Kinetics Of Enzyme Production Research Articles

Utilization of wild Cressa cretica biomass for pectinase production from a halo-thermotolerant bacterium.

Halophytes are the native inhabitants of saline environment. Their biomass can be considered as a potential substrate for the production of microbial enzymes. This study was intended at feasible utilization of a halophytic biomass, Cressia cretica, for pectinase production using a halo- and thermo-tolerant bacterium, Bacillus vallismortis MH 10. The data from fractionation of the C. cretica biomass revealed presence of 17% pectin in this wild biomass. Seven different factors (temperature, agitation, pH, inoculum size, peptone concentration, substrate concentration and incubation time) affecting pectinase production using C. cretica were assessed through a statistical tool, Plackett-Burman design. Consequently, two significant factors (incubation time and peptone concentration) were optimized using the Central Composite design. The strain produced 20 IU mL-1 of pectinase after 24h under optimized conditions. The enzyme production kinetics data also confirmed that 24h is the most suitable cultivation period for pectinase production. Fourier transform infrared spectroscopy and scanning electron microscopy of C. cretica biomass ascertained utilization of pectin and structural changes after fermentation. The purification of pectinase by using DEAE column yielded specific activity and purification fold of 88.26 IU mg-1 and 3.2, respectively. The purified pectinase had a molecular weight of>65kDa. This study offers prospects of large-scale production of pectinase by halotolerant strain in the presence of economical and locally grown substrate that makes the enzyme valuable for various industrial operations. This article is protected by copyright. All rights reserved.

Optimization and production kinetics for cellulases by wild and mutant strain of Thermomyces dupontii in stirred tank reactor

The main bottleneck in fermentation technology is scaling up procedure of industrial enzymes according to the biological characteristics of the organism. The current study describes the production kinetics of cellulases in stirred tank reactor by using mutant and wild strains of T. dupontii. The fermentation span of both the strains in bioreactor was examined. It is was found in mutant strain of T. dupontii fermentation time required for optimum production was reduced to 48h as compared to 72h in wild strain. The kinetic studies also exhibited greater value of µ (h-1) in case of mutated strain in comparison with wild strain. The effects of some other critical factors like agitation intensity dissolve oxygen, aeration, temperature, size of inoculum and pH was estimated on enzyme production kinetics. The results shows maximum activity of cellulases was attained at 220 rpm, 15% dissolve oxygen, aeration rate 1.5 vvm, 55°C, 8 % inoculum size and pH 5.5 for both strains respectively. The higher values of enzyme production kinetics i.e. Yp/x, Qp, Qx and qp in STR in case of mutant strain indicates its superiority over wild strain of T. dupontii. Thus mutant thermophilic T. dupontii might be a potential candidate for industrial applications.

Purification, Characterization of Alkaline Cold Active Lipase from Acinetobacter radioresistens PR8 and Development of a New Zymography Method for Lipase Detection.

Bacterial lipases find so many industrial applications. Therefore, new source of lipase suitable for industrial conditions is always required. Lipase zymography methods use costly chromogenic substrates and indicator dyes and are few in numbers. The objectives of this work include lipase purification and its characterization from Acinetobacter radioresiens PR8 and development of new zymography method for lipase detection. The lipase was purified using conventional method and cation exchange chromatography and it was characterized biochemically and analytically. Based on these characterization new in-gel lipase zymography method was developed. In this present work, an alkalophilic lipase producing bacterium was isolated from soil; screened for extracellular lipase activity and identified to be Acinetobacter radioresistens PR8 (Genbank accession ID: MF073322). Enzyme production kinetics showed maximum production (4.16 U/ml at pH 9) of enzyme after 72 h. The lipase activity was found to be highest in olive oil (1% v/v; 8.1 U/ml). Low molecular weight (27 kDa) alkaline (pH 9) cold active (20 °C) lipase was purified from Acinetobacter radioresistens PR8. Lipase was characterized using PMF, FT-IR and its high conformational stability (Transition temperature: 122.3 °C) was attributed from its DSC spectrum. The importance of magnesium and sodium ions for enhancing lipase activity was obtained from flux balance analysis. Based on the lipase activating role of Mn2+ and Na+ ions, optimum temperature, pH with no chromogenic substrates and indicator dyes, a new in gel zymography method for lipase detection was developed.

