Microbial Enzyme Production Research Articles

Statistics and mathematical modelling; A major recent modern tool in biotechnology and bioinformatics data analysis

Statistics and Mathematical models can be in many forms, including differential equations, or theoretic models. Lack of correlation between theoretical mathematical models and experimental measurements sometime leads to development of better theories in the area of biology. The mathematical design finds wide application in nutrient media optimization for microbial enzyme production. On the other hand these mathematical tools in the field of statistics play an important role with data analysis of proteomics and genomics studies. For the MS/MS spectroscopic data, micro array, RT-PCR data analysis, linkage study, gene-gene interaction studies are very difficult to interpret, but the statistical tools made it very easier from the conventional method of data analysis in both Biotechnology and Bioinformatics.

Reprint of “Plant regulation of microbial enzyme production in situ”

Soil extracellular enzymes regulate the rate at which complex organic forms of nitrogen (N) become bio-available. Much research has focused on the limitations to heterotrophic enzyme production via lab incubations, but little has been done to understand the limitations to enzyme production in situ. We created root and symbiotic mycelia exclusion treatments using mesh in-growth bags in the field to isolate the effect of roots and other portions of the microbial community on enzyme production. When fertilized with complex protein N we found increases in N-degrading enzyme concentrations only when root in-growth was allowed. No response was observed when complex N was added to root-free treatments. Expanding on economic rules of microbial element limitation theory developed from lab incubation data, we suggest this is due to active transport of labile carbon (C) from roots to associated microbial communities in root bags. Roots alleviate C limitation of microbial enzyme synthesis, representing a tradeoff between plants and microbes–plant C for microbially-derived N.

Fish processing wastes for microbial enzyme production: a review.

Fishery processing industries generate large amounts of by-products. The disposal of these wastes represents an increasing environmental and health problem. To avoid wasting these by-products, various disposal methods have been applied including, ensilation, fermentation, hydrolysate and fish oil production. Interestingly, fish by-products provide an excellent nutrient source for microbial growth useful in enzyme production process, which is largely governed by the cost related to the growth media. Fish wastes (heads, viscera, chitinous material, wastewater, etc.) were prepared and tested as growth substrates for microbial enzymes production such as protease, lipase, chitinolytic and ligninolytic enzymes. This new approach described in this review can reduce environmental problems associated with waste disposal and, simultaneously, lower the cost of microbial enzyme production.

Development of novel microbial systems for chitinolytic enzymes production

The nonlinearity of the biotechnological processes and the absence of cheap and reliable instrumentation require an enhanced modelling effort and estimation strategies for the state and the kinetic parameters. This work approaches nonlinear estimation strategies for microbial production of enzymes, exemplified by using a process of lipase production from olive oil by Candida rugosa. First, by using a dynamical mathematical model of this process, an asymptotic observer which reconstructs the unavailable state variables is proposed. The design of this kind of observers is based on mass and energy balances without the knowledge of kinetics being necessary; only minimal information concerning the measured concentrations is used. Second, a nonlinear high-gain observer is designed for the estimation of imprecisely known kinetics of the bioprocess. An important advantage of this high-gain estimator is that the tuning is reduced to the calibration of a single parameter. Numerical simulations in various scenarios are provided in order to test the behaviour and performances of the proposed nonlinear estimation strategies.

Influence of a diet rich in resistant starch on the degradation of non-starch polysaccharides in the large intestine of pigs

To investigate the effect of resistant starch to the degradation of other non-starch polysaccharides (NSPs) in the large intestine of pigs, two groups of pigs were fed either a diet containing digestible starch (DS) or a diet containing resistant starch (RS). Both diets contained NSPs from wheat and barley. Digesta from different parts of the large intestine were collected and analysed for sugar composition and carbohydrate-degrading-enzyme activities. Resistant starch, as well as β-glucans and soluble arabinoxylan, was utilised mainly in the caecum. The utilisation of β-glucans and soluble arabinoxylan in the caecum was higher in DS-fed pigs than in RS-fed pigs. Analyses on carbohydrate-degrading-enzyme activities demonstrated that microbial enzyme production was stimulated according to the diet composition, and the enzyme profile throughout the large intestine of RS-fed pigs indicated that the presence of resistant starch shifted the utilisation of NSPs to more distal parts of the colon.

