Maximum Enzyme Activity Research Articles

Pectinase Producing Bacteria Isolation from Halophilic Soil, Water Samples and Partial Purification of the Enzyme

Pectinases are protease enzymes capable of degrading pectin, which is one of the most important polysaccharide found in nature. Pectinase enzymes find uses in paper-pulp industry, textile industry, food industry and other industries where lignocellulosic material is utilized. Commercially pectinase enzymes are obtained from bacterial and fungal culture grown on decaying fruits and vegetables. In the present study, an attempt was made to isolate pectinase producing bacteria growing in halophilic conditions. Soil (sand) and water samples containing high concentrations of salts from beaches of Taiwan and Karwar, Karnataka were collected and screened for pectinase activity using modified M9 media. Among the obtained 16 bacterial strains, strain 15 showed the highest pectinase activity during screening and was identified to be a Bacillus sp. from morphological features and biochemical tests. This strain was selected and its cultural conditions were standardized for maximum production of pectinase enzyme. Maximum enzyme activity was obtained at pH 11, temperature 40°C, and incubation period of 24 hours. To enhance the productivity modification were tried out in culture media related to nitrogen source and carbon source in which the nitrogen source did not have any significance in activity of enzyme but glucose seemed to be the best carbon source for the growth of bacteria. Further the obtained enzyme was partially purified using the salting out method, dialysis followed by desalting, gel filtration chromatography and ion-exchange chromatography.

Thermostable arginase from Sulfobacillus acidophilus with neutral pH optimum applied for high-efficiency L-ornithine production.

This study aims to use neutral pH optimum arginase as the catalyst for high-efficiency L-ornithine production. Sulfobacillus acidophilus arginase was firstly cloned and overexpressed in Escherichia coli. The purified enzyme was obtained, and the molecular mass determination showed that this arginase was a hexamer. S. acidophilus arginase possessed similarities with the other arginases such as the conserved sequences, purification behavior, and the necessity for Mn2+ as a cofactor. The maximum enzyme activity was obtained at pH7.5 and 70°C. Thermostability and pH stability analysis showed that the arginase was stable at 30-60°C and pH7.0-8.5, respectively. The kinetic parameters suggested that S. acidophilus arginase could efficiently hydrolyze L-arginine. Bioconversion with this neutral pH optimum arginase had the advantages of avoiding producing by-product, high molar yield, and high-level production of L-ornithine. When the bioconversion was performed with a fed-batch strategy and a coupled-enzyme system involving S. acidophilus arginase and Jack bean urease, the final production of 2.87mol/L was obtained with only 1.72mmol/L L-arginine residue, and the molar yield was 99.9%. The highest production record suggests that S. acidophilus arginase has a great prospect in industrial L-ornithine production.

Neonicotinoid resistance in adults and nymphs of Bemisia tabaci (Genn., 1889) (Hemiptera: Aleyrodidae) populations in tomato fields from Tokat, Turkey

Bemisia tabaci (Genn., 1889) (Hemiptera: Aleyrodidae) is one of the most important agricultural pests in Turkey and in the world. This polyphagous pest is a highly efficient vectors of plant viruses and has the ability to rapidly develop resistance to diverse range of insecticides, hence controlling this pest is problematic. In this study, bioassays and biochemical tests were conducted to determine resistance to neonicotinoid in B. tabaci populations collected in 2017-2018 from Tokat (Turkey). According to the adult test results, resistance ratios for acetamiprid, imidacloprid and thiamethoxam were 5.64-16.8, 10.0-30.9 and 4.01-14.9, respectively. The highest resistance ratio for acetamiprid and thiamethoxam in the Pazar population were 16.8 and 14.9, respectively. The highest resistance ratio to imidacloprid was 30.9 in the TOGU campus population. According to the nymph test results, resistance ratios for acetamiprid, imidacloprid and thiamethoxam were 2.96-8.60; 4.29-8.74 and 2.48-4.88, respectively. Enzyme analysis revealed statistically higher metabolic resistance. Maximum enzyme activities were 4.37 and 3.79 pmol/min/mg protein for cytochrome P450 monooxygenase in TOGU campus and Pazar populations, respectively.

