Crude Enzyme Extract Research Articles

Ameliorating Direct Blue Dye Degradation Using Trametes versicolor Derived Laccase Enzyme Optimized through Box–Behnken Design (BBD) via Submerged Fermentation

The major intend of this study was to elucidate the laccase production by Trametes versicolor under submerged fermentation using fruit waste peel as substrate. The textile dye was decolorized by the procured crude enzymatic extract using the response surface methodology. The submerged media with organic fruit peel waste extract (jackfruit, pineapple & kaffir) supplemented with gypsum, calcium carbonate, and nutrient broth were considered superior for laccase production. The produced laccase enzyme was used in dye decolorization at the optimum conditions using the Box-Behnken design. Subsequently, the experiment was designed with four variables (dye concentration, pH, temperature & time) with three factors to achieve the maximum direct blue dye decolorization. The highest laccase activity level was obtained from jackfruit peel extract with 3.86U/ml on 15th day at 25oC with pH 5.0 when compared to the other two extracts. The maximum laccase activity with guaiacol was obtained at optimum pH 4 and 40oC. The predicted value was experimentally validated by attaining 81.25% of dye color removal. From the result, the optimum conditions for direct blue color removal were: dye concentration 40ppm, pH 4.0, temperature 40oC at 24 hours. From the results of this study, it was concluded that the jack fruit peel was a more suitable substrate for laccase production. The dye decolorization results were recommended that Box-Behnken design for parameters optimization. The T. versicolor laccase was more proficient for textile dye decolorization. The opportunity was created by using the laccase enzyme for the biological treatment of textile dyeing effluent before discharging into the environment.

Agro-industrial wastes revalorization as feedstock: production of lignin-modifying enzymes extracts by solid-state fermentation using white rot fungi

The purpose of the study was to evaluate the production of lignin-modifying enzyme extracts and delignified biomass from agro-industrial wastes using white rot fungi (Inonotus sp. Sp2, Stereum hirsutum Ru-104, Bjerkandera sp. BOS55, Pleurotus eryngii IJFM 169 and Phanerochaete chrysosporium BKM-F-1767). These were screened based on their adaptability and colonization ability on different substrates, as well as by the Laccase, Manganese peroxidase, and Lignin peroxidase enzymatic production. Native strains (Inonotus sp. Sp2 and S. hirsutum Ru-104) showed the highest growth kinetics under the solid-substrate fermentation conditions and the growth rate parameters of the kinetic logistic model for the different substrates were between 0.39–0.81 (1/d) and 0.42–0.83 (1/d), respectively; the determination coefficients were ≥0.99. Inonotus sp. Sp2 was subsequently cultured in static flasks to produce crude enzyme extracts, obtaining manganese peroxidase activity levels of 18.5 and 31.3 (U/g) when growing in corn cob husk and spent tea leaves, respectively. Besides, it was to establish that the best conditions for lignin-modifying enzymes production using corn cob husk are 70% of initial moisture and 2.12 mm of particle size; reaching after 30 incubation days a manganese peroxidase activity of 21 ± 6 (U/g) under these conditions; enzyme that showed a suitable thermostability.

Pectinases Secretion by Saccharomyces cerevisiae: Optimization in Solid-State Fermentation and Identification by a Shotgun Proteomics Approach.

A sequential design strategy was applied to optimize the secretion of pectinases by a Saccharomyces cerevisiae strain, from Brazilian sugarcane liquor vat, on passion fruit residue flour (PFRF), through solid-state fermentation (SSF). A factorial design was performed to determine the influence variables and two rotational central composite designs were executed. The validated experimental result was of 7.1 U mL−1 using 50% PFRF (w/w), pH 5, 30 °C for 24 h, under static SSF. Polygalacturonase, pectin methyl esterase, pectin–lyase and pectate–lyase activities were 3.5; 0.08; 3.1 and 0.8 U mL−1, respectively. Shotgun proteomics analysis of the crude extract enabled the identification of two pectin–lyases, one pectate–lyase and a glucosidase. The crude enzymatic extract maintained at least 80% of its original activity at pH values and temperatures ranging from 2 to 8 and 30 to 80 °C, respectively, over 60 min incubation. Results revealed that PFRF might be a cost-effective and eco-friendly substrate to produce pectinases. Statistical optimization led to fermentation conditions wherein pectin active proteins predominated. To the extent of our knowledge, this is the first study reporting the synthesis of pectate lyase by S. cerevisiae.