A trait‐based approach for modelling microbial litter decomposition

Trait-based models are an emerging tool in ecology with the potential to link community dynamics, environmental responses and ecosystem processes. These models represent complex communities by defining taxa with trait combinations derived from prior distributions that may be constrained by trade-offs. Herein I develop a model that links microbial community composition with physiological and enzymatic traits to predict litter decomposition rates. This approach allows for trade-offs among traits that represent alternative microbial strategies for resource acquisition. The model predicts that optimal strategies depend on the level of enzyme production in the whole community, which determines resource availability and decomposition rates. There is also evidence for facilitation and competition among microbial taxa that co-occur on decomposing litter. These interactions vary with community investment in extracellular enzyme production and the magnitude of trade-offs affecting enzyme biochemical traits. The model accounted for 69% of the variation in decomposition rates of 15 Hawaiian litter types and up to 26% of the variation in enzyme activities. By explicitly representing diversity, trait-based models can predict ecosystem processes based on functional trait distributions in a community. The model developed herein illustrates that traits influencing microbial enzyme production are some of the key controls on litter decomposition rates.

Non-supplemented aqueous extract from dry olive mill residue: A possible medium for fungal manganese peroxidase production

The low moisture content of the dry olive mill residue (DOR), solid waste from the two-phase olive oil extraction process, enables its long-term storage and its ready reconstitution with water thus leading to a candidate liquid growth medium for the microbial production of enzymes of commercial interest. To test this hypothesis, two white-rot fungi (WRF), namely Lentinus tigrinus CBS 577.79 and Phlebia sp. DABAC 9, were grown in shaken cultures on non-supplemented aqueous extracts of the dry olive mill residue (ADOR) and compared for their ability to grow therein and produce Mn-dependent peroxidase (MnP). Then, in order to assess the feasibility of scale transfer to the reactor level, the two fungi were grown in a 3-L bubble-column reactor on 25% (w/v) ADOR at 28°C and an aeration rate of 0.3vvm. Under these conditions, L. tigrinus and Phlebia sp. produced 42.60±1.74 and 38.88±1.68 nkatal mL−1 MnP with respective mean volumetric productivities of 222.9 and 324.1nkatalL−1h−1. These results clearly show that the use of non-supplemented ADOR as a production medium enables a significant MnP production thus representing an excellent basis for process optimization.

Influence of litter chemistry and stoichiometry on glucan depolymerization during decomposition of beech (Fagus sylvatica L.) litter

Glucans like cellulose and starch are a major source of carbon for decomposer food webs, especially during early- and intermediate-stages of decomposition. Litter quality has previously been suggested to notably influence decomposition processes as it determines the decomposability of organic material and the nutrient availability to the decomposer community. To study the impact of chemical and elemental composition of resources on glucan decomposition, a laboratory experiment was carried out using beech (Fagus sylvatica, L.) litter from four different locations in Austria, differing in composition (concentration of starch, cellulose and acid unhydrolyzable residue or AUR fraction) and elemental stoichiometry (C:N:P ratio). Leaf litter was incubated in mesocosms for six months in the laboratory under controlled conditions. To investigate the process of glucan decomposition and its controls, we developed an isotope pool dilution (IPD) assay using 13C-glucose to label the pool of free glucose in the litter, and subsequently measured the dilution of label over time. This enabled us to calculate gross rates of glucose production through glucan depolymerization, and glucose consumption by the microbial community. In addition, potential activities of extracellular cellulases and ligninases (peroxidases and phenoloxidases) were measured to identify effects of resource chemistry and stoichiometry on microbial enzyme production. Gross rates of glucan depolymerization and glucose consumption were highly correlated, indicating that both processes are co-regulated and intrinsically linked by the microbial demand for C and energy and thereby to resource allocation to enzymes that depolymerize glucans. At early stages of decomposition, glucan depolymerization rates were correlated with starch content, indicating that starch was the primary source for glucose. With progressing litter decomposition, the correlation with starch diminished and glucan depolymerization rates were highly correlated to cellulase activities, suggesting that cellulose was the primary substrate for glucan depolymerization at this stage of decomposition. Litter stoichiometry did not affect glucan depolymerization or glucose consumption rates early in decomposition. At later stages, however, we found significant negative relationships between glucan depolymerization and litter C:N and AUR:N ratio and a positive relationship between glucan depolymerization and litter N concentration. Litter C:N and C:P ratios were negatively related to cellulase, peroxidase and phenoloxidase activities three and six months after incubation, further corroborating the importance of resource stoichiometry for glucan depolymerization after the initial pulse of starch degradation.