Biosíntesis del agente fibrinolítico uroquinasa por Enterococcus gallinarum aislado de sardine

Fibrinogen degradation by thrombin leads to the formation of fibrin clots in the blood which results in cardiovascular diseases due to which certain fibrinolytic agents are used to treat cardiovascular complications. Urokinase is being favored as a fibrinolytic agent due to its high fibrin specificity and fewer side effects. Therefore, the main objective of this research was the isolation of potent urokinase producing bacteria from soil, seawater, and fermented food samples. Thirty-five isolates that were capable of producing urokinase were isolated using sixty samples and subjected to secondary screening for urokinase estimation. Bacterial isolate QGA-20 that showed maximum enzyme activity 52.6±0.03 FU/ml/min was identified by 18s RNA sequencing as Enterococcus gallinarum which has never been reported before for urokinase production. Different physical and cultural parameters such as fermentation medium, incubation time, temperature, pH, carbon source, nitrogen source, inoculum size, and trace elements were optimized for submerged fermentation to synthesize urokinase. Maximum production of urokinase 81.3±0.02 FU/ml/min was obtained with M-5 fermentation media at 96 hrs of incubation, 37oC temperature, 7 pH, sucrose as carbon, and soya flour as a nitrogen source, 3% inoculum size and CaCl2 as a trace element. In in vitro studies, urokinase enzyme was also applied for the disintegration of clot successfully. This study revealed a potent urokinase producer, reported first time, with appreciable clot lysis ability.

Antioxidant system status in threatened native fish Barbus meridionalis from the Osor River (Iberian Peninsula): I. Characterization and II. In vitro Zn assays

The evaluation of antioxidant system capacity is important in aquatic toxicology. It was aimed to characterize the liver antioxidant enzymes (SOD, CAT, GPX, GR, and GST) and to test the in vitro Zn effect (200 and 400 ZnSO4 μg/L) in native fish Barbus meridionalis obtained from the Osor River (NE, Spain) influenced by Zn contamination. The maximal enzyme activities were at pH 7.0–7.5 and 100 mM phosphate buffer. Barbel showed high catalytic activity (high Vmax and low Km) indicating the efficient antioxidant detoxification ability. Direct Zn effect caused an antioxidant system imbalance. Mostly upon lower Zn concentration, GPX activity decreased (95–100 %) though CAT, GR, and GST increased (36–1543 %). GSH values either stimulated (290 %) or inhibited (85–93 %) due to tissue differences. The first record of barbel antioxidant enzyme characterization and in vitro data presenting an unbalanced antioxidant pattern could be significant to evaluate the metal pollution in the Osor River for further in vivo studies.

Enhancing the Enzymatic Saccharification of Grain Stillage by Combining Microwave-Assisted Hydrothermal Irradiation and Fungal Pretreatment.

Grain stillage from the liquor industry was pretreated by using microwave-assisted hydrothermal pretreatment, fungal pretreatments, and their combination to enable efficient enzymatic hydrolysis for sugar production. The microwave-assisted hydrothermal (MH) pretreatment was optimized by using a response surface methodology, and the respective maximum reducing sugar yield and saccharification efficiency of 17.59 g/100 g and 33.85%, respectively, were achieved under the pretreatment conditions of microwave power = 120 W, solid-to-liquid ratio = 1:15 (g·mL–1), and time = 3.5 min. The fungal pretreatment with Phanerochaete chrysosporium digestion (PC) achieved the maximum ligninolytic enzyme activities in 6 days with 10% inoculum size at which the reducing sugar yield and saccharification efficiency reached 19.74 g/100 g and 36.29%, respectively. To further improve the pretreatment efficiency, MH and PC pretreatments were combined, but the sequence of MH and PC mattered on the saccharification efficiency. The MH + PC pretreatment (the MH prior to the PC) was better than PC + MH (the PC prior to the MH) in terms of saccharification efficiency. Overall, the MH + PC pretreatment achieved superior reducing sugar yield and saccharification efficiency (25.51 g/100 g and 66.28%, respectively) over all other studied pretreatment methods. The variations of chemical compositions and structure features of the raw and pretreated grain stillage were characterized by using scanning electron microscopy and Fourier transform infrared spectroscopy. The results reveal that both MH and PC pretreatments mainly functioned on delignification and decreasing cellulose crystallinity, thus enhancing the enzymatic saccharification of the pretreated grain stillage. The combined MH and PC pretreatment could be a promising method to enable cost-efficient grain stillage utilization for downstream applications such as biofuels.

