Marine-derived fungi: diversity of enzymes and biotechnological applications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

The filamentous fungus Paecylomices variotii was able to produce high levels of cell extract and extracellular invertases when grown under submerged fermentation (SbmF) and solid-state fermentation, using agroindustrial products or residues as substrates, mainly soy bran and wheat bran, at 40A degrees C for 72 h and 96 h, respectively. Addition of glucose or fructose (a parts per thousand yen1%; w/v) in SbmF inhibited enzyme production, while the addition of 1% (w/v) peptone as organic nitrogen source enhanced the production by 3.7-fold. However, 1% (w/v) (NH4)(2)HPO4 inhibited enzyme production around 80%. The extracellular form was purified until electrophoretic homogeneity (10.5-fold with 33% recovery) by DEAE-Fractogel and Sephacryl S-200 chromatography. The enzyme is a monomer with molecular mass of 102 kDa estimated by SDS-PAGE with carbohydrate content of 53.6%. Optima of temperature and pH for both, extracellular and cell extract invertases, were 60A degrees C and 4.0-4.5, respectively. Both invertases were stable for 1 h at 60A degrees C with half-lives of 10 min at 70A degrees C. Mg2+, Ba2+ and Mn2+ activated both extracellular and cell extract invertases from P. variotii. The kinetic parameters K-m and V-max for the purified extracellular enzyme corresponded to 2.5 mM and 481 U/mg prot(-1), respectively.

Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF

Sugarcane bagasse was characterized as a feedstock for the production of ethanol using hydrothermal pretreatment. Reaction temperature and time were varied between 160 and 200A degrees C and 5-20 min, respectively, using a response surface experimental design. The liquid fraction was analyzed for soluble carbohydrates and furan aldehydes. The solid fraction was analyzed for structural carbohydrates and Klason lignin. Pretreatment conditions were evaluated based on enzymatic extraction of glucose and xylose and conversion to ethanol using a simultaneous saccharification and fermentation scheme. SSF experiments were conducted with the washed pretreated biomass. The severity of the pretreatment should be sufficient to drive enzymatic digestion and ethanol yields, however, sugars losses and especially sugar conversion into furans needs to be minimized. As expected, furfural production increased with pretreatment severity and specifically xylose release. However, provided that the severity was kept below a general severity factor of 4.0, production of furfural was below an inhibitory concentration and carbohydrate contents were preserved in the pretreated whole hydrolysate. There were significant interactions between time and temperature for all the responses except cellulose digestion. The models were highly predictive for cellulose digestibility (R (2) = 0.8861) and for ethanol production (R (2) = 0.9581), but less so for xylose extraction. Both cellulose digestion and ethanol production increased with severity, however, high levels of furfural generated under more severe pretreatment conditions favor lower severity pretreatments. The optimal pretreatment condition that gave the highest conversion yield of ethanol, while minimizing furfural production, was judged to be 190A degrees C and 17.2 min. The whole hydrolysate was also converted to ethanol using SSF. To reduce the concentration of inhibitors, the liquid fraction was conditioned prior to fermentation by removing inhibitory chemicals using the fungus Coniochaeta ligniaria.

Physiology and Biotechnology of the Hydrogen Production with the Green Microalga Chlamydomonas reinhardtii