Enzyme production by a fungoid marine protist, Thraustochytrium striatum

Thraustochytrium striatum is a fungoid marine protist and was shown to be a promising enzyme producer for potential industrial applications. This research aimed at studying extracellular enzymes secreted by T. striatum under different conditions with specific objectives to qualitatively identify enzymes, quantify the cell growth and enzyme production, correlate enzyme production with extracellular polymeric substances (EPS), and examine the induction of enzyme by polysaccharide substrates. The qualitative analysis showed that T. striatum can produce at least seven extracellular enzymes including lipase and six polysaccharases (i.e., amylase, CMCase, xylanase, chitinase, pectinase, and κ-carrageenase). The carbon and nitrogen concentrations and salinity significantly affected the kinetics of enzyme production. T. striatum produced decent amount of polysaccharases at all conditions, but negligible lipase. Amylase was the predominant enzyme and reached the highest activity of 750 U/L with glucose = 30 g/L, nitrogen source = 6 g/L and salinity = 100% sea water. Enzymes appeared to correlate with the production and monosaccharide composition of EPS. Enzyme-specific polysaccharide substrates including starch, CMC, xylan, κ-carrageenan, pectin, and chitin did not induce the production of corresponding enzymes by T. striatum while carbon starvation condition resulted in comparable enzyme activities, which indicated that enzymes from T. striatum were constitutive.

O POTENCIAL DO STREPTOMYCES ISOLADOS NA REGIÃO DE MAUÉS NA PRODUÇÃO DE ENZIMAS HIDROLÍTICAS

The enzymes of microbial production area has grown in the last decades due to use in various industrial fields, and with that the research needs to innovations of existing products. The hydrolytic enzymes are biological catalysts of great commercial importance for low cost and high availability. This enzyme class has received special attention about his versatility in several areas of industrial biotechnology. The Brazil-nuts is a possible source of nutrients for the growth of micro-organisms in search of effective enzyme production and low cost. Were used two strains of Streptomyces sp. or production of protease, lipase, amylase and alkaline phosphatase using the residue Brazil-nut as carbon source in fermentation, semi-solid and liquid for the medium ISP-2 at pH 7.2 at a temperature 37°C for 15 days. The results showed that the best enzymatic activity was to protease secreted by Streptomyces sp. (1N) in semi solid fermentation. Concomitantly, was carried out an enzyme production kinetics with peak activity of 0.196 U / ml activity for 144 hours. The specific activity was observed a value of 2.17 U / mg protein for 240 hours. The production of enzymes excreted by Streptomyces sp. using the residue of Brazil-nuts is a resource for obtaining enzymes with high potential for solid state fermentation.

Mathematical Modeling of Biomass and Enzyme Production Kinetics by Aspergillus niger in Solid-State Fermentation at Various Temperatures and Moisture Contents

The effect of temperature and substrate moisture content on the growth and production of amylase, protease and phytase by Aspergillus niger during solid-state fermentation was investigated. A mathematical model regarding the kinetics of growth and enzyme production was performed to calculate the parameters at different temperatures and substrate moisture contents. The growth kinetics of A. niger could be described by the logistic growth model; the mathematical modeling parameters regarding maximum specific growth rate (μmax) and maximum biomass concentration (Xmax) were obtained by fitting the experimental data to the logistic model. The enzyme production kinetics could be described by the Luedeking-Piret model. The mathematical modeling parameters which included the growth-associated formation constant of the product i (αi) and the non-growth-associated formation constant of the product i (βi) were calculated. The production of amylase, protease and phytase was shown to be exclusively growth-associated. The effect of temperature on μmax, Xmax and αi could be described by the cardinal temperature model with inflection (CTMI). Both growth and enzyme formation were clearly influenced by temperature and the optimum culture conditions for growth and enzyme production by A. niger were determined to be approximately 34°C with a substrate moisture content ranging from 40 to 60%.

Mathematical modeling of enzyme production using Trichoderma harzianum P49P11 and sugarcane bagasse as carbon source

A mathematical model to describe the kinetics of enzyme production by the filamentous fungus Trichoderma harzianum P49P11 was developed using a low cost substrate as main carbon source (pretreated sugarcane bagasse). The model describes the cell growth, variation of substrate concentration and production of three kinds of enzymes (cellulases, beta-glucosidase and xylanase) in different sugarcane bagasse concentrations (5; 10; 20; 30; 40gL−1). The 10gL−1 concentration was used to validate the model and the other to parameter estimation. The model for enzyme production has terms implicitly representing induction and repression. Substrate variation was represented by a simple degradation rate. The models seem to represent well the kinetics with a good fit for the majority of the assays. Validation results indicate that the models are adequate to represent the kinetics for a biotechnological process.

