Maximum Enzyme Activity Research Articles

Improving decolorization of dyes by laccase from Bacillus licheniformis by random and site-directed mutagenesis.

BackgroundDye wastewater increases cancer risk in humans. For the treatment of dyestuffs, biodegradation has the advantages of economy, high efficiency, and environmental protection compared with traditional physical and chemical methods. Laccase is the best candidate for dye degradation because of its multiple substrates and pollution-free products.MethodsHere, we modified the laccase gene of Bacillus licheniformis by error-prone PCR and site-directed mutagenesis and expressed in E. coli. The protein was purified by His-tagged protein purification kit. We tested the enzymatic properties of wild type and mutant laccase by single factor test, and further evaluated the decolorization ability of laccase to acid violet, alphazurine A, and methyl orange by spectrophotometry.ResultsMutant laccase Lacep69and D500G were superior to wild type laccase in enzyme activity, stability, and decolorization ability. Moreover, the laccase D500G obtained by site-directed mutagenesis had higher enzyme activity in both, and the specific activity of the purified enzyme was as high as 426.13 U/mg. Also, D500G has a higher optimum temperature of 70 °C and temperature stability, while it has a more neutral pH 4.5 and pH stability. D500G had the maximum enzyme activity at a copper ion concentration of 12 mM. The results of decolorization experiments showed that D500G had a strong overall decolorization ability, with a lower decolorization rate of 18% for methyl orange and a higher decolorization rate of 78% for acid violet.ConclusionCompared with the wild type laccase, the enzyme activity of D500G was significantly increased. At the same time, it has obvious advantages in the decolorization effect of different dyes. Also, the advantages of temperature and pH stability increase its tolerance to the environment of dye wastewater.

Gonococcal Adaptation to Palmitic Acid Through farAB Expression and FadD Activity Mutations Increases In Vivo Fitness in a Murine Genital Tract Infection Model.

Neisseria gonorrhoeae is a bacterial pathogen that colonizes mucosal epithelia that are rich in antimicrobial molecules such as long-chain fatty acids. Here we studied the mechanisms involved in palmitic acid resistance and their impact on in vivo biological fitness in a murine genital tract infection model. A stable palmitic acid-resistant derivative was obtained by serial passage with incremental palmitic acid concentrations. This derivative outcompeted its parent strain for colonization and survival in the murine infection model. Subsequent whole-genome sequencing resulted in the identification of the 3 resistance-related SNPs ihfAC5T, fadDC772T, and farAG-52T (promoter) that were verified for resistance against palmitic acid. Subsequent characterization of the associated resistance determinants showed that ihfAC5T and farAG-52T induced gene expression of the FarAB efflux pump, whereas fadDC772T increased the maximum enzyme activity of the FadD long-chain fatty acid-coenzyme A ligase. Our results highlight the mechanisms involved in gonococcal adaptation to the murine host environment.

Earthworms increase the potential for enzymatic bio-activation of biochars made from co-pyrolyzing animal manures and plastic wastes

We assessed the enzymatic activation of four different biochars produced from pyrolyzing swine manure and poultry litter, and by co-pyrolyzing these livestock residues with agricultural spent mulch plastic film wastes (plastichars). Enzymatic activation consisted of incubating biochars in soil inoculated with earthworms (Lumbricus terrestris), which acted as biological vectors to facilitate retention of extracellular enzymes onto biochar surface. The activity of carboxylesterase ‒a pesticide-detoxifying enzyme‒ was measured in non-bioturbed soils (reference), linings of the burrows created by earthworms, casts (feces) and biochar particles recovered from the soil. Our results revealed that: 1) biochar increased soil carboxylesterase activity respect to biochar-free (control) soils, which was more prominent in the presence of earthworms. 2) The maximum enzyme activity was found in soils amended with plastichars. 3) The plastichars showed higher enzyme binding capacities than that of the biochars produced from animal manure alone, corroborating the pattern of enzyme distribution found in soil. 4) The presence of earthworms in soil significantly increased the potential of the plastichars for enzymatic activation. These findings suggest that the plastichars are suitable for increasing and stabilizing soil enzyme activities with no toxicity on earthworms.

