Secondary Structure Of Enzyme Research Articles

The effects of Cannabis sativa and cannabinoids on the inhibition of pancreatic lipase – An enzyme involved in obesity

IntroductionObesity is a chronic noncommunicable disease characterized by excessive body fat that can have negative health consequences. Obesity is a complex disease caused by a combination of genetic, environmental, and lifestyle factors. It is characterized by a discrepancy between caloric intake and expenditure. Obesity increases the risk of acquiring major chronic diseases, including heart disease, stroke, cancer, and Type 2 diabetes mellitus (T2DM). Currently, the inhibition of pancreatic lipases (PL) is a promising pharmacological therapy for obesity and weight management. In this study, the inhibition of pancreatic lipase by Cannabis sativa (C. sativa) plant extract and cannabinoids was investigated. MethodsThe inhibitory effect was assessed using p-nitrophenyl butyrate (pNPB), and the results were obtained by calculating the percentage relative activity and assessed using one-way analysis of variance (ANOVA). Kinetic studies and spectroscopy techniques were used to evaluate the mode of inhibition. Diet-induced; and diabetic rat models were studied to evaluate the direct effects of C. sativa extract on PL activity. ResultsKinetic analyses showed that the plant extracts inhibited pancreatic lipase, with tetrahydrocannabinol (THC) and cannabinol (CBN) being the potential cause of the inhibition noted for the C. sativa plant extract. CBN and THC inhibited the pancreatic lipase activity in a competitive manner, with the lowest residual enzyme activity of 52 % observed at a 10 μg/mL concentration of CBN and 39 % inhibition at a 25 μg/mL concentration of THC. Circular dichroism (CD) spectroscopy revealed that the inhibitors caused a change in the enzyme's secondary structure. At low concentrations, THC showed potential for synergistic inhibition with orlistat. C.sativa treatment in an in vivo rat model confirmed its inhibitory effects on pancreatic lipase activity. ConclusionThe findings in this study provided insight into the use of cannabinoids as pancreatic lipase inhibitors and the possibility of using these compounds to develop new pharmacological treatments for obesity.

Cloning, sequence analysis, and molecular docking of nuclease B from Bacillus paralicheniformis str. PMp/10

Bacterial biofilms cause serious problems in the industrial sector and human healthcare. Nucleases are characterized as biofilm-degrading enzymes. In this work, the extracellular nuclease nucB gene from a new isolate Bacillus paralicheniformis str. PMp/10 was isolated and cloned successfully into the cloning vector pET 29a+, then transformed into E. coli DH5α. The nucB gene has a molecular weight of 429 bp, its sequence was uploaded to the NCBI GenBank database under the accession number OP712506 which is the first report for B. paralicheniformis. The gene encoded a nuclease B enzyme consisting of 142 amino acids with an estimated molecular weight of 13 kDa. The 3D and 2D structure of nuclease B was predicted and the NucB enzyme's secondary structure prediction showed that it has five β-strands and three α-helices. Finally, the Molecular docking interaction between NucB and deoxyribonucleic acid was performed successfully with a high binding affinity (−7.1 kcal/mol).

Investigating the impact of common migration substances found in milk packaging on proteases: A multispectral and molecular docking approach

The effects of common migration substances in milk packaging on digestive protease were studied. We choose the common migrants found in eight types of multi-layer composite milk packaging. Enzyme activity experiments revealed that pepsin activity decreased by approximately 18 % at 500 μg/mL of stearic acid and stearamide treatment, while trypsin activity decreased by approximately 18 % only by stearic acid treatment (500 μg/mL). Subsequently, fluorescence spectroscopy, circular dichroism spectroscopy, and molecular docking technology were employed to investigate the inhibition mechanism of protease activity by migrating substances in three systems: stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin. Results showed that the inhibitory effect of stearic acid on trypsin is a reversible mixed inhibition, whereas the inhibitory effects of stearic acid and stearamide on pepsin are non-competitive. In all three systems, ΔH < 0, ΔS < 0, and ΔG < 0, indicating the binding process between the migrant and the protease is a spontaneous exothermic process primarily driven by hydrogen bonding and van der Waals forces. In addition, their binding constants are all around 104 L/moL, indicating that there are moderate binding affinities exist between migrants and proteases. The binding process results in the quenching of the protease's endogenous fluorescence and induces alterations in the enzyme's secondary structure. Synchronized fluorescence spectroscopy showed that stearic acid enhanced the hydrophobicity near the Tyr residue of trypsin. The molecular docking results indicated that the binding affinity of stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin was –22.51 kJ/mol, −12.35 kJ/mol, −19.28 kJ/mol respectively, which consistent with the trend in the enzyme activity results. This study can provide references for the selection of milk packaging materials and the use of processing additives, ensuring food health and safety.

