Increase In Enzyme Production Research Articles

Using Paper Waste and Local Bacterial Isolates to Produce Biomass and Cellulase Enzyme- A Study Waste Recycling

The study aims to isolate the cellulose-degradation bacteria that produce the cellulase enzyme as well as theproduction of SCP protein from Ramadi city soils and to obtain efficient local bacterial isolates in productionand efficient isolation was selected in the production of cellulase enzyme and single cell protein which isBacillus subtillis bacteria tested the efficiency of the isolated isolation to produce the enzyme and single cellprotein. The cell using local media contains the paper waste available in the environment and contaminatedwith it. Initial transactions were made for this paper waste and these factors included (heating to boilingpoint, dilute sulfuric acid and dilute sodium hydroxide). The paper waste used included the white cardboard,yellow cardboard and white paper Which was used as a single source of carbon and energy, after testingthe waste paper used, it was found that the white cardboard treated with heating gave the best production ofthe cellulase enzyme and single-cell protein. The bacterial isolation used showed the best growth and gavethe highest production of the enzyme and the single cell protein at pH 7, while the best temperature for theproduction of the enzyme was 45 ° C. The optimum temperature for the production of the single cell proteinwas 40 ° C. As for the carbon concentration, which is the white cardboard treated with heating, it was betterto produce than Concentration is 2%. As for the production of single cell protein, the highest output atconcentration was 4%. The best bacterial vaccine size for enzyme production was 3 ml / 100 ml medium. Asfor the production of single cell protein, the size of the inoculum gave 1.5 ml / 100 ml the best production.The addition of the organic nitrogen source (urea) gave a clear increase in enzyme production and singlecell protein, and when adding 0.5 g urea / 100 mL medium. Finally, the results showed that the best periodduration to obtain the highest cellulase enzyme production was after 96 hours, while it was found that thebest production of single-cell protein was after 24 hours incubation.

Impacts of UV Filters in Mytilus galloprovincialis: Preliminary Data on the Acute Effects Induced by Environmentally Relevant Concentrations

Ultraviolet (UV) filters are present in a broad range of personal hygiene products, which may be transported via aquatic environments and domestic wastewaters due to inefficient treating station sewage removal and direct human contact. The aim of the present study was to evaluate the potential effects of a UV filter, in particular benzophenone-3 (BP3) (also known as oxybenzone) on the mussel species Mytilus galloprovincialis. Mussels were exposed to this organic substance for 96 h in environmentally relevant concentrations (10, 100, and 1000 ng/L). After exposure, biomarkers related with the mussels’ metabolism and oxidative stress were evaluated. The results revealed significantly higher activity of electron transport system and energy reserves (glycogen and protein (PROT)) at the intermediate concentration of 100 ng/L, suggesting that at lower concentrations mussels’ metabolism was not activated due to low stress. Conversely, at the highest concentration (1000 ng/L), mussels were no longer able to continue to increase their metabolic activity. Higher metabolic capacity was accompanied by increased PROT content associated with increased enzyme production to activate their antioxidant system. Nevertheless, at the highest concentration, cellular damage occurred as a consequence of ineffective activation of antioxidant and biotransformation enzymes. The results of the present study address uncertainties that are fundamental to the environmental risk assessment and management of these economically important near-shore bivalves and other marine species. Although an acute exposure was performed, alterations observed indicate the negative impacts of BP3 towards marine bivalves, which could be enhanced after longer exposure periods or if mussels are simultaneously exposed to other stressors (e.g., other pollutants or climate change related factors). The present study may thus contribute to the definition of fundamental knowledge for the establishment of appropriate regulatory guidelines and practices that ensure the preservation and sustainability of biological resources, allowing for prediction and mitigation of the impacts from these compounds.