Optimum growth conditions of Lactobacillus brevis LIPI13-2-LAB131 in β-galactosidase enzyme production

Abstract. Indah AWN, Rohmatussolihat, Rahayu WP, Setyoningrum F, Priadi G, Afianti F. 2020. Optimum growth conditions of Lactobacillus brevis LIPI13-2-LAB131 in β-galactosidase enzyme production. Biodiversitas 21: 5403-5407. Deficiency of β-galactosidase enzyme causes lactose to become undigested in gastrointestinal system, therefore the system needs further addition of external β-galactosidase. The sources β-galactosidase enzyme varies from plants, animals, and microorganisms. In industrial applications, microorganisms have become a considered potential source of β-galactosidase. Lactobacillus brevis LIPI13-2-LAB131 had high β-galactosidase enzyme activity, which was 7.93 U/mL. The aim of this research was to optimize the growth condition of L. brevis LIPI13-2-LAB131 in order to produce maximum β-galactosidase enzyme activity. This research consisted of performing optimization processes using design expert 7.0 (DX7) program with response surface methodology (RSM) and partial purification of β-galactosidase enzyme. The results of this research showed that the optimum growth conditions of L. brevis LIPI13-2-LAB131 were in 1.48% lactose level, incubation temperature of 34.91 °C, incubation time of 48.48 hours, and 2.83% inoculum size with desirability value of 0.839. The result of enzyme purification showed that value of β-galactosidase enzyme activity increased up to 22.88 ± 0.29 U/mL with purification yield of 11.65%.

Efecto del surfactante Tween 80 en el crecimiento y la producción de esterasa de Fusarium culmorum en fermentación líquida

Tween 80 is a widely used non-ionic surfactant that is added to culture media to make hydrophobic substrate available to microorganisms. Because of this surfactant widespread use, it is important to understand how it affects microbial growth and enzyme production. In this work, the effect of different concentrations (100, 400 and 600 l/L; v/v) of Tween 80 (as the sole carbon source) on the biomass production, esterase activities (assessed through biochemical tests and zymographic assays) and protein content of Fusarium culmorum grown in liquid fermentation was determined. The specific growth rate (μ), biomass yield (YX/S), esterase yield (YE/X), esterase productivity (P), maximal enzymatic activity (Emax), and specific rate of enzyme production (qp) were also estimated. A control medium added with glucose was used. The highest μ was showed in the medium added with 100 l of Tween 80/L. However, the greatest esterase activity was observed in those media containing the highest concentrations of Tween 80 ( energy source and it also induced the esterase production by F. culmorum. Tween 80 concentration is positively correlated with the number of esterase isoforms produced by this fungus. The higher the Tween 80 concentration (400 μl/L and 600 μl/L, v/v), the more number of esterase isoforms will be induced. However, lower concentration (100 μl/L) of Tween 80/L did not show a significant effect on the induction of the esterase activity.400 l/L and 600 l/L; v/v). These results show that Tween 80 was used as the sole carbon and

Using Vinasse as a Source of Potassium Fertilization for Sugar Beet and a Sandy Soil Amendment

TWO field experiments were conducted in a sandy soil at Al-Hossein Agricultural Farm located at 64thkm, Cairo-Alexandria Desert Road, Egypt, in 2017/2018 and 2018/2019 seasons, to check the possibility of using cane vinasse as a source of potassium fertilization for sugar beet, and its effect as an amendment on some properties of soil. A randomized complete block design was used. The present work included 13 treatments as follows:- 100% K2O: 48kg K2O/fed “fed = 4200m2” as K2SO4 “48% K2O” (control).- Three treatments represent the sole application of 100% equivalent to 48kg K2O/fed, in the form of raw (V1), photo-chemically (V2) and biologically (V3) treated vinasse, i.e., 707.6, 707.6 and 817.7L/fed, respectively.- Nine treatments, using 75, 50 and 25% of the recommended rate as K2SO4 combined with 25, 50 and 75%, successively, equivalent to 48kg K2O/fed, from each of V1, V2 and V3.The lowest value of hydroxyl methyl furfural (HMF) was recorded with V3 and phenolic compounds with V2. The maximum activity of dehydrogenase enzyme was found with adding (50% K2O+50% V3)/ fed. Adding (50% K2O+50% V3) significantly increased root yield/fed. The different treatments including V3 markedly increased sugar yield/fed, as compared to that gained with 100% K2O/fed, in both seasons.The addition of [50% of the recommended rate of K-fertilizer (24 kg K2O) as K2SO4+50% of the biologically treated vinasse (408.85 liters)/fed] could be recommended to get economical root and sugar yields, which means saving 50% of mineral potassium sulphate required for sugar beet crop.

