Detergent Industry Research Articles

Purification and characterization of lipase enzyme from endophytic Bacillus pumilus WSS5 for application in detergent industry

Lipolytic enzymes from endophytic microbes have been the focus of intense and growing research in view of the applicability of such enzymes in various industrial applications and endophytic microorganisms may emerge as a potential source of lipases with distinct characteristics. The current work involved purification and characterization of the lipase from an endophytic-bacteria isolated from the stem of Withania somnifera L. Dunal. Based on the analysis of morphology and 16S rDNA sequence, the endophytic bacteria was identified as Bacillus pumilus and designated as B. pumilus WSS5. Purified lipase from the B. pumilus WSS5 was found to be of 27KDa as revealed by SDS-PAGE with optimum activity at 37 °C and pH 8. The stability of the enzyme in presence of various metal ions, surfactants, inhibitors, and organic solvents suggested that this enzyme could be exploited in leather and detergent industries. In addition, the stain removal potential of the crude and purified lipase was determined on cotton fabric pieces stained with vegetable oil. Lipase added to water along with detergent significantly enhanced the stain removal efficiency of a commercially available detergent. The current findings suggest the potential of B. pumilusWSS5 lipase owing to promising properties such as the alkali-thermostability, organic solvent-tolerance, and broad substrate specificity that makes it a strong candidate for application in detergent formulations, biotransformation, industries, and medicine. To our knowledge, this is the first report on the purification and characterization of lipase from bacterial endophyte isolated from the stem of Withania somnifera L. Dunal.

Heterologous Expression, Purification and Characterization of an Alkalic Thermophilic β-Mannanase CcMan5C from Coprinopsis cinerea.

A endo-1,4-β-mannanase (CcMan5C) gene was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified by Ni-affinity chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). CcMan5C hydrolyzed only locust bean gum galactomannan (LBG) but not α-mannan from S. cerevisiae or Avicel cellulose, oat spelt xylan, or laminarin from Laminaria digitata. CcMan5C exhibited distinctive catalytic features that were different from previously reported β-mannanases. (1) CcMan5C is the first reported fungal β-mannase with an optimal alkalic pH of 8.0-9.0 for hydrolytic activity under assay conditions. (2) CcMan5C is the first reported alkalic fungal β-mannase with an optimal temperature of 70 °C for hydrolytic activity under assay conditions. (3) The organic solvents methanol, ethanol, isopropanol, and acetone at concentrations of 10% or 20% did not inhibit CcMan5C activity, while 10% or 20% isopropanol and acetone even enhanced CcMan5C activity by 9.20-34.98%. Furthermore, CcMan5C tolerated detergents such as Tween 20 and Triton X-100, and its activity was even enhanced to 26.2-45.6% by 1% or 10% Tween 20 and Triton X-100. (4) CcMan5C solution or lyophilized CcMan5C exhibited unchanged activity and even increasing activity after being stored at -20 °C or -80 °C for 12 months and retained above 50% activity after being stored at 4 °C for 12 months. These features make CcMan5C a suitable candidate for the detergent industry and paper and pulp industry.

Application of the novel manta-ray foraging algorithm to optimize acidic peptidase production in solid-state fermentation using binary agro-industrial waste

Peptidases, which constitute about 20% of the global enzyme market, have found applications in detergent, food and pharmaceutical industries, and could be produced on a large scale using low-cost agro-industrial waste. An acidophilic Bacillus cereus strain produced acidic peptidase on binary-agro-industrial waste comprising yam peels and fish processing waste at pH 4.5 with high catalytic activity. A five-variable central composite rotatable design of a response surface methodology was used to model bioprocess conditions for improved peptidase production in solid-state fermentation. Data generated was leveraged as the basis for applying the novel Manta-ray foraging optimization-linked feed-forward artificial neural network to predict bioprocess conditions optimally. Results obtained from the optimization experiments revealed a significant coefficient of determination of 0.9885 with low-performance error. The bioprocess predicted a peptidase activity of 1035.32 U/mL under optimized conditions set as 54.8 g/100 g yam peels, 23.85 g/100 g fish waste, 0.31 g/100 g CaCl2, 47.54% (v/w) moisture content, and pH 2. Peptidase activity was improved 5-fold, and was stable for 240 min between pH 2.5 and 3.5. Michaelis-Menten kinetics revealed a Km of 0.119 mM and a catalytic efficiency of 45462.19 mM−1 min−1. The bioprocess holds promise for sustainable enzyme-driven applications.