Deciphering the immobilization of lipases on hydrophobic wrinkled silica nanoparticles

In this work, the adsorption of Candida antarctica B (CALB) and Rhizomucor miehei (RML) lipases into hydrophobic wrinkled silica nanoparticles (WSNs) is investigated. WSNs are hydrophobized by chemical vapor deposition. Both proteins are homogeneously distributed inside the pores of the nanoparticles, as confirmed by Transmission Electron Microscopy and Energy Dispersive X-ray measurements. The maximum enzyme load of CALB is twice that obtained for RML. Fourier Transform Infrared Spectroscopy confirms the preservation of the enzyme secondary structure after immobilization for both enzymes. Adsorption isotherms fit to a Langmuir model, resulting in a binding constant (KL) for RML 4.5-fold higher than that for CALB, indicating stronger binding for the former. Kinetic analysis reveals a positive correlation between enzyme load and RML activity unlike CALB where activity decreases along the enzyme load increases. Immobilization allows for enhancing the thermal stability of both lipases. Finally, CALB outperforms RML in the hydrolysis of ethyl-3-hydroxybutyrate. However, immobilized CALB yielded 20 % less 3-HBA than free lipase, while immobilized RML increases 3-fold the 3-HBA yield when compared with the free enzyme. The improved performance of immobilized RML can be explained due to the interfacial hyperactivation undergone by this lipase when immobilized on the superhydrophobic surface of WSNs.

Unraveling the Nano-Bio Interface Interactions of a Lipase Adsorbed on Gold Nanoparticles under Laser Excitation.

The complex nature and structure of biomolecules and nanoparticles and their interactions make it challenging to achieve a deeper understanding of the dynamics at the nano-bio interface of enzymes and plasmonic nanoparticles subjected to light excitation. In this study, circular dichroism (CD) and Raman spectroscopic experiments and molecular dynamics (MD) simulations were used to investigate the potential changes at the nano-bio interface upon plasmonic excitation. Our data showed that photothermal and thermal heating induced distinct changes in the secondary structure of a model nanobioconjugate composed of lipase fromCandida antarcticafraction B (CALB) and gold nanoparticles (AuNPs). The use of a green laser led to a substantial decrease in the α-helix content of the lipase from 66% to 13% and an increase in the β-sheet content from 5% to 31% compared to the initial conformation of the nanobioconjugate. In contrast, the differences under similar thermal heating conditions were only 55% and 11%, respectively. This study revealed important differences related to the enzyme secondary structure, enzyme-nanoparticle interactions, and the stability of the enzyme catalytic triad (Ser105-Asp187-His224), influenced by the instantaneous local temperature increase generated from photothermal heating compared to the slower rate of thermal heating of the bulk. These results provide valuable insights into the interactions between biomolecules and plasmonic nanoparticles induced by photothermal heating, advancing plasmonic biocatalysis and related fields.

Acoustomicrofluidic synthesis of ZIF-8/HRP metal–organic framework composites with enhanced enzymatic activity and stability

Encapsulating enzymes within metal–organic frameworks (MOFs) constitute a promising strategy to circumvent their fragile nature in harsh environments, although deleterious molecular conformational changes and decreased substrate accessibility within the pores of the MOF during enzyme loading can often result in decreased catalytic activity. Herein, we report a rapid (within seconds) and facile one-pot aerosol-based method for simultaneous MOF crystallisation and enzyme encapsulation for the synthesis of ZIF-8/horseradish peroxidase (HRP) biocomposites using a high frequency acoustomicrofluidic nebulisation platform. As a consequence of the enhanced mixing and fast crystallisation afforded by the acoustic coupling into the precursors, the resultant combination of enhanced porosity, missing-linker/metal-node defect creation, pore activation and favourable conformational changes to the enzyme secondary structure allows for more efficient enzyme loading and greater enzyme–substrate interactions, and hence stronger enzyme binding within the MOF structure. Together, these culminate in a biocomposite that possesses four times greater enzymatic activity compared to that for the same material synthesized using conventional bulk-solution techniques, and endows the enzyme with increased protective effect (by approximately three to seven times) against high temperature and organic solvents, even after several exposure cycles. Such a possibility facilitates better potential for the enzyme to be recycled—an important factor given their typically high costs and the difficulty in recovering free enzymes in solution.

