ASP-Enzymosomes with Saccharomyces cerevisiae Asparaginase II Expressed in Pichia pastoris: Formulation Design and In Vitro Studies of a Potential Antileukemic Drug.

The bacterial enzyme asparaginase is the main treatment option for acute lymphoblastic leukemia. However, it causes side effects, such as immunological reactions, and presents undesirable glutaminase activity. As an alternative, we have been studying asparaginase II from Saccharomyces cerevisiae, coded by ASP3 gene, which was cloned and expressed in Pichia pastoris. The recombinant asparaginase (ASP) presented antileukemic activity and a glutaminase activity 100 times lower in comparison to its asparaginase activity. In this work, we describe the development of a delivery system for ASP via its covalent attachment to functionalized polyethylene glycol (PEG) polymer chains in the outer surface of liposomes (ASP-enzymosomes). This new delivery system demonstrated antiproliferative activity against K562 (chronic myeloid leukemia) and Jurkat (acute lymphocytic leukemia) cell lines similar to that of ASP. The antiproliferative response of the ASP-enzymosomes against the Jurkat cells suggests equivalence to that of the free Escherichia coli commercial asparaginase (Aginasa®). Moreover, the ASP-enzymosomes were stable at 4 °C with no significant loss of activity within 4 days and retained 82% activity up to 37 days. Therefore, ASP-enzymosomes are a promising antileukemic drug.

Colorimetric protein determination in microalgae (Chlorophyta): association of milling and SDS treatment for total protein extraction.

The use of colorimetric methods for protein quantification in microalgae is hindered by their elevated amounts of membrane-embedded intracellular proteins. In this work, the protein content of three species of microalgae was determined by the Lowry method after the cells were dried, ball-milled, and treated with the detergent sodium dodecyl sulfate (SDS). Results demonstrated that the association of milling and SDS treatment resulted in a 3- to 7-fold increase in protein quantification. Milling promoted microalgal disaggregation and cell wall disruption enabling access of the SDS detergent to the microalgal intracellular membrane proteins and their efficient solubilization and quantification.

Synthesis of disaccharides using ß-glucosidases from Aspergillus niger, A. awamori and Prunus dulcis.

OBJECTIVE: Glucose conversion into disaccharides was performed with ß-glucosidases from Prunus dulcis (ß-Pd), Aspergillus niger (ß-An) and A. awamori (ß-Aa), in reactions containing initial glucose of 700 and 900 g l-1. RESULTS: The reactions' time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l-1, the final substrate conversions were 33, 38, and 23.5% for ß-An, ß-Aa, and ß-Pd, respectively. The use of ß-An yielded 103 g gentiobiose l-1 (15.5% yield), which is the highest reported for a fungal ß-glucosidase. The increase in glucose concentration to 900 g l-1 resulted in a significant increase in disaccharide synthesis by ß-Pd, reaching 128 g gentiobiose l-1 (15% yield), while for ß-An and ß-Aa, there was a shift toward the synthesis of higher oligosaccharides. CONCLUSION: ß-Pd and the fungal ß-An and ß-Aa ß-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while ß-Pd showed the highest productivity for gentiobiose synthesis, ß-An presented the highest specificity.

Cloning novel sugar transporters from Scheffersomyces (Pichia) stipitis allowing D-xylose fermentation by recombinant Saccharomyces cerevisiae.

OBJECTIVES: Since uptake of xylose limits its fermentation, we aimed to identify novel sugar transporters from Scheffersomyces stipitis that allow xylose uptake and fermentation by engineered Saccharomyces cerevisiae. RESULTS: An hxt-null S. cerevisiae strain, lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) but having high xylose reductase, xylitol dehydrogenase and xylulokinase activities, was transformed with a genomic DNA library from S. stipitis. Four plasmids allowing growth on xylose contained three genes encoding sugar transporters: the previously characterized XUT1 permease, and two new genes (HXT2.6 and QUP2) not previously identified as xylose transporters. High cell density fermentations with the recombinant strains showed that the XUT1 gene allowed ethanol production from xylose or xylose plus glucose as carbon sources, while the HXT2.6 permease produced both ethanol and xylitol, and the strain expressing the QUP2 gene produced mainly xylitol during xylose consumption. CONCLUSIONS: Cloning novel sugar transporters not previously identified in the S. stipitis genome using an hxt-null S. cerevisiae strain with a high xylose-utilizing pathway provides novel promising target genes for improved lignocellulosic ethanol production by yeasts.

Production of perillic acid from orange essential oil by Yarrowia lipolytica using a top-aerated bioreactor.