Increasing the Production of L-asparaginase from Bacillus substilis RRM-1 by UV-Mutation

L-asparaginase is an enzyme that can hydrolyze L-asparagine to L-glutamic acid and ammonia. This enzyme has essential usage for food and health. Bacillus subtilis RRM-1 produces enzyme L-asparaginase, yet the production of L-asparaginase from B. substilis RRM-1 still relatively low. Hence, the effort to increase the production of the enzyme is needed. For increasing the enzyme production, the bacteria were mutated using UV rays to increase the production of L-asparaginase. UV rays (15 watts) were exposed to bacteria with a distance of 15 cm and a contact duration of 30, 60, 90, 120 min. Survival rates of bacteria were calculated, and mutant bacteria were analyzed for their ability to produce L-asparaginase. The mutants examined were taken from a 120-minute mutation treatment, which produced six isolates (UV1, UV2, UV3, UV4, UV5, UV6). Mutant UV1 and UV6 showed better results in producing the enzyme L-asparaginase. Moreover, UV6 produces an activity of 8.02 ± 1.22 U/ml. UV6 production is higher than that of B. subtilis RM-1 wild type (3.18 ± 0.65 U/ml). Mutations with UV can produce mutants that can increase enzyme production by three times.

Purification of Keratinase from Bacillus sp. MD24 using Ammonium Sulfate Fractionation

A keratin degrading bacterium, Bacillus sp MD24, was isolated from soil. The crude keratinase produced by the bacterium has been reported to dehair goat skin. However, the dehairing process took 72 hours. In order to shorten the dehairing time it is necessary to increase the keratinase concentration. This could be done by optimizing keratinase production either finding the best fermentation media or optimizing fermentation condition. Another way to increase the concentration could be done by partially purifying the enzyme. Keratinase from Bacillus sp MD24 had been produced under submerged fermentation, however, it produced a relatively low amount of enzyme. Although an effort to increase enzyme production had been reported by solid state fermentation, the enzyme concentration was not enough for industrial purposes. This work aimed to increase enzyme concentration by partial purification through enzyme precipitation using ammonium sulphate. The research was conducted in three stages: (1) regeneration the bacterium, (2) production of keratinase, and (3) purification of keratinase with ammonium sulfate fractionation. Keratinase activity was measured by Anson method and protein concentration was measured by Lowry method. Enzyme purity was clarified using a combination of specific activity, purity level, and SDS-PAGE analysis. Based on the analysis result, ammonium sulphate did not act as a good precipitation agent for the keratinase. Two major bands were suggested as keratinase with an estimated molecular weight of 25 and 66 kDa as monomer and dimer form, respectively.

L-asparaginase production and enhancement by Sarocladium strictum: In vitro evaluation of anti-cancerous properties.

Utilization of l-asparaginase has been one of the effective strategies for the treatment of lymphoblastic leukaemia. Since the currently used bacterial l-asparaginase causes side effects, searching for new enzyme sources has been an active field of research. This study focuses on the characterization of an l-asparaginase-producing fungal strain. Sarocladium strictum was identified as a potent enzyme-producing strain. For the enhancement of enzyme production, we used two-level factorial design and response surface methodology. The optimization of significant factors showed a 1·84-fold increase in enzyme production. The Km and Vmax values of the enzyme were 9·74mmoll-1 and 8·19μmolmin-1 . The toxicity of the produced l-asparaginase was measured on K562 and HL60 cancer cell lines and L6 as normal cells. The IC50 values were calculated as 0·4 and 0·5IUml-1 for K562 and HL60 respectively and no significant effect was observed in L6. BrdU proliferation and caspase-3 activity assay in l-asparaginase treated HL60 and K562 cells indicated that cell proliferation rates and apoptotic cell death were reduced. The cytotoxic properties of the produced fungal enzyme indicated significant growth inhibition in cancer cells while having a little toxic effect on normal cells. The possibility of mass production alongside having suitable cytotoxic and kinetic properties suggest the probable use of the produced l-asparaginase for further researches as a potential chemotherapeutic agent. The lack of significant l-glutaminase activity and promising toxicity properties in S. strictum and the closer evolutionary relativeness of fungi enzymes to human enzymes compared to bacterial enzymes suggest a new source with lower toxicity and anti-cancerous properties, causing less side effect problems.