Enhancing SDS-PAGE Detection of Dilute Extracellular Polystyrene Degrading Enzymes Expressed by Bacillus megaterium Strain via Centrifugal Freeze Concentration Method

This study aims to enhance the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) detection and visualization of extracellular polystyrene degrading enzyme expressed by Bacillus megaterium as a result of utilizing polystyrene as a sole carbon source. Dilute polystyrene degrading enzyme expressed in the culture supernatant could not be directly visualized from SDS-PAGE stained with colloidal Coomassie blue stain as it falls below its sensitivity or detection limit level. To overcome this problem, the crude enzyme extract was concentrated via the centrifugal freeze concentration method (cryoconcentration). The extracellular enzyme profiles were determined by SDS-PAGE. The SDS-PAGE analysis of the concentrated enzyme extract revealed 6 visible protein bands ranging in size from 10 to 60 kDa. Three distinct protein bands at approximately 20, 45 and 60 kDa were observed to be highly expressed in the culture supernatant. Polystyrene degrading enzyme extracted from the culture supernatant was closely related to several polymer degrading enzymes which suggest that the extracted enzyme from Bacillus megaterium also belongs to the hydrolase’s enzyme family. GC-MS analysis of the extracted samples significantly contains benzene derivatives due to the breaking down of the long-chain aromatic hydrocarbon polymer. Hydroxylation of the aromatic ring formed phenolic substrate took place which suggests being facilitated by intracellular enzyme hydroxylases. Hence, the biodegradation of polystyrene by Bacillus megaterium was believed to incorporate both intracellular and extracellular enzymes.
 HIGHLIGHTS
 
 Bacillus megaterium strain was first reported to contribute to polystyrene biodegradation
 Polystyrene degrading enzyme expressed by Bacillus megaterium was detected through SDS-PAGE
 Cryoconcentration enhanced SDS-PAGE protein detection
 The protein bands were most distinct visualized at approximately 20, 45 and 60 kDa by SDS-PAGE
 GC-MS analysis detected benzene derivatives which confirmed enzymatic degradation activity
 
 GRAPHICAL ABSTRACT

Screening of Cellulolytic Bacteria from Various Ecosystems and Their Cellulases Production under Multi-Stress Conditions

Cellulose represents the most abundant component of plant biomass on earth; it is degraded by cellulases, specific enzymes produced by microorganisms. However, cellulases of bacterial origin attract more interest due to their natural diversity and ability to inhabit a variety of niches, allowing the selection of cellulolytic strains resistant to environmental stresses. The screening of the cellulolytic activity of 398 bacteria isolated from various ecosystems in Algeria (cave, ruins, chott, thermal station, and rhizosphere of arid and semi-arid regions) was performed by the appearance of a hydrolysis zone on carboxymethylcellulose (CMC) medium. The cellulase activity on CMC (1%) broth allowed to select 26 strains among which 12 had the best activity (0.3 U/mL to 2.2 U/mL). Optimization of physicochemical parameters (salinity: 0–1 M NaCl; pH: 3, 4, 7, 9, and 11; temperature: 30, 45, and 50 °C; PEG8000: 0 and 30%) involved in growth and cellulose production showed that the majority of strains were mesophilic, neutrophilic, or alkali- tolerant and tolerant to 30% of PEG8000. The cellulase activity and stability under different stress allowed to retain five strains, which the most efficient. Based on the 16S-rRNA sequencing results, they belonged to the genus Bacillus. The physicochemical properties of cellulases (crude extract) showed a CMCase active over a wide range of pH (4 to 11), optimal at 50 °C and 60 °C. The inhibiting salinity effect on the activity was not detected and was negligible on the enzymatic stability. The residual CMCase activity remained between 40 and 70% in a temperature range between 40 and 70 °C, was stable over a wide range of saline concentrations (0–2000 mM), and was weakly affected at 30% of PEG8000. The crude enzyme extract was able to hydrolyze both soluble and insoluble cellulosic substrates. The evaluation of the hydrolysis capacity of lignocellulosic waste revealed the ability of tested strains to degrade wheat bran, barley bran, and corncob. In addition, the enzyme showed significant multi-stress resistance on solid and liquid media. By these characteristics, these cellulolytic bacteria could be attractive to be used in various industrial and biotechnology applications.

