Commercial Enzyme Preparations Research Articles

Enzymatic dispersal of dual-species biofilms carrying Listeria monocytogenes and other associated food industry bacteria

Enzyme-based treatments could therefore be used to complement regular cleaning processes. Most studies using enzymes as anti-biofilms strategy are focused on their outcome in mono-species biofilms. Nevertheless, in real environments mixed biofilms are prevalent. In this work, seven types of dual-species biofilms were selected to serve as targets for enzymatic treatments carrying different environmental strains of L. monocytogenes and accompanying bacteria isolated from dairy, meat and seafood processing plants. The effectiveness of nine commercial enzymatic preparations, including pronase, cellulase, pectinase, DNase I, lysozyme, phospholipase, peroxidase, β-glucanase and chitinase, was evaluated. For this, residual attached viable cells of both L. monocytogenes and its partners were enumerated through swabbing and colony plate counting following the action of each enzyme. Moreover, Confocal Laser Scanning Microscopy (CLSM) images were analyzed pre and after enzymatic treatments in order to quantify changes in biofilm thickness, covered area and volume. The viable attached population of L. monocytogenes was almost unaffected by all of the enzymes here tested, being eliminated on average just the 90% of the initially attached population (around 1 Log10 cfu cm−2 reduction). Nevertheless, some of the partner species (Escherichia coli and Staphylococcus saprophyticus) were sometimes protected from enzymatic detachment when in dual-species biofilms, depending on the enzyme tested and the accompanying L. monocytogenes strain. CLSM images showed important changes in biofilm covered area and volume after DNase I, pronase and pectinase treatments. These results demonstrate that enzymes can greatly weaken dual-species biofilms structure. Nevertheless, it cannot be disregarded that detached cells from these treatments would still be viable. Thus, a control of cell viability after an enzymatic procedure in the food industry must be always considered before designing an efficient disinfection treatment.

Enzymes for Wine Fermentation: Current and Perspective Applications

Enzymes are used in modern wine technology for various biotransformation reactions from prefermentation through fermentation, post-fermentation and wine aging. Industrial enzymes offer quantitative benefits (increased juice yields), qualitative benefits (improved color extraction and flavor enhancement) and processing advantages (shorter maceration, settling and filtration time). This study gives an overview about key enzymes used in winemaking and the effects of commercial enzyme preparations on process engineering and the quality of the final product. In addition, we highlight on the presence and perspectives of beneficial enzymes in wine-related yeasts and lactic acid bacteria.

Production of ethanol fuel from enzyme-treated sugarcane bagasse hydrolysate using d-xylose-fermenting wild yeast isolated from Brazilian biomes.

In this study, we evaluated the potential of yeasts isolated from Amazon to produce second-generation ethanol from sugarcane bagasse delignified with alkaline hydrogen peroxide and hydrolysed with commercial enzyme preparation. The best efficiency savings in glucose and release of xylose were determined by considering the solids and enzyme loads. Furthermore, we selected Spathaspora passalidarum UFMG-CM-Y473 strain with the best fermentative parameters. Fermentations used bagasse hydrolysate without any nutritional supplementation, a significant difference from previous studies, which is closer to industrial conditions. Ethanol yield of 0.32g/g and ethanol productivity of 0.34g/Lh were achieved after the consumption of 78% of the sugar. This hydrolysis/fermentation technology package could represent the input of an additional 3180L of ethanol per hectare in areas of average sugarcane productivity such as 60ton/ha. Thus, we concluded that Sp. passalidarum UFMG-CM-Y473 has a clear potential for the production of second-generation ethanol from delignified and enzyme-hydrolysed bagasse.

Expression and purification of nuclease-free protocatechuate 3,4-dioxygenase for prolonged single-molecule fluorescence imaging

Single-molecule (SM) microscopy is a powerful tool capable of visualizing individual molecules and events in real time. SM imaging may rely on proteins or nucleic acids labelled with a fluorophore. Unfortunately photobleaching of fluorophores leads to irreversible loss of signal, impacting the collection of data from SM experiments. Trace amounts of dissolved oxygen (O2) are the main cause of photobleaching. Oxygen scavenging systems (OSS) have been developed that decrease dissolved O2. Commercial OSS enzyme preparations are frequently contaminated with nucleases that damage nucleic acid substrates. In this protocol, we purify highly active Pseudomonas putida protocatechuate 3,4-dioxygenase (PCD) without nuclease contaminations. Quantitation of Cy3 photostability revealed that PCD with its substrate protocatechuic acid (PCA) increased the fluorophore half-life 100-fold. This low cost purification method of recombinant PCD yields an enzyme superior to commercially available OSS that is effectively free of nuclease activity.