Protease from Bacillus subtilis ZMS-2: Evaluation of production dynamics through Response Surface Methodology and application in leather tannery

Proteolytic enzymes are the most versatile and commercially viable group of enzymes comprising over 65% share in the global enzyme market amongst which alkaline proteases have extensive applications in detergent and leather industry. Current study was designed to assess the potential of an alkaline serine protease from Bacillus subtilis ZMS-2 as a bating agent in leather processing. Initially, the production parameters were investigated through Response Surface Methodology (RSM) using Plackett-Burman Design, which identified substrate, agitation speed and incubation temperature as the most significant factors. The optimal levels of these factors were determined through the Box-Behnken experimental analysis as 0.436% substrate concentration, 36.5 °C incubation temperature and 56 rpm agitation speed. The statistical optimization experiments increased the volumetric production of enzyme by 3.94 times (2246 U mL−1) than the initial titer (571 U mL−1). The enzyme was partially purified and characterized as metal ions and detergent compatible serine protease having optimum activity at pH 8 and 60 °C. During the pilot-scale application as a bating agent, the enzyme (340 U mL−1) successfully removed the hair roots and other unwanted proteins from goat skins as observed during scudding and confirmed through Scanning Electron Microscopy. The processed skins displayed enhanced porosity, thumb impression, smoothness and pliability. These findings provide a strong basis for the use of this protease as an efficient and eco-friendly alternative for bating of animal skins in leather tanneries.

Isolation and characterization of detergent-compatible amylase-, protease-, lipase-, and cellulase-producing bacteria.

Detergent-compatible enzymes are the new trend followed by most in the detergent industry. Cellulases, lipases, proteases, and amylases are among the enzymes frequently used in detergents. Detergent-compatible enzymes can be obtained from many organisms, but the stability, cheapness, and availability of microbial enzymes make them preferable in industrial areas. In the present study, soil samples contaminated with household waste were collected from different regions of Trabzon (Turkey) for amylase-, cellulase-, protease-, and lipase-producing bacteria. A total of 55 bacterial isolates differing in colony morphology were purified from the samples and 25 of the isolates gave positive results in enzyme screening. The enzyme screening experiments revealed that 10 isolates produced amylase, 9 produced lipase, 7 produced cellulase, and 6 produced protease. While 2 isolates showed both protease and lipase activity, for 2 different isolates cellulose and amylase activity were detected together. It was also observed that one isolate, C37PLCA, produced all four enzymes. The morphological, physiological, and biochemical analyses of the bacteria from which we obtained the enzymes were performed and species close to them were determined using 16S rRNA sequences. Based on the results obtained, our enzymes show tremendous promise for the detergent industry.

Large-scale analysis of the genome of the rare alkaline-halophilic Stachybotrys microspora reveals 46 cellulase genes.

Fungi are of great importance in biotechnology, for example in the production of enzymes and metabolites. The main goal of this study was to obtain a high-coverage draft of the Stachybotrys microspora genome and to annotate and analyze the genome sequence data. The rare fungus S.microspora N1 strain is distinguished by its ability to grow in an alkaline halophilic environment and to efficiently secrete cellulolytic enzymes. Here we report the draft genome sequence composed of 3715 contigs, a genome size of 35 343 854 bp, with a GC content of 53.31% and a coverage around 20.5×. The identification of cellulolytic genes and of their corresponding functions was carried out through analysis and annotation of the whole genome sequence. Forty-six cellulases were identified using the fungicompanion bioinformatic tool. Interestingly, an S.microspora endoglucanase selected from those with a low isoelectric point was predicted to have a halophilic profile and share significant homology with a well-known bacterial halophilic cellulase. These results confirm previous biochemical studies revealing a halophilic character, which is a very rare feature among fungal cellulases. All these properties suggest that cellulases of S.microspora may have potential for use in the biofuel, textile, and detergent industries.