Síntesis benigna y sencilla de dímeros de tipo lignano usando peroxidasa cruda de rábano rojo (Raphanus sativus var sativus)

Alkyl esters of hydroxycinnamic acids and vanillin derivatives were used as model compounds in the oxidative coupling reactions catalyzed by a crude enzymatic extract of Raphanus sativus var sativus (red radish). Six products were isolated and characterized: three alkyl esters of hydroxycinnamic acids dimers and three vanilloids dimers. Herein, we report the enzymatic extract of Raphanus sativus var. sativus showed peroxidase activity catalyzing the oxidative coupling of phenols. Vanilloid dimers were coupled in an ortho-ortho way as expected, while alkyl ester dimers were formed through 5-8 and 8-8 bonds via a radical mechanism prior to cyclization

Isolation of Glucoamylase from Aspergillus Niger to Produce Liquid Sugar from Tapioca Flour

This study aims to isolate the glucoamylase enzyme from Aspergillus niger to produce liquid sugar from tapioca flour. Enzymes are produced in a batch system laboratory-scale bioreactor. The fermentation substrate in an agar medium modified with the addition of starch. Fermentation was carried out for 168 hours at room temperature with an agitation speed of 120 rpm. The manufacture of liquid sugar is done by adding an enzyme as much as 0.3; 0.4; and 0.5 ml while the substrate concentration was varied by 20%; 30%; and 40%. The results showed that A. niger produce extracellular enzymes such as amylase and glucoamylase, particulary at exponential phase and the initial stationary phase. Enzymatic hydrolysis of starch consists of two stages, namely liquification and saccharification. In the liquification process, the enzyme α-amylase is used to break down starch containing amylose and amylopectin into dextrins. Next in the second stage is the saccharification process, where dextrin is hydrolyzed into glucose with the help of the glucoamylase enzyme. Optimal liquid sugar production can be done by adding 0.5 mL of crude enzyme extract at 30% (w/v) tapioca concentration. Further research is recommended to carry out enzyme purification to produce liquid sugar products with higher sugar purity.

The Effect of Bacillus subtilis Bacteria Concentration on Production and Characterization of Pektinase Enzymes from Pineapple Peel Waste (Ananas comosus L. Merr)

This study aims to determine the effect of treatment concentrations (4%; 8%; 10%; 13%; 16%; 20%) of Bacillus subtilis bacterial isolates on the production of pectinase enzymes from pineapple peel waste, and the characteristics of the pectinase enzyme produced include (enzyme activity, optimum temperature, optimum pH, Km and Vmax values). The yield of crude enzyme extract production from the 6 treatments carried out, the highest yield was found in the 10% treatment at 1.59 g and the lowest in the 4% treatment at 1.53 g. In the results of enzyme characterization, the highest enzyme activity was found at a concentration of 20% at 1.883, and the pectinase enzyme produced was able to work optimally in environmental conditions with a temperature of 400C and pH 4 at all concentrations, but the highest activity at variations in temperature and pH was on enzymes with 20% bacterial concentration treatment, namely 0.680 at 400C and 0.650 at pH 4. Enzyme kinetics analysis obtained from the pectinase enzyme produced, showed the highest Km value of 0.021 at a concentration of 4% while the lowest value is 0.0095 at a concentration of 20%. Then at the Vmax value of the enzyme, the highest value was found at the 20% concentration treatment and the lowest value was at 4% treatment was 0.481.Keywords: Bacillus subtilis, Characterization, Enzyme Production, PektinaseEnzyme

Fluoxetine Removal from Aqueous Solutions Using a Lignocellulosic Substrate Colonized by the White-Rot Fungus Pleurotus ostreatus.