Immobilized polyphenol oxidase: Preparation, optimization and oxidation of phenolic compounds

Polyphenol oxidase (PPO) was immobilized on chitosan/montmorillonite (CTS/MMT) and chitosan-gold nanoparticles/montmorillonite (CTS-AuNPs/MMT) composites, respectively. Taguchi method was applied to determine the optimal immobilization conditions for achieving the maximum enzyme activity. PPO immobilized on CTS/MMT (IPPO) and CTS-AuNPs/MMT (IPPO-Au) showed the highest enzyme activity at 15.61 × 103 and 29.01 × 103 U/g, respectively. IPPO-Au exhibited the higher stability and reusability than that of IPPO. The bio-catalytic performance of immobilized PPO was evaluated for the removal of phenol (PH), 4-chlorophenol (4-CP) and 2,4-dichlorophenol (2,4-DCP) in aqueous solution. The effects of pH, temperature, enzyme/substrate ratio, substrate concentration, reaction time on the phenolic compounds removal were investigated in detail. The results showed that, for both immobilized PPO, the optimal pH was 7, 6 and 5 for PH, 4-CP and 2,4-DCP, respectively, and the optimal temperature was 30 °C for all substrate. The optimal enzyme/substrate ratio and reaction time for IPPO-Au was lower than that of IPPO proved the higher catalytic efficiency. Chlorophenols showed the improved catalytic efficiency in comparison with PH for both immobilized PPO, while the effect of substrate chemical structure on the IPPO-Au properties was limited. The results suggested AuNPs presented in support played an important role on the enzyme activity and stability of immobilized PPO.

Solid-state fermentation on poplar sawdust and corncob wastes for lignocellulolytic enzymes by different Pleurotus ostreatus strains

Solid state fermentation with different lignocellulolytic materials as inducers was used for lignocellulolytic enzyme production in this study. Pleurotus ostreatus strains were assessed by measuring laccase, CMCase, and xylanase activities. The secretion potential of the lignocellulolytic enzymes by wild and cultivated strains was analyzed for the first time. The wild and cultivated strain showed their unique capacities for secreting lignocellulolytic enzymes on solid-state fermentation with different lignocellulosic materials. The wild P. ostreatus strain preferred corncob for the secretion of laccase and xylanase activity, but the cultivated strain preferred poplar sawdust. The wild strain and cultivated strain showed a consistent preference for poplar sawdust for the secretion of CMCase activity. The wild strain was advantageous because it achieved the maximum hydrolytic enzyme activities within a short time period. Poplar sawdust and corncob were conducive to laccase secretion by the wild or cultivated strains and the rapid accumulation of laccase on solid-state fermentation. Additionally, continuous, stable laccase production was an extremely important advantage by solid-state fermentation of poplar sawdust, particularly in the wild strain. These findings are helpful in selecting the appropriate strain that corresponds to suitable lignocellulosic materials. The optimization of integrated industrial lignocellulolytic enzyme production can also be achieved.

Screening of Process Parameters to Produce Xylanase from Aspergillus niger for Secondary Bioethanol Production

In recent years, the biotechnological use of xylanases has grown remarkably. Xylanase is a hydrolytic enzyme with a broad industrial application. In specific, xylanase can convert xylan into xylose, a fermentable sugar source for secondary bioethanol production. The objective on this study is to investigate the significance of different parameter effects for an efficient xylanase production from Aspergillus niger (A. niger). In this study, four factors: incubation temperature, medium pH, incubation time, and agitation speed were screened by performing One-factor-at-a-time (OFAT) analysis. Xylanase production with the maximal enzyme activity was successfully obtained from OFAT analysis under condition of 32°C, pH 5.0, 5 days, and 150 rpm.

