Use Of Microbial Enzymes Research Articles

Isolation and Screening of Microorganisms from Municipal Solid Waste for Production of Amylase

One of the major problems of the present world is the management of municipal solid waste which in produced in enormous amount. For sustainable management of the solid waste, the use of microbial enzymes can be a better alternative over chemical conventional methods. Amylase is of great importance in textile, food and pharmaceutical industry. It can be used for biotransformation of wastes. The municipal solid waste can be used to isolate amylase producing bacteria that can help in bio-transformation of irritant municipal waste. Here, we have isolated two potent amylase producing strains from municipal solid waste, OW1 and OW2. These isolates were studied for their morphological, cultural and biochemical characters. The isolates were screened for the production of enzyme amylase. Optimization of growth of the isolates with respect to pH and temperature. The potential of the isolates for waste degradation was checked by weight loss method. The isolates were then tentatively identified as Pseudomonas aeruginosa and Bacillus subtilis.

Comparison Study of Two Fumonisin-Degrading Enzymes for Detoxification in Piglets.

Fumonisins (FBs), particularly fumonisin B1 (FB1) and fumonisin B2 (FB2) produced mainly by Fusarium verticillioide and Fusarium proliferatum, are common contaminants in animal feed and pose a serious threat to both animal and human health. The use of microbial enzymes to efficiently and specifically convert fumonisins into non-toxic or low-toxic metabolites has emerged as the most promising approach. However, most of the available enzymes have only been evaluated in vitro and lack systematic evaluation in vivo. In this study, the detoxification efficacy of two carboxylesterases, FumD (FUMzyme®) and FumDSB, was evaluated comparatively in piglets. The results show that feeding piglets 4.4 mg/kg FBs-contaminated diets for 32 days did not significantly affect the average daily gain, organ indices, and immunoglobulins of the piglets. However, a significant reduction (21.2%) in anti-inflammatory cytokine interleukin-4 was observed in the FBs group, and supplementation with FUMzyme® and FumDSB significantly increased interleukin-4 by 62.1% and 28.0%, respectively. In addition, FBs-contaminated diets resulted in a 3-fold increase in the serum sphinganine/sphingosine (Sa/So) ratio, which is a specific biomarker that has been used to accurately reflect fumonisin levels. The serum Sa/So ratio was significantly reduced by 48.8% after the addition of FUMzyme®, and was insignificantly reduced by 8.2% in the FumDSB group. These results suggested that FUMzyme was more effective than FumDSB in mitigating FBs toxicity in piglets by down-regulating the Sa/So ratio.

Wastewater treatment using microbial enzymatic mixture

The effectiveness of the use of microbial enzymes as a wastewater treatment method depends on factors such as temperature, pressure, and oxygenation level, among others. However, very few scientific studies have provided real data regarding the evaluation of the applicability of this type of treatment. This research was therefore carried out to evaluate the effect of temperature and aerobic treatment time on the level of treatment of wastewater pollutants, with a mixture composed of enzymes and aerobic microorganisms under environmental conditions in the city of Huancayo, Peru. For this purpose, synthetic wastewater samples were prepared, considering a value of CODinitial= 800 mg L-1. One group of experiments was conducted at a 20°C controlled temperature and another group without control. The following average kinetic constants were obtained as results (Ko= 1.662 d-1, Kc=2.53 mg COD mg-1 VSS) when the T°= 20°C. For the experiments where the T° was not controlled, the average constants were: Ko= 1.217 d-1, Kc=1.24 mg COD mg-1 SSV. We observed from the measurements that the pollutant removal was adjusted to the kinetic model proposed by Orozco, and the biomass production was adjusted to the stoichiometric equation of biomass production with endogenous respiration. Therefore, we concluded that the effect of temperature control and treatment time on the level of purification caused by the microbial enzymatic product is significant at a 95% confidence level.
 Keywords: aerobic purification, biomass, digesting bacteria, kinetics, microorganisms.

An environment-benign approach of bamboo pulp bleaching using extracellular xylanase of strain Bacillus stratosphericus EB-11 isolated from elephant dung.