One of the main challenges in both the design of new wastewater treatment plants and the expansion and improvement of existing ones is the removal of emerging pollutants. Therefore, the search for economic and sustainable treatments is needed to enhance the removal of pharmaceuticals. The potential of a lignocellulosic substrate colonized by Pleurotus ostreatus, a waste from mushroom production, to remove fluoxetine from aqueous solutions was studied. Batch assays were performed to remove 600 µg∙L−1 fluoxetine from aqueous solutions using the colonized mushroom substrate (CMS) and crude enzyme extracts. The removal efficiencies achieved were, respectively, ≥83.1% and 19.6% in 10 min. Batch assays with sterilized CMS and 1-aminobenzotriazole (to inhibit cytochrome P450 enzymes) showed that the higher removal efficiencies achieved in the CMS assays may be attributed to the synergistic contribution of biosorption onto the CMS and lignin modifying enzymes activity, namely laccase activity. A column assay was performed with the CMS, fed with 750 µg∙L−1 fluoxetine aqueous solution. The removal efficiency was 100% during 30 min, decreasing to a final value of 70% after 8 h of operation. The results suggested that CMS can be a promising eco-friendly alternative to remove fluoxetine from aqueous solutions.

Laccase-Driven Transformation of High Priority Pesticides Without Redox Mediators: Towards Bioremediation of Contaminated Wastewaters.

In this study, Pleurotus dryinus was grown on municipal biosolids (BS) as the substrate to produce laccase for the removal of pesticides (fungicides, herbicides, and insecticides) from wastewater. Among the various types of BS tested, sterilized biosolids were the most promising substrate for laccase production by P. dryinus with a maximal laccase activity (162.1 ± 21.1 U/g dry substrate), followed by hygenized biosolids (96.7 ± 17.6 U/g dry substrate), unsterilized biosolids (UBS) (31.9 ± 1.2 U/g dry substrate), and alkali-treated biosolids (8.2 ± 0.4 U/g dry substrate). The ultrasound-assisted extraction of this enzyme from fermented UBS was carried out with 0.1 M phosphate buffer at pH 7.0, which increased the enzyme activity of the crude extract by 30%. To test the catalytic potential of the biocatalyst in real matrices, 1 U/ml of recovered crude laccase extract was applied for 24 h for the removal of 29 pesticides (nine fungicides, 10 herbicides, and 10 insecticides) either separately or as a mixture from spiked biologically treated wastewater effluent. When treated with crude enzyme extract, high-priority herbicides metolachlor and atrazine were completely removed, while 93%–97% of the insecticides aldicarb, spinosad, and azinphos-methyl and up to 91% of kresoxim-methyl were removed. Promising results were obtained with BS-derived crude enzyme extract exhibiting improved pesticides removal, which may be due to the mediator effect resulting from the catalytic transformation of other molecules in the cocktail. The results demonstrated a promising integrated bioprocess for the removal of pesticides in wastewater using crude laccase obtained from BS.

A Multicomponent THF Hydroxylase Initiates Tetrahydrofuran Degradation in Cupriavidus metallidurans ZM02.

Tetrahydrofuran (THF) has been recognized as a water contaminant because of its human carcinogenicity, extensive use, and widespread distribution. Previously reported multicomponent monooxygenases (MOs) involved in THF degradation were highly conserved, and all of them were from Gram-positive bacteria. In this study, a novel THF-degrading gene cluster (dmpKLMNOP) encoding THF hydroxylase was identified on the chromosome of a newly isolated Gram-negative THF-degrading bacterium, Cupriavidus metallidurans ZM02, and functionally characterized. Transcriptome sequencing and RT-qPCR demonstrated that the expression of dmpKLMNOP was upregulated during the growth of strain ZM02 on THF or phenol. The deletion of oxygenase alpha or beta subunit or the reductase component disrupted the degradation of THF but did not affect the utilization of its hydroxylated product 2-hydroxytetrahydrofuran. Cupriavidus pinatubonensis JMP134 heterologously expressing dmpKLMNOP from strain ZM02 could grow on THF, indicating that the THF hydroxylase DmpZM02KLMNOP is responsible for the initial degradation of THF. Furthermore, the THF and phenol oxidation activities of crude enzyme extracts were detected, and the highest THF and phenol catalytic activities were 1.38 ± 0.24 μmol min-1 mg-1 and 1.77 ± 0.37 μmol min-1 mg-1, respectively, with the addition of NADPH and Fe2+. The characterization of THF hydroxylase associated with THF degradation enriches our understanding of THF-degrading gene diversity and provides a novel potential enzyme for the bioremediation of THF-containing pollutants. IMPORTANCE Multicomponent MOs catalyzing the initial hydroxylation of THF are vital rate-limiting enzymes in the THF degradation pathway. Previous studies of THF degradation gene clusters have focused on Gram-positive bacteria, and the molecular mechanism of THF degradation in Gram-negative bacteria has rarely been reported. In this study, a novel THF hydroxylase encoded by dmpKLMNOP in strain ZM02 was identified to be involved in both THF and phenol degradation. Our findings provide new insights into the THF-degrading gene cluster and enzymes in Gram-negative bacteria.