Expression of d-psicose-3-epimerase from Clostridium bolteae and Dorea sp. and whole-cell production of d-psicose in Bacillus subtilis

Purposed-psicose-3-epimerase (DPEase) catalyses the isomerisation of d-fructose to d-psicose, a rare sugar in nature with unique nutritional and biological functions. An effective industrial-scale method is needed for d-psicose production. Herein, the expression of a neutral and a slightly acidic pH DPEase in Bacillus subtilis was evaluated.MethodsTwo DPEase genes from Clostridium bolteae and Dorea sp. were separately expressed in B. subtilis via plasmid pSTOP1622, and an extra P43 promoter was employed to the expression cassette. The fermentation conditions of the engineered B. subtilis strains were also optimised, to facilitate both cell growth and enzyme production.ResultThe introduction of P43 promoter to the two DPEase genes increased enzyme production by about 20%. Optimisation of fermentation conditions increased DPEase production to 21.90 U/g at 55 °C and 24.01 U/g at 70 °C in B. subtilis expressing C. bolteae or Dorea sp. DPEase, equating to a 94.67% and 369.94% increase, respectively, relative to controls.ConclusionEnhanced DPEase production was achieved in B. subtilis expressing C. bolteae or Dorea sp. DPEase genes.

Recombinant xylanase production by Escherichia coli using a non-induced expression system with different nutrient sources

The application of enzymes for sustainable and low-environmental impact industrial processes requires high-level enzyme production at low-cost. A promising strategy is the use of a high efficiency heterologous protein expression system using E. coli and the pT7BsXA vector encoding the GH11 xylanase from Bacillus subtilis with promoter, replication origin and signal peptide sequences from B. subtilis (Ruller et al. 2006). This expression system produces high amounts of enzyme that are secreted to the culture broth. The present study aimed to maximize the xylanase production by this system through evaluation of culture medium composition. Different culture media previously described in the literature together with compositions derived from agro-industrial residues were evaluated. A culture medium derived from agro-industrial residues using sugarcane molasses as carbon source showed a 9-fold increase in enzyme production (195,000 U/L) in relation to LB medium and the lowest production cost, which was 8.5-fold lower than LB medium using sugarcane molasses as carbon source and brewer’s yeast as vitamin source in shaker experiments. In a bioreactor experiment the best production medium promoted an 8.5-fold higher production at a 10.8-fold lower cost as compared to shaker LB cultivation.

Ligno(hemi)cellulolytic Enzyme Profiles during the Developmental Cycle of the Royal Oyster Medicinal Mushroom Pleurotus eryngii (Agaricomycetes) Grown on Supplemented Agri-Wastes.

We have determined the production profiles of major ligno(hemi)cellulolytic enzymes at different stages of the mushroom development cycle during industrial scale cultivation of Pleurotus eryngii on supplemented agri-wastes. Endo-1,4-β-glucanase, cellobiohydrolase and endoxylanase levels remained relatively low during substrate colonization, increased sharply when small fruit bodies appeared, and peaked at maturation. β-Glucosidase and β-xylosidase levels decreased when substrate colonization was complete, increased with the appearance of small fruit bodies and primordia, respectively, and reached maxima at maturation. Laccase peaked along with substrate colonization but, after falling sharply in the upper substrate layers, remained relatively low until postinduction. Levels increased slightly when primordia appeared, fell to minimal values during the small and mature fruit body stages, and increased again postharvest. Manganese peroxidase (Mn-P) exhibited a similar pattern initially but high enzyme levels also coincided with primordia formation. Laccase and Mn-P activity patterns were compatible with a lignin-degradation function associated with substrate colonization and, in the former case, a putative role in fruit body morphogenesis. Based on the relatively low levels of polysaccharidases recorded during the initial stages of substrate colonization, we conclude that reducing sugar levels in noncolonized substrate were adequate for sustainable vegetative growth at that stage. We further conclude that the increase in enzyme production later in the developmental cycle was consistent with the replenishment of depleted reducing sugar from cellulose in the growth substrate to levels required for fruit body formation. These data provide new information describing combined temporal and spatial enzyme production profiles throughout the mushroom development cycle under a set of conditions used in industrial scale production.