Production of manganese peroxidase and laccase in a solid-state bioreactor and modeling of enzyme production kinetics

Lignin-modifying enzymes have various promising applications such as biobleaching, biopulping, the functionalization of lignocellulosic materials, the modification of wood fibers, the remediation of contaminated soil and effluents, as well as improvement of the enzymatic hydrolysis of lignocellulosic substrates. In this study, the production of laccase and manganese peroxidase (MnP) in solid-state cultivation was examined. Oat husks were used as an inexpensive substrate for the white-rot fungus Cerrena unicolor PM170798 (FBCC 387). The addition of a fines fraction (consisting of oat flour and finely ground husks) enhanced MnP production fivefold and laccase production almost threefold. The enzyme production was studied first on a 100g scale, and the cultivation experiments were then repeated at a larger laboratory-scale (4kg) in a solid-state bioreactor. High enzyme activity levels were obtained (MnP: 340nkatg(-1)DM, laccase: 470nkatg(-1)DM). In addition, the correlation between the CO2 evolution rate and enzyme production was mathematically modeled from the bioreactor experimental data. The model parameters could be used to predict enzyme production.

Some Investigations on Protease Enzyme Production Kinetics UsingBacillus licheniformisBBRC 100053 and Effects of Inhibitors on Protease Activity

Due to great commercial application of protease, it is necessary to study kinetic characterization of this enzyme in order to improve design of enzymatic reactors. In this study, mathematical modeling of protease enzyme production kinetics which is derived fromBacillus licheniformisBBRC 100053 was studied (at 37°C, pH 10 after 73 h in stationary phase, and 150 rpm). The aim of the present paper was to determine the best kinetic model and kinetic parameters for production of protease and calculatingKi(inhibition constant) of different inhibitors to find the most effective one. The kinetic parametersKm(Michaelis-Menten constant) andVm(maximum rate) were calculated 0.626 mM and 0.0523 mM/min. According to the experimental results, using DFP (diisopropyl fluorophosphate) and PMSF (phenylmethanesulfonyl fluoride) as inhibitors almost 50% of the enzyme activity could be inhibited when their concentrations were 0.525 and 0.541 mM, respectively.Kifor DFP and PMSF were 0.46 and 0.56 mM, respectively. Kinetic analysis showed that the Lineweaver-Burk model was the best fitting model for protease production kinetics DFP was more effective than PMSF and both of them should be covered in the group of noncompetitive inhibitors.

Performance improvement of Bacillus aryabhattaiITBHU02 for high‐throughput production of a tumor‐inhibitory L‐asparaginase using a kinetic model based approach

AbstractBACKGROUNDAn unstructured model for batch culture production of L‐asparaginase by Bacillus aryabhattai ITBHU02, by means of the Luedeking–Piret incorporated logistic equation, was developed explaining the correlation between growth dynamics and enzyme production kinetics with respect to glucose depletion at different levels. The main goal was to build up a means for process optimization, design, control and analysis of L‐asparaginase production. Various thermodynamic parameters were estimated to comprehend enzyme stability and affiliated industrial applicability.RESULTSSpecific growth rate maxima were increased with increasing initial glucose concentration, whereas the specific productivity was best supported at a glucose concentration 5.0 g L‐1. The fermentative production of L‐asparaginase was greatly influenced by oxygen supply, reaching a maximum level at an aeration rate of 0.6 vvm. The activation energies for growth and death rate were 33.8±6.0 and 99.8±9.0 kJ mol‐1, respectively, with activation enthalpy values of enzyme formation and thermal deactivation 70.2±9.0 and 46.1±11.0 kJ mol‐1, respectively.CONCLUSIONThe economic production of L‐asparaginase by B. aryabhattai was achieved efficiently at low glucose concentration and mild aeration. Endogenous metabolism of the strain for L‐asparaginase synthesis was thermostable up to 40°C, which makes the strain commercially important as it can be utilized for cost‐effective L‐asparaginase production within countries such as India, where 35–40°C temperatures are quite common. © 2013 Society of Chemical Industry

Seleção de diferentes meios para produção de lipase a partir de Bacillis licheniformis (UCP 1014)