Modeling of enzymatic waste water treatment

Processing and recycling of waste water is an important environmental problem. One possible and promising way to use the generated sludge is production biofertilizers for agriculture. However, in view of the risks of chemical and bacteriological contamination, it is necessary to develop technologies that ensure a high level of conditioning and stabilization of sludge. Maximum enzyme activity is observed in a microbubble medium under the conditions of a mesophilic process. To accelerate the biochemical transformation of pollutants, an enzyme-cavitation method has been developed, based on the stimulation of growth processes of microorganisms in bioreactors by low and high intensity cavitation created by turbojets. It has been established that the quality and duration of sludge treatment depends on the pressure of the substrate at the entrance to the oxidizing jets. It has been proven that at a pressure p = 0.30-0.35 MPa, the processing time of the sludge before stabilization of the chemical oxygen consumption at 16% is 8 hours. Microscopic studies found that the processed substrate is an accumulation of microorganisms with a total surface of up to 100 m2 per 1 gram of dry matter that provides high sorption properties with respect to moisture.

Optimization of On-Site Xylanase Production from Aspergillus niger via Central Composite Design (CCD)

Xylanases have stimulated considerable interest due to their potential application in several industries, especially in the bioethanol sector. Since the vitality of this enzyme is undeniable, this research is focused on optimization of on-site xylanase production from Aspergillus niger (A. niger). This initiative could reduce the dependence of commercial xylanase. Central Composite Design (CCD) was implemented in the process of xylanase production optimization. Incubation temperature and medium pH were two parameters selected to statistically optimized using Response Surface Methodology (RSM) in order to improve the xylanase production. From the data analyzed by Design of Experiment (DoE), maximal xylanase production was predicted to produce under condition of 32.67 °C and pH 4.56 with desirability of 0.936. A validation test with triplicate was done to verify the predicted result. The maximum enzyme activity of 0.5638 U/mL was obtained from the validation test.

Estrone degrading enzymes of Spirulina CPCC-695 and synthesis of bioplastic precursor as a by-product

Estrone, a steroidal estrogen that is persistently contaminating the surface water has been classified as an endocrine disruptor and as Group-1 carcinogen by the World Health Organization. Long-term exposure to estrone-contaminated water disrupt physiology, behaviour and sexual development of living organisms that lead to many disorders. So, it has to be eliminated from our surrounding. Its biological degradation is a cost effective and eco-friendly approach. The present study targets to predict the degradation pathway and understand the role of cyanobacterial enzymes: oxidoreductases (laccase, peroxidase) and esterase in estrone degradation. Poly-β-hydroxy butyrate (PHB) was also quantified as a by-product of estrone biodegradation. The estrone degradation pathway was predicted using EAWAG-BBD/PPS database. Spirulina CPCC-695 was grown in different concentration of estrone (20 mg/l, 50 mg/l, 100 mg/l and 200 mg/l). The culture without estrone was considered as control. The culture supernatant was used for testing laccase and esterase activity whereas the biomass was used to test peroxidase activity and quantify by-product (PHB). The enzymes showed concentration-dependent activities. Maximum enzyme activities were seen at 20 mg/l estrone. Spirulina CPCC-695 utilizes estrone as a carbon source and degrades it to produce pyruvate which forms acetyl CoA that undergo condensation, reduction and polymerization to form PHB. Maximum PHB (169 μg) was also produced at 20 mg/l as a by-product during degradation.