Computational modelling approach for the optimization of apple juice clarification using immobilized pectinase and xylanase enzymes

Apple juice is typically marketed as a clear juice, and hence enzymatic treatments are common practices in juice industry. However, enzymatic treatments have been shown to face some challenges when process efficiency, and cost effectiveness are concerned. Therefore, it is necessary to optimize the enzymatic treatment process to maximize efficiency, and reuse enzymes to minimize the overall cost via immobilization. In this context, the present work features the immobilization of pectinase and xylanase from M. hiemalis on genipin-activated alginate beads, with subsequent evaluation of its efficacy in apple juice clarification. A central composite rotatable design (CCRD), coupled with artificial neural network (ANN) for modeling and optimization was used to design the experiments. Deploying a coupling time up to 120 min, and an agitation rate of 213 rpm (pectinase) - 250 rpm (xylanase), a maximum fractional enzyme activity recovered was observed to be about 81–83% for both enzymes. Optimum enzyme loading and genipin concentration were found to be 50 U/ml and 12% (w/v), respectively. The immobilized enzyme preparations were suitable for up to 5 repeated process cycles, losing about 45% (pectinase) - 49% (xylanase) of their initial activity during this time. The maximum clarity of apple juice (%T660, 84%) was achieved at 100 min when pectinase (50 U/ml of juice) and xylanase (20 U/ml of juice) were used in combination at 57 °C. The immobilized enzymes are of industrial relevance in terms of biocompatibility, recoverability, and operational-storage stability.

Immobilization of keratinase on chitosan grafted-β-cyclodextrin for the improvement of the enzyme properties and application of free keratinase in the textile industry

Immobilization of enzymes is an effective and potential technique for improving the enzyme characteristics and plays an important role in reducing the final cost of enzymatic reactions. However, the method of enzyme immobilization should be easy, cost-effective and environment friendly when applicable at industrial scale. In present study, the successful biochemical characterization of free and immobilized keratinase was evaluated. The enzyme was effectively immobilized on chitosan and chitosan grafted-β-cyclodextrin beads. Enzyme yield of immobilized biocatalyst on chitosan alone and chitosan-β-CD-E was determined to be 90 and 93% respectively. Keratinase was able to act in highly alkaline conditions (optimum pH 11) both in free and immobilized form and showed maximum enzyme activity at 70 and 75 °C respectively. The free and immobilized enzyme exhibited remarkable thermo stability at 70 °C implying that it is capable for its usage in textile industry. The storage stability and reusability of the immobilized keratinase (chitosan-E and chitosan-β-CD-E) was significantly enhanced, with 25 and 53.5% activity, respectively, retained at 4 °C after 30 days of storage. In the preliminary experiments it was found that free keratinase have the potential to improve the quality of woollen fabrics and suitable for application in textile industries.

Impact of land configuration and organic nutrient management on productivity, quality and soil properties under baby corn in Eastern Himalayas