Enhancement of catalytic activity and thermostability of a thermostable cellobiohydrolase from Chaetomium thermophilum by site-directed mutagenesis

Enzymatic saccharification of lignocellulosic biomass is increasingly applied in agricultural and industrial applications. Nevertheless, low performance in the extreme environment severely prevents the utilization of commercial enzyme preparations. To obtain cellobiohydrolases with improved catalytic activity and thermostability, structure-based rational design was performed based on a thermostable cellobiohydrolase CtCel6 from Chaetomium thermophilum. In the present study, four conserved and noncatalytic residue substitutions were generated via site-directed mutagenesis. Mutations were heterologously expressed in yeast Pichia pastoris, purified, and ultimately assayed for enzymatic characteristics. The mutant Y119F increased the catalytic activity 1.82-, 1.65- and 1.43-fold against β-d-glucan, phosphoric acid swollen cellulose (PASC) and carboxymethylcellulose sodium (CMC-Na), respectively. In addition, S131 W effectively enhanced the enzyme's heat resistance to elevated temperatures. The half-life (t1/2) of this mutant enzyme was increased 1.42- and 2.40-fold at 80 °C and 90 °C, respectively, compared to the wild-type. This study offers initial insight into the biological function of the conserved and noncatalytic residues of thermostable cellobiohydrolases and provides a valid approach to the improvement of enzyme redesign proposal.

Characterization of thermoadaptive serine metalloprotease and application in waste management

Thermoadaptive enzymes offer new opportunities for bio-catalysis and bio-transformations owing to their ability to function over wide temperature ranges. Microbial alkaline proteases are important constituents of commercial enzyme preparations, accounting for more than 60% of the total industrial enzyme market. Fibrous proteins, such as horn, feathers etc. are abundantly available in nature as recalcitrant solid wastes. Extracellular alkaline proteases derived from microorganisms can be used for degradation of proteinaceous wastes and convert them into value-added products e.g. peptide concentrates or amino acids. The present study relates to the isolation of an extracellular, bacterial thermoadaptive serine metalloprotease from the soil. The purified protease showed a remarkable increase in thermostability (4 °C–100 °C) that was enhanced in the presence of Ca2+. The protease (+Ca2+) was utilized to efficiently degrade fish scales and feather to produce amino acids, as declogging agent and for removing blood stains from cloth with a short reaction time. The ability of the protease could be utilized in bulk for solid waste management providing for an eco-friendly and effective process.

Lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier rather than by inducing nonproductive adsorption of enzymes

BackgroundLignin is known to hinder efficient enzymatic conversion of lignocellulose in biorefining processes. In particular, nonproductive adsorption of cellulases onto lignin is considered a key mechanism to explain how lignin retards enzymatic cellulose conversion in extended reactions.ResultsLignin-rich residues (LRRs) were prepared via extensive enzymatic cellulose degradation of corn stover (Zea mays subsp. mays L.), Miscanthus × giganteus stalks (MS) and wheat straw (Triticum aestivum L.) (WS) samples that each had been hydrothermally pretreated at three severity factors (log R0) of 3.65, 3.83 and 3.97. The LRRs had different residual carbohydrate levels—the highest in MS; the lowest in WS. The residual carbohydrate was not traceable at the surface of the LRRs particles by ATR-FTIR analysis. The chemical properties of the lignin in the LRRs varied across the three types of biomass, but monolignols composition was not affected by the severity factor. When pure cellulose was added to a mixture of LRRs and a commercial cellulolytic enzyme preparation, the rate and extent of glucose release were unaffected by the presence of LRRs regardless of biomass type and severity factor, despite adsorption of the enzymes to the LRRs. Since the surface of the LRRs particles were covered by lignin, the data suggest that the retardation of enzymatic cellulose degradation during extended reaction on lignocellulosic substrates is due to physical blockage of the access of enzymes to the cellulose caused by the gradual accumulation of lignin at the surface of the biomass particles rather than by nonproductive enzyme adsorption.ConclusionsThe study suggests that lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier blocking the access of enzymes to cellulose rather than by inducing retardation through nonproductive adsorption of enzymes.