Extracellular Tannase from Aspergillus ochraceus: Influence of the Culture Conditions on Biofilm Formation, Enzyme Production, and Application.

Aspergillus ochraceus biofilm, developed on an inert support, can produce tannase in Khanna medium containing 1.5% (w/v) tannic acid as the carbon source, at an initial pH of 5.0, for 72 h at 28 °C. Addition of 0.1% yeast extract increased enzyme production. The enzyme in the crude filtrate exhibited the highest activity at 30 °C and pH 6.0. At 50 °C, the half-life was 60 min and 260 min at pH 6.0. In general, addition of detergents and surfactants did not affect tannase activity significantly. Tannase has potential applications in various biotechnological processes such as the production of propyl gallate and in the treatment of tannin-rich effluents. The content of tannins and total phenolic compounds in effluents from leather treatment was reduced by 56-83% and 47-64%, respectively, after 2 h of enzyme treatment. The content of tannins and total phenolic compounds in the sorghum flour treated for 120 h with tannase were reduced by 61% and 17%, respectively. Interestingly, the same A. ochraceus biofilm was able to produce tannase for three sequential fermentative process. In conclusion, fungal biofilm is an interesting alternative to produce high levels of tannase with biotechnological potential to be applied in different industrial sectors.

Transcription Factor Atf1 Regulates Expression of Cellulase and Xylanase Genes during Solid-State Fermentation of Ascomycetes.

Transcriptional regulation of cellulolytic and xylolytic genes in ascomycete fungi is controlled by specific carbon sources in different external environments. Here, comparative transcriptomic analyses of Penicillium oxalicum grown on wheat bran (WB), WB plus rice straw (WR), or WB plus Avicel (WA) as the sole carbon source under solid-state fermentation (SSF) revealed that most of the differentially expressed genes (DEGs) were involved in metabolism, specifically, carbohydrate metabolism. Of the DEGs, the basic core carbohydrate-active enzyme-encoding genes which responded to the plant biomass resources were identified in P. oxalicum, and their transcriptional levels changed to various extents depending on the different carbon sources. Moreover, this study found that three deletion mutants of genes encoding putative transcription factors showed significant alterations in filter paper cellulase production compared with that of a parental P. oxalicum strain with a deletion of Ku70 (ΔPoxKu70 strain) when grown on WR under SSF. Importantly, the ΔPoxAtf1 mutant (with a deletion of P. oxalicumAtf1, also called POX03016) displayed 46.1 to 183.2% more cellulase and xylanase production than a ΔPoxKu70 mutant after 2 days of growth on WR. RNA sequencing and quantitative reverse transcription-PCR revealed that PoxAtf1 dynamically regulated the expression of major cellulase and xylanase genes under SSF. PoxAtf1 bound to the promoter regions of the key cellulase and xylanase genes in vitro This study provides novel insights into the regulatory mechanism of fungal cellulase and xylanase gene expression under SSF.IMPORTANCE The transition to a more environmentally friendly economy encourages studies involving the high-value-added utilization of lignocellulosic biomass. Solid-state fermentation (SSF), that simulates the natural habitat of soil microorganisms, is used for a variety of applications such as biomass biorefinery. Prior to the current study, our understanding of genome-wide gene expression and of the regulation of gene expression of lignocellulose-degrading enzymes in ascomycete fungi during SSF was limited. Here, we employed RNA sequencing and genetic analyses to investigate transcriptomes of Penicillium oxalicum strain EU2101 cultured on medium containing different carbon sources and to identify and characterize transcription factors for regulating the expression of cellulase and xylanase genes during SSF. The results generated will provide novel insights into genetic engineering of filamentous fungi to further increase enzyme production.