Biochemical and molecular characterization of proteolytic bacterial strains isolated from Jazan region, KSA with the application as an antibacterial agent

Objectives: Biochemical and molecular characterization of proteolytic bacterial strains isolated from Jazan region, KSA with the application as an antibacterial agent. Materials and Methods: Three samples were collected from extreme environment, Jazan, KSA. Skim milk nutrient agar medium was used for protease screening for several colonies by streaking method at 37°C. API biochemical kit was used to characterize the three isolates using some selective media. The genetic identification was done using 16S rRNA gene sequencing. The sensitivity of the tested strains;Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae against the extracellular crude protease enzyme produced from the three isolated bacteria and different antibiotics was done. Results: The isolates were identified as Bacillus subtilis, Bacillus licheniformis, and Bacillus cereus. B. cereus and B. licheniformis recorded high sensitivity (71%) against most antibiotics, in addition, B. subtilis showed resistance to Aztreonam only. It was found a considerable increase in the level of both of protease activity (units/ml) and bacterial growth (colony-forming units/ml) of the cultures that were directed by the B. subtilis and B. licheniformis up to 37°C then decreased at 45°C. On the contrary, the growth of B. cereus and its activity gradually increased up to 45°C. The enzyme activity and bacterial growth of B. subtilis and B. cereus strains were increased at alkaline medium. However, B. licheniformis gave the highest growth and activity at neutral pH. In addition, it was found that the enzyme activity and bacterial growth of B. subtilis were reached to the maximum at 5% NaCl. However, the maximum bacterial growth and enzyme activity for B. licheniformis and B. cereus was at 2% NaCl. It was found high effect on inhibiting the growth of pathogenic bacteria using 5 μl of crude enzyme with specific enzyme activity 73, 76, and 92 (units/ml)/(mg protein/ml) for B. subtilis, B. licheniformis, and B. cereus, respectively. All pathogenic bacteria were totally inhibited with 10 μl of crude enzyme. Conclusion: The potential Bacillus proteases can promote new industry as antimicrobial agents.

Comparative study on four amylosucrases from Bifidobacterium species

Amylosucrase (ASase) is α-glucan-producing enzyme. Four putative ASase genes (bdas, blas, bpas, and btas) were cloned from Bifidobacterium sp. and expressed in Escherichia coli. All ASases from Bifidobacterium sp. (BAS) displayed typical ASase properties with slightly different characteristics. Among the BASs studied, BdAS and BpAS showed maximal enzyme activities at 35 and 30 °C, respectively, whereas BlAS and BtAS were maximally active at higher temperatures, i.e., 45 and 50 °C, respectively. BpAS exhibited optimum pH under slightly basic conditions (pH 8.0), while BdAS, BlAS, and BtAS preferred weakly acidic conditions (pH 5.0–6.0). All BASs showed higher isomerization activities. Particularly, BlAS produced more trehalulose than turanose. Although polymerization was the highest for BtAS, BtAS synthesized α-1, 4-glucans with a lower degree of polymerization than that of the other BASs. The versatile properties of the BASs described could contribute to the efficient production of highly valuable biomaterials for the agriculture, food, and pharmaceutical industries.

Advanced biological sequential treatment of mature landfill leachate using aerobic activated sludge SBR and fungal bioreactor.

This study utilized Penicillium spp. to treat mature landfill leachate (MLL) in a continuous bioreactor and batch experimental tests under non-sterile conditions. MLL characteristics such as chemical oxygen demand (COD), soluble COD (sCOD), total carbon (TC), total organic carbon (TOC), and color removal efficiency were determined. The lignocellulosic enzymatic activity of laccase (Lac), lignin-peroxidase (LiP), and manganese-peroxidase (MnP) was also determined. The batch experimental test was carried out with raw and pretreated MLL containing the initial NH4 +-N concentrations of 0, 105, 352, and 914mg/L. A maximum COD reduction of 41% and maximum enzymatic activity of 193, 37, and 25U/L for Lac, LiP and MnP was recorded for the MLL containing 352mg/L NH4 +-N. The continuous bioreactor exhibited maximum values of 52, 54, 60, 58, and 75 percentage of COD, sCOD, TC, TOC, and color removal efficiency with MLL containing 352mg/L NH4 +-N that was pretreated at HRT 120h, while the maximum detected lignocellulosic enzymatic activities were 149, 27, and 16U/L for Lac, LiP, and MnP, respectively. A total of 64% COD reduction was achieved from the raw MLL considering 12% COD and 100% NH4 +-N reduction in the aerobic activated sludge sequencing batch reactor pretreatment process. The steady and higher removal efficiency of the bioreactor over the entire study period is promising for further exploration to enhance removal of refractory contaminants from the MLL.