Appropriate land configuration and assured nutrient supply are prerequisites for quality organic baby corn (Zea mays L.) production in high rainfall areas of the delicate Eastern Himalayan Region of India. A long term (5-year) study was conducted during 2012–2016 on a sandy loam soil in the mid attitude of Sikkim, Eastern Himalayan Region of India to evaluate the productivity, produce quality, the profitability of baby corn, and soil properties under different land configurations comprising flatbed, ridge and furrow, and broad bed and furrow, and organic nutrient management practices comprising un-amended control, farmyard manure 12 t ha−1, vermicompost 4 t ha−1 and farmyard manure 6 t ha−1 + vermicompost 2 t ha−1. The baby corn sown on broad bed and furrow had the tallest plant (149.25 cm), maximum dry matter (64.33 g plant−1), highest leaf area index (3.5), maximum cob length (8.10 cm), cob girth (6.13 cm) and cob weight (8.14 g) leading to significantly higher fresh baby corn yield (1.89 t ha−1), and net returns (US$ 906.1 ha−1) than those of other treatments. Mineral composition (phosphorus, potassium, iron, and zinc), protein, and ascorbic acid content were also the highest in baby corn grown under the broad bed and furrow system. The soil of broad bed and furrow had a higher pH, organic carbon content, organic carbon pools, microbial biomass carbon, and enzymatic activities (dehydrogenase, fluorescein diacetate, and acid phosphatase) compared to soils of other land configurations. A combined application of farmyard manure (6 t ha−1) + vermicompost (2 t ha−1) improved the crop growth and produced 117.8% higher fresh baby corn and 99.7% higher fodder yield over control (0.9 t fresh corn and 13.02 t fodder yield ha−1), respectively. This treatment also registered significantly higher gross return (US$ 1746.9 ha−1), net return (US$ 935.8 ha−1), and benefit–cost ratio (2.15) than other nutrient management practices. Fresh cob quality in terms of protein (22.91%) and ascorbic acid content (101.6 mg 100 g−1) was observed to be significantly superior under combined application of farmyard manure (6 t ha−1) + vermicompost (2 t ha−1) than those of other nutrient management systems. However, fresh baby corn cobs produced with vermicompost 4 t ha−1 had the highest concentration of phosphorus, potassium, iron, and zinc. Application of farmyard manure 12 t ha−1 registered the maximum increment in soil organic carbon content (1.52%), its pool (40.6 t ha−1) and carbon sequestration rate (0.74 t ha−1 year−1) followed by integrated application of farmyard manure (6 t ha−1) and vermicompost (2 t ha−1). The maximum soil microbial biomass carbon and enzymatic activities [dehydrogenase (22.1 µg TPF g−1 soil h−1) and fluorescein diacetate (67.1 µg FDA g−1 soil h−1)] were noted with the combined use of farmyard manure (6 t ha−1) + vermicompost (2 t ha−1). Thus, the study suggests that the broad bed and furrow land configuration along with the combined application of farmyard manure + vermicompost could be an economically feasible practice for quality organic baby corn production and soil health improvement in the Eastern Himalaya and other similar eco-regions elsewhere.

Extraction and Purification of Lipases Enzyme from Germinating Seeds of Four Crops

Lipase enzyme has attracted a lot of attention in recent years because of its diverse biotechnological applications. The present study was conducted to screen germinated seeds of four crops, namely sunflower (Helianthus annuus), flaxor linseed (Linum usitatissimum ), peanut (Arachis hypogaea ) and castor bean (Ricinus communis), for the activity of their lipases. to the study also included the extraction and purification of lipase from the seeds of the most promising crop using different solvents.
 The results indicated that the maximum enzymatic activity (0.669 U/ml) was obtained when 0.1 M Tris-HCl buffer extract was used after 3 days of seed germination of all the tested species, as compared to the other test solvents (acetone and water). Sunflower germinated seeds showed the highest lipase activity, which was higher by 159.67, 185.32, and 285.90 % over the activities of castor bean, flax, and peanut seeds, respectively. Among the used ranges of saturation of ammonium sulfate, the ratio of 70% was the best in precipitating the crude enzyme, showing a highest specific activity of 2.576 U/ mg protein.
 The first stage of gel filtration chromatography column by Sephadex G-200 indicated the presence of two non-identical peaks, one for protein and another for lipase activity, between the fractions of 18 to 23. The active fractions were pooled and loaded again in the Sephadex G-200 column and the eluted fractions showed two identical peaks, one for protein and another for lipase activity, between the fractions of 19 to 23. The final purification step by gel filtration showed a specific activity of 6.482 U/mg proteins with a yield of 38.75 % and 11.33 folds of time of purification. The study revealed that sunflower seeds are a better source of lipase as compared to the other used plant seeds, which can be used in different industries.

