the possibility of large-scale production. An extracellular lipase of Pseudozyma hubeiensis (strain HB85A) was immobilized by adsorption onto a polystyrene-divinylbenzene support. Furthermore, response surface methodology (RSM) was employed to optimize enzyme immobilization and evaluate the optimum temperature and pH for free and immobilized enzyme. The optimal immobilization conditions observed were 150 min incubation time, pH 4.76, and an enzyme/support ratio of 1282 U/g support. Optimal activity temperature for free and immobilized enzyme was found to be 68°C and 52°C, respectively. Optimal activity pH for free and immobilized lipase was pH 4.6 and 6.0, respectively. Lipase immobilization resulted in improved enzyme stability in the presence of nonionic detergents, at high temperatures, at acidic and neutral pH, and at high concentrations of organic solvents such as 2-propanol, methanol, and acetone.

Roberta Bussamara

Luciane Dall'Agnol

Augusto Schrank

Kátia Flávia Fernandes

Marilene Henning Vainstein

Optimal Conditions for Continuous Immobilization of Pseudozyma hubeiensis (Strain HB85A) Lipase by Adsorption in a Packed-Bed Reactor by Response Surface Methodology

The optimization of physiochemical parameters for alkaline protease production using Bacillus licheniformis NCIM 2042 were carried out by Plackett-Burman design and response surface methodology (RSM). The model was validated experimentally and the maximum protease production was found 315.28 U using optimum culture conditions. The protease was purified using ammonium sulphate (60%) precipitation technique. The HPLC analysis of dialyzed sample showed that the retention time is 1.84 min with 73.5% purity. This enzyme retained more than 92% of its initial activity after preincubation for 30 min at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:msup><mml:mrow><mml:mn>37</mml:mn></mml:mrow><mml:mo>∘</mml:mo></mml:msup><mml:mtext>C</mml:mtext></mml:mrow></mml:math>
 in the presence of 25% v/v DMSO, methanol, ethanol, ACN, 2-propanol, benzene, toluene, and hexane. In addition, partially purified enzyme showed remarkable stability for 60 min at room temperature, in the presence of anionic detergent (Tween-80 and Triton X-100), surfactant (SDS), bleaching agent (sodium perborate and hydrogen peroxide), and anti-redeposition agents (Na2CMC, Na2CO3). Purified enzyme containing 10% w/v PEG 4000 showed better thermal, surfactant, and local detergent stability.

Biswanath Bhunia

Apurba Dey

Statistical Approach for Optimization of Physiochemical Requirements on Alkaline Protease Production from Bacillus licheniformis NCIM 2042

The phosphatidylinositol 3-kinase (PI3K) pathway plays a pivotal role in the maintenance of processes such as cell growth, proliferation, survival, and metabolism in all cells and tissues. Dysregulation of the PI3K/Akt signaling pathway occurs in patients with many cancers and other disorders. This aberrant activation of PI3K/Akt pathway is primarily caused by loss of function of all negative controllers known as inositol polyphosphate phosphatases and phosphoprotein phosphatases. Recent studies provided evidence of distinct functions of the four main phosphatases—phosphatase and tensin homologue deleted on chromosome 10 (PTEN), Src homology 2-containing inositol 5′-phosphatase (SHIP), inositol polyphosphate 4-phosphatase type II (INPP4B), and protein phosphatase 2A (PP2A)—in different tissues with respect to regulation of cancer development. We will review the structures and functions of PTEN, SHIP, INPP4B, and PP2A phosphatases in suppressing cancer progression and their deregulation in cancer and highlight recent advances in our understanding of the PI3K/Akt signaling axis.

Qingxiu Zhang

Francois X. Claret

Phosphatases: The New Brakes for Cancer Development?

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Enzyme Research ceased publication in 2019 and is no longer accepting submissions.

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Enzyme Research publishes original research articles as well as review articles that advance our understanding of enzymes and the way in which they work, both in vivo and in vitro.

Enzyme Research