The use of microbial enzymes is highly encouraged in paper and pulp industries to reduce the excessive use of hazardous chemicals. During the study, xylanase of BacillusstratosphericusEB-11 was characterized for pulp bleaching applications. The extracellular xylanase was produced under submerged fermentation using bamboo wasteas a natural carbon source. There was fast cell division and enzyme production under optimized fermentation conditions in the bioreactor. The highest activity was 91,200Uafter30h of growth with Km and Vmax of 3.52mg/mL and 391.5μmol/min per mgrespectively. The purified enzyme with molecular mass ~ 60kDa had conferred positive activity on native PAGE. The strong inhibition by ethylenediaminetetraacetate and SDS showed the metallo-xylanase nature of the purified enzyme.The bacterial xylanase reduces the use of hydrogen peroxide by 0.4%. Similarly, biological oxygen demand and chemical oxygen demand were reduced by 42.6 and 35.2%. The xylanase-hydrogen peroxide combined treatment and conventional chlorine dioxide-alkaline (CDE1D1D2) bleaching showed almost similar improvement in physicochemical properties of bamboo pulp. Xylanase-peroxide bleaching reduces the lignin content to 4.95% from 13.32% unbleached pulp. This content after CDE1D1D2treatment was 4.21%. The kappa number decreased from 15.2 to 9.46 with increasing the burst factor (15.51),crystallinity index (60.25%), viscosity (20.1 cp), and brightness (65.4%). The overall finding will encourage the development of new cleaner methods of bleaching in the paper and pulp industry.

Potential Use of Microbial Enzymes for the Conversion of Plastic Waste Into Value-Added Products: A Viable Solution.

The widespread use of commercial polymers composed of a mixture of polylactic acid and polyethene terephthalate (PLA-PET) in bottles and other packaging materials has caused a massive environmental crisis. The valorization of these contaminants via cost-effective technologies is urgently needed to achieve a circular economy. The enzymatic hydrolysis of PLA-PET contaminants plays a vital role in environmentally friendly strategies for plastic waste recycling and degradation. In this review, the potential roles of microbial enzymes for solving this critical problem are highlighted. Various enzymes involved in PLA-PET recycling and bioconversion, such as PETase and MHETase produced by Ideonella sakaiensis; esterases produced by Bacillus and Nocardia; lipases produced by Thermomyces lanuginosus, Candida antarctica, Triticum aestivum, and Burkholderia spp.; and leaf-branch compost cutinases are critically discussed. Strategies for the utilization of PLA-PET’s carbon content as C1 building blocks were investigated for the production of new plastic monomers and different value-added products, such as cyclic acetals, 1,3-propanediol, and vanillin. The bioconversion of PET-PLA degradation monomers to polyhydroxyalkanoate biopolymers by Pseudomonas and Halomonas strains was addressed in detail. Different solutions to the production of biodegradable plastics from food waste, agricultural residues, and polyhydroxybutyrate (PHB)-accumulating bacteria were discussed. Fuel oil production via PLA-PET thermal pyrolysis and possible hybrid integration techniques for the incorporation of thermostable plastic degradation enzymes for the conversion into fuel oil is explained in detail.

High-resolution method for isocratic HPLC analysis of inulin-type fructooligosaccharides

In recent decades, strategies to improve human health by modulating the gut microbiota have developed rapidly. One of the most prominent is the use of prebiotics, which can lead to a higher abundance of health-promoting microorganisms in the gut. Currently, oligosaccharides dominate the prebiotic sector due to their ability to promote the growth and activity of probiotic bacteria selectively. Extensive efforts are made to develop effective production strategies for the synthesis of prebiotic oligosaccharides, including the use of microbial enzymes. Within the genus Lactobacillus, several inulosucrases have been identified, which are suitable for the synthesis of prebiotic inulin-type fructooligosaccharides (inulin-FOS). In this study, a truncated version of the inulosucrase from Lactobacillus gasseri DSM 20604 was used for the efficient synthesis of inulin-FOS. Product titers of 146.2 ± 7.4 g inulin-FOSL−1 were achieved by the catalytic activity of the purified recombinant protein InuGB-V3. A time and resource-saving HPLC method for rapid analysis of inulin-FOS in isocratic mode was developed and optimized, allowing baseline separated analysis of inulin-FOS up to a degree of polymerization (DP) of five in less than six minutes. Long-chain inulin-FOS with a DP of 17 can be analyzed in under 45 min. The developed method offers the advantages of isocratic HPLC analysis, such as low flow rates, high sensitivity, and the use of a simple, inexpensive chromatographic setup. Furthermore, it provides high-resolution separation of long-chain inulin-FOS, which can usually only be achieved with gradient systems.

