Kinetic Analysis of Crude Enzyme Extract Produced Via Solid State Fermentation of Banana Pseudo Stem Waste

Banana Pseudo stem waste after each harvest contributes about 70–80 Milli Tons Per hector. The banana pseudo stem will be thrown as waste biomass after each harvest as it is unstable for the upcoming harvest. The biggest challenge in banana cultivation is the utilization of biomass of banana pseusostem waste into valuable products. In this study, Xylano-pectinase enzyme extract was produced from banana pseudo stem waste under solid-state fermentation by Enterobacter cloacae PMC04. The highest pectinase and xylanase activities obtained using banana pseudo stem as carbon source were 124.62 U/ml and 173.81 U/ml respectively. Thermodynamics stated that range 40-50oC were considered to be the optimal temperature for xylano-pectinase enzyme production and subsequent degumming of banana fibers. The crude enzyme extract were then used in the degumming of banana fibers for textile application. Textile processing of banana fiber necessitates the removal of hemicellulose substance which can be achieved by crude xylano-pectinase enzyme. It was found that crude xylano-pectinase was efficient in the removal of hemicellulose substance from the fibers. Results obtained from this study demonstrate that the proposed bioprocess could be successfully applied for the degumming of banana fibers sustainably.

Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

Valorization of Lignocellulosic Wastes to Produce Phytase and Cellulolytic Enzymes from a -Thermophilic Fungus, Thermoascus aurantiacus SL16W, under Semi-Solid State Fermentation

Agricultural wastes are lignocellulosic biomasses that contain high mineral and nutrient contents. This waste can be used as a raw material in industrial enzyme production by microbial fermentation. Phytase is an important enzyme used in animal feed to enhance the amount of phosphorus available for the growth and overall health improvement of monogastric animals. Fungi offer high potential as an effective source in the production of various extracellular enzymes. In this study, the production of lignocellulolytic enzymes (endoglucanase and xylanase) and phytase by a thermophilic fungus, namely Thermoascus aurantiacus strain SL16W, was evaluated using sixteen different Thai agricultural forms of waste under conditions of high temperature (45 °C). Semi-solid state fermentation was used in the production experiments. The results of this study reveal that the highest phytase activity (58.6 U/g substrate) was found in rice bran, whereas the highest degrees of activity of endoglucanase and xylanase were observed in wheat bran and red tea leaves at 19 and 162 U/g substrate, respectively. Consequently, the optimal conditions for phytase production of this fungus using rice bran were investigated. The results indicate that the highest phytase yield (58.6 to 84.1 U/g substrate) was observed in rice bran containing 0.5% ammonium sulfate as a nitrogen source with 10 discs of inoculum size at a cultivation period of 9 days at 45 °C and moisture content of 95%. Notably, the phytase yield increased by 1.71-fold, while endoglucanase and xylanase were also increased by 1.69- and 1.12-fold, respectively. Furthermore, the crude enzyme obtained from the optimal condition was extracted. The crude enzyme extract was then separately added to red tea leaves, rice straw, corncobs, palm residue, and peanut husks. Subsequently, total reducing sugar and phosphorus contents were determined. The results indicate that the highest level of reducing sugar (122.6 mg/L) and phosphorus content (452.6 mg/L) (p < 0.05) were obtained in palm residue at 36 and 48 h, respectively, after the addition of the crude enzyme extract. This study has provided valuable information on a potentially eco-friendly way to valorize agricultural waste into value-added products as industrial enzymes.

Enzymatic Electroanalytical Biosensor Based on Maramiellus colocasiae Fungus for Detection of Phytomarkers in Infusions and Green Tea Kombucha

In this work, we developed an enzymatic voltammetric biosensor for the determination of catechin and gallic acid in green tea and kombucha samples. The differential pulse voltammetry (DPV) methodology was optimized regarding the amount of crude enzyme extract, incubation time in the presence of the substrates, optimal pH, reuse of the biosensor, and storage time. Samples of green tea and kombucha were purchased in local markets in the city of Goiânia-GO, Brazil. High performance liquid chromatography (HPLC) and Folin-Ciocalteu spectrophotometric techniques were performed for the comparison of the analytical methods employed. In addition, two calibration curves were made, one for catechin with a linear range from 1 to 60 µM (I = −0.152 * (catechin) − 1.846), with a detection limit of 0.12 µM and a quantification limit of 0.38 µM and one for gallic acid with a linear range from 3 to 60 µM (I = −0.0415 * (gallic acid) − 0.0572), with a detection limit of 0.14 µM and a quantification limit of 0.42 µM. The proposed biosensor was efficient in the determination of phenolic compounds in green tea.