The Recent Advances in the Utility of Microbial Lipases: A Review

Lipases are versatile biocatalysts and are used in different bioconversion reactions. Microbial lipases are currently attracting a great amount of attention due to the rapid advancement of enzyme technology and its practical application in a variety of industrial processes. The current review provides updated information on the different sources of microbial lipases, such as fungi, bacteria, and yeast, their classical and modern purification techniques, including precipitation and chromatographic separation, the immunopurification technique, the reversed micellar system, aqueous two-phase system (ATPS), aqueous two-phase flotation (ATPF), and the use of microbial lipases in different industries, e.g., the food, textile, leather, cosmetics, paper, and detergent industries. Furthermore, the article provides a critical analysis of lipase-producing microbes, distinguished from the previously published reviews, and illustrates the use of lipases in biosensors, biodiesel production, and tea processing, and their role in bioremediation and racemization.

Biochemical characteristics of Myceliophthora thermophila recombinant β-glucosidase (MtBgl3c) applicable in cellulose bioconversion

The GH3 β-glucosidase gene of Myceliophthora thermophila (MtBgl3c) has been cloned and heterologously expressed in E. coli for the first time. This study highlights the important characteristics of recombinant MtBgl3c (rMtBgl3c) which make it a promising candidate in industrial applications. Optimization of the production of rMtBgl3c led to 28,000 U L−1. On purification, it has a molecular mass of ∼100 kDa. It is a broad substrate specific thermostable enzyme that exhibits pH and temperature optima at 5.0 and 55 °C, respectively. The amino acid residues Asp287 and Glu514 act as nucleophile and catalytic acid/base, respectively in the enzyme catalysis. Its low Km value (1.28 mM) indicates a high substrate affinity as compared to those previously reported. The rMtBgl3c displays a synergistic action with the commercial enzyme cocktail in the saccharification of sugarcane bagasse suggesting its utility in the cellulose bioconversion. Tolerance to solvents, detergents as well as glucose make this enzyme applicable in wine, detergent, paper and textile industries too.

The structure-directing role of heterologous seeds in the synthesis of zeolite

Zeolites have been widely used as catalysts, ion-exchangers, and adsorbents in chemical industries, detergent industry, steel industry, glass industry, ceramic industry, medical and health field, and environmental field, and recently applied in energy storage. Seed-assisted synthesis is a very effective approach in promoting the crystallization of zeolites. In some cases, the target zeolite cannot be formed in the absence of seed zeolite. In homologous seed-assisted synthesis, the structure of the seed zeolite is the same to that of the target zeolite, while the structure of the seed zeolite is different to that of the target zeolite in the heterologous seed-assisted synthesis. In this review, we briefly summarized the heterologous seed-assisted syntheses of zeolites and analyzed the structure-directing effect of heterologous seeds and surveyed the “common composite building units (CBUs) hypothesis” and the “common secondary building units (SBUs) hypothesis”. However, both hypotheses cannot explain all observations on the heterologous seed-assisted syntheses. Finally, we proposed that the formation of the target zeolite does need nuclei with the structure of target zeolite and the formation of the nuclei of the target zeolite can be promoted by either the undissolved seed crystals with the same CBUs or SBUs to the target zeolite or by the facilitated appropriate distribution of the specific building units due to the presence of the heterologous seed that does not have any common CBUs and SBUs with the target zeolite.

Enzyme Immobilization Technologies and Industrial Applications.