Plasma-mediated enhancement of enzyme secretion in Aspergillus oryzae.

Technical bottlenecks in protein production and secretion often limit the efficient and robust industrial use of microbial enzymes. The potential of non-thermal atmospheric pressure plasma to overcome these technical barriers was examined. Spores of the fermenting fungus Aspergillus oryzae (A. oryzae) were submerged in potato dextrose broth (PDB) (5×106 per ml) and treated with micro dielectric barrier discharge plasma at an input voltage of 1.2kV and current of 50 to 63mA using nitrogen as the feed gas. The specific activity of α-amylase in the broth was increased by 7.4 to 9.3% after 24 and 48h of plasma treatment. Long-lived species, such as NO2 - and NO3 - , generated in PDB after plasma treatment may have contributed to the elevated secretion of α-amylase. Observations after 24h of plasma treatment also included increased accumulation of vesicles at the hyphal tip, hyphal membrane depolarization and higher intracellular Ca2+ levels. These results suggest that long-lived nitrogen species generated in PDB after plasma treatment can enhance the secretion of α-amylase from fungal hyphae by depolarizing the cell membrane and activating Ca2+ influx into hyphal cells, eventually leading to the accumulation of secretory vesicles near the hyphal tips.

English

Industrialization has come with environmental challenges. Industries like paper, printing, textile, leather and so on widely use chemical dyes whose waste treatment or degradability is difficult. Among various methods employed, the use of microbial enzymes is the most effective. The study aimed at producing laccase from identified yeast strains for potential industrial use in dye decolourization. Laccase produced by Kluyveromyces dobzhanskii DW1 and Pichia manshurica DW2 were purified and immobilized up to 65.2 and 73.1%, respectively. The crude, purified and immobilized forms of the enzymes were used to decolourize malachite green and methyl red dyes each at concentrations of 0.05 and 0.1 g/L. The highest percentage decolourization by K. dobzhanskii DW1 was 81.50% (immobilized) and 87.50% (purified), respectively for malachite green and methyl red dyes while P. manshurica DW2b (crude) had 84.40 and 76.89%, respectively. The Fourier transform infrared (FTIR) spectrum of the dyes was collected within a scanning range of 4000 to 400 cm-1. The spectrum of methyl red dye by the purified K. dobzhanskii DW1 laccase showed disappearance of some chemical groups (peak), while the crude P. manshurica DW2 laccase removed the main azo-group, alcohol/phenol and higher alkane (1487.17) groups. The spectrum of untreated malachite green also showed 25 peaks with one disulphide, 2 aliphatic halogens, 2 thio ethers, 3 sulphones, 4 imino groups among other chemical groups. The decolourization of the dye with the immobilized K. dobzhanskii DW1 laccase showed a spectrum of 17 peaks with the removal of the disulphide (420.5), one aliphatic halogen (C-I), thio ether, 2 sulphone, 4 imino and 2 higher alkanes, while the crude P. manshurica DW2 laccase removed the 2 aliphatic halogen, 4 imino, 1 amine, 2 alkane and 2 of the 3 alcohol/phenol chemical groups. The removal of the main components (azo chemical group) of the dyes proved their effectiveness in decolourisation and bioremediation of the textile wastes.   Key words: Decolourisation, azo-dye, laccase, immobilized, FT-IR spectra.