R-(+)-Perillic acid, a promising anticancer and immunomodulatory agent, is the major product from the biotransformation of R-(+)-limonene-rich orange essential oil by the yeast Yarrowia lipolytica. Due to the abundance and low cost of orange essential oil, which is a byproduct of the citrus industry, we attempted to improve the biotransformation process by optimizing yeast cell mass production. Then, the whole process was transposed and adapted to a 2-L instrumented bioreactor. Cell mass production was optimized in shaker flasks using a statistical experimental design. The optimized medium (g·L-1: 22.9 glucose, 7.7 peptone, 4.1 yeast extract and 1.0 malt extract) resulted in a 13.0 g·L-1 final cell concentration and 0.18 g cell·L-1·h-1 productivity. A further increase to 18.0 g·L-1 was achieved in a 2-L bioreactor upon fed-batch culture. High-purity limonene bioconversion was performed in the same bioreactor utilizing top aeration to diminish terpene volatilization; as a result, 839.6 mg·L-1 perillic acid accumulated after 48 h. Under the same conditions, industrial orange essential oil afforded 806.4 mg·L-1 perillic acid. The yeast growth medium optimization resulted in a twofold increase in biomass accumulation and a reduction in growth medium nitrogen sources, which lowered the catalytic biomass production cost. Compared with conventional bottom aeration, the bioreactor top aeration strategy resulted in higher bioconversion rates. The conditions developed for high-purity limonene bioconversion were successfully applied to low-cost orange essential oil, showing the robustness of Y. lipolytica yeast.

Dry extrusion pretreatment of cassava starch aided by sugarcane bagasse for improved starch saccharification.

The enzymatic hydrolysis of native starch lacks efficiency because starch is mostly confined in semi-crystalline granules. To address the challenges associated with gelatinization and render native cassava starch (CS) amenable to enzymatic hydrolysis (enzyme cocktail from Aspergillus awamori and Trichoderma reesei), dry-extrusion pretreatment of CS mixed with sugarcane bagasse (SB) was studied. Results showed that among the CS:SB mass ratios studied (1:1; 1:0.5 and 1:0.25), extruded CS:SB (1:0.25) gave the highest 3-hour glucose yield (71.5%) after enzymatic hydrolysis. Extrusion reduced CS:SB (1:0.25) crystallinity by 78% and increased the intensity of all major FTIR absorption bands by 67-202%. The optimum 3-hour glucose yield from extruded CS:SB (1:0.25) hydrolysis was 74.1%, which was 330% higher than from untreated CS. The water absorption and solubility indices of the treated biomass increased by 145% and 12,640%, respectively under the optimum conditions, aiding the hydrolysis process. The dry extrudates were easy to manipulate and store.

Cellulolytic potential of a novel strain of Paenibacillus sp. isolated from the armored catfish Parotocinclus maculicauda gut.

A cellulolytic bacterial strain, designated P118, isolated from the gut of the tropical fish Parotocinclus maculicauda was identified as belonging to the genus Paenibacillus based on phenotypic and chemotaxonomic characteristics and the 16S rRNA gene sequence. The novel strain was Gram-positive, spore-forming and rod-shaped. Catalase but not oxidase was produced. Carboxymethylcellulose was hydrolyzed but starch or gelatin was not. Acetoin production was negative whereas nitrate reduction and urease production were positive. Many carbohydrates served as carbon sources for growth. MK-7 was the predominant isoprenoid quinone. Anteiso-C15:0 (38.73%) and C16:0 (20.85%) were the dominant cellular fatty acids. Strain P118 was closely related to Paenibacillus amylolyticus NRRL NRS-290, P. pabuli HSCC 492, P. tundrae Ab10b, P. xylanexedens B22a, and P. tylopili MK2 with 98.3-98.8% 16S rRNA gene sequence similarity. The results presented here suggest that strain P118 represents a novel species of the genus Paenibacillus and it is a potential strain for further studies concerning its role in the production of industrially important products from cellulosic biomass.

Constraints and advances in high-solids enzymatic hydrolysis of lignocellulosic biomass: a critical review.

The industrial production of sugar syrups from lignocellulosic materials requires the conduction of the enzymatic hydrolysis step at high-solids loadings (i.e., with over 15% solids [w/w] in the reaction mixture). Such conditions result in sugar syrups with increased concentrations and in improvements in both capital and operational costs, making the process more economically feasible. However, this approach still poses several technical hindrances that impact the process efficiency, known as the "high-solids effect" (i.e., the decrease in glucan conversion yields as solids load increases). The purpose of this review was to present the findings on the main limitations and advances in high-solids enzymatic hydrolysis in an updated and comprehensive manner. The causes for the rheological limitations at the onset of the high-solids operation as well as those influencing the "high-solids effect" will be discussed. The subject of water constraint, which results in a highly viscous system and impairs mixing, and by extension, mass and heat transfer, will be analyzed under the perspective of the limitations imposed to the action of the cellulolytic enzymes. The "high-solids effect" will be further discussed vis-à-vis enzymes end-product inhibition and the inhibitory effect of compounds formed during the biomass pretreatment as well as the enzymes' unproductive adsorption to lignin. This review also presents the scientific and technological advances being introduced to lessen high-solids hydrolysis hindrances, such as the development of more efficient enzyme formulations, biomass and enzyme feeding strategies, reactor and impeller designs as well as process strategies to alleviate the end-product inhibition. We surveyed the academic literature in the form of scientific papers as well as patents to showcase the efforts on technological development and industrial implementation of the use of lignocellulosic materials as renewable feedstocks. Using a critical approach, we expect that this review will aid in the identification of areas with higher demand for scientific and technological efforts.