The hydrogen production in the green microalga Chlamydomonas reinhardtii was evaluated by means of a detailed physiological and biotechnological study. First, a wide screening of the hydrogen productivity was done on 22 strains of C. reinhardtii, most of which mutated at the level of the D1 protein. The screening revealed for the first time that mutations upon the D1 protein may result on an increased hydrogen production. Indeed, productions ranged between 0 and more than 500 mL hydrogen per liter of culture (Torzillo, Scoma et al., 2007a), the highest producer (L159I-N230Y) being up to 5 times more performant than the strain cc124 widely adopted in literature (Torzillo, Scoma, et al., 2007b). Improved productivities by D1 protein mutants were generally a result of high photosynthetic capabilities counteracted by high respiration rates. Optimization of culture conditions were addressed according to the results of the physiological study of selected strains. In a first step, the photobioreactor (PBR) was provided with a multiple-impeller stirring system designed, developed and tested by us, using the strain cc124. It was found that the impeller system was effectively able to induce regular and turbulent mixing, which led to improved photosynthetic yields by means of light/dark cycles. Moreover, improved mixing regime sustained higher respiration rates, compared to what obtained with the commonly used stir bar mixing system. As far as the results of the initial screening phase are considered, both these factors are relevant to the hydrogen production. Indeed, very high energy conversion efficiencies (light to hydrogen) were obtained with the impeller device, prooving that our PBR was a good tool to both improve and study photosynthetic processes (Giannelli, Scoma et al., 2009). In the second part of the optimization, an accurate analysis of all the positive features of the high performance strain L159I-N230Y pointed out, respect to the WT, it has: (1) a larger chlorophyll optical cross-section; (2) a higher electron transfer rate by PSII; (3) a higher respiration rate; (4) a higher efficiency of utilization of the hydrogenase; (5) a higher starch synthesis capability; (6) a higher per cell D1 protein amount; (7) a higher zeaxanthin synthesis capability (Torzillo, Scoma et al., 2009). These information were gathered with those obtained with the impeller mixing device to find out the best culture conditions to optimize productivity with strain L159I-N230Y. The main aim was to sustain as long as possible the direct PSII contribution, which leads to hydrogen production without net CO2 release. Finally, an outstanding maximum rate of 11.1 ± 1.0 mL/L/h was reached and maintained for 21.8 ± 7.7 hours, when the effective photochemical efficiency of PSII (ΔF/F'm) underwent a last drop to zero. If expressed in terms of chl (24.0 ± 2.2 µmoles/mg chl/h), these rates of production are 4 times higher than what reported in literature to date (Scoma et al., 2010a submitted). DCMU addition experiments confirmed the key role played by PSII in sustaining such rates. On the other hand, experiments carried out in similar conditions with the control strain cc124 showed an improved final productivity, but no constant PSII direct contribution. These results showed that, aside from fermentation processes, if proper conditions are supplied to selected strains, hydrogen production can be substantially enhanced by means of biophotolysis. A last study on the physiology of the process was carried out with the mutant IL. Although able to express and very efficiently utilize the hydrogenase enzyme, this strain was unable to produce hydrogen when sulfur deprived. However, in a specific set of experiments this goal was finally reached, pointing out that other than (1) a state 1-2 transition of the photosynthetic apparatus, (2) starch storage and (3) anaerobiosis establishment, a timely transition to the hydrogen production is also needed in sulfur deprivation to induce the process before energy reserves are driven towards other processes necessary for the survival of the cell. This information turned out to be crucial when moving outdoor for the hydrogen production in a tubular horizontal 50-liter PBR under sunlight radiation. First attempts with laboratory grown cultures showed that no hydrogen production under sulfur starvation can be induced if a previous adaptation of the culture is not pursued outdoor. Indeed, in these conditions the hydrogen production under direct sunlight radiation with C. reinhardtii was finally achieved for the first time in literature (Scoma et al., 2010b submitted). Experiments were also made to optimize productivity in outdoor conditions, with respect to the light dilution within the culture layers. Finally, a brief study of the anaerobic metabolism of C. reinhardtii during hydrogen oxidation has been carried out. This study represents a good integration to the understanding of the complex interplay of pathways that operate concomitantly in this microalga.

The production of the enzyme dextransucrase by batch and continuous fermentation techniques

A review of the literature of work carried out on dextransucrase production, purification, immobilization and reactions has been carried out. A brief review has also been made of the literature concerning general enzyme biotechnology and fermentation technology. Fed-batch fermentation of the bacteria Leuconostoc mesenteroides NRRL B512 (F) to produce dextransucrase has formed the major part of this research. Aerobic and anaerobic fermentations have been studied using a 16 litre New Brunswick fermenter which has a 3-12 litre working volume. The initial volume of broth used in the studies was 6 litres. The results of the fed-batch fermentations showed for the first time that yields of dextransucrase are much higher under the anaerobic conditions than during the aerobic fermentations. Dextransucrase containing 300-350 DSU/cm3 of enzyme activity has been obtained during the aerobic fermentations, while in the anaerobic fermentations, enzyme yields containing 450-500 DSU/cm3 have been obtained routinely. The type of yeast extract used in the fermentation medium has been found to have significant effects on enzyme yield. Of the different types studied, the Gistex Standard was found to be the type that favoured the highest enzyme production. Studies have also been carried out on the effect of agitation rate and antifoam on the enzyme production during the anaerobic experiments. Agitation rates of up to 600 rpm were found not to affect the enzyme yield, however, the presence of antifoam in the medium led to a significant reduction in enzyme activity (less than 300 DSU/cm3). Scale-up of the anaerobic fermentations has been performed at up to the 1000 litre level with enzyme yields containing more than 400 DSU/cm3 of activity being produced. Some of the enzyme produced at this scale was used for the first time to produce dextran on an industrial scale via the enzyme route, with up to 99% conversion of sucrose to dextran being obtained. An attempt has been made at continuous dextransucrase production. Cell washout was observed to occur at dilution rates of greater than 0.4 h-1. Dextransucrase containing up to 25 DSU/cm3/h has been produced continuously.