Modification of the interfacial properties of sodium caseinate using a commercial peptidase preparation from Geobacillus stearothermophilus

Sodium caseinate was hydrolyzed with the commercial enzyme preparation Sternzym BP 25201, containing a thermolysin-like peptidase from Geobacillus stearothermophilus as the only peptidase. The hydrolysis was carried out at 65 °C with an enzyme activity of 1 nkat mL−1 or 15 nkat mL−1, leading to various degrees of hydrolysis (DH) ranging between 0.1 and 8.5%. The hydrolysates obtained were analyzed in a multi-scale approach, covering the hydrolysate properties (viscosity, hydrophobicity, peptide composition) and their interaction at oil–water (emulsion) and air–water (foam) interphases. The viscosity and surface hydrophobicity generally decreased with an increasing DH. Longer, more hydrophobic peptides, which self-assembled into network-like supramolecular particles, were detected up to a DH of 2.2%. Compared to untreated sodium caseinate, these structures could increase the half-life of emulsions (+400%) and foams (+31%). This was most probably caused by an increase in particle size (45.2-fold). By contrast, a higher DH led to a less hydrophobic product and smaller, spherical-shaped supramolecular structures. Foams and emulsions prepared with those hydrolysates were not stable and phase separated within minutes (for example, emulsion half-life = 5 min; foam half-life = 4.6 min at DH of 8.5%).

Application of a recombinant laccase-chlorogenic acid system in protein crosslink and antioxidant properties of the curd

Milk protein crosslink through the action of enzymes represents a feasible strategy to impart new functionalities to cheese. In this work we reported the effects of a laccase mediator system (LMS) on protein crosslink and antioxidant property of curd. The crosslinking activity of a purified recombinant laccase Ery4 and a commercial enzyme preparation (cLC), with three mediators was firstly evaluated in milk and then applied before curd manufacture. Only Ery4-LMS significantly increased curd weight compared to that of the control sample. SDS-PAGE revealed that similar high molecular weight bands produced by both LMSs in milk were also retained in curds. The antioxidant activity recorded in curds with Ery4-LMS was the highest among all samples both before and after gastro-pancreatic digestion. This is the first time that a CGA-based LMS is used in manufacture of curd with improved antioxidant properties. These results open new perspectives for dairy applications.

High activity CAZyme cassette for improving biomass degradation in thermophiles

BackgroundThermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms.ResultsTo advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own.ConclusionsWe have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.

Deactivation of Cellulase at the Air-Liquid Interface Is the Main Cause of Incomplete Cellulose Conversion at Low Enzyme Loadings

Amphiphilic additives such as bovine serum albumin (BSA) and Tween have been used to improve cellulose hydrolysis by cellulases. However, there has been a lack of clarity to explain their mechanism of action in enzymatic hydrolysis of pure or low-lignin cellulosic substrates. In this work, a commercial Trichoderma reesei enzyme preparation and the amphiphilic additives BSA and Tween 20 were applied for hydrolysis of pure Avicel cellulose. The results showed that these additives only had large effects on cellulose conversion at low enzyme to substrate ratios when the reaction flasks were shaken. Furthermore, changes in the air-liquid interfacial area profoundly affected cellulose conversion, but surfactants reduced or prevented cellulase deactivation at the air-liquid interface. Not shaking the flasks or adding low amounts of surfactant resulted in near theoretical cellulose conversion at low enzyme loadings given enough reaction time. At low enzyme loadings, hydrolysis of cellulose in lignocellulosic biomass with low lignin content suffered from enhanced enzyme deactivation at the air-liquid interface.

Using an Inducible Promoter of the Glucoamylase Gene to Construct New Multienzyme Complexes from Penicillium verruculosum

New recombinant strains of Penicillium verruculosum are created using a new plasmid construct based on an inducible promoter of glucoamylase gene (gla1) that secretes heterologous xylanase E (XylE) from P. canescens. New biocatalysts are produced that contain cellulolytic enzyme preparations (EPs) enriched with XylE. The amount of XylE in the recombinant EPs varies in the optimum range of 11–24% of the total protein while generally preserving the P. verruculosum cellulose complex. The hydrolytic activity of the new EPs with respect to polymeric plant-derived substrates exceeds that of EPs produced using other expression systems and commercial preparations. The new EP glaX-17 surpasses in particular a control EP based on the recipient strain by 13% in the efficiency of hydrolyzing aspen wood and is 20% more efficient than the commercial EP Accelerase Duet. The new EP glaX-17 displays 25% greater efficiency (35–43%) during the hydrolysis of wheat bran than the commercial EP Accelease Duet. The effectiveness is demonstrated of using the new gla1 promoter for the production of EPs (biocatalysts) while preserving the balanced cellulose complex of the strain and optimum yield of heterologous XylE required for the deep hydrolysis of xylan-containing plant biomass.