Investigation of the interaction behavior between quercetin and pepsin by spectroscopy and MD simulation methods

The ability of a therapeutic compound to bind to proteins is critical for characterizing its therapeutic impacts. We have selected quercetin (Qu), a most common flavonoid found in plants and vegetables among therapeutic molecules that are known to have anti-inflammatory, antioxidant, anti-genotoxic, and anti-cancer effects. The current study aimed to see how quercetin interacts with pepsin in an aqueous environment under physiological conditions. Absorbance and emission spectroscopy, circular dichroism (CD), and kinetic methods, as well as molecular dynamic (MD) simulation and docking, were applied to study the effects of Qu on the structure, dynamics, and kinetics of pepsin. Stern-Volmer (Ksv) constants were computed for the pepsin-quercetin complex at three temperatures, showing that Qu reduces enzyme emission spectra using a static quenching. With Qu binding, the Vmax and the kcat/Km values decreased. UV–vis absorption spectra, fluorescence emission spectroscopy, and CD result indicated that Qu binding to pepsin leads to microenvironmental changes around the enzyme, which can alter the enzyme's secondary structure. Therefore, quercetin caused alterations in the function and structure of pepsin. Thermodynamic parameters, MD binding, and docking simulation analysis showed that non-covalent reactions, including the hydrophobic forces, played a key role in the interaction of Qu with pepsin. The findings conclude of spectroscopic experiments were supported by molecular dynamics simulations and molecular docking results.

Insight into fermentable sugar recovery process from sugarcane bagasse: in silico elucidation of enzymatic hydrolysis and techno-economic assessment

A technological scheme was developed to produce 2G fermentable sugar from sugarcane bagasse and was assessed for the process feasibility. This paper describes recovery of fermentable sugar, characterization of pretreated hydrolysed bagasse, in-silico elucidation of enzymatic hydrolysis and techno-economic analysis of the recovery of sugar from bagasse. The in-silico approach included characterization of both the enzymes, estimation of physiochemical and functional properties through ProtParam. Secondary structures of enzymes were represented using SOPMA. Modelling of enzymes was done using MODELLER 9.24. The developed model was validated through PROCHECK and was evaluated using Verify3D. The active sites of both the enzymes were predicted using 3D LigandSite – Ligand binding site prediction server and molecular docking of the predicted Models were performed using Swiss Dock. The techno-economic analysis showed 0.516 kg (2G fermentable sugar)/kg bagasse).

Investigation of interaction between dexamethasone/pheniramine and trypsin by fluorescence, UV–vis, CD, and molecular docking

Antihistamines and glucocorticoids are commonly used to treat allergy symptoms and the inflammatory conditions. In present study, the in-vitro binding interactions a glucocortikoid, dexamethasone/an antihistamine, pheniramine with TSN (TSN) secreted from pancreas to small intestine for protein digestion were investigated by fluorescence emission spectroscopy (FES), UV-Vis spectroscopy, synchronous fluorescence spectroscopy (SFS), CD spectroscopy, FT-IR and molecular modeling methods. Also, the effect of these drugs on the catalytic activity of trypsin (TSN) was determined. The fluorescence quenching experiments indicated that each drugs quenched the intrinsic fluorescence of TSN with their increased concentrations. The results of SFS and UV-Vis spectra proved the interaction of dexamethasone and pheniramine with TSN. CD spectra showed that the secondary structure of enzyme was altered in the presence of the drugs. All these spectroscopy results were validated and explained by molecular docking and molecular dynamic simulation (MD) studies. The IC50 values were determined as 0.0049 mM and 0.0038 mM for dexamethasone and pheniramine, respectively. So, both drugs have inhibition effect on the catalytic activity of TSN. The results of this study can provide valuable information in the field of pharmacokinetics and pharmacodynamics. Communicated by Ramaswamy H. Sarma