Use of grape pomace for the production of hydrolytic enzymes by solid-state fermentation and recovery of its bioactive compounds.

A cocktail of biomass hydrolytic enzymes was produced by solid-state fermentation (SSF) by the mutant strain Aspergillus niger 3T5B8, using as substrate a mixture of grape pomace and wheat bran, and compared to the production when wheat bran was used as the sole substrate. The two enzymatic cocktails were subsequently used for the extraction of bioactive compounds from grape pomace and the relationship between the activities of the cocktail and the release of phenolic compounds was evaluated. Although the wheat bran SSF process was more effective for enzyme production, the enzymatic cocktail produced by the grape pomace - wheat bran mixture was more effective for the extraction of compounds with higher proanthocyanidins content and higher antioxidant potential (p < 0.05). A significant correlation between the bioactive compounds and enzyme activity was observed.

High-yield Bacillus subtilis protease production by solid-state fermentation.

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on pro tease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7-2.0 mg g(-1). A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5-10), but the same optimum temperature (37 degrees C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g(-1) and 15.4 U g-1 h-1 for SSF, and 12 U mL-1 and 1.3 U mL-1 h-1 for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.

Combining biomass wet disk milling and endoglucanase/ß-glucosidase hydrolysis for the production of cellulose nanocrystals.

Cellulose nanocrystals (CNCs), a biomaterial with high added value, were obtained from pure cellulose, Eucalyptus holocellulose, unbleached Kraft pulp, and sugarcane bagasse, by fibrillating these biomass substrates using wet disk milling (WDM) followed by enzymatic hydrolysis using endoglucanase/ß-glucosidase. The hydrolysis experiments were conducted using the commercial enzyme OptimashBG or a blend of Pyrococcus horikoshii endoglucanase and Pyrococcus furiosus ß-glucosidase. The fibrillated materials and CNCs were analyzed by X-ray diffraction, atomic force microscopy, scanning electron microscopy, and the specific surface area (SSA) was measured. WDM resulted in the formation of long and twisted microfibers of 1000-5000 nm in length and 4-35 nm in diameter, which were hydrolyzed into shorter and straighter CNCs of 500-1500 nm in length and 4-12 nm in diameter, with high cellulose crystallinity. Therefore, the CNC's aspect ratio was successfully adjusted by endoglucanases under mild reaction conditions, relative to the reported acidic hydrolysis method.

4-chlorophenol degradation by chloroperoxidase from Caldariomyces fumago: formation of insoluble products.

This study investigated the degradation of 4-chlorophenol (4-CP) by Caldariomyces fumago chloroperoxidase (CPO). Enzymatic oxidations were studied in reaction mixtures at pH 3.0, 4.0, and 6.0 in the presence and absence of Cl- containing 3.5 IU of CPO and 4-CP and hydrogen peroxide concentrations within the range of 0.5-50 and 0.005-50 mM, respectively. Distinct patterns of products regarding color, concentration, and solubility were observed. Reaction mixtures at pH 6.0 containing 3.5 IU of CPO and 5.0 mM 4-CP and H2O2 (1:1 stoichiometry) showed the highest 4-CP removal of 95% and the highest formation of a dark precipitate.

Effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A.

The effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A was studied in a bench-scale bioreactor using a previously optimized growth medium (0.65% yeast extract and 0.1% corn oil, pH 7.0) at 37 degrees C and natural pH. Airflow rates of 0.3, 1.0, and 1.5 vvm and a fixed agitation of 200 rpm were initially studied followed by 1.0 vvm and 200, 300, 400, and 500 rpm. The use of 1.0 vvm and 400 rpm increased enzyme concentration 1.8-fold (100-180 U/L) and process productivity 4.8-fold (1.4-6.7 U/[L h]) in comparison with the use of 200 rpm and 0.3 vvm. The inexpensive corn oil, used as carbon source, besides its antifoam properties, proved to be nonrepressive for enzyme production.

Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium.

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.