Molecular diversity and hydrolytic enzymes production abilities of soil bacteria.

Soil is an integral part of ecosystem which is niche for varieties of microflora. The present study was investigated to isolate varied strains of bacteria from soil samples of three different geographical regions of Tamil Nadu (India) and evaluate their hydrolytic enzymes (amylase, cellulase, and inulinase) producing potentialities. Among 72 bacterial cultures isolated from Ambattur Industrial Estate, Neyveli Lignite Corporation, and Arignar Anna Zoological Park regions, 41.66, 38.88, and 36.11% of isolates were observed amylase, cellulase, and inulinase producers, respectively. On the other hand, 20.83% of total bacteria isolated from all three regions exhibited concurrent production of amylase, cellulase, and inulinase. Potent isolates depicting maximum enzyme activities were identified as Bacillus anthracis strain ALA1, Bacillus cereus strain ALA3, Glutamicibacter arilaitensis strain ALA4, and Bacillus thuringiensis strain ALA5 based on molecular characterization tools. Further, the thermodynamics parameters, open reading frames (ORFs) regions, and guanine-cytosine (GC) content were determined by distinct bioinformatics tools using 16S rRNA sequences of strains. Minimum free energy values for strain ALA1, strain ALA3, strain ALA4, and strain ALA5 were calculated as −480.73, −478.76, −496.63, and −479.03 kcal/mol, respectively. Mountain plot and entropy predicted the hierarchical representation of RNA secondary structure. The GC content of sequence for strain ALA1, strain ALA3, strain ALA4, and strain ALA5 was calculated as 53.06, 52.94, 56.78, and 53.06%, respectively. Nine ORFs were obtained for strain ALA1, strain ALA3, and strain ALA5 while 10 ORFs were observed for strain ALA4. Additionally, bootstrap tree demonstrated close resemblance of strains with existing bacteria of similar genus. Findings showed higher variability of bacterial diversity as hydrolytic enzymes producers in the investigated geographical regions.

Microbial Conversion of Shrimp Heads to Proteases and Chitin as an Effective Dye Adsorbent.

As a green and effective technique in the production of a large number of valuable products, the microbial conversion of chitinous fishery wastes is receiving much attention. In this study, protease production using the Paenibacillus mucilaginosus TKU032 strain was conducted on culture media containing several common types of chitinous fishery by-products serving as the carbon and nitrogen (C/N) nutrition source. Among the chitinous wastes, 1.5% (w/v) shrimp head powder (SHP) was found to be the most appropriate nutritional source for protease production when a maximal enzyme activity of 3.14 ± 0.1 U/mL was observed on the 3rd day of the culture period. The molecular mass of P. mucilaginosus TKU032 protease was estimated to be nearly 32 kDa by the polyacrylamide gel electrophoresis method. The residual SHP obtained from the culture medium was also considered to be utilized for chitin extraction. The deproteinization rate of the fermentation was estimated to be 45%, and the chitin obtained from fermented SHP (fSHP) displayed a similar characteristic Fourier-transform infrared spectroscopy (FTIR) profile as that from SHP. In addition, SHP, fSHP, and chitins obtained from SHP and fSHP were investigated for their adsorptive capacity of nine types of dyes, and chitin obtained from fSHP displayed a good adsorption rate on Congo Red and Red No. 7, at 99% and 97%, respectively. In short, the results provide potential support for the utilization of SHP in the production of P. mucilaginosus TKU032 protease via the fermentation as well as the preparation of chitin from fSHP as an effective dye adsorbent.

Biochemical and Taxonomic Characterization of Novel Haloarchaeal Strains and Purification of the Recombinant Halotolerant α-Amylase Discovered in the Isolate.