Enzymes play vital roles in diverse industrial sectors and are essential components of many industrial products. Immobilized enzymes possess higher resistance to environmental changes and can be recovered/recycled easily when compared to the free forms. The primary benefit of immobilization is protecting the enzymes from the harsh environmental conditions (e.g., elevated temperatures, extreme pH values, etc.). The immobilized enzymes can be utilized in various large-scale industries, e.g., medical, food, detergent, textile, and pharmaceutical industries, besides being used in water treatment plants. According to the required application, a suitable enzyme immobilization technique and suitable carrier materials are chosen. Enzyme immobilization techniques involve covalent binding, encapsulation, entrapment, adsorption, etc. This review mainly covers enzyme immobilization by various techniques and their usage in different industrial applications starting from 1992 until 2022. It also focuses on the multiscale operation of immobilized enzymes to maximize yields of certain products. Lastly, the severe consequence of the COVID-19 pandemic on global enzyme production is briefly discussed.

Purification and enzymatic properties of a new thermostable endoglucanase from Aspergillus oryzae HML366.

Aspergillus oryzae HML366 is a newly screened cellulase-producing strain. The endoglucanase HML ED1 from A. oryzae HML366 was quickly purified by a two-step method that combines ammonium sulfate precipitation and strong anion exchange column. SDS-PAGE electrophoresis indicated that the molecular weight of the enzyme was 68kDa. The optimum temperature of the purified endoglucanase was 60 ℃ and the enzyme activity was stable below 70 ℃. The optimum pH was 6.5, and the enzyme activity was stable at pH between 4.5 and 9.0. The analysis indicated that additional Na+, K+, Ca2+, and Zn2+ reduced the catalytic ability of enzyme to the substrate, but Mn2+ enhanced its catalytic ability to the substrate.The Km and Vmax of the purified endoglucanase were 8.75mg/mL and 60.24μmol/min·mg, respectively. In this study, we report for the first time that A. oryzae HML366 can produce a heat-resistant and wide pH tolerant endoglucanase HML ED1, which has potential industrial application value in bioethanol, paper, food, textile, detergent, and pharmaceutical industries.

Production Optimization and Industrial Applications of Amylase From Indigenous Bacterial Species Using Banana Peels

Introduction: Alpha amylases are starch hydrolyzing enzymes that possess high industrial demand. Various strategies have been adopted to enhance cost-effective enzyme production of which utilization of agro-industrial waste is very promising. Methodology: In this study two amylase producers Bacillus megaterium and Exiguobacterium auranticum were isolated and identified by 16sRNA sequencing their growth conditions were optimized via submerged fermentation using banana peels as carbon source. Bacillus megaterium found to be mesophilic alkaline (37℃, pH of 9) strain giving 472 U/ml while Exiguobacterium auranticum found to be acidic thermophilic (50°C, pH 5) that gave 391 U/ml. Results and Conclusion: Production of amylase by acidic thermophilic E. auranticum is reported here for the first time. The crude enzyme also showed better chocolate and curry stain removal capacity when combined with commercial detergent. Also juice clarification assay showed promising results which indicates that the enzymes could be potentially used in detergent and food industry.

Purification and Characterization of Bromelain from Waste Parts of Ananas comosus for its Application in Detergent Industry

Purification and Characterization of Bromelain from Waste Parts of Ananas comosus for its Application in Detergent Industry

Growth Media Optimization for Production of Alkaline Protease from Industrial Wastewater using Bacillus subtilis PTCC 1254

Alkaline proteases are widely used in industrial processes due to their high pH tolerance and thermal stability. In present work, the protease producing ability of Bacillus strains (Bacillus subtilis PTCC 1254, B. subtilis PTCC 1156 and B. subtilis PTCC 1715) was studied. B. subtilis PTCC 1254 showed the highest proteolytic activity and therefore, the strain was selected as the biological agent in the submerged fermentation. Cell growth kinetic model was investigated using Malthus and Logistic equations, which were relatively well fitted to the experimental data. The maximum specific growth rate for Malthus and Logistic models were 0.187 and 0.377 h-1, respectively. The optimum culture conditions were defined as follows: pH 9, temperature 37°C, fermentation time 72 h, agitation speed 150 rpm and 4% inoculum with medium contained 1 g/l CaCl2, 0.6 g/l K2HPO4, 1 g/l KH2PO4, 0.2 g/l MgSO4.7H2O, 2 g/l sugarcane bagasse and 4 g/l corn bran as carbon and nitrogen sources. A 25% v/v industrial wastewater containing starchy waste was used as main substrate. Under optimum conditions, maximum alkaline protease activity of 117.43 U/ml was achieved. Also, the obtained protease was able to remove blood stain from cotton fabric and hydrolyze gelatin of X-ray film. Thus, this protease showed potential applications in detergent and photographic industries.