Production of xylanase and β-xylosidase from autohydrolysis liquor of corncob using two fungal strains

Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and β-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 °C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of β-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of β-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of β-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of β-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.

Selected Papers from ENZITEC 2010

The fundamental need for environmental preservation has been calling, around the world, for the development and deployment of friendly industrial processes. Moreover, presently, development has to take into account sustainability. Within this context, enzyme technology is of outmost importance, having a major role for the achievement of the goals of sustainable development. This technology is of particular significance to Brazil due to the need to preserve Brazil's singular ecosystems, biodiversity, and quality and availability of the country's water resources. Furthermore, Brazil has an unrivalled availability of natural wealth, in both diversity and quantity, to be processed, via biocatalysis, into a wide range of diverse and innovative products as well as into biofules. Enzymes are the logical tool to process renewable resources as both of them have naturally evolved to match each other. It is now, therefore, time to develop dexterity and expand the use of enzymes in a fully efficient manner. As biochemical processes are cleaner than its chemical counterparts and generate higher-quality products, this shift would benefit the country's socioeconomic development. However, going from traditional chemistry into enzymatic processes is more than a technical and economical challenge, it is a change in the way we think technology—scientific and technical knowledge— and thoughtfulness are much needed. The Brazilian Seminar on Enzyme Technology (ENZITEC) has been taking place every two years since 2003. Its main objective has been to further the knowledge on the wide range of industrial, technical, and specialty enzymes alongside promoting its economic viability. The event encompasses the worldwide interest in industrial enzyme research and uses vis-a-vis the necessary move toward biocatalysis. Eight papers, which were selected from a wealth of submitted manuscripts, were accepted for publication in this special issue of Enzyme Research. They relate to fundamental areas of enzyme research and technology such as microbial screening, enzymes production and characterization, fine chemistry, biorefinery, and biofuels. Denise M. G. Freire Elba P. S. Bon Gustavo Viniegra-Gonzalez Francisco Girio Robert F. H. Dekker

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.

Plant regulation of microbial enzyme production in situ

Soil extracellular enzymes regulate the rate at which complex organic forms of nitrogen (N) become bio-available. Much research has focused on the limitations to heterotrophic enzyme production via lab incubations, but little has been done to understand the limitations to enzyme production in situ. We created root and symbiotic mycelia exclusion treatments using mesh in-growth bags in the field to isolate the effect of roots and other portions of the microbial community on enzyme production. When fertilized with complex protein N we found increases in N-degrading enzyme concentrations only when root in-growth was allowed. No response was observed when complex N was added to root-free treatments. Expanding on economic rules of microbial element limitation theory developed from lab incubation data, we suggest this is due to active transport of labile carbon (C) from roots to associated microbial communities in root bags. Roots alleviate C limitation of microbial enzyme synthesis, representing a tradeoff between plants and microbes–plant C for microbially-derived N.