Combined enzymatic and crossflow microfiltration process to assure the colloidal stability of beer

In this work, two beers of the industrial and craft types were stabilized and clarified by combining a commercial enzyme preparation of fungal origin (Brewers Clarex®), which is commonly used for the production of gluten-free beers, with crossflow microfiltration.By resorting to some European Brewing Convention indicative forcing tests (i.e., sensitive proteins, alcohol chill haze, and chill haze development), it was possible to confirm the efficacy of the enzyme preparation for both beers examined. By combining the enzymatic and polyvinylpolypyrrolidone (PVPP) treatments, each beer exhibited a greater colloidal stability with respect to that achievable with any single treatment. Independently of the stabilizing pretreatment used, final membrane clarification gave rise to permeated beers with almost the same chill haze development. By coupling the enzymatic treatment to crossflow microfiltration, it would be possible to replace PVPP, and avoid its expensive recovery and regeneration processes, thus leading to a simpler one-step beer conditioning process.

New assay of α-l-rhamnosidase

Free rutinose was prepared by enzymatic hydrolysis of rutin using defatted seed meal from tartary buckwheat. This disaccharide was used as substrate in spectrophotometric assay of α-l-rhamnosidase. The assay is based on hydrolysis of rutinose and subsequent determination of released glucose by a standard glucose oxidase assay kit. The method is easy to perform and requires no expensive equipment. The assay was applied in α-l-rhamnosidase estimation in ten commercial enzyme preparations and compared with standard assay on chromogenic substrate.

Immobilization and some properties of commercial enzyme preparation for production of lactulose-based oligosaccharides

The commercial enzymatic preparation, Pectinex Ultra SP-L, was successfully immobilized on activated chitosan particles for production of lactulose-based galacto-oligosaccharide (LuOS). The main degrees of polymerization (DP) of LuOS produced in this bioreactor were DP3 and DP4. The immobilization yield in term of expressed activity and recovery activity were 71% and 36.05%, respectively. The optimum temperature (60°C) was not affected by immobilization process, while the optimum pH was shifted from pH 5.5 to pH 4.5. In addition, a thermal stability of the immobilized enzyme was better than that of free enzyme. The half-life time of hydrolytic activity increased from 2.5days (free enzyme) to 14days (immobilized enzyme). Hydrolytic KM values were 651.3mM and 187.9mM lactulose for free enzyme and immobilized enzyme, respectively. Furthermore, the immobilized enzyme showed high reusability. The yield of LuOS formation was more than 60% after 33 cycles of reuses. In the case of trans-galactosylation, a model with 8 reaction rates was build using five-step reaction pathways with different initial lactulose concentrations. This catalytic model was validated well for a long-term period (27 days). These results strongly support to develop a continuous bioconversion to synthesize functional food ingredients, prebiotic LuOS.

Changes in the level and antioxidant activity of polyphenols during storage of enzymatically treated raspberry juices and syrups.

Berry juices are a rich source of phenolic compounds exhibiting antioxidant activity. Unfortu- nately, polyphenols and especially anthocyanins are degraded during storage. The levels of total phenolic compounds, phenolic acids, flavonoids, and antho- cyanins as well as antioxidant activity (radical scavenging ability against DPPH and ABTS+• and chelating power Fe2+) were determined in raspberry juices (obtained after enzymatic treatment with three commercial pectinolytic enzyme preparations) and syrups (obtained by the addition of sucrose at concentrations of 30% and 70%) during storage. During the five-month storage of juices and syrups at room temperature, there was significant re- duction in the level of phenolic compounds, in particular anthocyanins (up to 95% in relation to the initial content). Storage of raspberry juices and syrups also resulted in a reduction in antioxidant activity. The enzymatic treatment of the raspberry mash generally increased the losses of anthocyanins. The addition of sugar to fruit juices only slightly reduced these losses.