Characterization of alkaline metalloprotease isolated from halophilic bacterium Bacillus cereus and its applications in various industrial processes.

Microbial proteases are one of the most demanding enzymes for various industries with diverse applications in food, pharmaceutics, and textile industries to name the few. An extracellular alkaline metalloprotease was produced and purified from moderate halophilic bacterial strain, Bacillus cereus TS2, with some unique characteristics required for various industrial applications. The protease was produced in basal medium supplemented with casein and was partially purified by ion exchange chromatography followed by ammonium sulphate precipitation. The alkaline metalloprotease has molecular weight of 35 kDa with specific activity of 535.4 µM/min/mg. It can work at wide range of pH from 3 to 12, while showing optimum activity at pH 10. Similarly, the alkaline metalloprotease is stable till the temperature of 80 °C and works at wide range of temperature from 20 to 90 °C with optimum activity at 60 °C. The turnover rate increases in the presence of NaCl and Co+2 with k cat/KM of 1.42 × 103 and 1.27 × 103 s-1.M-1 respectively, while without NaCl and Co+2 it has a value of 7.58× 102. The alkaline metalloprotease was relatively resistant to thermal and solvent mediated denaturation. Applications revealed that the metalloprotease was efficient to remove hair from goat skin, remove blood stains and degrade milk, thus can be a potential candidate for leather, detergent, and food industry.

An Overview on Microbial Enzymes and their Industrial Applications

The application of enzymes for commercial interests is a very well-known practice over centuries. A diverse range of new enzymes have been discovered with the progression of technologies and some of these are yet unexplored. The environment-friendly nature of microbial enzymes gained interest of researchers because they reduce the production of greenhouse gases, during industrial processing. There are so many applications for microbial enzymes in a variety of industries for example (textiles, leather, paper and pulp, pharmaceutical, agriculture, detergent, waste, biorefineries, photography and food industries. There is a preference for microbial enzymes over plants and animals’ sources because of some of their specific characteristics e.g.- inexpensive production value, short time taken procedure and high yield. This review focuses on to reveal some industrial enzymes listed in comprehensive manner with their microbial origins and a diverse range of commercial implementation.

English

Lipases are important enzymes involved in hydrolytic processes that bring about the breakdown of triacylglycerol to free fatty acids and glycerol. Microbial lipases have diverse applications in the various biotechnological fields such as tannery, food, detergent and pharmaceutical industries. The main purpose of this study was to isolate and characterize the lipase-producing bacteria from the effluent of a tannery located in Kasur, Pakistan. Eight bacterial strains L1, L3, L5, L6, L9, L10, L11, and L12 were isolated, followed by screening on Tributyrin Agar Medium supplemented with mustard oil. These strains were morphologically and biochemically characterized. Growth kinetic studies of these bacteria showed optimum growth at 37°C and pH 7. Sucrose, yeast extract, and olive oil were found best as carbon, nitrogen, and oil source, respectively. The bacterial strains were non- halophilic as they preferred to grow at a low salt concentration (3%). Both of the strains L5 and L10 showed good lipase activity (99 and 92U/mL) and lipase enzyme of these strains was thermostable till 67ºC with a high protein content of 321.32 and 307.02 mg/mL, respectively. The molecular weight of lipase produced by L5 and L10 varied from 32-52kDa. Among the isolated bacterial strains, two strains L5 and L10 showed high lipase activity. 16S rRNA sequencing revealed that L5 and L10 strains had 99% homology with Bacillus altitudinis. This study signifies that the tanneries can be a good source of lipase-producing bacteria and Bacillus altitudinis can be a good lipase producer.