Haloarchaea are salt-loving archaea and potential source of industrially relevant halotolerant enzymes. In the present study, three reddish-pink, extremely halophilic archaeal strains, namely wsp1 (wsp-water sample Pondicherry), wsp3, and wsp4, were isolated from the Indian Solar saltern. The phylogenetic analysis based on 16S rRNA gene sequences suggests that both wsp3 and wsp4 strains belong to Halogeometricum borinquense while wsp1 is closely related to Haloferax volcanii species. The comparative genomics revealed an open pangenome for both genera investigated here. Whole-genome sequence analysis revealed that these isolates have multiple copies of industrially/biotechnologically important unique genes and enzymes. Among these unique enzymes, for recombinant expression and purification, we selected four putative α-amylases identified in these three isolates. We successfully purified functional halotolerant recombinant Amy2, from wsp1 using pelB signal sequence-based secretion strategy using Escherichia coli as an expression host. This method may prove useful to produce functional haloarchaeal secretory recombinant proteins suitable for commercial or research applications. Biochemical analysis of Amy2 suggests the halotolerant nature of the enzyme having maximum enzymatic activity observed at 1 M NaCl. We also report the isolation and characterization of carotenoids purified from these isolates. This study highlights the presence of several industrially important enzymes in the haloarchaeal strains which may potentially have improved features like stability and salt tolerance suitable for industrial applications.

Biomass and Bio-butanol Production from Borodinellopsis texensis CCALA 892 in Synthetic Wastewater: Determination of Biochemical Composition

Microalgae can generally maintain the high amounts of biomass in the wastewater and they can be converted from biomass to bio-butanol. Bio-butanol is a liquid biofuel and it has significant physical and chemical properties. In this study, we carried out bio-butanol production from Borodinellopsis texensis CCALA 892 grown in various concentrations of the municipal wastewater. Also, we determined biochemical composition ratios of microalgae samples and studied the some antioxidant enzymes such as catalase, superoxide dismutase and ascorbate peroxidase. In the current study, bio-butanol was produced by the acetone-butanol-ethanol (ABE) fermentation method. The microalgae sample grown in 25% of wastewater had the highest biomass productivity among five wastewater samples with 0.114 ± 0.002 g L-1d-1. The carbohydrate and protein concentrations of control group increased day by day and their values reached stationary phases at seven days. The sample grown in 25% of wastewater had the highest carbohydrate concentration with 0.30 g L-1 and protein concentration with 0.35 g L-1 at the stationary phase. The maximum enzyme activities for catalase, superoxide dismutase and ascorbate peroxidase were 15.33 ± 0.88, 8.67 ± 0.67 and 33 ± 1.53 µmole/mg, respectively at 25% of wastewater. In addition, bio-butanol content of B. texensis CCALA 892 was 3.63 ± 0.21 g L-1 and its bio-butanol yield was found as 0.18 ± 0.011 g/g sugar. In the next study, we can examine large scale butanol production.

Improving laccase production from Trametes versicolor using lignocellulosic residues as cosubstrates and evaluation of enzymes for blue wastewater biodegradation

Biocatalytic degradation of recalcitrant pollutants employing ligninolytic enzymes is a promising approach for wastewater treatment. However, enzymes production must be improved to make biodegradation a more cost-effective treatment. In this research, laccase production from Trametes versicolor using lignocellulosic residues (agave bagasse, coconut fibers and wheat bran) as cosubstrates was improved using a central composite face-centered design, and the application of the enzymes-rich culture supernatant was evaluated for blue wastewater biodegradation. Findings revealed that the optimal conditions for laccase production were found at 35 °C and 5 g/L of wheat bran as cosubstrate, reaching about 200 U/mL in 11 days in a batch submerged fermentation. These conditions were scaled up for a submerged fermentation using an airlift reactor, and a maximum enzymatic activity of 1200 U/mL was achieved in 9 days at 30 °C. This enzymes-rich culture supernatant was tested for the degradation of blue wastewater from aircraft in an airlift reactor. Results showed a COD removal efficiency of 43% and an increase of the biodegradability index from 0.64 to 1.36, both results applying an enzymatic activity of supernatant of 300 U/mL. In conclusion, the enzymatic biodegradation becomes a viable strategy for the pretreatment of a real effluent such as the blue wastewater collected in public transportation.