Formulation of an optimized synergistic enzyme cocktail, HoloMix, for effective degradation of various pre-treated hardwoods

In this study, two selected hardwoods were subjected to sodium chlorite delignification and steam explosion, and the impact of pre-treatments on synergistic enzymatic saccharification evaluated. A cellulolytic core-set, CelMix, and a xylanolytic core-set, XynMix, optimised for glucose and xylose release, respectively, were used to formulate HoloMix cocktail for optimal saccharification of various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of 75%:25% protein dosage, CelMix: XynMix, while for untreated and steam exploded biomass the HoloMix consisted of 93.75%:6.25% protein dosage. Saccharification by HoloMix (27.5mgprotein/gbiomass) for 24h achieved 70–100% sugar yields. Pre-treatment of the hardwoods (especially those with a higher proportion of lignin) with a laccase, improved saccharification by HoloMix. This study provided insights into enzymatic hydrolysis of various pre-treated hardwood substrates and showed the same lignocellulolytic cocktail comparable to/if not better than commercial enzyme preparations can be used to efficiently hydrolyse different hardwood species.

Combination of ultrasound, enzymes and mechanical stirring: A new method to improve Vitis vinifera Cabernet Sauvignon must yield, quality and bioactive compounds

In this work the effects of ultrasound (US), mechanical stirring (MS) and nine commercial enzyme preparations (EP) were investigated on Vitis vinifera Cabernet Sauvignon must extraction (GME). Yield, quality parameters and bioactive compounds were analyzed for individual and combined extraction techniques. The EP Zimopec PX5® presented the best results at the concentration of 1.0Ug−1 of pectinase, 50°C and 30min. Compared to MS, US improved the extraction yield, anthocyanin content, color and total soluble solids, while the combined techniques (US+MS) with enzyme improved yield, antioxidant activity and color in 7.1%, 30.2% and 9.6%, respectively, when compared to control samples. The extraction process resulted from the combination of ultrasound, mechanical stirring and enzyme, enhanced the extraction quality and also showed a synergistic and positive effect to grape must extraction.

Improved chromatic and sensory characteristics of Plavac Mali wines - efficiency of maceration enzymes

Two commercial enzyme preparations were used in the production of wine from the Croatian autochthonous red grape variety Plavac Mali in order to improve the extraction of polyphenolic components from grapes, chromatic parameters, and sensory quality. During two vintages, the conventional maceration without enzymes was compared with the maceration using products with different characteristics: pectinase with additional cellulase and hemicellulase activity and pectinase with inactive yeast cells. Both products affected polyphenolic extraction and colour parameters: intensity and hue, and ratio between the yellow, red, and blue colour in young wines (2 months after fermentation) and at the moment of bottling (9 months after fermentation). The correlation between anthocyanins and colour intensity was very strong. The expected reduction of quantitative chromatic parameters during aging was confirmed. Significantly better results were observed in wines produced with pectinase, in relation to all analysed physical and chemical parameters. The sensory analysis showed that wines produced with pure pectolytic enzymes were significantly better than those produced without the enzymes. A product of the combination of pectolytic enzymes and inactive yeast cells had a partial influence on the improvement of the phenolic and sensory quality. The overall quality was significantly more expressed in wines produced with pectolytic enzymes, especially in young wines.

Using an artificial neural network to predict the optimal conditions for enzymatic hydrolysis of apple pomace

The enzymatic degradation of lignocellulosic biomass such as apple pomace is a complex process influenced by a number of hydrolysis conditions. Predicting optimal conditions, including enzyme and substrate concentration, temperature and pH can improve conversion efficiency. In this study, the production of sugar monomers from apple pomace using commercial enzyme preparations, Celluclast 1.5L, Viscozyme L and Novozyme 188 was investigated. A limited number of experiments were carried out and then analysed using an artificial neural network (ANN) to model the enzymatic hydrolysis process. The ANN was used to simulate the enzymatic hydrolysis process for a range of input variables and the optimal conditions were successfully selected as was indicated by the R2 value of 0.99 and a small MSE value. The inputs for the ANN were substrate loading, enzyme loading, temperature, initial pH and a combination of these parameters, while release profiles of glucose and reducing sugars were the outputs. Enzyme loadings of 0.5 and 0.2 mg/g substrate and a substrate loading of 30% were optimal for glucose and reducing sugar release from apple pomace, respectively, resulting in concentrations of 6.5 g/L glucose and 28.9 g/L reducing sugars. Apple pomace hydrolysis can be successfully carried out based on the predicted optimal conditions from the ANN.