Crude Enzyme Extract Research Articles

Heterologous Expression of Ketoreductase ChKRED20 Mutant in Pichia pastoris and Bioreductive Production of (R)-1, 3-Butanediol.

(R)-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The ChKRED20 is a robust NADH-dependent ketoreductase identified from Chryseobacterium sp. CA49. We obtained a ChKRED20 mutant (M12) through directed evolutionary screening of ChKRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to (R)-1, 3-BDO. So far, both ChKRED20 and its mutants have been expressed in intracellular in E. coli, the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in P. pastoris, and the target protein yield was 302 mg/L in shake-flask fermentation and approximately 3.5 g/L in high-density fermentation. The recombinant M12 showed optimal enzyme activity at 30 °C and had high activity within a broad pH range of 6.0-8.0, and also showed high thermal stability. The recombinant M12 was further used for the reduction of 4H2B to (R)-1, 3-BDO, and 98.9% yield was achieved at 4540 mM 4H2B. The crude M12 enzyme extract was found to catalyze the bioreductive production of (R)-1, 3-BDO with excellent stereoselectivity (ee > 99%) and meet the production requirements. Our research shows that the M12 mutant can be used for the synthesis of (R)-1, 3-BDO, and the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of ChKRED20 or its mutants, which may have applications in industrial settings.

Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.

Enhancing circular economy: eco-friendly starch extraction and modification using fungal crude enzymatic extract from cocoa shell waste

Enhancing circular economy: eco-friendly starch extraction and modification using fungal crude enzymatic extract from cocoa shell waste

Assessment of Fungal Lytic Enzymatic Extracts Produced Under Submerged Fermentation as Enhancers of Entomopathogens’ Biological Activity

The application of enzymes in agricultural fields has been little explored. One potential application of fungal lytic enzymes (chitinases, lipases, and proteases) is as an additive to current biopesticides to increase their efficacy and reduce the time of mortality. For this, a screening of lytic overproducer fungi under submerged fermentation with a chemical-defined medium was performed. Then, the enzymatic crude extract (ECE) was concentrated and partially characterized. This characterization consisted of measuring the enzymatic activity (lipase, protease and, chitinase) and determining the enzyme stability after storage at temperatures of − 80, − 20 and, 4 °C. And lastly, the application of these concentrated enzymatic crude extracts (C-ECE) as an enhancer of spores-based fungal biopesticide was proven. Beauveria were not as good producers of lytic enzymes as the strains from Trichoderma and Metarhizium. The isolate M. robertsii Mt015 was selected for the co-production of chitinases and proteases; and the isolate T. harzianum Th180 for co-production of chitinases, lipases, and proteases. The C-ECE of Mt015 had a protease activity of 18.6 ± 1.1 U ml−1, chitinase activity of 0.28 ± 0.01 U ml−1, and no lipase activity. Meanwhile, the C-ECE of Th180 reached a chitinase activity of 0.75 U ml−1, lipase activity of 0.32 U ml−1, and protease activity of 0.24 U ml−1. Finally, an enhancing effect of the enzymatic extracts of M. robertsii (66.7%) and T. harzianum (43.5%) on the efficacy of B. bassiana Bv064 against Diatraea saccharalis larvae was observed. This work demonstrates the non-species-specific enhancing effect of enzymatic extracts on the insecticidal activity of conidial-based biopesticides, which constitutes a contribution to the improvement of biological control agents’ performance.

A systematic review on products derived from nematophagous fungi in the biological control of parasitic helminths of animals.

Nematophagous fungi have been widely evaluated in the biological control of parasitic helminths in animals, both through their direct use and the use of their derived products. Fungal bioproducts can include extracellular enzymes, silver nanoparticles (AgNPs), as well as secondary metabolites. The aim of this study was to conduct a systematic review covering the evaluation of products derived from nematophagous fungi in the biological control of parasitic helminths in animals. In total, 33 studies met the inclusion criteria and were included in this review. The majority of the studies were conducted in Brazil (72.7%, 24/33), and bioproducts derived from the fungus Duddingtonia flagrans were the most commonly evaluated (36.3%, 12/33). The studies involved the production of extracellular enzymes (48.4%, 16/33), followed by crude enzymatic extract (27.2%, 9/33), secondary metabolites (15.1%, 5/33) and biosynthesis of AgNPs (9.1%, 3/33). The most researched extracellular enzymes were serine proteases (37.5%, 6/16), with efficacies ranging from 23.9 to 85%; proteases (31.2%, 5/16), with efficacies from 41.4 to 95.4%; proteases + chitinases (18.7%, 3/16), with efficacies from 20.5 to 43.4%; and chitinases (12.5%, 2/16), with efficacies ranging from 12 to 100%. In conclusion, extracellular enzymes are the most investigated derivatives of nematophagous fungi, with proteases being promising strategies in the biological control of animal helminths. Further studies under in vivo and field conditions are needed to explore the applicability of these bioproducts as tools for biological control.

Nutritive value of feed substrate waste from black soldier fly larvae production, and its potential as an aquafeed ingredient

Abstract Black soldier fly larvae (BSFL, Hermetia illucens) are an alternative source of protein whereas little research has yet been conducted to investigate the potential application of feed substrate waste (FSW) from larval production in aquaculture. The nutritive value and physicochemical properties of FSW from BSFL were here investigated in vitro. Ten-day-old first-instar larvae were cultivated on two feed substrates: laying hen feed (FS I) or a laying hen feed-rice bran blend (1:1 w/w, FS II). FSW samples were collected at day 4.5 (0.5FSW) and day 9 (1FSW) of larval rearing, and compared to the original feed substrate (0FSW). Crude protein increased depending on FS type, harvesting time (HT), and their interaction. Crude lipid, crude fiber, acid detergent fiber, neutral detergent fiber, crude ash, nitrogen-free extract, and gross energy decreased (). Analysis of the physicochemical properties of FSW by various techniques (scanning electron microscopy, differential scanning colorimetry, X-ray diffractometry, and Fourier transform infrared spectroscopy) revealed qualitative and quantitative changes in FSW, driven by biological processes of BSFL. The digestibility of protein in FSW was determined by an in vitro protein digestibility (IVPD) test using crude digestive enzyme extracts from Nile tilapia (Oreochromis niloticus) and striped catfish (Pangasianodon hypophthalmus). 1FSW from FS I showed significantly better availability of protein relative to 0.5FSW (), while the IVPD of FSW from FS II was stable () or reduced (). Findings from this study suggest FSW from BSFL cultivation could be a protein source in aquafeed.

Pectinases production using an exotic caatinga passion fruit waste and wheat bran as a substrate for clarification of grape juice

Caatinga passion fruit waste (CPFW):Wheat bran has shown reliable performance for enzyme production. However, the mixture of wheat bran and CPFW may provide all nutrient sources needed by Aspergillus niger for pectinase generation. This work investigates the production of pectinolytic enzymes using a substrate composed of CPFW:Wheat bran using Aspergillus niger FSDE16 via semi-solid-state fermentation (SSSF). A blend of wheat bran:CPFW (1:1, g/g) with 60% moisture content was inoculated with 106 spores/mL for 144 h. Substrates were fermented using the 7 combinations of temperature (25–75 °C) and pH (3–6) dispose in an experimental design. Upon the completion of fermentation, all analyses were conducted including the enzymatic production, optimal activity, and enzymatic thermostability. Afterwards, grape juice clarification was conducted, and the clarified juice was evaluated considering viscosity reduction, yield increase, turbidity reduction, yield increase, colour, and phenolic profile. Results demonstrate that enzyme stability was achieved within the temperature range of 30–40 °C, with optimal activity recorded at 50 °C (42.7 U/g). The application of crude enzymatic extract in grape juice clarification led to an increase of yield (>280%), and a viscosity (>80%) and turbidity (>98%) reduction. The clarification treatment revealed a significant increase in the phenolic compounds evaluated, except epicatechin gallate, Pro-B2, and resveratrol, which reduced their concentrations. The clarification resulted in a colour intensity reduction. This study underscores the potential of using food waste for enzyme production, presenting a sustainable approach with implications for cost reduction and waste management in the food industry.

Stemphylium lycopersici immobilized in polyurethane (PU) treated in pressurized fluid CO2 as biocatalyst for ω-transamination reactions

Lyophilized whole cells and the crude enzymatic extract from the fungus Stemphylium lycopersici were successfully immobilized in situ in rigid polyurethane foam, as an alternative biocatalyst for the kinetic resolution of rac-1-phenylethylamine, an interesting building block in the synthesis of pharmaceuticals. The enzymatic activity of immobilized whole cells and the crude enzymatic extract was 2.52 and 5.05 U/g, respectively. The immobilization yield in polyurethane of both forms was at least above 100%, and the process did not change the affinity of the biocatalyst for the substrate. The optima reaction conditions for the kinetic resolution of rac-1-phenylethylamine into acetophenone were: 32.5 °C, 120 rpm by 24 h containing 10 mL (20% m/v) of phosphate buffer (pH 7.5) and 20% biocatalyst mass, resulting in conversions of 45% and 34% using the immobilized lyophilized fungus and crude enzymatic extract, respectively. Stemphylium lycopersici was also subjected to high pressure using CO2. The best conditions provided conversions of 49 (99% ee) and 26.21 (without ee) using the immobilized lyophilized fungus and the immobilized lyophilized crude enzyme extract, respectively, with 20 cycles of reuse and recovery greater than 50% (fungus). Interestingly, the compounds were satisfactorily converted to the corresponding ketones with up to 90% ee for the R-enantiomer. The capacity for conversion of the immobilized lyophilized fungus to different amines: rac-1,2,3,4-tetrahydro-1-naphthylamine, rac-1-phenylpropylamine, and rac-phenylbutylamine was also evaluated. ω-transaminase activity changed significantly depending on the experimental conditions applied, allowing the selection of proper operating conditions for advantageous application of this biocatalyst in transamination reactions.

Characterization and application of a microemulsion as model system for lipophilic phytochemicals in high‐pressure processing

AbstractHigh‐pressure processing (HPP) is considered as gentle preservation technique for especially heat‐sensitive food ingredients. So far, the focus has been on the fact that it is called a nonthermal process and high pressure can affect bioavailability, but it is questionable whether the high pressure affects the ingredients themselves. By using an o/w‐microemulsion (ME) as a model system, it was possible to investigate the influence of pressure, especially on lipophilic compounds (e.g., carotenoids and vitamin E), without the complexity of a food matrix. The ME consisted of Capryol® TM 90, Tween® 80 or 20, Transcutol® HP and distilled water. Lipophilic and hydrophilic compounds were introduced to the oil phase and to the aqueous phase, respectively. Storage experiments confirmed the applicability for β‐carotene and α‐tocopherol. HPP of MEs, performed for 10 min at room temperature (RT) and up to 600 MPa, resulted in pressure stability of β‐carotene (exceptional at 400 MPa; −11%) and α‐tocopherol. Multicomponent ME showed that both had a positive effect on the stability of chlorophyll a/b during HPP. An ME environment was used to facilitate co‐oxidation of β‐carotene via lipoxygenase (LOX) from an Edamame‐based crude enzyme extract and lyophilized LOX‐1 from soybeans during storage (RT and 4°C, dark conditions) and HPP treatment. A loss of β‐carotene occurred after addition of linoleic acid, whereas effects of added α‐tocopherol could be related to β‐carotene protection. Overall, the introduced ME for studying HPP effects on lipophilic food ingredients showed promising results as versatile model system for future investigations regarding interactions of phytochemicals.

Quantitative proteomic analysis reveals the mechanism and key esterase of β-cypermethrin degradation in a bacterial strain from fermented food

Beta-cypermethrin (β-CY) residues in food are an important threat to human health. Microorganisms can degrade β-CY residues during fermentation of fruits and vegetables, while the mechanism is not clear. In this study, a comprehensively investigate of the degradation mechanism of β-CY in a food microorganism was conducted based on proteomics analysis. The β-CY degradation bacteria Gordonia alkanivorans GH-1 was derived from fermented Pixian Doubanjiang. Its crude enzyme extract could degrade 77.11% of β-CY at a concentration of 45 mg/L within 24 h. Proteomics analysis revealed that the ester bond of β-CY is broken under the action of esterase to produce 3-phenoxy benzoic acid, which was further degraded by oxidoreductase and aromatic degrading enzyme. The up-regulation expression of oxidoreductase and esterase was confirmed by transcriptome and quantitative reverse transcription PCR. Meanwhile, the expression of esterase Est280 in Escherichia coli BL21 (DE3) resulted in a 48.43% enhancement in the degradation efficiency of β-CY, which confirmed that this enzyme was the key enzyme in the process of β-CY degradation. This study reveals the degradation mechanism of β-CY by microorganisms during food fermentation, providing a theoretical basis for the application of food microorganisms in β-CY residues.

Characterization of Extracellular Chitinase from Bacillus cereus SAHA 12.13 and Its Potency as a Biocontrol of Curvularia affinis

Bacillus cereus SAHA 12.13 can produce chitinase, an enzyme that digests chitin in the main compounds of cell walls, mycelia, and spores in pathogenic fungi that cause leaf spots on oil palm plants such as Curvularia affinis. This study aims to determine the properties of the chitinase enzyme B. cereus SAHA 12.13 that can inhibit the growth of C. affinis. Chitinase enzyme production and characterization were measured using the Spindler method. Antagonism test against pathogenic fungi using dual culture method by testing cell culture and enzyme crude extract. This result showed that the isolate produced a high level of specific chitinase activity at 37 °C for 45 h of incubation with 8.45 U mg-1 proteins with a growth rate (k) of 0.25 generation/h, and the generation time was 3.96 h/generation. The optimum chitinase activity was achieved at pH 7.0 and 45 °C and was stable for 3 h with a half-life (t1/2) of 770 min. The crude enzyme and cell culture of strain can inhibit the growth of C. affinis by 36.27±0.043% and 34.25±0.041%, respectively. These characteristics indicate that B. cereus strain SAHA12.13 can be used to inhibit C. affinis, which causes leaf blight of oil palm, under varying pH and temperature conditions.

In vitro assessment of thrombolytic potential of red and white ginger (Zingiber officinale)

Background: Cardiovascular diseases (CVDs) are a leading cause of death, and their pathogenesis is commonly attributed to thrombosis. Although existing medications are effective and fast-acting for thrombosis management, they tend to be expensive and cause severe side effects. Plant-based thrombolytic agents are actively sought after as inexpensive and safe alternatives for the treatment and prevention of thrombosis. Red ginger (Zingiber officinale var. rubrum) and white ginger (Zingiber officinale var. officinale) are widely used in foods and beverages and are believed to confer a wide variety of health benefits. Objective: This study aims to elucidate the thrombolytic and fibrinolytic potential of red and white ginger extracts in vitro.Methods: In this study, in vitro analyses were performed using erythrocyte liberation, euglobulin degradation, fibrin degradation, and fibrin zymography assays. The ability of crude enzyme extracts from both red (rgEx) and white ginger (wgEx) to degrade blood clots was analyzed using the erythrocytes liberation assay. Then, the thrombolytic and fibrinolytic activities of rgEx and wgEx were evaluated using euglobulin and fibrin degradation assays, both of which were visualized using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Finally, fibrinolytic enzymes were identified using a fibrin zymography assay. Results: Red and white ginger extracts were found to have strong thrombolytic properties via high total liberated erythrocyte count from the erythrocyte liberation assay. The ginger extract proteases can rapidly degrade euglobulin and fibrin, with their priority order beginning with A

Optimizing extract recovery from sorghum using combinations of exogenous crude amylase and pullulanase isolated from yeast strains from cassava mill factory

Crude enzyme extracts of yeasts isolated from a cassava mill factory were utilized in optimizing extract recovery from sorghum or malted sorghum. Microbial enzymes are known to be very beneficial in industrial processes due to various advantages derived from them. Micro-organisms multiply very rapidly and produce diverse enzymes during the process. Such micro-organisms with potential to produce industrial crude enzymes abound in many natural environments. This study examined the performance of crude enzyme extracts (pullulanase and amylase) from yeasts strains (SP40 and SS12) isolated from a cassava mill factory. The crude enzyme extracts were used in mashing grain sorghum and malted sorghum using various enzyme combinations to study their effects on quality of wort specific gravity (SG), reducing sugars (RS) and free amino nitrogen (FAN). The decantation mashing process which protected the enzymes and gelatinized sorghum starch was more effective in producing wort of higher SG, RS and FAN products. Control enzyme (lower doses) gave wort SG of 1.034 (infusion mashing) and 1.056 (decantation mashing). Test enzymes (lower doses) also gave good wort SG of 1.018 (infusion mashing) and 1.034 (decantation mashing). The activities of pullulanase and amylase from SP40 and SS12 were similar because both microorganisms produced mainly starch hydrolysing enzymes. From this study, the crude enzymes compared well with commercial enzymes in using lower doses of the crude enzyme extract to produce wort of higher SG, RS and FAN products and this will save costs for the industry. The mashing regime is also an important factor to consider.

Biological evaluation of mangrove endophytic fungi Aspergillus terreus derived from Sonneratia alba

Mangroves represent highly diverse marine ecosystems, encompassing various vegetation such as shrubs, plants, trees, palms, and other plant forms that have adapted to thrive in both freshwater and saline environments. Within these mangrove ecosystems, secondary metabolites are present, contributing to various pharmacological functions and holding significant ecological importance. Endophytic fungi constitute a substantial and quantifiable portion of fungal biodiversity and are acknowledged for their impact on the composition and diversity of plant communities. This study aims to investigate the extracellular proteins and enzyme activity exhibited by endophytic fungi discovered in the leaves of Sonneratia alba mangroves collected in Buton, Southeast Sulawesi, Indonesia. The primary objective is to evaluate the compatibility of marine fungi with antibacterial and antioxidant activity. The research involves analyzing the biological activities of marine fungi containing cellulase enzyme through methods such as DNS for reducing sugar, protein concentration determination, antioxidant analysis, and total phenol content assessment. Sample A (shaker) has highest enzyme activity at Day 4 and Sample B has highest enzyme activity at Day 6 (static condition). The protein concentration in the purified cellulase from Aspergillus terreus surpassed that of crude enzyme extracts.

Polyphenol Oxidase Enzyme Activity of Mulberries Grown in Iğdir Ecological Conditions

The presence of polyphenol oxidase enzyme (PFO) in black and white mulberry varieties grown in Iğdır province were determined. In the study, no polyphenol oxidase enzyme was detected in the white mulberry varietie, therefore the study was conducted only with black mulberry varieties. The mulberry samples were collected from Iğdır Center, Melekli and Karakoyunlu districts during June and July, both in 2015 and 2016, when the fruits were fully ripe. Mulberries were stored at -20 °C, and the crude enzyme extract was stabilized with pH 5.0 sodium-acetate buffer containing 1% Polyethylene glycol (PEG). To weigh with a precision scale, 20 grams of frozen mulberries were taken into mortars. After the mulberries were thoroughly crushed in these mortars, 40 ml of crude enzyme extract preparation solution was added to each variety and mixed homogeneously. These mixtures were filtered through a cheesecloth and rotated at 4 °C, 10,000 rpm for 30 minutes. The obtained supernatants were used for enzyme solubilization, and the enzyme activity of the solution was determined spectrophotometrically. The PFO content depended on the genotype. There was a significant difference among the tested mulberry genotypes.

Extraction of B-galactosidase Enzyme from Lacticacid Bacteria

The dairy industry has employed the B-galactosidase enzyme (EC 3.2.1.23) to reduce lactose intolerance. The ability of probiotic Lactobacillus spp. to produce B-galactosadase enzyme and hydrolyze 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal) and ONPG was investigated. Through the use of sontication to lyse the cell, crude enzyme extract was produced. Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) was used as an enzyme substrate in order to measure the B-galactosidase enzyme activity. B-galactosidase activity is demonstrated by blue-colored colonies of Lactobacillus spp. on X-Gal plates. The ideal temperature and pH for the synthesis of B-galactosidase were pH 7 and 40 °C. People who are lactose intolerant may benefit from Lactobacillus spp., which produces B-galactosidase enzyme as a probiotic for dairy products.

Enzymatic Properties of Red Beet (Beta vulgaris L.) Leaf, Root Pulp, and Peel.

Beetroot (Beta vulgaris</i> L.) is botanically classified as a herbaceous biennial belonging to the Chenopodiaceae family and has several varieties with bulb colors ranging from yellow to red. Peroxidases are widely occurring in organisms including microorganisms, plants, and animals, and have been involved in various physiological and biochemical functions. The study was conducted to investigate the characteristics of enzyme extracts from red beet leaves, root pulp, and peel. The enzyme extraction involved the homogenization of the sample and filtrate in cold acetone and then the filtrate was homogenized in 0.1M sodium acetate buffer, pH 7. The protein content was determined using the Lowry assay using bovine serum albumin (BSA) as a standard protein. Then, enzymatic activity was determined by peroxidase, polyphenol oxidase, and catalase assays. The patent for biological activity of enzymes was obtained from the Office of Career Development, Haramaya University. The antioxidant activities of the enzyme extract were conducted by using DPPH and hydrogen peroxide free radical scavenging activities. The result indicated that the Enzymatic activity of crude enzyme extract of red beet leaf, root pulp and peel indicated that significantly the highest total soluble protein (16.68 mg/ml), peroxidase activity (PODA, 111.50 U/ml), polyphenol oxidase activity (PPOA, 170.90 U/ml), polyphenol oxidase specific activity (PPOspa, 10.25 U/mg), catalase activity (CATA, 180.50 U/ml) and catalase specific activity (CATspa, 10.82 U/mg), were recorded for red beet leaf enzyme extract. The antioxidant activity of the enzyme extracts demonstrated that significantly higher DPPH radical scavenging activity of leaf extract (59.16) and peel extract (61.92) were recorded. The Pearson correlation coefficient of enzyme activity parameters and free radical scavenging activities presented that protein content was significantly and positively correlated with CATA, PPOA, and PPOspa. Catalase- specific activity (CATspa) was significant and positively correlated only with HPSA. Peroxidase-specific activity (PODspa) was significant and positively correlated with PODA and DPPH. Based on the plot for principal component PC2 vs. PC1 for D statistics DPPH, PODA, and PODspa have close PC1 and PC2 scores (with vector angle < 90° showing similar/correlated effects. In this study, B. vulgaris</i> has shown promising peroxidase enzyme activity. Beetroot peel contained higher antioxidant compounds thus promising a more intense utilization of the peels in food and nutraceuticals.

Biocontrol of biofilm forming Burkholderia cepacia using a quorum quenching crude lactonase enzyme extract from a marine Chromohalobacter sp. strain D23.