Statistical optimization and biochemical characterization of bioactive Bacillus megaterium 314 caseinase produced on egg shell and molokihya stalks

Abstract Egg shell (ES) was a good inducer for caseinase enzyme production by Bacillus megaterium 314 without adding supplements (only distilled water). Mixture of ES with molokihya stalk (MS) was the most suitable substrate that increased enzyme production by 4.7-fold. Optimized HCl pretreatment of ES using Central composite design enhanced enzyme production by 3.3-fold. Moreover, caseinase production was enhanced by 2.5-fold using Plackett-Burman (PB) followed by Central composite (CC) design. Enzyme showed maximum activity at temperature 50 °C and pH 9.0. Activation energy (Ea) and activation energy for the thermal denaturation (Ed) were 11.2 and 135.9 kJ/mol, respectively. Caseinase was highly tolerant to heat inactivation retained 93.6% of its initial activity after 1 h heating at 50 °C. In addition, the calculated half-life time (t0.5) and decimal reduction time (D-value) at 50 °C were 26.9 and 89.3 h, respectively. Moreover, thermodynamic parameters (enthalpy, Gibbs free energy and entropy) indicated that the enzyme was highly tolerant to heat inactivation. Sesame protein was the most suitable for hydrolysis by caseinase. Optimization of Sesame proteolysis using CC design causing 16.9-fold increases compared with the unoptimized. In vitro results showed promising biological activities as anticoagulation and fibrinolytic for caseinase preparation.

Enhanced production of alkaline protease by Neocosmospora sp. N1 using custard apple seed powder as inducer and its application for stain removal and dehairing

Alkaline proteases find extensive applications ranging from detergent industries to therapeutics due to their broad alkaline range activity. The present work describes the first report on optimization of alkaline protease production from Neocosmospora sp. N1 and explores its utility in detergent and leather industries. Wheat bran was found to be a suitable substrate for protease production under solid state fermentation amongst several low cost agro-based materials used in this study. Protease production was enhanced when wheat bran was supplemented with a novel inducer, custard apple seed powder in the ratio of 4:1 which increased enzyme production up to 1.88 fold. One factor at a time approach was used to select parameters important for production and response surface methodology was used for further optimization. The optimum level was attained at 123 h fermentation time, 63% moisture content, 1 × 10 8 spores/ml inoculum size and 1.4 mm particle size which resulted in 3.12 fold increase in protease production. The partially purified enzyme exhibited maximum activity at 60 °C and was active over a wide pH range of 8–12. Protease was compatible with various laundry detergents viz . Tide, Surf Excel, Ariel, Wheel etc. showing more than 80% stability even after 3 h of incubation and was efficient in removing blood stain from the cotton cloth. The enzyme was also more efficient in dehairing goat skin as compared to conventional leather processing treatment. • Statistical optimization of alkaline protease production in SSF from Neocosmospora species is studied. • The protease production increased 1.88 fold by use of custard apple seed powder as a novel inducer. • 3.12 fold enhancement in protease production was achieved by using Box-Behnken Design. • Neocosmospora sp. alkaline protease is efficient in removing blood stains having 80% compatibility with commercial detergents. • Protease produced cleaner, whiter, smoother skin as compared to sulphide treatment.

Amylase Production by Solid State Fermentation of Agro-industrial Wastes Using Bacillus species

This study evaluated amylase production by Bacillus species employing the solid state fermentation (SSF) method using five agro-industrial wastes namely corn cobs, potato peel and maize straw, groundnut husk and corn chaff. Five Bacillus species were tested for amylase production abilities and Bacillus subtilis showed the highest amylase production ability after incubation. Corn chaff gave maximum enzyme production (3.25 U/ml) while the least enzyme was recorded on groundnut husk (2.35 U/ml) at 25. Potato peel had maximum enzyme production by Bacillus subtilis (3.05 U/ml) at pH 7.0 while the least enzyme production was from groundnut husk (2.84 U/ml) at pH 4.0.Thus there was an increase in enzyme production with corresponding increase in substrate concentration. The results obtained in this study support the suitability of using agro-industrial wastes as solid state fermentation substrates for high production of amylase. It’s also a means of solving pollution problems thus making solid state fermentation an attractive method.