Microbial Fructosyltransferase: Production by Submerged Fermentation and Evaluation of pH and Temperature Effects on Transfructosylation and Hydrolytic Enzymatic Activities

Fructosyltransferases (FTase, E.C. 2.4.1.9) are enzymes that catalyze transfructosylation reactions obtaining, as final product, fructose oligomers. In terms of industrial production, the use of microbial enzymes is interesting, especially those produced by Aspergillus sp. The aim of the work was to study the production of FTase by submerged fermentation and evaluation of pH and temperature effects on fructosyltransferase activity. The enzyme was produced by Aspergillus oryzae IPT 301 in a 10-liter bioreactor with growth medium containing sucrose as main carbon source. The assay was performed at 800 rpm, 30 °C, 0.75 vvm and pH 4.5. Transfructosylation (At) and hydrolytic (Ah) activities were determined in the temperature range from 35 to 65 °C and pH range from 3.5 to 6.0. It was observed that mycelial is increases with temperature, holding the maximum value at 50 – 65 oC, while the optimum pH value were 5.0. The optimum temperature for extracellular At ranged from 55 to 65 °C and the optimum pH ranged from 5.0 to 6.0. Furthermore, the optimum temperature for mycelial Ah was 65 °C and optimum pH 4.5 – 5.0. For extracellular Ah, the optimum temperature and optimum pH were 55 to 65 °C and 3.5 to 6.0, respectively.

Decolorization of Remazol Brilliant Blue R by laccase of newly isolated Leiotrametes flavida Strain ZUL62 from Bangka Heath Forest, Indonesia

Falah S, Sari NM, Hidayat A. 2018. Decolorization of Remazol Brilliant Blue R by laccase of newly isolated Leiotrametes flavida Strain ZUL62 from Bangka Heath Forest, Indonesia. Biodiversitas 19: 633-639. The use of microbial enzymes seems to be an appropriate approach to neutralize synthetic dyes, providing an eco-friendly and cost-competitive alternative solution for treating industrial effluents. The objective of this research was to select the most potential fungal isolate with high laccase activity to decolorize Remazol Brilliant Blue R (RBBR) synthetic dyes. The isolation of fungal body and sampling of wood decay specimens were conducted in six different locations in heath (kerangas) forest of Bangka Belitung, Indonesia. In the present study, a total of 13 isolates were screened by using indicator plate method and for their enzyme activity. The preliminary screening was done to screen ligninolytic fungi. Then the primary screening using various indicator compounds was conducted to select laccases-producing fungi. The enzyme activity assay was performed to select fungal isolate with the highest activity. We found that the most potential fungi belonged to Leiotrametes flavida Strain ZUL62, which had been confirmed by molecular identification using 5.8S rDNA/ITS analysis. In addition, the laccase from Leiotrametes flavida Strain ZUL62 could decolorize RBBR, exhibiting a high rate of decolorization rate of 62% without any mediator within 24 h of incubation. To our best of knowledge, this study represented the first report about Leiotrametes flavida Strain ZUL62 and its potential laccase enzyme for dyes effluent treatment.

Degradation of Synthetic Azo Dyes of Textile Industry: a Sustainable Approach Using Microbial Enzymes

By releasing of azo dye through textile effluent, textile industry is the main cause of water pollution resulting into acute effect on environment and human health. Development of any eco-friendly and cost-effective method that may address the drawbacks to physical or chemical methods of dye removal is the recent global priority. Physical or chemical methods for textile wastewater pretreatment are of high cost, extremely energy consuming, and environmentally low efficient and generate toxic sludge. Thus, the use of microbial technique for textile dye degradation will be eco-friendly and is probably a lucrative alternative to physico-chemical processes. Microbial enzymes, viz. laccase and azoreductase, are cost-efficient, easy to harvest, easily downstream processable, and effortlessly mobilizable. Recent research trends on nanoparticle-microbial enzyme conjugates are also highly efficient to remove the azo dye from textile waste within a few minutes. But unfortunately, due to some gap between academia and industry, these methods remain only limited up to laboratory and its industrialization is still a challenge. The present review is an illustrated compilation of the use of microbial enzymes in removal of textile dyes.

Optimization of the Medium for the Production of Extracellular Amylase by the Pseudomonas stutzeri ISL B5 Isolated from Municipal Solid Waste.