A novel L‐asparaginase from the symbiotic Enterobacter aerogenes isolated from Eucheuma sp.

This study was aimed to purify and characterize the enzyme L-asparaginase from endophytic microorganisms of Eucheuma sp. in Puntondo Island, South Sulawesi, Indonesia. Research stages were ammonium sulfate precipitation, dialysis, and Sephadex G-75 gel filtration chromatography and Sephadex CMC-50 ion-exchange chromatography matrices. The result of test activities of ammonium sulfate fraction showed the highest range of 40%–60% the activity value of 6.71 IU/ml. The highest protein content was obtained on a 0%–20% fraction of 2.76 mg/ml. Temperature and pH assessments indicated that L-asparaginase had optimum activity at 37°C and pH 8, respectively. At the stage of dialysis, the highest protein content was 5.17 IU/ml in a 0%–20% fraction, and the highest enzyme activity was 1.23 IU/ml in a 20%–40% fraction. The results of gel filtration chromatography fractions G-75 were F5–F10, with a specific activity of 0.60 U/mg, 2.05 times higher purity level than the crude extract enzyme. The results of CMC-50 ion-exchange chromatography showed that F54 and F55 fractions had a specific activity of 106.97 and 295.38 U/mg, respectively, with a purity level of 366.34 and 1,011.57 times higher than the crude extract enzyme. Novelty impact statement Enterobacter aerogenes strain P5, an endophytic bacterium from Eucheuma sp., can produce pure L-asparaginase enzyme with a high specific activity applied in pharmaceuticals nutraceutical applications.

Partial pretreatment of ingredient mixture effectively improved feed chemical composition, physicochemical properties and in vitro digestibility

Physical pretreatment, using microwave, has been performed to qualitatively improve feed ingredients prior to inclusion in aquafeed. Both partial and full microwaving pretreatments of solid ingredient mixture were investigated. Three feeds containing either pretreated protein (PP; fish meal, meat and bone meal, and soybean meal), pretreated carbohydrate (PC; broken rice and rice bran) or pretreated completely of both protein and carbohydrate (PPPC) were formulated and compared with a control feed containing non-pretreated ingredient mixture (NP). Significantly improved proximate chemical compositions were observed in both partial and full pretreatments of the ingredient mixture (P < 0.05). Qualitative changes in nutritive profiles assessed through Fourier transform infrared spectroscopy were also observed. Decreased pH and water absorption capacity, increased turbidity, with more irregular, aggregation and fusion in microstructure, and modified secondary protein structure (height and area of amides I and II, and quantities of ß-turns and random coils) indicated desirable characteristics for the pretreated feeds over the NP feed. In vitro digestibility based on crude digestive enzyme extracts from economically important fishes, Nile tilapia (Oreochromis niloticus) and striped catfish (Pangasianodon hypophthalmus), exhibited significantly improved availability of both protein and carbohydrate digestibility in all pretreated feeds (P < 0.05), and indicated that striped catfish could utilize the four feeds better than Nile tilapia did (P < 0.05). Surprisingly, the partial microwaving of only carbohydrate in the ingredient mixture (PC feed) improved aquafeed with the significantly highest bioavailability of both protein and carbohydrate (P < 0.05). This production process can be directly applied in aquafeed production.