Production of highly active fungal milk-clotting enzyme by solid-state fermentation

Cheese production is projected to reach 20 million metric tons by 2020, of which 33% is being produced using calf rennet (EC 3.4.23.4). There is shortage of calf rennet, and use of plant and microbial rennets, hydrolyze milk proteins non-specifically resulting in low curd yields. This study reports fungal enzymes obtained from cost effective medium, with minimal down streaming, whose activity is comparable with calf and Mucor rennet. Of the fifteen fungi that were screened, Mucor thermohyalospora (MTCC 1384) and Rhizopus azygosporus (MTCC 10195) exhibited the highest milk-clotting activity (MCA) of 18,383 ± 486 U/ml and 16,373 ± 558 U/ml, respectively. Optimization exhibited a 33% increase in enzyme production (30 g wheat bran containing 6% defatted soy meal at 30 °C, pH 7) for M. thermohyalospora. The enzyme was active from pH 5–10 and temperature 45–55 °C. Rhizopus azygosporus exhibited 31% increase in enzyme production (30 g wheat bran containing 4% defatted soy meal at 30 °C, pH 6) and the enzyme was active from pH 6–9 at 50 °C. Curd yields prepared from fungal enzyme extract decreased (5–9%), when compared with calf rennet and Mucor rennet. This study describes the potential of fungal enzymes, hitherto unreported, as a viable alternative to calf rennet

Extracellular Expression of L-Aspartate-α-Decarboxylase from Bacillus tequilensis and Its Application in the Biosynthesis of β-Alanine.

L-aspartate-α-decarboxylase was extracellularly expressed to enhance its production for β-alanine biosynthesis. L-aspartate-α-decarboxylase and cutinase were coexpressed in Escherichia coli; more than 40% of the L-aspartate-α-decarboxylase was secreted into the medium. Selection of best conditions among tested variables enhanced L-aspartate-α-decarboxylase production by the recombinant strain. The total L-aspartate-α-decarboxylase activity reached 20.3U/mL. Analysis of the enzymatic properties showed that the optimum temperature and pH for L-aspartate-α-decarboxylase were 60°C and 7.5, respectively. Enzyme activity was stable at pH4.0-8.5 and displayed sufficient thermal stability at temperatures < 50°C. In addition, enzymatic synthesis of β-alanine was performed using extracellularly expressed L-aspartate-α-decarboxylase, and a mole conversion rate of > 99% was reached with a substrate concentration of 1.5M. Extracellular expression of L-aspartate-α-decarboxylase resulted in increased enzyme production, indicating its possible application in the enzymatic synthesis of β-alanine.

Valorization of Parthenium hysterophorus Biomass by its Utilization in Endoglucanase Production by Penicillium citrinum NAF5

The primary aim of the present study was to evaluate the potential of P. hysterophorus weed biomass valorization through its utilization in the production of cellulase enzyme. The work involved bio-prospecting of fungal microbes and selecting the most potent fungal isolate for its cellulolytic potential. Thereafter, endoglucanase production by the fungal isolate was enhanced by utilizing P. hysterophorus biomass as a substrate. The optimization study involved the production of the enzyme under both stationary as well as shaking conditions. A scale-up for the enzyme production was also carried out under batch mode of fermentation in Erlenmeyer flasks as well as in a 2 l-bioreactor. Penicillium citrinum NAF5, isolated from garden soil, was found as a potent cellulase producer. Under optimized conditions, P. citrinum NAF5 produced 4.224 ± 0.035 U/ml and 3.13 ± 0.026 U/ml endoglucanase in the stationary and shaking conditions of fermentation respectively, in the presence of steam pretreated P. hysterophorus biomass as the substrate. Scale-up in the flasks resulted in 3.86% and 12.11% increase in enzyme production under stationary and shaking conditions respectively; while 24.12% enhancement in enzyme production was achieved in a 2 l fermenter. The study reveals that the biomass from P. hysterophorus can be utilized for cellulase production, and this can be an effective approach for the valorization of this weed’s biomass. Also, cellulase production by P. citrinum NAF5 fungus is appreciable and holds potential for utilization in various applications.