The management of municipal solid waste is one of the major problems of the present world. The use of microbial enzymes for sustainable management of the solid waste is the need of the time. In the present study, we have isolated a potent amylase producing strain (ISL B5) from municipal solid waste. The strain was identified as Pseudomonas stutzeri (P. stutzeri) both biochemically and by 16S rDNA sequencing. The optimization studies revealed that the strain ISL B5 exhibited maximum activity in the liquid media containing 2% starch (2.77 U/ml), 0.8% peptone (2.77 U/ml), and 0.001% Ca2+ ion (2.49 U/ml) under the pH 7.5 (2.59 U/ml), temperature 40°C (2.63 U/ml), and 25 h of incubation period (2.49 U/ml). The highest activity of crude enzyme has also been optimized at the pH 8 (2.49 U/ml).

Antiviral Effect of Ribonuclease from <i>Bacillus pumilus</i> against Phytopathogenic Rna-Viruses

Background: Viruses can cause different diseases in plants. To prevent viral infections, plants are treated with chemical compounds and antiviral agents. Chemical antiviral agents usually have narrow specificity, which limits their wide application. Alternative antiviral strategy is associated with the use of microbial enzymes, which are less toxic and are readily decomposed without accumulation of harmful substances. The aim of this work is to study the effect of Bacillus pumilus ribonuclease on various phytopathogenic viruses with specific focus on the ability of enzyme to eliminate them from plant explants in vitro. Materials and methods: Extracellular ribonuclease of B. pumilus is tested as an antiviral agent. To study the antiviral effect of RNase, depending on concentration and the time of application several plant-virus model systems are used. Virus detection is conducted by serological testing and RT-PCR. Results: Bacillus pumilus ribonuclease possesses antiviral activity against plant Rna-viruses RCMV (red clover mottle virus), PVX (Potato Virus X) and AMV (Alfalfa Mosaic Virus). The maximum inhibitory effect against actively replicating viruses is observed when plants are treated with the enzyme in the concentration of 100 ug/ml prior to infection. In case of local necrosis ribonuclease in the concentration of 1 ug/ml completely inhibits the development of RCMV virus on bean plants. The enzyme is able to penetrate plants and inhibit the development of viral infection, inhibiting effect for untreated surfaces decreased on average for 20%. It is also found that B. pumilus ribonuclease protects apical explants of sprouts of potato tubers from PVM and PVS viruses. Conclusion: B. pumilus ribonuclease possesses antiviral activity against plant Rna-viruses and produces viruses-free plants in the apical meristem culture.

Isolation and Characterization of Proteases Enzyme from Locally Isolated Bacillus sp.

A bacterium was isolated from the natural source. Gram staining & spore staining showed that the organism is gram positive and forms spore during adverse condition in the growth medium. After various tests it was suggested and the features agreed with the description of Bacillus sp in Bergey’s Manual of Systematic Bacteriology [24]. It was also identified as Bacillus sp with 99.9% identity by API 50 CHB. In growth curve determination showed that the growth of the organism is increased with the increase of incubation period and the growth reached maximum at around 24 hours of incubation and the protease activity was the maximum of the 26 hours culture. This microbe has grown at high temperature and pressure. Its optimum pH and temperature were 8.5 and 60°C. It secretes an extracellular protease in the growth medium. The enzyme hydrolyses a number of proteins including azocasein which suggests that that it is an extracellular protease. The enzyme seems to be alkaline protease which is capable of De-Hairing from skin and hides. A number of companies such as NOVO chemicals started to produce NOVOzymes for tannery industries. The potential for use of microbial enzymes in leather processing lies mainly in areas in which pollution-causing chemicals are being used.