Plant regulation of microbial enzyme production in situ

Plant regulation of microbial enzyme production in situ

Plant regulation of microbial enzyme production in situ

AbstractSoil extracellular enzymes regulate the rate at which complex organic forms of nitrogen (N) become bio-available. Much research has focused on the limitations to heterotrophic enzyme production via lab incubations, but little has been done to understand the limitations to enzyme production in situ. We created root and symbiotic mycelia exclusion treatments using mesh in-growth bags in the field to isolate the effect of roots and other portions of the microbial community on enzyme production. When fertilized with complex protein N we found increases in N-degrading enzyme concentrations only when root in-growth was allowed. No response was observed when complex N was added to root-free treatments. Expanding on economic rules of microbial element limitation theory developed from lab incubation data, we suggest this is due to active transport of labile carbon (C) from roots to associated microbial communities in root bags. Roots alleviate C-limitation of microbial enzyme synthesis, representing a trade off between plants and microbes- plant C for microbial derived N.

Pectinase enzyme-complex production by Aspergillus spp. in solid-state fermentation: A comparative study

A comparative evaluation of three Aspergillus species according to their pectinase production in solid-state fermentation was performed. Solid-state fermentation offers several potential advantages for enzyme production by fungal strains. Utilization of agricultural by-products as low-cost substrates for microbial enzyme production resulted in an economical and promising process. The pectinolytic enzyme activities of two Aspergillus sojae strains were compared to a known producer, Aspergillus niger IMI 91881, and to A. sojae ATCC 20235, which was re-classified as Aspergillus oryzae. Evaluation of polymethylgalacturonase and polygalacturonase activity was performed as well as exo- vs. endo-enzyme activity in the crude pectinase enzyme-complex of the mentioned strains. Furthermore, a plate diffusion assay was applied to determine the presence and action of proteases in the crude extracts. A. sojae ATCC 20235 with highest polymethylgalacturonase activity and highest polygalacturonase activity both exo- and endo-enzyme activity, is a promising candidate for industrial pectinase production, a group of enzymes with high commercial value, in solid-state fermentation processes. Beside the enzymatic assays a protein profile of each strain is given by SDS-PAGE electrophoresis and in addition species-specific zymograms for pectinolytic enzymes were observed, revealing the differences in protein pattern of the A. sojae strains to the re-classified A. oryzae.

Biotechnological Potential of Agro-Industrial Wastes as a Carbon Source to Thermostable Polygalacturonase Production inAspergillus niveus

Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. The aim of this work was to study polygalacturonase (PG) production by Aspergillus niveus cultured on liquid or solid media supplemented with agro-industrial wastes. Submerged fermentation (SbmF) was tested using Czapeck media supplemented with 28 different carbon sources. Among these, orange peel was the best PG inducer. On the other hand, for solid state fermentation (SSF), lemon peel was the best inducer. By comparing SbmF with SSF, both supplemented with lemon peel, it was observed that PG levels were 4.4-fold higher under SSF. Maximum PG activity was observed at 55°C and pH 4.0. The enzyme was stable at 60°C for 90 min and at pH 3.0–5.0. The properties of this enzyme, produced on inexpensive fermentation substrates, were interesting and suggested several biotechnological applications.

Investigation of Liquid and Solid Fermentation Processes of the Fungus Aspergillus fumigatus for Protease Production