Xylitol production from DEO hydrolysate of corn stover by Pichia stipitis YS-30

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a d-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3-a dagger 1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. d-Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g(xylitol)/g(xylose)) and volumetric productivity (0.18 g(xylitol)/g(xylose) l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe(3+)-reductants. Phenolates were the major compounds with Fe(3+)-reducing activity in both fungi and displayed Fe(3+)-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe(3+) and H(2)O(2) (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum-a model brown rot fungus-other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.

Biological detoxification of different hemicellulosic hydrolysates using Issatchenkia occidentalis CCTCC M 206097 yeast

This work had as its main objective to contribute to the development of a biological detoxification of hemicellulose hydrolysates obtained from different biomass plants using Issatchenkia occidentalis CCTCC M 206097 yeast. Tests with hemicellulosic hydrolysate of sugarcane bagasse in different concentrations were carried out to evaluate the influence of the hydrolysate concentration on the inhibitory compounds removal from the sugarcane bagasse hydrolysate, without reduction of sugar concentration. The highest reduction values of inhibitors concentration and less sugar losses were observed when the fivefold concentrated hydrolysate was treated by the evaluated yeast. In these experiments it was found that the high sugar concentrations favored lower sugar consumption by the yeast. The highest concentration reduction of syringaldehyde (66.67%), ferulic acid (73.33%), furfural (62%), and 5-HMF (85%) was observed when the concentrated hydrolysate was detoxified by using this yeast strain after 24 h of experimentation. The results obtained in this work showed the potential of the yeast Issatchenkia occidentalis CCTCC M 206097 as detoxification agent of hemicellulosic hydrolysate of different biomass plants.

Inhibitory action of toxic compounds present in lignocellulosic hydrolysates on xylose to xylitol bioconversion by Candida guilliermondii

The inhibitory action of acetic acid, ferulic acid, and syringaldehyde on metabolism of Candida guilliermondii yeast during xylose to xylitol bioconversion was evaluated. Assays were performed in buffered and nonbuffered semidefined medium containing xylose as main sugar (80.0 g/l), supplemented or not with acetic acid (0.8-2.6 g/l), ferulic acid (0.2-0.6 g/l), and/or syringaldehyde (0.3-0.8 g/l), according to a 2(3) full factorial design. Since only individual effects of the variables were observed, assays were performed in a next step in semidefined medium containing different concentrations of each toxic compound individually, for better understanding of their maximum concentration that can be present in the fermentation medium without affecting yeast metabolism. It was concluded that acetic acid, ferulic acid, and syringaldehyde are compounds that may affect Candida guilliermondii metabolism (mainly cell growth) during bioconversion of xylose to xylitol. Such results are of interest and reveal that complete removal of toxic compounds from the fermentation medium is not necessary to obtain efficient conversion of xylose to xylitol by Candida guilliermondii. Fermentation in buffered medium was also considered as an alternative to overcome the inhibition caused by these toxic compounds, mainly by acetic acid.

Optimal recovery process conditions for manganese-peroxidase obtained by solid-state fermentation of eucalyptus residue using Lentinula edodes

Enzyme production is a growing field in biotechnology and increasing attention has been devoted to the solid-state fermentation (SSF) of lignocellulosic biomass for production of industrially relevant lignocellulose deconstruction enzymes, especially manganese-peroxidase (MnP), which plays a crucial role in lignin degradation. However, there is a scarcity of studies regarding extraction of the secreted metabolities that are commonly bound to the fermented solids, preventing their accurate detection and limiting recovery efficiency. In the present work, we assessed the effectiveness of extraction process variables (pH, stirring rate, temperature, and extraction time) on recovery efficiency of manganese-peroxidase (MnP) obtained by SSF of eucalyptus residues using Lentinula edodes using statistical design of experiments. The results from this study indicated that of the variables studied, pH was the most significant (p < 0.05%) parameter affecting MnP recovery yield, while temperature, extraction time, and stirring rate presented no statistically significant effects in the studied range. The optimum pH for extraction of MnP was at 4.0-5.0, which yielded 1500-1700 IU kg (1) of enzyme activity at extraction time 4-5 h, under static condition at room temperature. (C) 2011 Elsevier Ltd. All rights reserved.

Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with kappa-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA(app)) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g(-1) of gel for GLU, 7.76 mg g(-1) of gel for GLY, and 7.65 mg g(-1) of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g(-1) of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H(2)SO(4) with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, beta-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2(3) central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19-4.81%, w/w) and loadings of enzymes (1.9-38.1 FPU/g bagasse) and Tween 20 (0.0-0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.

The behavior of key enzymes of xylose metabolism on the xylitol production by Candida guilliermondii grown in hemicellulosic hydrolysate

A variety of raw materials have been used in fermentation process. This study shows the use of rice straw hemicellulosic hydrolysate, as the only source of nutrient, to produce high added-value products. In the present work, the activity of the enzymes xylose reductase (XR); xylitol dehydrogenase (XD); and glucose-6-phosphate dehydrogenase (G6PD) during cultivation of Candida guilliermondii on rice straw hemicellulosic hydrolysate was measured and correlated with xylitol production under different pH values (around 4.5 and 7.5) and initial xylose concentration (around 30 and 70 g l(-1)). Independent of the pH value and xylose concentration evaluated, the title of XD remained constant. On the other hand, the volumetric activity of G6PD increased whereas the level of XR decreased when the initial xylose concentration was increased from 30 to 70 g l(-1). The highest values of xylitol productivity (Q (P) a parts per thousand 0.40 g l(-1)) and yield factor (Y (P/S) a parts per thousand 0.60 g g(-1)) were reached at highest G6PD/XR ratio and lowest XR/XD ratio. These results suggest that NADPH concentrations influence the formation of xylitol more than the activity ratios of the enzymes XR and XD. Thus, an optimal rate between G6PD and XR must be reached in order to optimize the xylitol production.

Biotransformation using Mucor rouxii for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens

The goal of this study is to produce oleanolic acid derivatives by biotransformation process using Mucor rouxii and evaluate their antimicrobial activity against oral pathogens. The microbial transformation was carried out in shake flasks at 30A degrees C for 216 h with shaking at 120 rpm. Three new derivatives, 7 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, 7 beta,21 beta-dihydroxy-3-oxo-olean-12-en-28-oic acid, and 3 beta,7 beta,21 beta-trihydroxyolean-12-en-28-oic acid, and one know compound, 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, were isolated, and the structures were elucidated on the basis of spectroscopic analyses. The antimicrobial activity of the substrate and its transformed products was evaluated against five oral pathogens. Among these compounds, the derivative 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid displayed the strongest activity against Porphyromonas gingivalis, which is a primary etiological agent of periodontal disease. In an attempt to improve the antimicrobial activity of the derivative 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, its sodium salt was prepared, and the minimum inhibitory concentration against P. gingivalis was reduced by one-half. The biotransformation process using M. rouxii has potential to be applied to the production of oleanolic acid derivatives. Research and antimicrobial activity evaluation of new oleanolic acid derivatives may provide an important contribution to the discovery of new adjunct agents for treatment of dental diseases such as dental caries, gingivitis, and periodontitis.

Extending teaching and learning initiatives in the cross-disciplinary field of biotechnology

Our AUTC Biotechnology study (Phases 1 and 2) identified a range of areas that could benefit from a common approach by universities nationally. A national network of biotechnology educators needs to be solidified through more regular communication, biennial meetings, and development of methods for sharing effective teaching practices and industry placement strategies, for example. Our aims in this proposed study are to: a. Revisit the state of undergraduate biotechnology degree programs nationally to determine their rate of change in content, growth or shrinkage in student numbers (as the biotech industry has had its ups and downs in recent years), and sustainability within their institutions in light of career movements of key personnel, tightening budgets, and governmental funding priorities. b. Explore the feasibility of a range of initiatives to benefit university biotechnology education to determine factors such as how practical each one is, how much buy-in could be gained from potentially participating universities and industry counterparts, and how sustainable such efforts are. One of many such initiatives arising in our AUTC Biotech study was a national register of industry placements for final-year students. c. During scoping and feasibility study, to involve our colleagues who are teaching in biotechnology – and contributing disciplines. Their involvement is meant to yield not only meaningful insight into how to strengthen biotechnology teaching and learning but also to generate ‘buy-in’ on any initiatives that result from this effort.