Improving Chocolate Flavor in Poor‐Quality Cocoa Almonds by Enzymatic Treatment

This paper proposes a method to enzymatically treat poor-quality cocoa almonds (known as "slate") to ensure the formation of chocolate flavor precursors. The production of flavor precursors improves the quality of these almonds, which are usually responsible for the low quality of the liquor produced. Proteases and carboxypeptidases from different sources were tested under various conditions. The different treatments were evaluated by chemical analysis (hydrolysis efficiency) and sensory analysis of the treated material compared to good-quality cocoa almonds. The results show that it is possible, through the use of microbial enzymes, to generate the mixture of compounds that will release, after roasting, the characteristic chocolate flavor in poor-quality almonds. However, it is necessary to optimize the conditions of enzymatic treatment to obtain better results and thus establish a process that can be used for industrial purposes for manufacturing cocoa and chocolate. The basidiomycete Moniliophtora perniciosa is the causative agent of witches' broom disease (WBD) of the cocoa tree, whose seeds are the source of chocolate. It is the most important phytopathological problem of cocoa-producing areas of the American continent, and has decimated the Brazilian cocoa industry. In Bahia (Brazil), M. perniciosa was identified in 1989 and, as a consequence of its spreading, the annual production of cocoa almonds dropped from 450,000 to 90,000 tons within 12 y, reducing export values from an all-time high of about US$ 1 billion to 110 million. The high incidence of WBD incapacitates Brazil to produce enough cocoa almonds even for the internal market, leading the country to import low-quality cocoa almonds mainly from African countries. Our work proposes an enzymatic treatment to increase the quality of that cocoa almonds and, consequently, to improve the quality of the chocolate produced and consumed in the country.

Medical bioremediation of age-related diseases.

Catabolic insufficiency in humans leads to the gradual accumulation of a number of pathogenic compounds associated with age-related diseases, including atherosclerosis, Alzheimer's disease, and macular degeneration. Removal of these compounds is a widely researched therapeutic option, but the use of antibodies and endogenous human enzymes has failed to produce effective treatments, and may pose risks to cellular homeostasis. Another alternative is "medical bioremediation," the use of microbial enzymes to augment missing catabolic functions. The microbial genetic diversity in most natural environments provides a resource that can be mined for enzymes capable of degrading just about any energy-rich organic compound. This review discusses targets for biodegradation, the identification of candidate microbial enzymes, and enzyme-delivery methods.

Use of Microbial Enzymes as Antistaling Agents for Corn Tortillas

Use of Microbial Enzymes as Antistaling Agents for Corn Tortillas

微生物酵素の生産と臨床分析への応用

This article reviews recent developments in use of microbial enzymes for clinical analysis and new trends in production of the enzymes by use of genetic engineering techniques. The increased availability of microbial enzymes has led to many analytical applications in the areas of clinical chemistry. Especially during the last decade enzymatic assays have rapidly spred in clinical laboratories through efforts of the search of useful enzymes. The applications of cholesterol oxidase to the determination of serum cholesterol showed that the enzymatic method was more specific, precise and sensitive than the chemical method. This success stimulated developments and adoptions of other enzymatic assays for triglycerides, phospholipids and free fatty acids. The principle of enzymatic methods are mainly based on either the colorimetric assay of hydrogen peroxide formed during the reaction by oxidase or the spectrophotometric determination of the change of NAD (P) linked with dehydrogenase. Whether the enzymatic method can prove its worth or not depends on properties of microbial enzymes used in the assay. The enzymes have to be equipped with enough qualifications in specificity, affinity, optimum pH and stability. The advance in gene-splicing technology made it possible to produce microbial enzymes at high levels and the cloned N-acetylneuraminate lyase has become the first enzyme to be used in clinical laboratories. Protein engineering also gives us possibilities that properties of the enzymes are improved to be more suitable for clinical analysis.

The potential of microbial enzymes as diagnostic reagents.

There has been a rapid growth in the application of microbial enzymes for diagnosis in the area of clinical chemistry. The reasons behind this trend are discussed. Descriptions and illustrations are given of the use of microbial enzymes in the determination of metabolic marker analytes; in the assay of antibiotics, analgesic and chemotherapeutic drugs, and in the quantitation of antibody-antigen interactions.

Clinical analysis of lipids using microbial enzymes: The last decade has seen the increasing use of microbial enzymes for clinical analysis. Glycerol oxidase, recently discovered, has been applied to the analysis of serum triglycerides

Clinical analysis of lipids using microbial enzymes: The last decade has seen the increasing use of microbial enzymes for clinical analysis. Glycerol oxidase, recently discovered, has been applied to the analysis of serum triglycerides