The production of microbial enzymes obeys the proper conditions of each microorganism species and the means applied to fermentation, being subject to the differences between liquid and solid fermentations. This way, filamentous fungi have been widely exploited as a productive source of proteases due to their growth capacity and a wide production range. The fungus A. fumigatuswas isolated from the soil having decaying wood. Production studies were conducted through liquid-state fermentation in Erlenmeyer flasks of 250mL with 50mL containing KH2PO4, 0,7%; K2HPO4, 0,2%; MgSO4.7H2O, 0,01%; citrate 2H2O, 0,05%; yeast extract, 0,1%; CaCl2.2H2O, 0,01% and peptone 1% with supplementation of casein and glucose. The solid fermentation was performed in Erlenmeyer flasks of 250mL using wheat bran as a substrate adding protein supplementation, e.g. ovalbumin. The partial characterization was donewithpartially-purified enzyme throughethanol precipitation. The partial characterization was done by using azocasein as a substrate. The peak protease production in liquid fermentation was of 96hours, the same production period obtained for the solid one. The protease resulted from liquid fermentation presented pH and optimumtemperatureof 8,0 and50 ◦C, respectively and thermostability of 50% after a 60-minute incubation at 45 ◦C, and belongs to the class of the serine proteases. The protease obtained from solid fermentation presented pH and optimum temperature of 8,0 and 50 ◦C, respectively and thermostability of 75% at 45 ◦C for 60minutes.Weconclude that theproteasesproducedby the fungus A. fumigatus in liquid and solid fermentation processes present the same biochemical characteristics, except for the thermostability. Future experiments of purification and biochemical characterizationwill be carried out for protease obtained by solid fermentation. Financial support: FAPESP N◦ 2008/11303-6.

The RNPP family of quorum-sensing proteins in Gram-positive bacteria

Quorum sensing is one of several mechanisms that bacterial cells use to interact with each other and coordinate certain physiological processes in response to cell density. This mechanism is mediated by extracellular signaling molecules; once a critical threshold concentration has been reached, a target sensor kinase or response regulator is activated (or repressed), facilitating the expression of quorum sensing-dependent genes. Gram-positive bacteria mostly use oligo-peptides as signaling molecules. These cells have a special kind of quorum-sensing systems in which the receptor protein interacts directly with its cognate signaling peptide. The receptors are either Rap phosphatases or transcriptional regulators and integrate the protein family RNPP, from Rap, Npr, PlcR, and PrgX. These quorum-sensing systems control several microbial processes, like sporulation, virulence, biofilm formation, conjugation, and production of extracellular enzymes. Insights of the mechanism of protein-signaling peptide binding as well as the molecular interaction among receptor protein, signaling peptide, and target DNA have changed some earlier perceptions. In spite of the increased knowledge and the potential biotechnological applications of these quorum-sensing systems, few examples on engineering for biotechnological applications have been published. Real applications will arise only when researchers working in applied microbiology and biotechnology are aware of the importance of quorum-sensing systems for health and bioprocess applications.

Pectin and Pectinases: Production, Characterization and Industrial Application of Microbial Pectinolytic Enzymes

Departamento de Bioquimica e Microbiologia Instituto de Biociencias Universidade Estadual Paulista, UNESP, Avenida 24A, 1515, CEP 13506-900 Rio Claro, SP

Brewer’s spent grain and corn steep liquor as substrates for cellulolytic enzymes production byStreptomyces malaysiensis

To evaluate cellulase production by Streptomyces malaysiensis in submerged fermentation using brewer's spent grain (BSG) and wheat bran (WB) as carbon source, and corn steep liquor (CSL) as nitrogen source, as compared to yeast extract (YE), and partial characterization of the crude enzyme. Maximum cellulase production by Streptomyces malaysiensis (720 U l(-1)) occurred within 4 days incubation when using a growth medium containing BSG 0.5% (w/v) and CSL1.2% (w/v). CMCases activity showed to be stable over an acidic pH range (2.0-7.0) and in temperatures of 40-60 degrees C. Zymogram indicated three bands of CMCase activity, with different molecular masses. S. malaysiensis was able to grow and produce good levels of CMCases using solely brewer's spent grain and corn steep liquor as low-cost substrates, making this strain and these low cost by-product worthy for further investigation, and potentially feasible for biotechnological applications in different areas. To our knowledge, this is the first study reporting the use of the low-cost by-products brewer's spent grain and corn steep liquor, as sole substrates for microbial enzyme production.