Poly(l-lactide)-Degrading Enzyme Production by Laceyella sacchari LP175 Under Solid State Fermentation Using Low Cost Agricultural Crops and Its Hydrolysis of Poly(l-lactide) Film

The aim of this study was to investigate the enhancement of poly(l-lactide) (PLLA)-degrading enzyme production by Laceyella sacchari LP175 under solid state fermentation (SSF) using low-cost agricultural crops as substrates and its hydrolysis of poly(l-lactide) film. Cassava chip, soybean meal and corncob were used to investigate the enzyme production using a statistical mixture design method. The effect of various inducers and SSF growth conditions parameters affecting PLLA-degrading enzyme production were also investigated. The fermentation of solid substrates was up-scale in a static tray bioreactor. The crude enzyme extracted from the fermented solid substrate was used to evaluate the biological degradation of PLLA film at 50 °C for 24 h. The results of substrate combination showed that 5 g of the substrate mixture, consisting of 4.3 g cassava chips and 0.7 g soybean meal, yielded 320 U/g dry solid. The addition of 0.1 g peptone to the mixture of solid materials increased enzyme production up to 456 U/g dry solid. The production of enzyme in a static tray bioreactor with optimized physical conditions yielded the maximum enzyme production, 472 U/g dry solid. The 20 mL reaction consisting of 8600 mg/L PLLA film was degraded by the crude enzyme extracted from the fermented solid substrate. Agricultural crops and wastes contain significant amounts of nutrients for microbial growth and products. This is the first reported of PLLA degrading enzyme production under SSF using low-cost substrates showing the possibility for application in large-scale biological recycling of bio-plastic as a future sustainable process.

Environmental drivers and stoichiometric constraints on enzyme activities in soils from rhizosphere to continental scale

Microbial activity and functioning in soils are strongly limited by carbon (C) availability, of which a great proportion is released by living roots. Rhizodeposition and especially root exudates stimulate microbial activity and growth, and may shift the stoichiometric balance between C, N, and P. Thereby, exudates heighten microbial nutrient demand and acquisition of N and P from organic matter, leading to an increase in enzyme production. Aim of this study was to determine environmental controls of extracellular enzyme production, and hence on potential enzyme activities (Vmax) and substrate affinities (Km). To determine the controlling factors, we worked on four spatial scales from the microscale (i.e. rhizosphere) through the mesoscale (i.e. soil depth) and landscape scale (relief positions), and finally to the continental scale (1200 km transect within the Coastal Cordillera of Chile). Kinetics of seven hydrolyzing enzymes of the C, N, and P cycles (cellobiohydrolase, β‑glucosidase, β‑xylosidase, β‑N‑acetylglucosaminidase, leucine‑aminopeptidase, tyrosine‑aminopeptidase, and acid phosphatase) were related to soil texture, C and N contents, pH, and soil moisture via redundancy analysis (RDA). Potential activities of C, N, and P acquiring enzymes increased up to 7-times on the continental scale with rising humidity of sites and C and N contents, while substrate affinities simultaneously declined. On the landscape scale, neither Vmax nor Km of any enzyme differed between north and south slopes. From top- to subsoil (down to 120 cm depth) potential activities decreased (strongest of aminopeptidases under humid temperate conditions with up to 90%). Substrate affinities, however, increased with soil depth only for N and P acquiring enzymes. Affinities of cellobiohydrolase and β‑xylosidase, on the contrary, were 1.5- to 3-times higher in top- than in subsoil. Potential activities of N and P acquiring enzymes and β‑glucosidase increased form bulk to roots. Simultaneously, substrate affinities of N and P acquiring enzymes declined, whereas affinities of β‑glucosidase increased. These trends of activities and affinities in the rhizosphere were significant only for acid phosphatase. The RDA displayed a strong relation of potential activities of C and P acquiring enzymes and β‑N‑acetylglucosaminidase to C and N contents in soil as well as to the silt and clay contents. Aminopeptidase activity was mainly dependent on soil moisture and pH. We conclude that substrate availability for microorganisms mainly determined enzyme activity patterns on the continental scale by the humidity gradient. Patterns on the meso- and microscale are primarily controlled by nutrient limitation, which is induced by a shift of the stoichiometric balance due to input of easily available C by roots in the rhizosphere.