Enhancement of saccharification of corn stover by cellulolytic enzyme produced from biomass-degrading bacteria

Enzymatic saccharification of corn stover can be enhanced by partially replacing commercial enzymes with bacterial crude enzyme extracts. Thus, in this study, three bacteria (Bacillus sp. A0, Bacillus sp. CH20S1, and Exiguobacterium sp. AS2B) were cultured in a media with corn stover as the substrate to produce crude enzyme extract and saccharify corn stover. The cultural conditions were monitored and optimized to maximize CMCase and xylanase activity in the crude enzyme extracts. After 72 h of hydrolysis of corn stover with diluted crude enzymes (DCE) from the three strains, reducing sugars ranging from 48.2 to 71.7 mg g-1 were released from non-pretreated and pretreated corn stover. Furthermore, the maximum reducing sugars of 316 and 321 mg g-1 were observed when 12 and 4 FPU g-1 of commercial cellulase were added to the DCE of the CH20S1 strain, respectively. It was shown that an effective combination of bacterial DCE with commercial enzymes could achieve higher saccharification of lignocellulosic biomass, which might be cost-efficient compared to their single-use. Overall, this study aims to show the enhanced enzymatic saccharification of corn stover.

Statistical approach of Ca2+ effect on the amylases production by Saccharomyces diastaticus ATCC 13007 using corn and cassava starch as inducers

Amylase production from starch sources is associated with the nature of this carbon source, particle size and crystalline architecture of natural starch granules. The aim of this work was to evaluated the amylase production by Saccharomyces diastaticus ATCC 13007, using different CaCO3 concentration and corn starch (Zea mays) and cassava starch (Manihot esculenta) as inducers. A 22 Central Composite Rotatable Design with three replications at the center point was used to determine the influence of calcium and starch concentrations on the production process. Besides, the partial characterization of the crude enzymatic extracts obtained was investigated. Ca2+ influenced the α-amylase production by both evaluated carbon sources. The production of glucoamylase was not influenced by the variables under study. Partial characterization of crude extracts indicated that, for corn substrate, the enzyme showed maximum activity at pH 5.0, and no influence of temperature was observed. Regarding cassava starch, the results showed that conditions at central point (pH 6.5 and 50ºC) should be avoided in order to maximize enzyme activity.

New application of SptA protease: A cleaner and potential water-conserving approach to dehairing of skin

Leather making technology is undergoing a profound change from chemical process to enzyme-assisted dehairing. In this work, the SptA protease from halophilic archaeon Natrinema sp. J7 was expressed by Bacillus subtilis SCK6. The crude enzyme extract was applied into enzymatic dehairing process with the enzyme activity of 1800 U/mL. Histologic analysis, scanning electron microscope and wastewater test indicated this SptA protease could serve as a good alternative to sodium sulphide in goatskins dehairing technique. Noteworthily, 8% sodium chloride was introduced in SptA protease dehairing system and achieved a comparable result after 6 h of treatment. This would not only reduce the pollution load significantly, but also bring about 16–20% water-conserving benefit by addressing the issue of high salt in the preservation of hides and skins. In addition, based on bioinformatics, molecular modelling and docking analysis were made as an attempt to illustrate how SptA protease contribute to the loosing and removal of hair from the micro and molecule perspective. It was concluded that this SptA protease could efficiently dehair goatskins without any damage to the grain and collagen structure, ensuring its use as a promising candidate for leather making.

Bio-scouring of Non-spinnable Cotton by a Crude Enzyme of a New Fungal Strain Aspergillus sp. VM-1, Isolated from Banana Pseudostem Waste

In the present study, we isolated and identified a new fungus, Aspergillus sp VM-1 from banana pseudostem waste. The fungal strain, Aspergillus sp. VM-1 was grown on a substrate (banana pseudostem, cottonseed hulls and cottonseed meal in the ratio of 60:30:10, respectively) for 7 days and the crude enzyme was extracted from the fermented substrate. The pectinase activity in crude enzyme extract was 551 (U/ml). The crude enzyme extracted from Aspergillus sp. VM-1 was evaluated for bio-scouring of non-spinnable cotton. The non-spinnable cotton taken in the study was short staple cotton (SSC), cotton linters (CL) and non-woven cotton fabric (NWCF). The maximum absorbency (2 s) in non-spinnable cotton was achieved under optimized process conditions: enzyme extract level (40%), temperature (40 ± 2 °C) and time (40 min). The quality parameters of bio-scoured cotton meet the Indian Pharmacopoeia (IP) standards. A solid state fermenter was designed to scale-up the crude enzyme production up to 30 l. This is the first report on bio-scouring of under-utilized short cotton fibres at lower temperature (40 ± 2 °C). The present study offers a simple and eco-friendly bio-scouring process as an alternative to toxic chemical scouring of under-utilized short staple cotton fibres.Graphic Supplementary InformationThe online version contains supplementary material available at 10.1007/s12649-021-01621-9.