Characterization of β-Galactosidase from Lactose Utilizing Yeast Isolated from the Dairy Sample

Objectives: The objective of the study was to isolate lactose positive yeasts from dairy samples collected from local markets of Kathmandu, to extract crude β-galactosidase from the lactose positive yeast and to characterize the enzyme for optimum time duration, pH, temperature, Michaelis-Menten constant (Km) and maximum activity (Vmax). Methods: Four lactose positive yeast strains were isolated from dairy samples collected from local market of Kathmandu by pour plate method. Single strain having maximum lactose positive activity was selected for the study. The mass culture of the lactose positive yeast strain was lysed by 2% Chloroform and the yeast cell lysate containing β-galactosidase (i.e. crude enzyme extract) was characterized by using ONPG (Ortho-Nitrophenyl-β-D-galactopyranoside) as substrate. ONPG is a colorless substrate for the enzyme assay which is hydrolyzed by the enzyme into yellow colored product ONP (Ortho-Nitrophenol). The concentration of product formed was monitored spectrophotometrically at 420 nm to determine the enzyme activity and to characterize the enzyme. Results: The enzyme had wide range of working temperature from 0-50ºC, with optimal temperature of 37ºC. However, greater than 50% hydrolyzing ability was maintained in the range of 14-40ºC. Optimum time of reaction was 70 min. The enzyme had maximum activity in the near neutral pH of 6.8. Michaelis-Menten constant of the enzyme was found to be 2.23 mM of ONPG and Vmax was 58.82 nmol/min/ml. Enzyme activity was 27.88 nmol/min/ml, Specific enzyme activity was 59.97 nmol/ min/mg and total enzyme activity was 3346.33 nmol/min. Conclusion: The activity over a wide range of temperature 0-50ºC with low Km value shows that the enzyme has a commercial application in clearance of lactose pollution in waste water in different environmental conditions.

Bioconversion of Sweet Sorghum Residues by Trichoderma citrinoviride C1 Enzymes Cocktail for Effective Bioethanol Production

Improved cost-effective bioethanol production using inexpensive enzymes preparation was investigated. Three types of waste lignocellulosic materials were converted—for the production of enzyme preparation, a mixture of sugar beet pulp and wheat bran, while the source of sugars in hydrolysates was sweet sorghum biomass. A novel enzyme cocktail of Trichoderma citrinoviride C1 is presented. The one-step ultrafiltration process of crude enzyme extract resulted in a threefold increase of cellulolytic and xylanolytic activities. The effectiveness of enzyme preparation, compared to Cellic® CTec2, was tested in an optimized enzymatic hydrolysis process. Depending on the test conditions, hydrolysates with different glucose concentrations were obtained—from 6.3 g L−1 to 14.6 g L−1 (representing from 90% to 79% of the CTec2 enzyme yield, respectively). Furthermore, ethanol production by Saccharomyces cerevisiae SIHA Active Yeast 6 strain DF 639 in optimal conditions reached about 120 mL kg d.m.−1 (75% compared with the CTec2 process). The achieved yields suggested that the produced enzyme cocktail C1 could be potentially used to reduce the cost of bioethanol production from sweet sorghum biomass.

Purification and Characterization of Aspartic Protease Produced from Aspergillus oryzae DRDFS13 under Solid-State Fermentation

Aspartic proteases (E.C.3.4.23.) are endopeptidases with molecular masses ranging between 30–45 kDa. They depend on aspartic acid residues for their catalytic activity and show maximal activity at low pH. Thus the main objective of the present study was to purify and characterize aspartic protease from locally identified fungi by solid-state fermentation. The aspartic protease in the current study was obtained from A. oryzae DRDFS13 under SSF. The crude enzyme extract was purified by size-exclusion (SEC) and ion-exchange (IEC) chromatography. The protein contents of crude enzyme and IEC fractions were determined by BCA methods while the presence of N-glycosylation was checked using Endo-H. Inhibition studies were conducted using protease inhibitors. The milk-clotting activity (MCA), protease activity (PA); molecular weight and enzyme kinetics were determined using standard methods. Optimum temperature and stability, optimum pH and stability, and the effect of cations on MCA were assessed using standard methods. The maximum MCA (477.11 U/mL) was recorded from IEC fraction A8. The highest specific activity (183.50 U/mg), purification fold (6.20) and yield (9.2%) were also obtained from the same fraction (IEC A8). The molecular weight of 40 kDa was assigned for the purified enzyme (IEC A8). However, its molecular weight was decreased to 30 KDa upon deglycosylation assay which infers that the protein is glycosylated. Incubation of the pure enzyme (IEC A8) with pepstatin A caused a 94 % inhibition on MCA. The dialyzed enzyme showed a Km and Vmax values of 17.50 mM and 1369 U, respectively. The enzyme showed maximum MCA at 60 °C and pH 5.0 with stability at pH 4.5-6.5 and temperature 35-45 °C. Most cat-ions stimulate the activity of the enzyme; moreover, the highest MCA was detected at 50 mM of MnSO4. Furthermore, the results obtained in the present study confirmed that the aspartic protease enzyme produced from A. oryzae DRDFS13 and purified in ion-exchange chromatography could be used as a substitute source of rennet enzyme for cheese production.ImportanceThe production of pure aspartic protease enzyme from local microbes which is useful to substitute shortage of calf rennet enzyme and valuable to diversify cheese production throughout the world.