Cloning and characterization of low-temperature adapted GH5-CBM3 endo-cellulase from Bacillus subtilis 1AJ3 and their application in the saccharification of switchgrass and coffee grounds

Endocellulase is a key cellulase for cellulosic material pretreatment in the industry by hydrolyzing long cellulose chains into short chains. To investigate the endocellulase characteristics from Bacillus subtilis 1AJ3, and increase its production yield, this paper cloned an endocellulase gene denoted CEL-5A from strain 1AJ3 and expressed in E. coli BL21 (DE3). The CEL-5A gene was sequenced with a full-length of 1500 bp, encoding a totally of 500 amino acids, and containing two domains: the GH5 family catalytic domain (CD) and the CBM3 family cellulose-binding domain (CBD). Recombinant endocellulase Cel-5A with a His-tag was purified of the Ni-NTA column, and SDS-PAGE results demonstrated that Cel-5A exhibited a molecular weight of 56.4 kDa. The maximum enzyme activity of Cel-5A was observed at pH 4.5 and 50 °C. Moreover, it was active over the broad temperature region of 30–60 °C, and stable within the pH range of 4.5–10.0. In addition, Co2+ was able to increase enzyme activity, while the majority of metal ions demonstrated stable enzyme activity under low- concentration. The substrate specificity of Cel-5A exhibited a high specific activity on the β-1,3-1,4 glucan linkage from barley. The Michaelis–Menten constant and the maximum velocity of the recombinant Cel-5A for CMC-Na were determined as 14.87 mg/mL and 19.19 μmol/min/mg, respectively. When Cel-5A was applied to the switchgrass and coffee grounds, its color became lighter and the biomass was observed to loosen following hydrolyzation. The saccharification rate reached 12% of the total weight of switchgrass in 20 h. These properties highlight the potential application of Cel-5A as an endocellulase in the pretreatment of biomass, for example, in the coffee grounds/waste, and related industries.

Optimization of cultural conditions for enhanced keratinase production by Bacillus cereus N14 obtained from the poultry farm of Himachal Pradesh (India)

To enhance the keratinolytic potential of bacterial strain Bacillus cereus N14, isolated from poultry farm of Sundernagar, District Mandi, Himachal Pradesh, different process parameters like temperature, pH, incubation time, inoculum size, inoculum age etc. were optimized using One Variable at a Time (OVAT) approach and Response Surface Methodology (RSM). Bacillus cereus N14 showed maximum enzyme activity (19.5 U/mL) after 3rd day of incubation at 35°C, pH 9.0, 12.5 (%) of inoculum size using 3 days old culture and 2.5 and 2.0 per cent of maltose and yeast extract as best carbon and nitrogen source, respectively in the presence of Mn2+ as divalent metal ion and EDTA as best media additive. Central composite design (CCD) of RSM was successfully applied to investigate the effect of 5 independent variables viz., incubation time (days), inoculums size (%), inoculum age (days), concentration of carbon and nitrogen source (%) on keratinase production. The maximum enzyme activity (74.86 U/mL) was attained by the B. cereus N14 at 22nd run. The main highlight of the present study is that a significant increase of 27.5 per cent in keratinase production after optimizing different process parameters, by Bacillus cereus N14 was observed.

Isolation of Thermostable Lignocellulosic Bacteria From Chicken Manure Compost and a M42 Family Endocellulase Cloning From Geobacillus thermodenitrificans Y7.