Statistical optimization of chitinase production by Streptomyces rubiginosus SP24 and efficacy of purified chitinase to control root-knot nematode infection in Vigna radiata under controlled conditions

BackgroundActinomycetes are known to produce various lytic enzymes such as chitinase which have different biotechnological aspects. Plackett–Burman design (PBD) was applied to evaluate significant components of medium to improve chitinase enzyme production. Response surface methodology was implemented to select those variables which are highly fitted in the model and helped in increasing enzyme production. A second experiment was performed as one of the major applications of statistically optimized and purified chitinase (method and result of purification not shown) for reducing root-knot nematode infection in roots known for causing diseases in plants. Among them, Meloidogyne sp. is one which is responsible for decreasing plant growth and fruit yield. For this, Meloidogyne sp. (1000 J2 stage) was used to infect Vigna radiata (mung) plant which was previously coated with formulations of purified chitinase and organic manure ratio—1:1 (w/w).ResultsFrom ANOVA results of PBD data analysis, it was confirmed that colloidal chitin, xylose, peptone and dihydrogen potassium phosphate were found to be significant components for chitinase enzyme production. The best combination of factors (gm) to accomplish the optimum response was found to be colloidal chitin: 1.0, xylose: 2.0, peptone: 0.6 and dihydrogen potassium phosphate: 0.25 for predicted response of 3.81. Seeds coated with purified chitinase (5 ml) isolated from Streptomyces rubiginosusSP24 helped in enhancing plant growth promotion and decreasing root-knot nematode infection.ConclusionStatistically optimized seeds of mung coated with purified chitinase was used for suppressing plant-parasitic nematodes which cause severe harm to the production of crops in terms of plant growth and fruit yield.

A combined treatment using ethylmethane sulfonate and ultraviolet light to compare amylase production by three Bacillus sp. isolates

In this study, three Bacillus sp.-producing amylase enzymes were isolated from soil samples and identified using 16S rDNA sequence analysis. Amylase production and total protein productions were spectrophotometrically measured. The following media were tested to increase enzyme production: LB medium and molasses. Three Bacillus sp. were identified as follows: Bacillus subtilis subtilis, Bacillus thuringiensis, and Bacillus cereus. Amylase production levels were in the range of 10 U/mL, whereas total protein production levels were at 15 mg/mL. Higher amylase activity was found in the Bacillus subtilis isolate. Ethylmethane sulfonate (EMS) and ultraviolet (UV) mutagenesis in combination were applied to compare amylase production. Amylase activity was increased to around 58% in the treatment with 0.03 mL of EMS and UV when compared to the control group. A pilot scale bioreactor with a total working volume of 10 liters was used to produce amylase by B. subtilis subtilis. In conclusion, B. subtilis subtilis can be used to produce amylase enzyme for various industrial purposes, and, for the first time, the amylase activities of B. subtilis can be enhanced with EMS and UV treatment.