Kinetics of asparaginase II fermentation in Saccharomyces cerevisiae ure2dal80 mutant: effect of nitrogen nutrition and pH.

Although the quality of nitrogen source affects fermentation product formation, it has been managed empirically, to a large extent, in industrial scale. Laboratory-scale experiments successfully use the high-cost proline as a nonrepressive source. We evaluated urea as a substitute for proline in Saccharomyces cerevisiae ure2dal80 fermentations for asparaginase II production as a model system for nitrogen-regulated external enzymes. Maximum asparaginase II levels of 265 IU/L were observed in early stationary-phase cells grown on either proline or urea, whereas in ammonium cells, the maximum enzyme level was 157 IU/L. In all cases, enzyme stability was higher in buffered cultures with an initial pH of 6.5.

Xylose and xylose/glucose co-fermentation by recombinant Saccharomyces cerevisiae strains expressing individual hexose transporters.

Since the uptake of xylose is believed to be one of the rate-limiting steps for xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) with an integrative plasmid to overexpress the genes for xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using xylose, glucose, or xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose/glucose co-fermentations, but was incapable to allow xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient xylose fermentation, but during xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing xylose/glucose blends, whereas the HXT7 permease would be a better choice for xylose-enriched sugar streams.

Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrystalline cellulose and sugarcane bagasse pretreated by either ball milling or ionic liquid [Emim][Ac].

This study investigated the requirement of cellobiohydrolases (CBH) for saccharification of microcrystalline cellulose and sugarcane bagasse pretreated either by ball milling (BM) or by ionic liquid (IL) [Emim][Ac]. Hydrolysis was done using CBH-free blends of Pyrococcus horikoshii endoglucanase (EG) plus Pyrococcus furiosus ß-glucosidase (EGPh/BGPf) or Optimash™ BG while Acremonium Cellulase was used as control. IL-pretreated substrates were hydrolyzed more effectively by CBH-free enzymes than were the BM-pretreated substrates. IL-treatment decreased the crystallinity and increased the specific surface area (SSA), whereas BM-treatment decreased the crystallinity without increasing the SSA. The hydrolysis of IL-treated cellulose by EGPh/BGPf showed a saccharification rate of 3.92 g/Lh and a glucose yield of 81% within 9h. These results indicate the efficiency of CBH-free enzymes for the hydrolysis of IL-treated substrates.

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica.

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 °C) in a reaction medium containing 0.5% v/v limonene and 10 g/L of stationary phase cells (dry weight). The best results, corresponding to 564 mg/L of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 °C for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mg/L, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.

Major improvement in the rate and yield of enzymatic saccharification of sugarcane bagasse via pretreatment with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim] [Ac]).

In this study, sugarcane bagasse was pretreated by six ionic liquids (ILs) using a bagasse/IL ratio of 1:20 (wt%). The solubilization of bagasse in the ILs was followed by water precipitation. On using 1-ethyl-3-methylimidazolium acetate [Emim] [Ac] at 120 °C for 120 min, 20.7% of the bagasse components remained dissolved and enzymatic saccharification experiments resulted on 80% glucose yield within 6h, which evolved to over 90% within 24 h. Moreover, FE-SEM analysis of the precipitated material indicated a drastic lignin extraction and the exposure of nanoscopic cellulose microfibrils with widths of less than 100 nm. The specific surface area (SSA) of the pretreated bagasse (131.84 m2/g) was found to be 100 times that of untreated bagasse. The ability of [Emim] [Ac] to simultaneously increase the SSA and to decrease the biomass crystallinity is responsible for the improved bagasse enzymatic saccharification rates and yields obtained in this work.

Production of cellulolytic enzymes by fungi Acrophialophora nainiana and Ceratocystis paradoxa using different carbon sources.

Although a number of filamentous fungi, such as Trichoderma and Aspergillus, are well known as producers of cellulases, xylanases, and accessory cellulolytic enzymes, the search for new strains and new enzymes has become a priority with the increase in diversity of biomass sources. Moreover, according to the type of pretreatment applied, biomass of the same type may require different enzyme blends to be efficiently hydrolyzed. This study evaluated cellulases, xylanases, and beta-glucosidases produced by two fungi, the thermotolerant Acrophialophora nainiana and Ceratocystis paradoxa. Cells were grown in submerged culture on three carbon sources: lactose, wheat bran, or steam-pretreated sugarcane bagasse, a commonly used cattle feed in Brazil. Xylanase and endo-1-4-beta-glucanase (CMCase) highest production were found in A. nainiana growing on lactose and reached levels of 2,200 and 2,016 IU/L, respectively. C. paradoxa showed highest activity for xylanase when grown on wheat bran and for beta-glucosidase when grown on steam-treated bagasse, at levels of 12,728 and 1,068 IU/mL, respectively.