Extraction, Purification and Characterization of Transglutaminase From Some Plants

The present study aimed to Isolate trans-glutaminase EC:2.3.2.13 from some plants sources and Purified it and Studied it’s Charctarestics as well as it’s practical applications in the production of sausage.The enzyme was extracted from four types of plants (rosemary, chard, radish, arugula) using nine extraction solutions that included distilled water,Sodium chloride 3% solution, sodium chloride 5% solution, sodium phosphate solution 0.1 M and an pH 6.5, sodium phosphate 0.1 M and an pH of 7.5, Tris - Hcl solution 0.2 M and pH 7, Tris - Hcl solution 0.2 M and an pH 8, Tris - Hcl solution 0.1 M and an pH 7 and Tris - Hcl solution 0.1 M and an pH 8 in order to find out the best source of enzyme and the best extraction solution. chard was the best source of enzyme compared with other sources, Tirs -HCl 0.1M, pH 8 solution was the best extraction solution which gave the highest specific activity 8.104 unit/mg.Protein content for the crude enzyme extracts were concentrated using saturated ammonium sulfate in arrange 20-60%, Dialysis was done using distilled water. Then, the purification steps of the enzyme were completed using the gel filtration in the Sephadex G-100 Purification Folds 13.91 time and the yield was 20.04% %. Electrophoresise process using poly acryl amid gel in the absence of SDS observe the presence of one protein band which indicates the complete purification of transglutaminase. transglutaminase molecular weight was 42,660 Dalton when it was evaluated using poly acryl amide electrophoresis in the presence of SDS. the optimum pH for enzyme activity and enzyme stability was 7, while the optimum temperature for enzyme activity was 55°C, and the optimum temperature for enzyme stability was between 25-45°C.

Anaerobic digestion of cornmeal – the effect of crude enzyme extract and co‐digestion with cow manure

AbstractThis study examined the effect of a crude enzyme extract, containing mainly starch‐degrading enzymes, on cornmeal (Zea mays) hydrolysis. This was followed by an investigation of the effect of enzymatic treatment for the anaerobic digestion of this biomass. Cornmeal and cow manure were also co‐digested, and both batch and semi‐continuous experiments were performed. The enzymatic pretreatment of cornmeal resulted in a yield of 65 ± 5% reducing sugars, with 1:10 w/v (grams of dry substrate per mililiters of enzyme extract) enzyme load at 45 °C for 48 h. There was an 8% enhancement of methane production observed during the batch assays, both when cornmeal hydrolysate was digested and when enzymes were added directly to the digester. Synergetic effects were found when co‐digesting cornmeal and cow manure, leading to higher methane yield (280 NmL gVS–1) than that (200 NmL gVS–1) calculated based on the methane potential of the individual substrates. Regarding long‐term effects, the laboratory‐scale semi‐continuous experiments also demonstrated that the co‐digestion of cornmeal and cow manure (1:1 volatile solid (VS) basis) led to a stable process reaching an organic loading rate of 3 g VS L day–1 and achieving a daily methane production of 1280.12 ± 99.4 NmL CH4/day. However, when cornmeal was investigated in mono‐digestion, and the enzyme extract was directly added during semi‐continuous digestion of cornmeal, volatile fatty acid (VFA) accumulation was observed, leading to a decrease in pH, and no significant enhancement of the conversion into methane was observed. © 2021 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Thermostable trypsin-like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting.

The increased demand for cheese and the limited availability of calf rennet justifies the search for milk-clotting enzymes from alternative sources. Trypsin-like protease by Penicillium roqueforti was produced by solid-state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert-type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10-12) and an optimum temperature of 80°C, being stable at temperatures above 60°C. Enzymatic activity was maximized in the presence of Na+ (192%), Co2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin-like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses.