Crude Enzyme Research Articles

The Effects of Agaro-Oligosaccharides Produced by Marine Bacteria (Rheinheimera sp. (HY)) Possessing Agarose-Degrading Enzymes on Myotube Function

Agarase and its metabolites are reported to have applications in a variety of fields, but there have been few studies of the effects of agaro-oligosaccharide hydrolysate on muscle function. In this study, we analyzed the functionality of agarase and its metabolites in bacteria isolated from seawater. A bacterium with agar-degrading activity was isolated from Shimabara, Nagasaki, Japan. Through 16S rRNA sequence alignment, it was identified as being closely related to Rheinheimera sp. WMF-1 and was provisionally named Rheinheimera sp. (HY). Crude enzymes derived from this bacterium demonstrated an ability to hydrolyze various polysaccharides, including agar, agarose, and starch, with the highest specificity observed for agarose. The optimum pH and temperature were pH 10 and 50 °C. A glycoside bond specificity analysis of enzymatic activity indicated the cleavage of the α-linkage. Next, we investigated the functional effects of agaro-oligosaccharides on C2C12 myotubes. Treatment with 10–30 kDa oligosaccharides significantly increased the hypertrophy rate, diameter, and expression of myosin heavy-chain genes in C2C12 myotubes. These results indicate that the agaro-oligosaccharides produced by the enzymes identified in this study improve muscle mass, suggesting their potential contribution to muscle function.

Optimizing extraction of collagen from hazardous leather shaving dusts by crude protease enzyme: Chrome recovery and reuse for leather rechroming

This study explores an eco-friendly method to achieve 99.98 % dechroming (reducing Cr from 7700 to 4.71 ppm) of Chrome-tanned leather shaving dust (CSD), through enzymatic hydrolysis using crude protease. The enzyme exhibited strong activity (>40 U mL−1) across a temperature range of 50 °C to 65 °C and a pH range of 5 to 11. The pH of the mixture containing CSD, water, MgO, and enzyme was maintained at 8.9 during hydrolysis. The temperature, hydrolysis time, and enzyme concentration were optimized using response surface methodology (RSM). Under optimal conditions of 17.57 % enzyme concentration, 62.01 °C, and a hydrolysis time of 7 h, the predicted collagen yield was 31.66 %. Laboratory experiments conducted with these predicted parameters resulted in a collagen recovery of 30.9 %, achieving an accuracy of 97.6 % compared to the predictions. In contrast, hydrolysis with MgO alone achieved only 87.01 % dechroming with about 2 % collagen yield, highlighting the enzyme's superior effectiveness. The enzymatic cleavage of peptide bonds in the leather matrix enhanced collagen solubilization. Characterization techniques, including FT-IR, NMR, XRD, DSC, and TGA, confirmed successful extraction and purity, with the collagen being rich in glycine and proline. Cytotoxicity results demonstrated its non-toxic nature, indicating potential applications in biomedical fields, tissue engineering, food, pharmaceuticals, and cosmetics. The recovered chromium sulfate was reused for rechroming goat wet blue leather, with chrome content in crust leather treated with recovered basic chromium sulfate (rBCS) and fresh basic chromium sulfate (BCS) being 3.23 % and 3.35 %, respectively, both meeting industry standards for goat crust leather. SynopsisAn eco-friendly method for dechroming chrome-tanned leather shaving dust (CSD) and extracting collagen was established. The process achieved 99.98 % dechroming and 30.9 % collagen yield using crude protease enzyme. Characterization confirmed the collagen's purity, and recovered chromium sulfate was reused for rechroming goat leather, meeting industry standards.

Phenolic Content, Antioxidant and Antimicrobial Properties of Hawthorn (Crataegus orientalis) Fruit Extracts Obtained via Carbohydrase-Assisted Extraction

The enzyme-assisted approaches for plant phenolics extraction are more eco-friendly methods compared to acid or alkaline hydrolysis. Carbohydrase enzymes can release free phenolics from plant materials by cleaving the glycosidic bonds between phenolic compounds and cell wall polymers. In this study, the efficiency of carbohydrase-assisted treatment approaches was evaluated to extract bioactive phenolics from hawthorn (Crataegus orientalis) fruit residues. Enzymatic treatment of the fruits was operated by using a crude cellulolytic enzyme cocktail from Rhizomucor miehei NRRL 5282 and a pectinase preparate from Aspergillus niger. Both cellulase and combined cellulase–pectinase treatments improved the total phenolic content (TPC) and antioxidant activity of extracts. The TPC increased to 1899 ± 27 mg gallic acid equivalents/100 g dry matter during the combined enzyme treatment, showing a strong correlation with the average antioxidant capacity determined by ferric-reducing antioxidant power (1.7-fold increment) and 2,2-diphenyl-1-picrylhydrazyl (1.15-fold increment) reagents. The major phenolics in enzyme-treated extracts were vanillic and ferulic acids, the concentrations of which increased 115.6-fold and 93.9-fold, respectively, during carbohydrase treatment. The planktonic growth of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Chromobacterium violaceum was slightly inhibited by the extracts with minimum inhibitory concentration values between 15.0 and 77.9 mg/mL, while the yeasts tested were quite resistant to the samples. B. subtilis and yeast biofilms were sensitive to the enzyme-treated extracts, which also showed quorum-sensing inhibitory effects against C. violaceum. The obtained bioactive hawthorn extracts hold potential as a natural source of antioxidants and antimicrobials.

Production Physiology and Biochemical properties of Crude Thermophile Protease by Mild - Halophilic Bacillus thuringiensis aizawai HD 865

Objective: The current study aims to enrich the research library of Bacillus thuringensis, the most famous bacteria as a biopesticide, with a product that may enhance its ability as a biopesticide as well as its many other applications, namely the heat-tolerant alkaline protease. Methods: The strain Bacillus thuringiensis aizawai HD 865, obtained from HD Culture Collection was grown on a liquid growth medium of nutrient broth and yeast extract, and colonies were counted on agar plates. The zygote is then transferred to a skim milk environment to estimate the enzymatic activity under the different growth conditions of the growth environment (sodium chloride concentration, pH, aeration, fermentation duration), then testing the optimal conditions for enzymatic activity in the enzymatic reaction mixture (sodium chloride concentration, salinity resistance, optimum temperature, thermal stability, optimum pH and stability). Then analyze the data statistically. Results: Optimum alkaline protease medium production conditions were, 2 % NaCl, so Bacillus thuringiensis aizawai HD865 a mild - halophilic microorganism, aeration level 80%, pH 7 and 8 days of fermentation period. On the other hand, crude alkaline protease properties were found that, the optimum temperature for its activity was relatively high (70ºC), while heat-stabilities after 5 min of heating crude enzyme in boiling water bath then incubated for 15min on 30, 40, 50, 60, 70ºC, were 184.23, 178.07, 164.29, 165.51 and 121.46 Units, respectively; whereas the optimum pH was at pH 7.6 (151.76 Units) with tris buffer then at pH 9.6 with glycine - NaOH buffer; While pH stability was at pH 7.6 with tris buffer then at pH 8.4 with glycine - NaOH buffer. Thus, activities have decreased at different concentrations (0, 1, 2, 3, 4, 5 %) of sodium chloride in reaction mixture with enzyme activities (151.25, 137.35, 135.12, 135, 135.89, 114.38 Units) respectively, also decreased when rinse crude enzyme for 5 min in NaCl (0, 1, 2, 3, 4, 5 %), with (151.04, 175.57, 168.69, 154.61, 155.39 and 143.39 Units) respectively. Conclusion: Bacillus thuringiensis aizawai HD 865 strain is considered a low - salt tolerant strain, while the alkaline protease extracted from it is heat - resistant.

Expression and degradation function analysis of the key genes from Arthrobacter nitroquajacolicus HW08 in Lactococcus lactis for the degradation of swainsonine

In order to mitigate the adverse effects of madrassa poisoning disease on our livestock industry and to fully utilize the potential pasture resources, biodegradation of locoweed can remove swainsonine, the major toxic component of locoweed, so that the locoweed can be used as high-quality forage. Arthrobacter nitroquajacolicus HW08 can stably and efficiently degrade swainsonine. In this study, Lactococcus lactis, as a food-grade microorganism, was used as a vector to express four key degradation genes from A. nitroquajacolicus HW08. Subsequently, liquid chromatography was employed to evaluate the swainsonine-degrading performance. The crude enzyme solution extracted from the L. lactis strain transformed with the ethanol dehydrogenase gene A1R6C3 degraded 323.4 μg of swainsonine in 24 h at 30 ℃. The crude enzyme solutions from the L. lactis strains transformed with the genes encoding glutathione synthase, esterase/acyl hydrolase, and glycosyltransferase did not show any degradation ability for swainsonine when being used alone but degraded about 140.5 μg of swainsonine when being used in mixture. The findings will help the clinical promotion of swainsonine-degrading engineering strains and provide new research ideas for the prevention and treatment of swainsonine poisoning in animals and the detoxification and utilization of locoweed.

Bhargavaea beijingensis a promising tool for bio-cementation, soil improvement, and mercury removal

Microbially Induced Calcite Precipitation (MICP) has emerged as a promising technique for bio-cementation, soil improvement, and heavy metal remediation. This study explores the potential of Bhargavaea beijingensis, a urease-producing bacterium, for these applications. Six ureolytic bacteria were isolated from calcareous bricks mine soil and screened for urease and calcite production. B. beijingensis exhibited the highest urease activity and calcite precipitation. Urease activity, calcite precipitation, sand solidification, heavy metal removal efficiency, and compressive strength were evaluated. It showed significant heavy metal removal efficiency, particularly highest for HgCl2. Mortar blocks treated with B. beijingensis or its crude enzyme exhibited improved compressive strength, suggesting its potential for bio-cementation. Crack remediation tests demonstrated successful crack healing in mortar blocks using the bacterium or its enzyme. This study identifies B. beijingensis as a novel and promising MICP agent with potential applications in bio-cementation, soil improvement, and heavy metal remediation. Hence, B. beijingensis diversified abilities prove superior performance compared to commonly used strains like Bacillus subtilis and Shewanella putrefaciens in bio-cementation applications. Its high urease activity, calcite precipitation, and heavy metal removal abilities make it a valuable candidate for sustainable and eco-friendly solutions in various fields.

Strategies for enhanced acetone-butanol-ethanol production by Clostridium beijerinckii Y10 from glucose and xylose and exploration of its physiological mechanisms

To enhance the production of butanol and its ratio to the total solvent, the impacts of exogenous amino acids supplementation on acetone-butanol-ethanol (ABE) fermentation were studied. With this effort, the highest butanol and total solvent production of 14.18 ± 0.24 g/L and 16.29 ± 0.31 g/L were achieved when 5.0 mg/L of phenylalanine was added at 18 h. Additionally, to explore the physiological reasons for the efficient synthesis of butanol under the optimal condition, the changes of key enzyme activities in the butanol synthesis pathway and the intracellular microenvironment were investigated. The findings show that phenylalanine addition could enhance crude enzyme activities of acetate kinase and butyrate kinase activities, strengthen NADH synthesis and expend more ATP to promote the growth of Clostridium beijerinckii Y10. This study suggests that the method of a tiny amount of amino acid addition was a simple and efficient approach to enhance bio-butanol production, thus providing a new strategy to improve the performance of other similar fermentation.

Process development and environmental impact assessment of sustainable poly(3-hydroxybutyrate) separation and purification via Paraburkholderia sacchari cell lysis using crude enzymes

Downstream separation and purification of poly(3-hydroxybutyrate) has been developed via combined crude enzyme bacterial cell lysis followed by solvent extraction and circular utilisation of bacterial lysate as nutrient supplement for PHB production. Enzymatic lysis of Paraburkholderia sacchari cells was initially evaluated using crude enzymes produced via solid state fermentation of Aspergillus oryzae. The optimum protease activity (156 U per g cell dry weight) resulted in 66.9% lysis of residual cell weight. PHB purification with 1,3-dioxolane, dimethyl carbonate, anisole, ammonium laurate and chloroform were evaluated at different processing parameters (extraction duration, temperature) using wet and dry bacterial cells as well as enzymatically lysed wet bacterial cells. The highest recovery yield (94.5%) and purity (98.1%) were obtained with 1,3-dioxolane at 80°C and 6h processing time using enzymatically disrupted bacterial cells. The bacterial cell lysate was used as nutrient supplement for circular PHB production in fed-batch P. sacchari bioreactor cultures leading to the production of 78.7 gPHB/L, 56.9% (w/w) PHB content and 2.3g/(Lꞏh) productivity. Low global warming potential (1.3 CO2-eq/kgPHB) and abiotic depletion fossil (14.4 MJ/kgPHB) were estimated for PHB purification via enzymatic cell lysis and PHB extraction with 1,3-dioxolane demonstrating the development of a sustainable and circular process for PHB production.

Prospects and Application of Solid-State Fermentation in Animal Feed Production – A Review

Abstract Animal feed production has recently received significant attention in the food and livestock sectors due to its high nutritional value and substantial environmental potential. Extensive studies have been conducted to explore the effects of solid fermented feeds on different growth stages of animals but also on the health status of animals, the quality of animal products, and the composition of intestinal micro-flora to replace non-fermented feed production. The purpose of this review is to provide up-to-date existing trends, recent developments, and prospects of solid-state fermentation (SSF) practices for the production of animal feeds. Studies on enhancing nutritional factors by increasing the crude protein content, enzymes, and antioxidant activity of feed using physical processing methods on agro-industrial waste such as rapeseed meal, cottonseed, wheat bran, soybean meal, and legumes by mainly SSF are reviewed and discussed thoroughly.

Bioethanol production from groundnut shell using microorganisms derived from sugarcane root soil

Bioethanol is a form of renewable energy that can be produced from fuel or energy crops. Ethanol is produced by the fermentation of sugars present in agricultural feed stocks and crop residues. This study investigates the use of agro wastes like groundnut shells for ethanol production. Initially, the groundnut shells were washed, dried and grinded into powder. Then it was subjected to ethanol production using yeast. After 20 days of incubation the ethanol was estimated using potassium dichromate method. The maximum ethanol yield (1.55%) was obtained when 1% yeast was used. To increase the efficiency of ethanol production, the cellulolytic bacteria was isolated from the cow dung dumped soil site. The 10 bacterial isolates were screened for cellulase enzyme production. Among them one bacterium showed maximum decolourization of congored which was subjected for enzyme production in nutrient broth. The organism showed maximum enzyme activity of 558.12 U/ml. The isolated cellulolytic bacteria were identified as Bacillus anthracis using 16s rDNA sequencing. Ethanol production from the groundnut shells was again carried out with various concentration of crude cellulase enzyme isolated from the bacteria. The estimation result showed 3.8% of ethanol as maximum. Then the ethanol was condensed using a rotary evaporator and when again estimated, showed 7.3% of ethanol. Finally, the presence of ethanol was confirmed by iodoform test. Thus, groundnut shells can efficiently be used for the production of ethanol, which can be used as a high potential fuel source for transportation in the future.

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.

Isolation and characterization of a novel bacterium that promotes the degradation of poly(glycolic acid) by its extracellular esterase under thermophilic conditions

Poly(glycolic acid) (PGA) is widely utilized in the shale oil and gas industry owing to its biodegradable nature, as well as superior mechanical and barrier properties. However, PGA degradability is limited by environmental conditions such as temperature and pH, and using acids to optimize the degradation can have adverse environmental impact. Thus, it is essential to identify methods that can effectively promote the degradation of PGA under mild conditions, such as microbial degradation. In the present study, strain DB14, a bacterium that promotes PGA degradation, was isolated from drain water of a steam pipeline. Sequence analysis of the 16S rRNA gene of the bacterium revealed that it is mostly closely related to Geobacillus icigianus; however, it also differed from Geobacillus icigianus in several physiological properties. To investigate PGA degradation by strain DB14, a PGA film and disc were incubated with the strain. The residual weight of the PGA film (thickness: 170 μm) significantly reduced after incubation, whereas the decrease in the thickness of the PGA disc (thickness: 3 mm) was relatively small. The penetration of water, the bacterium, and the extracellular enzymes into the interior from the reaction-erosion front of the PGA disc may be inhibited by the high barrier performance of PGA. Strain DB14 was also found to change the pH of the surrounding environment to approximately 8–9. To investigate the effect of pH on PGA degradability, degradation tests with crude extracellular enzymes derived from strain DB14 were conducted in various buffers. The results showed that the degradation activity was highest at pH 8, which implied that DB14 efficiently maximized the hydrolytic capacity of its enzyme for degrading PGA. Thus, this study provides a basis for developing environmentally friendly technologies that can promote the degradation of PGA molding articles, especially those used in wellbores for oil and gas recovery.

Biocontrol of fungal pathogens and growth promotion in the Korean fir (Abies koreana E.H.Wilson) seedling using Bacillus velezensis CE 100

The restoration of the endangered Korean fir (Abies koreana E.H.Wilson) forests requires mass production of high-quality and well-developed seedlings for outplanting. However, phytopathogenic fungal infections lower the survival rate and growth vigor of A. koreana seedlings, frustrating the re-afforestation efforts. This study isolated and identified the phytopathogenic fungi causing seedling blight and wilt diseases in A. koreana seedlings and investigated the potential of Bacillus velezensis CE 100 in controlling the fungal pathogens and promoting seedling growth. Phomopsis mali and Fusarium oxysporum were confirmed as the causal agents of blight and wilt diseases, respectively, and both fungi reduced the survival rate of A. koreana seedlings. B. velezensis CE 100 produced cell wall-degrading enzymes: chitinase, protease, and β-1,3-glucanase, and the bacterial crude enzyme fraction inhibited the spore germination and mycelial growth of P. mali and F. oxysporum in a concentration-dependent manner, causing severe morphological deformations. Consequently, treatment with B. velezensis CE 100 improved the survival rate of seedlings infected with either P. mali or F. oxysporum compared to the control and PB media treatment. The bacterium also increased nutrient availability through ammonia–nitrogen production and phosphate solubilization and produced indole-3-acetic acid (IAA), and its inoculation remarkably improved seedling growth (root and shoot dry weight) compared to the control and PB media treatment group. This is the first study to report P. mali and F. oxysporum as the pathogens causing seedling blight and wilt diseases in A. koreana nursery seedling, and the possibility of using B. velezensis CE 100 to control the fungal pathogens and improve the seedling growth without chemical use.

Prospects of crude enzymes in replacing pure enzymes for dissolving pulp production.

High-purity cellulose from paper pulp can be obtained after appropriate treatments involving pure xylanases and cellulases/endoglucanases. This study investigated the efficacy of using crude xylanase and cellulase instead of commercial ones to improve process economics. Kraft paper grade pulp produced from veneer waste, hardwood, and non-wood sources was utilized as a more sustainable option. Crude xylanase and cellulase from isolated soil bacteria Bacillus pumilus 3GAH and Bacillus subtilis PJK6 were used for process optimization. The correlation between Fock reactivity, chain scission, and crystallinity after crude-cellulase treatment was established through chemical, FTIR, and XRD analyses. Pentosans in kraft pulp were reduced from an initial 18.7% to 4.9% through sequential treatments with crude xylanase and alkali. Subsequent crude-cellulase treatment, even at 8U/g o.d. pulp, improved Fock reactivity from 28.2% to 61.2%, fulfilling a major criterion for viscose. Thus, crude enzymes can be effectively used for the efficient and economical upgrading of paper pulp to dissolving pulp.

Biophysical Properties of Thermostable Amidase Produced by Aspergillus fumigatus in Submerged Fermentation

Amidases, also known as amidohydrolases (EC 3.5.1.4), are enzymes within the nitrilase superfamily or amidase signature family. They hydrolyze amides and nitriles into their corresponding acids while releasing ammonia. These enzymes are widely used in biotechnology and industry. However, their production is limited to a few organisms, which cannot meet industrial demand. Therefore, exploring new amidase sources is crucial. This study focuses on purifying and characterizing amidase from Aspergillus fumigatus using cold acetone precipitation and column chromatography with Sephadex G-100. The effects of temperature, pH and metal ions, on the enzyme activity were examined for both crude and purified amidase. The thermal and pH stability of the enzyme alongside the enzyme kinetics were also assessed. The purified amidase exhibited optimal activity of 13.1 U/mL at 70°C, with 65% residual activity after 5 hours at this temperature. The pH studies showed optimal activity at pH 5.0 for the purified enzyme (7.8 U/mL) and at pH 4.0 for the crude enzyme (5.5 U/mL). Metal ion effects indicated that Zn²⁺, Mg²⁺, and EDTA enhanced, while Cu²⁺ inhibited, the activities of both crude and purified amidase. Kinetic analysis showed that the enzyme followed Michaelis-Menten kinetics, with Km and Vmax values of 0.104 mM and 1.884 U/mL for crude amidase, and 0.017 mM and 1.872 U/mL for purified amidase. These findings suggest that amidase from Aspergillus fumigatus holds potential for industrial applications where amidases are essential.

A novel method for non-destructive efficient green recovery of multiple high value products from microalgal cells with enzymatically enhanced permeability

A method is developed for simultaneous extraction of lipids, hydrocarbons and carotenoids from primary algal biomass without the need for cell sacrifice. Enzymatically predigested cells (from previous experiments) with enhanced permeability were used, for ‘milking’ with biocompatible solvent. The continuous non-destructive extraction of lipids and carotenoids from an improved permeabilized microalgal cell using crude enzyme concoctions and biocompatible solvents has not been reported so far. Oocystis sp. cells showed considerable viability following enzymatic pretreatment and extraction using dodecane which was verified through cell viability assays, and chlorophyll content. HPLC analysis confirmed the presence of lutein and zeaxanthin. GC-MS analysis showed the presence of omega-3 alpha linolenic acid, PUFAs, and phenolics, in enzymatically digested microalgal cells. The content of fatty acids and esters in enzyme treated samples was 57.18 %, and the enzyme with dodecane treated samples showed 31.4 % fatty acids and esters content. Omega-3 linolenic acid comprised 11.66 % in enzyme treated cells and showed a marginal increase of 12.5 % in enzyme with dodecane treated cells. The method enables recovery of multiple compounds from a single biomass. In the present study, a cultivation strategy of microalgal cells with enhanced permeability, grown in biocompatible solvent is proposed, whereby maximum yield of multiple valuable metabolites can be obtained from a single biomass. The method simplifies downstream processing operations by circumventing cell harvesting and drying. The use of green extraction solvents reduces the overall carbon footprint of the process.

Optimization and purification of L-methioninase enzyme purified from clinical samples of Pseudomonas spp.

Background: This study focused on Pseudomonas aeruginosa, which secrete important enzymes in medical and pharmaceutical applications and are isolated from clinical samples. One of these enzymes is L-methioninase, the most desirable enzyme in the medical and industrial fields today since it converted L-methionine into methanethiol, ammonia, and alpha-ketobutyrate. Objectives: Evaluated the optimum conditions of L-methioninase production and then purified it. Methods: Pseudomonas samples were identified by microscopic and biochemical tests and confirmed with the VITEK2 system. After the L-methioninase production was examined, the optimal conditions for the production of L-methioninase (MGL) were established using clinical samples of P. aeruginosa. Additionally, MGL was purified from the supernatant of P.aeruginosa using ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G150 accordingly. Results: There were 33 isolates of P.aeruginosa according to microscopic characteristics and biochemical tests, followed by the VITEK2 system to ensure identification. The optimization conditions were achieved when supplement media with sucrose 1% w\v, casein 1% w\v, pH 7, temperature 37oC, and incubation for 48hrs within the specific activity of 1.6, 3.3, 3.4, 3.4, and 3.5U\mg protein, respectively, and 4.6 fold the amount of the purified enzyme as compared to the crude enzyme. A single 55 kDa band on the SDS-PAGE indicates the enzyme is purified. Conclusions: P.aeruginosa can be an efficient source of L-methioninase, and this enzyme had better and higher specific activity after applying optimum conditions for it and purifying it.

Expression of a novel hydrolase MhpC in Brevibacillus parabrevis BCP-09 and its characteristics for degrading synthetic pyrethroids

Synthetic pyrethroids are widely used insecticides which may cause chronic diseases in non-target organisms upon long-term exposure. Microbial degradation offers a reliable method to remove them from the environment. This study focused on Brevibacillus parabrevis BCP-09 and its enzymes for degrading pyrethroids. The predicted deltamethrin-degrading genes phnA and mhpC were used to construct recombinant plasmids. These plasmids, introduced into Escherichia coli BL21(DE3) cells and induced with L-arabinose. The results indicated that the intracellular crude enzyme efficiently degraded deltamethrin by 98.8 %, β-cypermethrin by 94.84 %, and cyfluthrin by 73.52 % within 24 h. The hydrolytic enzyme MhpC possesses a catalytic triad Ser/His/Asp and a typical “Gly-X-Ser-X-Gly” conservative sequence of the esterase family. Co-cultivation of induced E. coli PhnA and E. coli MhpC resulted in degradation rates of 41.44 ± 3.55 % and 60.30 ± 4.55 %, respectively, for deltamethrin after 7 d. This study states that the degrading enzymes from B. parabrevis BCP-09 are an effective method for the degradation of pyrethroids, providing available enzyme resources for food safety and environmental protection.

Enhanced extracellular production of maltotetraose amylase from Pseudomonas saccharophila in Bacillus subtilis through regulatory element optimization

Maltotetraose amylase (Mta) catalyzes the hydrolysis of amylaceous polysaccharides into maltotetraose, which is an important functional sugar used in the food industry. However, the lack of efficient expression systems for recombinant Mta has hindered its scale-up production and application. In this study, a codon-optimized mta gene from Pseudomonas saccharophila was efficiently produced in Bacillus subtilis by optimizing the regulatory elements. First, a plasmid library containing 173 different signal peptide sequences placed upstream of mta gene was constructed, and transformed into B. subtilis strain WB800N(amyEΔ1) for high-throughput screening. The signal peptide yhcR was found to significantly enhance the secretion of Mta, reaching an activity of 75.4 U/mL in the culture medium. After optimization of the promoters, the Mta activity was further increased to 100.3 U/mL using a dual-promoter PHpaIIPamyE. Finally, the carbon sources and nitrogen sources for recombinant Mta production were optimized, yielding a highest Mta activity of 288.9 U/mL under the optimal culture conditions. The crude enzyme solution containing recombinant Mta produced a highest maltotetraose yield of 70.3% with 200 g/L of maltodextrin as the substrate. Therefore, the present study have demonstrated a high yield of Mta produced in B. subtilis, laying the foundation for large-scale Mta production and application.

Optimization of Papain Enzyme Activity Using a Response Surface Methodology Approach

Enzyme-based leather dehairing has become widely recognized as a more environmentally friendly alternative to conventional chemical processes. In recent decades, the demand for plant-based enzymes has grown, leading to the need to improve catalytic efficiency and meet industrial requirements through advanced extraction and isolation techniques. Therefore, the objective of this study was to extract plant papain enzyme from <i>Carica papaya</i> and evaluate their potential as a dehairing agent in the leather industry. The experimental study was conducted in a laboratory setting from July to August 2023. <i>Carica papaya</i> samples were collected from various locations in Arba Minch town. Papain enzyme was extracted from <i>Carica papaya</i> by grinding it in a blender with extraction media such as distilled water and 50 mM phosphate buffers. The optimization of proteolytic activity was performed using the response surface method and Box-Behnken Design (BBD), considering parameters like pH, temperature, and reaction time. The optimized conditions for temperature, pH, and reaction time were found to be 55°C, 7.5, and 38.5 minutes, respectively, resulting in an optimum protease activity of 32 U/ml. The crude protease enzyme derived from papaya was successfully used to dehair goat hides at 25°C for 16 hours without the addition of Na<sup>2</sup>S. Microscopic analysis revealed hides that were free of epidermis and hair, with clean pores and no significant damage to the grain surface. In conclusion, the papain enzyme derived from <i>Carica papaya</i> can be a valuable tool for implementing cleaner technologies in tanneries, particularly for lime and sulphide-free dehairing processes.