Investigation on the interaction behavior between safranal and pepsin by spectral and MD simulation studies

The ability of therapeutic molecules to bind to enzymes is critical for determining their therapeutic effects. We've chosen safranal, a monoterpene aldehyde found in saffron petals among therapeutic molecules that are known to have antioxidant, anti-inflammatory, tumor-killing, anti-genotoxic, and anti-aging effects. The purpose of this study was to see how safranal interacts with pepsin in an aqueous solution under physiological circumstances. UV–Visible spectroscopy, fluorescence spectra, circular dichroism (CD) analysis, and kinetic approaches, as well as molecular simulation and docking, were used to investigate the effects of safranal on the structure, kinetics, and dynamics of pepsin. Stern-Volmer (Ksv) constants were calculated for the pepsin-safranal complex at different temperatures, indicating that safranal decrease enzyme emission using a static method. With safranal binding, the Vmax and the kcat/Km values increased. UV–Visible and fluorescence spectroscopy and CD findings indicated that safranal binding to pepsin causes micro-environmental alterations around pepsin, which can modify the enzyme's secondary structure. Therefore, safranal causes changes in the structure and function of pepsin. Analysis of MD bonding, simulation methods, and thermodynamic parameters showed that non-covalent reactions, including van der Waals forces, and hydrogen bonds play a critical role in the interaction of safranal with pepsin. The results conclude of CD, and spectroscopic experiments were further supported by molecular dynamics simulations and molecular docking data.

Mechanistic investigations in sonoenzymatic synthesis of n-butyl levulinate

n-Butyl levulinate (n-BL) has numerous applications in biofuel and green solvents. In this paper, we have reported mechanistic accounts of sonoenzymatic synthesis of n-BL using Novozym 435. Esterification parameters were optimized with statistical design of experiments. Application of 35 kHz sonication boosted n-BL yield from 70.9% to 92.22%. Mechanistic investigation using Ping-Pong Bi-Bi kinetics model and molecular docking simulations revealed interesting influence of sonication on esterification reaction. Exposure to sonication induced significant changes in the secondary structure of enzyme, as revealed in ATR-FTIR analysis. Sonication increased α-helix content of enzyme that increased enzyme activity by opening of flapping lid and widening the catalytic cavity. Sonication also caused unfolding of secondary structural motifs with rise in random coil content that favored formation of enzyme-ligand complexes. These effects enhanced reaction velocity and substrate affinity with reduction in inhibition and unfavorable dissociation of intermediate complexes, which ultimately enhanced n-BL yield.

Inactivation kinetics and cell envelope damages of foodborne pathogens Listeria monocytogenes and Salmonella Enteritidis treated with cold plasma

In recent years, more attention has been paid to the application of cold plasma (CP) in eliminating foodborne pathogenic bacteria. This work investigated CP effects on inactivation kinetics and cell envelopes of Listeria monocytogenes (L. monocytogenes) and Salmonella Enteritidis (S. Enteritidis). Bacterial suspensions were treated with dielectric barrier discharge atmospheric CP at 75 kV for different treatment time. Three regression models were tested for estimating inactivation kinetics. Reactive species generated in plasma, the appearance and integrity of bacterial cells, the activity and secondary structure of enzymes in the cell envelope, and molecular docking, were measured for evaluating the envelope damages. Results indicated that Log-linear model was suitable for L. monocytogenes and the Weibull model was suitable for S. Enteritidis. S. Enteritidis was more sensitive to short-lived reactive species (such as OH radicals) in plasma than L. monocytogenes, and the cell envelope of S. Enteritidis was more severely damaged (the increased membrane permeability and leakage of intracellular substances) after plasma treatment. Interestingly, compared with S. Enteritidis, the decrease in the activity of enzymes existing in the cell envelope of L. monocytogenes did not contribute significantly to the death of bacteria. Molecular docking further suggested that the decrease in the enzyme activity might be due to the modification of the enzyme, by the interaction between reactive species in plasma (H2O2) and amino acid residues of the enzyme through the hydrogen bond.