Izbor tehnike kultivacije za proizvodnju fungalnih enzima na čvrstom otpadu vinarije

The wine industry generates significant amounts of waste, the management and disposal of which represents a major environmental problem due to its seasonal character and polluting characteristics. Solid waste generated in wineries in Serbia is mostly used as a biological fertilizer. The production of fungal enzymes could be a potential solution for the valorization of this waste. Fungal enzymes are used in numerous industries, including the detergent industry. The aim of this study was to select the most successful cultivation technique for the production of fungal enzymes by cultivating a wild-type isolate of Trichoderma sp. on media that contained solid waste generated in the winery by processing black and white grapes. The success of the bioprocess was evaluated based on the protein content and proteolytic, lipolytic, amylolytic, and cellulolytic activity of the obtained crude enzyme preparations at the temperature conditions of detergent application, i.e. at 30 °C and 60 °C. The submerged cultivation technique with external mixing and spontaneous aeration proved to be the cultivation technique that obtained the highest protein content in the crude enzyme preparations. The submerged cultivation technique with intensive aeration proved to be the most appropriate for achieving the highest enzyme activity of the crude enzyme preparations. On the other side, the solid-state cultivation technique with intensive aeration resulted in the lowest protein content and the lowest enzymatic activity of crude enzyme preparations. Liquid medium cultivation techniques have proven to be more successful when compared to solid-state techniques.

Genome sequence analysis and characterization of Bacillus altitudinis B12, a polylactic acid- and keratin-degrading bacterium.

Keratin-rich wastes, mainly in the form of feathers, are recalcitrant residues generated in high amounts as by-products in chicken farms and food industry. Polylactic acid (PLA) is the second most common biodegradable polymer found in commercial plastics, which is not easily degraded by microbial activity. This work reports the 3.8-Mb genome of Bacillus altitudinis B12, a highly efficient PLA- and keratin-degrading bacterium, with potential for environmental friendly biotechnological applications in the feed, fertilizer, detergent, leather, and pharmaceutical industries. The whole genome sequence of B. altitudinis B12 revealed that this strain (which had been previously misclassified as Bacillus pumilus B12) is closely related to the B. altitudinis strains ER5, W3, and GR-8. A total of 4056 coding sequences were annotated using the RAST server, of which 2484 are core genes of the pan genome of B. altitudinis and 171 are unique to this strain. According to the sequence analysis, B. pumilus B12 has a predicted secretome of 353 proteins, among which a keratinase and a PLA depolymerase were identified by sequence analysis. The presence of these two enzymes could explain the characterized PLA and keratin biodegradation capability of the strain.

Purification and characterization of thermostable alpha‐amylase from Geobacillus sp. DS3 from Sikidang Crater, Central Java, Indonesia

Amylases are considered the most essential enzymes in biotechnology since they are widely utilized in the textile, food processing, and detergent industries. It is necessary to explore extracellular enzymatic activity in several microorganisms to discover a new potential application from amylases. In a previous study, thermophilic bacteria Geobacillus sp. DS3 isolated from Sikidang Crater, Dieng Plateau, Central Java, Indonesia showed amylase activity in starch medium at 70 °C. This study aimed to purify and characterize the thermostable alpha‐amylase from Geobacillus sp. DS3. The alpha‐amylase was produced and purified using ammonium sulfate and DEAE Sephadex A‐25 column. The enzyme activity was determined using the 3,5‐dinitrosalicylic acid (DNS) method. Geobacillus sp. DS3 optimally produced the alpha‐amylase at 60 °C for 15 h. The alpha‐amylase exhibited high enzymatic activity in 40–60% saturated ammonium sulfate extract. The molecular weight of the enzyme was estimated to be 58 kDa. The thermostable alpha‐amylase showed activity at the optimum temperature of 50 °C in 200 mM sodium phosphate buffer pH 7.0. The enzyme was inhibited by EDTA, PMSF, 2‐ME, and mostly by HgCl2. The Km and Vmax of the pure enzyme were 235.43 mM and 1428.57 U/mL, respectively. The result suggested that the purified thermostable alpha‐amylase from Geobacillus sp. DS3 offers potential application in areas of the food industry, such as the bakery industry.