Optimization of enzymatic hydrolysis for bioethanol production by semi-simultaneous saccharification and fermentation using mature coconut fibre

Alternative substrates to produce useful chemicals such as biofuel have been attractive, in particular, for cellulosic ethanol production. In this context, the objective of this work was optimized the synergistic mixture of enzymes and bioethanol production. The enzymes of Trichoderma reesei and crude enzyme extract from Lichtheimia ramosa were used in the hydrolysis of mature coconut fibre pretreated by sequential process of alkaline hydrogen peroxide (Alk-H2O2)-sodium hydroxide (NaOH). Furthermore, these enzymes and pretreated vegetable biomass were applied in the bioethanol production by Saccharomyces cerevisiae in semi-simultaneous saccharification and fermentation strategy (SSSF). Resulting in the yields and conversions of delignified mature coconut fibre into reducing sugars between 12.7-82.14% and 0.09-0.64 g reducing sugars/g dry biomass, respectively, with an initial hydrolysis rate at 12 h between 0.10-0.89 g/(L.h). Yields and conversions of delignified mature coconut fibre into glucose between 10.16-83.78% and 0.06-0.43 g glucose/g dry biomass, in that order, with an initial hydrolysis rate at 12 h between 0.03-0.35 g/(L.h). Bioethanol production by S. cerevisiae using delignified mature coconut fibre, enzymes from T. reesei and crude enzyme extract from L. ramosa resulted in the production of 4.62 g/L, yield of 0.41 g ethanol/g glucose and volumetric productivity of ethanol of 0.13 g/(L.h), respectively. The results showed synergistic effects between enzymes from T. reesei and crude enzyme extract from L. ramosa, without promoting inhibition in the alcoholic fermentation. Therefore, allowing to formulate an optimized enzymatic preparation aiming cellulosic ethanol production.

Metabolite Identification from Biodegradation of Congo Red by Pichia sp.

Azo dyes are commonly used in textile and paper industries. However, its improper disposal often results in polluting water bodies. Azo dyes can cause adverse health effects because of its carcinogenic properties. Various methods to remove azo dyes from water have been proposed, including biological methods such as biosorption and biodegradation. Biosorption and biodegradation were done by using bacteria, yeast or mold. In general, yeasts have some advantages for azo dyes degradation due to its faster growth compared to mold and better resistance against unfavorable environment compared to bacteria. Previously, we observed that yeast Pichia sp. have the ability to degrade Congo red, an azo dye. However, information regarding biodegradation of azo dyes by Pichia sp. are still limited. Therefore, in this study, we showed degradation of Congo red by Pichia sp. crude enzyme extract obtained from separating Pichia cells from medium by centrifugation, followed by identification of its biodegradation products. Biodegradation product was separated from enzyme by ethyl acetate and then Gas Chromatography-Mass Spectroscopy (GC-MS) method was employed to identify biodegradation product. Chromatogram results of GC-MS showed that Congo red were degraded into various products such as biphenyl, naphthalene and smaller molecules with 94 m/z and 51 m/z. These results suggest involvement of azo reductase and laccase-like enzymes which cleaves azo bonds and oxidize the dye molecules to smaller molecules. This study implies the use of Pichia sp. as a bioremediation agent for the removal of azo dyes. Keywords: Biodegradation, Congo red, Pichia sp., metabolite identification, GC-MS

Evaluation of nutraceuticals including vitamin B12 and xenobiotic degradation capacity of Pleurotus species

Diverse edaphic zone (namely: usar, wastelands, forest area, wetlands, flood prone area and fertile lands) were identified in the eastern part of Uttar Pradesh and collected species of Pleurotus for present study. A total of 39 purified isolates were evaluated for the presence of neutraceuticals like proteins, carbohydrates, phenolic and vitamin B12 along with xenobiotic dye degradation capacity for textile dyes (MG and BPB) and production of laccase enzyme simultaneously. Isolate no. 06, appeared most distant in dendrogram having two major clusters, which also showed highest MG degradation capacity, however, other isolates also showed excellent degradation of BPB; and the laccase activity was found in the range of 4.03 to 19.13 IU/ml of crude enzyme extract from mycelia. All the isolates used in the present study, were also mounted for their genetic diversity analysis through RAPD. Diversity study revealed gene frequency from 0.012 to 0.987 and the average gene diversity for all RAPD loci were 0.244. The Shannon Information Index was 0.397. The unbiased genetic similarity among all pairs of isolates was 0.36 to 0.93 with a mean of 0.64. Significant genetic diversity, nutraceuticals and laccase enzyme availability and dye degradation capacity within the studied genus Pleurotus was found, which makes necessary to carry out a selection process in each one for superior selection not only for human being but also many aquatic as well as other terrestrial flora and fauna. Present investigation suggest that due to wide range of variation within species, the adaptation of strains to different edaphic zones must be taken into account in selection processes.