Insights into the Molecular-Level details of betaine interactions with Laccase under various thermal conditions

Laccase is a blue enzyme with a remarkable ability to eliminate environmental pollutants. The use of the eco-friendly enzyme for removing ecological contaminants is extensive. In addition, there are some restrictions like its deactivation in various environmental conditions. Researchers have applied many strategies to improve the Laccase stability in different conditions, but those ways are expensive or demanding. Herein, we used betaine, a co-solvent, to increase the enzyme stability in various temperatures and pHs as an appropriate alternative strategy to improve the Laccase stability. The experimental results showed that the enzyme's stability would increase in the presence of betaine at high temperatures. Although no significant changes were detected for the enzyme secondary structure contents, betaine at high temperatures exhibited a protective effect on the enzyme's alpha-helical contents. The simulation results indicated that betaine and ion molecules could be excluded from the enzyme surface and help Laccase maintain its stability and activity. Increasing the betaine concentration up to 0.5 M increased the Tm value of laccase. The molecular dynamics (MD) simulation results also demonstrated that the enzyme's polar amino acids play essential roles in making hydrogen bonds with betaine molecules. Lys377 was also recognized as the most critical residue for electrostatic interactions with the osmolyte at low and high temperatures. At the same time, the higher temperatures could not considerably affect the electrostatic interactions between the carboxyl group of betaine and the basic residues. The results indicated that the negatively charged amino acids could strengthen electrostatic interactions with betaine at high temperatures. The computational analyses also demonstrated that the hydrated state of betaine would not change even at high temperatures. This study's outcomes could be valuable to researchers who seek to use a more affordable approach to increase Laccase activity in severe environmental conditions.

Catalytic Dyad Residues His41 and Cys145 Impact the Catalytic Activity and Overall Conformational Fold of the Main SARS-CoV-2 Protease 3-Chymotrypsin-Like Protease.

Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145–His41 catalytic dyad. We here characterized the catalytic roles of the cysteine–histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine–histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.

Investigation of binding interaction behavior between antiemetic drugs and Trypsin by spectroscopy and molecular docking

Antiemetic drugs are used to control excessive vomiting and nausea and generally absorbed through gastrointestinal tract. In present study, the in-vitro binding interactions two of the antiemetic drugs (dimenhydrinate and ondansetron) between Trypsin (Tsn) secreted from pancreas to small intestine for protein digestion were investigated by fluorescence emission spectroscopy (FES), UV–VIS spectroscopy, synchronous fluorescence spectroscopy (SFS), FT-IR spectroscopy and molecular modeling methods. Also, the effect of these drugs on the catalytic activity of Tsn was determined. The fluorescence quenching experiments indicated that each drugs quenched the intrinsic fluorescence of Tsn with their increased concentrations. The results of SFS and UV–VIS spectra proved the interaction of dimenhydrinate and ondansetron with Tsn. FT-IR spectra showed that the secondary structure of enzyme was altered in the presence of the drugs. All these spectroscopy results were validated and explained by molecular docking studies. Both drugs have inhibition effect on the catalytic activity of Tsn and the IC50 values were determined as 2.6 × 10−4 M and 6.4 × 10−4 M for dimenhydrinate and ondansetron, respectively. Docking results revealed that the hydrogen bond interaction of dimenhydrinate with active-site residue Ser195 and ondansetron with active-site residues His57 and Ser195 hydrogen bonds might be cause the inhibition of enzyme activity. The results of this study can provide valuable information in the field of pharmacokinetics and pharmacodynamics.

Substrate Binding Stiffens Aspartate Aminotransferase by Altering the Enzyme Picosecond Vibrational Dynamics

Protein dynamics on various time scales from femtoseconds to milliseconds impacts biological function by driving proteins to conformations conducive to ligand binding and creating functional states in enzyme catalysis. Neutron vibrational spectroscopy carried out by measuring inelastic neutron scattering from protein molecules in combination with molecular simulations has the unique ability of detecting and visualizing changes in the picosecond protein vibrational dynamics due to ligand binding. Here we present neutron vibrational spectra of a homodimeric pyridoxal 5′-phosphate-dependent enzyme, aspartate aminotransferase, obtained from the open internal aldimine and closed external aldimine conformational states. We observe that in the external aldimine state the protein structure stiffens relative to the internal aldimine state, indicating rigidified vibrational dynamics on the picosecond time scale in the low-frequency regime of 5–50 cm–1. Our molecular dynamics simulations indicate substantial changes in the picosecond dynamics of the enzyme secondary structure elements upon substrate binding, with the largest contributions from just two helices and the β-sheet.