Plant invasion alters the Michaelis–Menten kinetics of microbial extracellular enzymes and soil organic matter chemistry along soil depth

Microbial extracellular enzymes decompose distinct components of soil organic matter (SOM), thus influencing its stability. However, we lack the knowledge about how the kinetics of individual enzymes vary when multiple substrates change simultaneously. Here we used Japanese knotweed (Polygonum cuspidatum) invasion as a model system to explore how the Michaelis–Menten kinetics (Vmax and km) of microbial extracellular enzymes vary with corresponding SOM components across soil depth (0–5, 5–10, and 10–15 cm). We hypothesized that invasion will increase the Vmax (maximum enzyme activity) and km (substrate concentration at half Vmax) of oxidative enzymes but decrease the Vmax and km of hydrolytic enzymes, and that increasing soil depth will alleviate the invasion effects on the enzyme kinetics. The invasion of knotweed, which input litter rich in recalcitrant compounds, altered soil chemistry including an increase in lignin and fungal biomass compared to the adjacent non-invaded soils. The Vmax of peroxidase, the oxidative enzyme that degrades lignin, increased in the invaded soils (0–5 cm) compared to the non-invaded soils. Among the hydrolytic enzymes, the Vmax of N-acetyl-glucosaminidase which degrades chitin from fungal cell walls increased in the invaded soils (0–5 cm). However, there was no associated change in the km of peroxidase and N-acetyl-glucosaminidase under invasion, suggesting that microbes modified the enzyme production rates, not the types (isozyme) of enzymes under invasion. The Vmax of all enzymes decreased with depth, due to the reduced substrate availability. These results highlight that the addition of relatively recalcitrant substrates due to plant invasion altered the kinetics of microbial extracellular enzymes with implications for SOM chemistry in the invaded soils.

World biocatalysis & biocatalyst market analysis to 2024 - market forecast to grow at a CAGR of 15% from 2019 to 2024

The purpose of the study is isolation and application of novel Hot spring bacterial enzymes. It also reports on purification and characterization of thermostable α-amylase from a newly hot spring isolate, Exiguobacterium sp. This thermostable amylase is Ca2+-independent an added improvement in starch saccharification process at a higher temperature because it removes the addition of Ca2+ for improving the stability of amylases. Maximum enzyme activity was obtained at 45 °C at pH 8.5 and stability at concentration of 3.0% NaCl. Thermostable α-amylase from Exiguobacterium sp was purified by 3.9 fold with 54.6% recovery and specific activity was 1083 U/ml. The molecular weight of α-amylase was 54 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apparent Km and Vmax value was 5.88 mg/ml and 250µmol/min/ml, respectively for the hydrolysis of soluble starch. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the amylase is predominantly turn structure and a detailed structural composition showed alpha helix 10.8%, Beta sheet 27.1%, Turn 29.8% and Randomness 32.3% respectively. The amylase combined with soap-nut extract was able to de stain blood stained cloth within 30 min

Process optimization for simultaneous production of cellulase, xylanase and ligninase by Saccharomyces cerevisiae SCPW 17 under solid state fermentation using Box-Behnken experimental design

Multienzyme complex has attracted increased attention in biofuel technology. They offer solutions to effective degradation of complex plant material into fermentable sugars. Microorganisms, especially bacteria and fungi, are well studied for their ability to produce enzymes complex unlike yeast. Yeast strain isolated from mushroom farm was studied for simultaneous production of cellulase, xylanase and ligninase enzymes using lignocellulose waste as substrates. A response surface methodology (RSM) involving Box-Behnken design (BBD) was used to investigate interaction between variables (moisture content, inoculum size, initial pH, incubation time) that affect enzyme production. Crude filtrate was partially purified and characterised. Yeast strain identified as Saccharomyces cerevisiae SCPW 17 was finally studied. Evaluation of lignocellulose waste for enzyme complex production revealed corn cob to be most effective substrate for cellulase, xylanase and ligninase production with enzyme activity of 17.63 ± 1.45 U/gds, 29.35 ± 1.67 U/gds and 150.75 ± 2.01 μmol/min respectively. Time course study showed maximum enzyme complex production was obtained by day 6 with cellulase activity of 12.5 U/gds, xylanase 48.3 U/gds and ligninase 90.8 μmol/min. Using RSM involving BBD, maximum enzyme activity was found to be 19.51 ± 0.32 U/gds, 56.86 ± 0.38 U/gds, 408.17 ± 1.04 μmol/min for cellulaase, xylanase and ligninase respectively. The developed models were highly significant at probability level of P = 0.0001 and multiple correlation co-efficient (R2) was 0.9563 for cellulase, 0.9532 for xylanase and 0.9780 for ligninase. Enzyme complex was stable at varying pH and temperature conditions. Saccharomyces cerevisiae (SCPW 17) studied produced enzyme complex which can be used for bioconversion of biomass to value-added chemicals.