The composting ecosystem provides a potential resource for finding new microorganisms with the capability for cellulose degradation. In the present study, Congo red method was used for the isolating of thermostable lignocellulose-degrading bacteria from chicken manure compost. A thermophilic strain named as Geobacillus thermodenitrificans Y7 with acid-resident property was successfully isolated and employed to degrade raw switchgrass at 60°C for 5 days, which resulted in the final degradation rates of cellulose, xylan, and acid-insoluble lignin as 18.64, 12.96, and 17.21%, respectively. In addition, GC-MS analysis about aromatic degradation affirm the degradation of lignin by G. thermodenitrificans Y7. Moreover, an endocellulase gene belong to M42 family was successfully cloned from G. thermodenitrificans Y7 and expressed in Escherichia coli BL21. Recombinant enzyme Cel-9 was purified by Ni-NTA column based the His-tag, and the molecular weight determined as 40.4 kDa by SDA–PAGE. The characterization of the enzyme Cel-9 indicated that the maximum enzyme activity was realized at 50°C and pH 8.6 and, Mn2+ could greatly improve the CMCase enzyme activity of Cel-9 at 10 mM, which was followed by Fe2+ and Co2+. Besides, it also found that the β-1,3-1,4, β-1,3, β-1,4, and β-1,6 glucan linkages all could be hydrolyzed by enzyme Cel-9. Finally, during the application of enzyme Cel-9 to switchgrass, the saccharification rates achieved to 1.81 ± 0.04% and 2.65 ± 0.03% for 50 and 100% crude enzyme, respectively. All these results indicated that both the strain G. thermodenitrificans Y7 and the recombinant endocellulase Cel-9 have the potential to be applied to the biomass industry.

Discovery and Biochemical Characterization of a Methanol Dehydrogenase From Lysinibacillus xylanilyticus.

Bioconversion of C1 chemicals such as methane and methanol into higher carbon-chain chemicals has been widely studied. Methanol oxidation catalyzed by methanol dehydrogenase (Mdh) is one of the key steps in methanol utilization in bacterial methylotrophy. In bacteria, few NAD+-dependent Mdhs have been reported that convert methanol to formaldehyde. In this study, an uncharacterized Mdh gene from Lysinibacillus xylanilyticus (Lxmdh) was cloned and expressed in Escherichia coli. The maximum alcohol oxidation activity of the recombinant enzyme was observed at pH 9.5 and 55°C in the presence of 10 mM Mg2+. To improve oxidation activity, rational approach-based, site-directed mutagenesis of 16 residues in the putative active site and NAD+-binding region was performed. The mutations S101V, T141S, and A164F improved the enzyme’s specific activity toward methanol compared to that of the wild-type enzyme. These mutants show a slightly higher turnover rate than that of wild-type, although their KM values were increased compared to that of wild-type. Consequently, according the kinetic results, S101, T141, and A164 positions may related to the catalytic activity in the active site for methanol dehydrogenation. It should be further studied other mutant variants with high activity for methanol. In conclusion, we characterized a new Lxmdh and its variants that may be potentially useful for the development of synthetic methylotrophy in the future.

From Agricultural Waste to Biofuel: Enzymatic Potential of a Bacterial Isolate Streptomyces fulvissimus CKS7 for Bioethanol Production

To avoid a negative environmental and economic impact of agricultural wastes, and following the principles of circular economy, the reuse of agricultural wastes is necessary. For this purpose, isolation of novel microorganisms with potential biotechnological application is recommended. The current researches in bioethanol production are aimed to reduce the production costs using low-cost substrates and in-house produced enzymes by novel isolated microorganisms. In line with this, in this study valorization of these agricultural by-products by novel isolate S. fulvissimus CKS7 to biotechnological value added products was done. Standard microbiological methods were used for the isolation and characterization of strain. Enzymes activities were determinated using DNS method while, the ethanol concentration was determined based on the density of the alcohol distillate at 20 °C. The maximal enzymatic activities for amylase, cellulases (carboxymethyl cellulase and Avicelase), pectinase and xylanase were achieved using rye bran as a waste substrate for CKS7 growth. Obtained crude bacterial enzymes were used for enzymatic hydrolysis of lignocellulosic materials including horsetail waste, yellow gentian waste, corn stover, cotton material and corona pre-treated cotton material. The maximum yield of reducing sugars was obtained on horsetail waste and corona pre-treated cotton material. Waste brewer’s yeast Saccharomyces cerevisiae was successfully used for the production of bioethanol using horsetail waste hydrolysate and corona pre-treated cotton material hydrolysate. The obtained results showed that bacterial strain CKS7 has a significant, still unexplored enzymatic potential that could be used to achieve a cleaner, environmental friendly and economically acceptable biofuel production.