O gênero Bacillus tem se destacado como um excelente produtor de diversos produtos biotecnológicos no mercado mundial, principalmente as enzimas. As lipases microbianas, apresentam um grande potencial para aplicações comerciais devido sua estabilidade, seletividade e larga especificidade por substratos. A utilização e a elaboração de meios de produção para as enzimas microbianas têm sido bastante utilizada nas últimas décadas. Foram testados três meios de composições diferentes: Meio A (5,0% de peptona, 0,1% de NaNO3, 0,1% de MgSO4 e 1,0% de óleo de soja), Meio B (peptona 1%, NaCl 0,5%, CaCl2.2H2O 0,1%, Tween 20 1%) e o Meio C (glicose 1,0%, peptona, 2,0%, extrato de levedura 0,5%, óleo de oliva, 1,0%, NaNO3 0,1%, KH2PO4 0,1%, MgSO4. 7H2O 0,05%). Os resultados obtidos indicaram que o B. licheniformis obteve um melhor crescimento no Meio C, apresentando valores de pH variando de 5,8 a 7,5, e atividade lipolítica de 256 U/mL.

Growth and enzyme production kinetics of a heparinase-producing fungal isolate

Heparinases are microbially produced enzymes that degrade heparins, the sulfated glycosaminoglycans, to generate low-molecular-weight heparins (LMWHs). A heparinase-producing fungus, Aspergillus oryzae, was isolated from soil sample. The fungus produced heparinase, both extracellularly and intracellularly, in its late log phase of growth. Heparinase production and growth kinetics was studied in complex protein digest medium, with heparin employed as the inducer. Heparinase activity was found to be dependent on the rate of utilization of heparin during growth. Heparin was taken up rapidly in the initial exponential phase of the growth at a rate of 0.16 g cell−1 h−1. Enzyme activity increased at a rate of 1.53 Units h−1 with the exhaustion of heparin in the medium. Enhancement of enzyme production by 92% was achieved by manipulation of the fermentation conditions and medium ingredients.

Exo-polygalacturonase production by Penicillium roqueforti on pumpkin oil cake in solid state fermentation

The feasibility of using pumpkin oil cake (PuOC), individual and in combination with wheat bran (WB), as substrate for the production of Exo-polygalacturonase (Exo-p) by starter culture Penicillium roqueforti in solid state fermentation (SSF) has been evaluated. The kinetics of enzyme production was investigated using PuOC alone in the range from 13 to 168 h, with moisture contents varying from 44% the ability to grow and produce Exo-p activity on this substrate, reaching a maximum value of 1451.75 U/g.d.w PuOC by the 5th day of fermentation. Fermentation experiments indicated that the water activity (aw) influenced the enzyme production. A medium with aw 0.932 and the fermentation time of 5 days were selected, as these conditions resulted in the highest pectolytic activity and were used for further investigation. A next step in this research was to examine the effect of the substrate combination, PuOC with wheat bran (WB), in different ratios. The addition of WB as carbon sources was found to have a significant influence on the enzymes yields. Exo-p activities were the highest with initial water activity of a w 0.932 and PuOC supplementation with WB (1:0.67).

Modeling of enzyme production kinetics

Models of single cells, cell populations, and cultures can be most useful in organizing information in a comprehensive system description, as well as in optimizing and controlling actual production operations. Models discussed in this article are of various degrees of biological structure and mathematical complexity. The models are developed based on the biomass formation, substrate consumption, and product formation. the potentials asn the limitations of all the models have been reported. The parameter estimation by different methods has been discussed in this communication. These parameters will be helpful to explore the areas where future-modeling studies may be especially valuable.

Screening strains for directed biosynthesis of β- d-mono-glucuronide-glycyrrhizin and kinetics of enzyme production

One fungus, tentatively named Penicillium sp. Li-3, was screened to biosynthesize β- d-mono-glucuronide-glycyrrhizin (GAMG), directly. Using glycyrrhizin as elicitor and the sole carbon source, this strain was capable of expressing β- d-glucuronidase, one intracellular enzyme with high substrate specificity. And glycyrrhizin was hydrolyzed directly into GAMG by enzyme from Penicillium sp. Li-3 with high production. It was found that the mol conversion ratio of this reaction was up to 88.45%. Research about kinetics of β- d-glucuronidase production showed that the cell growth and enzyme production of this strain was partial coupled. During the expressing of target enzyme, carbon catabolite repression existed, so only glycyrrhizin was the best carbon source as well as the elicitor. It was found that the surfactant (Tween 80 0.12%) could improve the ability of enzyme production markedly. Under the condition of initial pH 4.8 of the medium and 32 °C of the culture temperature, the maximum enzyme activity of 181.53 U ml −1 was obtained.