Extracellular Thermostable Laccase-Like Enzymes from Bacillus licheniformis Strains: Production, Purification and Characterization

This study presents the exploration of laccase-like enzyme produced by thermophilic bacterial strains present in the Tattapani hotspring located in Chhattisgarh, India. Two bacterial isolates namely TPNR1 and TPNR6 were found to be positive for laccase-like enzyme production on screening with guaiacol as a substrate. The biochemical and 16S rRNA gene sequence analysis indicated that the isolates have similarity with Bacillus licheniformis strains and, thus, named as B. licheniformis TPNR1 and B. licheniformis TPNR6. The activity of crude enzymes was estimated using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as a substrate and measured as 3.1 U/mL and 7.1 U/mL for TPNR1 and TPNR6, respectively. The SDS-PAGE of purified enzymes showed that the enzymes are monomers with molecular weight of 44 (TPNR1) and 38 (TPNR6) kDa. The native-PAGE technique followed by activity staining using ABTS confirms the presence of purified active enzyme with laccase-like activity in both cases. Kinetic studies displayed that catalytic efficiency of the enzymes was higher for the substrate ABTS than 2,6-dimethoxyphenol for both enzymes. The maximum enzyme activity was observed at 50°C for both enzymes while the optimal pH for TPNR1 and TPNR6 was 5.0 and 6.0, respectively. The TPNR1 exhibited half-life of 4 h at 70°C and was stable at 60°C for 180 min with a residual activity of 79%. Similarly, the TPNR6 possessed half-life of 3.1 h at 70°C and retained 88% of its activity at 60°C for 180 min. In addition, the catalytic efficiency of the enzymes was tested by decolourization of toxic dyes which showed that the both enzymes are highly potential to degrade them.

Screening for Optimal Parameters of Nattokinase Synthesis by Bacillus subtilis natto in Solid-State Fermentation

Nattokinase, which is an extracellular enzyme synthesized by Bacillus subtilis natto, is a medication for cardiovascular disease treatment. In this study, soybean seed was used as a substrate for culturing Bacillus subtilis natto in solid-state fermentation to produce nattokinase. The optimal culture parameters for solid-state fermentation to synthesize nattokinase by Bacillus subtilis natto were 2:50 (v:w) of the ratio of Bacillus subtilis natto pre-culture to the substrate, 42 h of fermentation time, and 3 cm of the thickness of the substrate. At this optimal culture parameter of solid-state fermentation by using Bacillus subtilis natto, the enzymatic activity of crude nattokinase was 7.13 ± 0.2, 16.24 ± 0.33, and 16.55 ± 0.06 (specific activity/mL), respectively. Furthermore, we aim to apply this study with large scale production initially. Thus, the ratio of reusing Bacillus subtilis natto in fermentation product to substrate for a new process of solid-state fermentation and circulation time of reusing Bacillus subtilis natto in new solid fermentation process were screened. The maximal enzymatic activity of crude nattokinase of 14.10 ± 0.18 (specific activity/mL) was found at 3:100 (w:w) of reusing Bacillus subtilis natto in fermentation product to substrate for a new process of solid-state fermentation. The suggestion for the circulation time of reusing Bacillus subtilis natto for new solid fermentation process was 2nd. The results of this study had provided the necessary information for further research on nattokinase.