Biofilm plays advantageous role in Burkholderia cepacia by exerting multi-drug resistance. As quorum sensing (QS) system regulates biofilm formation and pathogenicity in B. cepacia strains, quorum quenching (QQ) may be a novel strategy to control persistent B. cepacia infections. In these regards, 120 halophilic bacteria were isolated from marine sample and tested using Chromobacterium violaceum and C. violaceum CV026-based bioassays initially, showing reduced violacein synthesis by QQ enzyme by 6 isolates. Among them, Chromohalobacter sp. D23 significantly degraded both C6-homoserinelactone (C6-HSL) and C8-HSL due to potent lactonase activity, which was detected by C. violaceum CV026 biosensor. Further high-performance liquid chromatography (HPLC) study confirmed degradation of N-acyl homoserine lactones (N-AHLs) particularly C6-HSL and C8-HSL by crude lactonase enzyme. Chromohalobacter sp. D23 reduced biofilm formation in terms of decreased total biomass and viability in biofilm-embedded cells in B. cepacia significantly which was also evidenced by fluorescence microscopic images. An increase in antibiotic susceptibility of B. cepacia biofilm was achieved when crude lactonase enzyme of Chromohalobacter sp. strain D23 was combined with chloramphenicol (1-5 × MIC). Chromohalobacter sp. D23 also showed prominent decrease in QS-mediated synthesis of virulence factors such as extracellular polymeric substances (EPS), extracellular protease, and hemolysin in B. cepacia. Again crude lactonase enzyme of Chromohalobacter sp. strain D23 inhibited B. cepacia biofilm formation inside nasal oxygen catheters in vitro. Finally, antibiotic susceptibility test and virulence tests revealed sensitivity of Chromohalobacter sp. strain D23 against a wide range of conventional antibiotics as well as absence of gelatinolytic, hemolytic, and serum coagulating activities. Therefore, the current study shows potential quorum quenching as well as anti-biofilm activity of Chromohalobacter sp. D23 against B. cepacia.

Design strategy for effective passion fruit waste bioconversion with crude fungal enzyme extracts

Design strategy for effective passion fruit waste bioconversion with crude fungal enzyme extracts

Valorizing Shrimp Shell as a Source of Chitin with Crude Enzymes from Germinated Winter Wheat and Buckwheat

Chitin is a valuable resource found in shrimp shells. Typically, the industrial process for isolating chitin from crustacean shells involves using chemical methods that require large amounts of acid and alkaline. While commercial protease application is a more environmentally friendly method, it can be costly. Germinated grains can be a source of low-cost protease for chitin isolation. This study aimed to assess the effectiveness of crude enzymes extracted from germinated winter wheat (Triticum aestivum) and buckwheat (Fagopyrum esculentum) to isolate crude chitin from white shrimp shells. The proteolytic and chitinolytic activities of the crude enzyme extracts were determined and applied in the deproteinization step during chitin isolation from shrimp shells. The deproteinization rate was assessed, the crude chitin yield was compared, and the isolated chitins were characterized through Fourier Transform Infrared (FTIR) spectroscopy analysis and x-ray diffraction (XRD) analysis. Crude enzymes extracted from germinated winter wheat and buckwheat exhibited proteolytic activity of approximately 0.49 U/mL and 0.46 U/mL, respectively. Using winter wheat extract and buckwheat extract acted on demineralized shrimp shells exerted a deproteinization rate of 53.6 ± 1.0% and 58.0 ± 2.3% and yielded 30.3 ± 5.31% and 29.2 ± 3.99% crude chitin, respectively. FTIR spectra of the isolated chitins showed amide I, amide II, amide III, asymmetric stretching of the C-O-C bridge, O-H stretching, N-H stretching and asymmetric C-H stretching, typical functional groups for chitin. The crystallinity index for commercial chitin, chemical-isolated chitin, winter wheat extract-isolated chitin and buckwheat extract-isolated chitin was 86.49%, 88.74%, 88.82% and 75.87%, respectively. Buckwheat extract-isolated chitin, deacetylated with lower crystallinity, warrants further investigation. Crude enzymes from winter wheat and buckwheat to remove protein from demineralized shrimp shells can be a more environmentally friendly method to valorize shrimp shells as a chitin source.