Integration of elastin-like polypeptide fusion system into the expression and purification of Lactobacillus sp. B164 β-galactosidase for lactose hydrolysis

An elastin-like polypeptide (ELP) sequence fused with Lactobacillus sp. B164 β-galactosidase modified with 6x-Histidine (β-Gal-LH) to produce recombinant β-Gal-Linker-ELP-His (β-Gal-LEH) was expressed in E. coli and purified via inverse thermal cycling (ITC) and nickel-nitrilotriacetic acid (Ni-NTA) resin. The β-galactosidase integrated with ELP-system showed an improved purification at 1.75 M (NH4)2SO4 after 1 round ITC (95.66% recovery rate and 13.04 purification fold) with better enzyme activity parameters compared to Ni-NTA. The enzyme maintained an optimal temperature (40 °C) and pH (7.5) for both β-Gal-LEH and β-Gal-LH. The results further showed that the ELP-fusion system improved the enzyme’s thermal and storage stability. Moreover, the enzyme secondary structure was not changed by ELP-tag. Enzyme activity was completely inactivated by Hg2+, Cd2+ and Cu2+, unaffected by Ca2+, EDTA and urea, but partially activated by Mn2+ at lower concentration. Compared to commercial β-galactosidases, β-Gal-LEH exhibited similar biocatalytic efficiency on lactose and could potentially catalyze transgalactosylation.

ANALYTICAL APPROACHES IN UNVEILING THE COMPATIBILITY OF COLLAGENASE WITH ADDITIVES IN VIEW OF ITS COLLOIDAL DELIVERY

The core objective of present work was to investigate the chemical interactions of selected additives with therapeutic enzyme Collagenase (COLG) and determination of its compatibility for futuristic colloidal formulation. Initially, solubility of COLG with additives was determined and emulsification efficiency of surfactants was examined. FTIR spectra of COLG with its binary blends were compared. Non-denaturing SDS-PAGE and Circular Dichroism (CD) used to investigate primary and secondary structure of enzyme in binary mixture. Finally, the control and accelerated studies was performed by spectroscopy.

Matrix-assisted pulsed laser evaporation of β-glucosidase from a dopa/quinone target

β-glucosidase (BG) plays a key role in determining the efficiency of the enzymatic complex cellulase for the degradation of cellulose into sugars. It hydrolyses the cellobiose, an inhibitor of the enzymatic complex. Therefore, the immobilization of BG is a great challenge for the industrial application of cellulases. Cellulases usually contains a BG amount insufficient to avoid inhibition by cellobiose. Here the BG was immobilized by matrix assisted pulsed laser evaporation (MAPLE) technique. The frozen matrix was composed of water, water/m-DOPA and water/m-DOPA/quinone. The effect of the excipients on the final conformation of the enzyme after the MAPLE processing was determined. The enzyme secondary structure was studied by FTIR analysis. The catalytic performances of the deposited films were tested in the cellobiose hydrolysis reaction. The results demonstrate that the presence of the oxidized form of m-DOPA, the O-quinone form, can protect the protein native structure, with the laser inducing little or no damage. In fact, only the samples deposited from this target preserved the secondary structure of the polypeptide chain and allowed a complete hydrolysis of cellobiose for four consecutive runs, showing a high operational stability of the biocatalyst.

Inhibition of the amylolytic hydrolysis of starch by ethanol

Gelatinized native corn starch dispersion (GSD, 5 g/100 mL water) was diluted with ethanol/water mixtures (EWM, 0.0, 1.0, 2.5, and 5.0 mL ethanol/100 mL) to a final concentration of 2.0 g GSD/100 mL EWM. The GSD were subjected to digestion by porcine pancreas α-amylase (0.33 and 0.66 IU/mL) at 37 °C for 90 min. The digested starch was reduced as an inverse function of ethanol concentration. The digested starch reduction can be as high as 15–30%, depending on the α-amylase concentration. Log-of-slope (LOS) method was used for analyzing the digestion kinetics curve, showing the presence of two (fast and slow) time scales. The fast and slow digestion kinetics were linked to rapidly (RDS) and slowly (SDS) starch fractions. It was found that ethanol reduced the RDS fraction, while increasing the resistant starch (RS) fraction. FTIR analysis suggested that modification of the enzyme secondary structure is behind the inhibition of the α-amylase activity by ethanol solvation.