Production of lipases by Rhizopus oligosporous by solid-state fermentation

The present investigation describes the production of lipase by solid-state fermentation. Ten mould cultures were screened for the production of lipases. Rhizopus oligosporous GCBR-3 supported maximum production of lipases (48.0 U/g substrate) under optimum environmental and cultural conditions such as selection of efficient organism from the available microorganisms, selection of almond meal as the best substrate among different substrates, inoculum size, extractants/diluents and incubation period. The organism secreted the enzyme extracellularly. To study the kinetics of enzyme production, the organism was grown under optimum growth conditions for enzyme production. The lipase value supported by the organism and calculated as units per gram substrate per hour was greater than the values reported by other workers. This organism can be exploited for large-scale production of enzyme for commercial purposes.

Protease production during growth and autolysis of submerged Metarhizium anisopliae cultures

The growth and autolysis of two strains of the entomopathogenic deuteromycete fungus Metarhizium anisopliae var. anisopliae were evaluated in medium containing casein or glucose as carbon source. Parameters such as economic coefficient and degree of autolysis were determined for each strain. Protease production was determined throughout the growth and autolysis phases of the cultures on medium under conditions of protease induction (in the presence of casein as sole source of carbon and nitrogen). The fungus was shown to utilize casein as a carbon/energy source in a more efficient manner than glucose. The autolysis shown by the strains was intense under both types of growth conditions, reaching up to 62.7% of the dry mass produced and started soon after the depletion of the exogenous carbon source. The relationship between the proteolytic activities of the two strains evaluated varied significantly (a maximum of 19.78 on the 5th day and a minimum of 2.03 on the 16th day of growth) during the various growth and autolysis phases, clearly showing that the difference between the growth curves and the difference in the kinetics of enzyme production may decisively affect the process of strain selection for protease production.

Enhanced production of Serratia marcescens chitinase in PEG/dextran aqueous two-phase systems

Extractive bioconversion was carried out in 2% (w/w) PEG 20,000 and 5% (w/w) dextran T500 aqueous two-phase systems (ATPS) for the production of extracellular chitinase from Serratia marcescens. Fermentation results were compared in the polymer-free reference system, the ATPS, and a polymer solution of PEG or dextran simulating the top and bottom phases of the ATPS. The cell growth and enzyme production kinetics were similar in all systems. However, enhanced enzyme production was observed in the presence of either polymer. Compared to the maximum enzyme activity in the reference system, the activities were 2.5, 1.9, and 3.1 times as high in the dextran solution, the PEG solution, and the ATPS, respectively. The mechanism responsible for the enhanced production of chitinase in ATPS was investigated. It is not caused by increased activity and stability of the enzyme, less adsorption of enzyme to chitin, or less protease production by the cells. PEG was found to cause cell autolysis, but this may represent only one of the effects of polymers on fermentation. Semicontinuous production of chitinase was carried out with repeated batch fermentation by intermittently removing the bottom phase, replenishing the fresh bottom phase containing chitin, and recycling the cell-containing top phase. This resulted in increasing enzyme activity and productivity in successive batches as a result of the removal of the inhibitory metabolite.

Simultaneous production and partition of chitinase during growth of Serratia marcescens in an aqueous two-phase system

Partition and production of the extracellular chitinase from Serratia marcescens were studied in PEG/dextran aqueous two-phase systems. The enzyme partitions into the bottom phase and the cells segregate into the top phase. The best system is 2% (w/v) PEG 20000 and 5% (w/v) dextran T500. The cell growth and enzyme production kinetics are similar in the aqueous two-phase system and in the polymer-free reference system. However, the maximum enzyme concentration in the former system is 1.5 times that in the latter one.

Isolation and characterization of a lambdapolA transducing phage.

A plaque-forming lambdapolA phage was isolated from a population of transducing phage made in vitro from Escherichia coli DNA and a phage vector digested with restriction endonuclease HindIII. Amber mutations, in genes whose products are necessary for late protein synthesis (Q) and cell lysis (S), were crossed into the lambdapolA phage. Infection of either polA+ or polA- bacteria with this phage, under conditions permitting DNA replication but preventing phage production and lysis, elevated the levels of DNA polymerase I to between 75- and 100-fold that detected in a wild-type strain. The kinetics of enzyme production suggest that the polA gene is transcribed from its own promoter rather than from any of the well-characterized phage promoters. The fragment of E. coli DNA within the lambdapolA phage comprises approximately 5000 base pairs, sufficient to accommodate the polA gene and one, or two, coding sequences for smaller proteins.