Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

Immobilization of fibrinolytic protease from Mucor subtilissimus UCP 1262 in magnetic nanoparticles.

This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.

A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA.

Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.

Application of lipase immobilized on a hydrophobic support for the synthesis of aromatic esters.

The present study aimed at preparing three biocatalysts via physical adsorption of lipases from Candida rugosa (CRL), Mucor javanicus, and Candida sp. on a hydrophobic and mesoporous support (Diaion HP-20). These biocatalysts were later applied to the synthesis of aromatic esters of apple peel and citrus (hexyl butyrate), apple and rose (geranyl butyrate), and apricot and pineapple (propyl butyrate). Scanning electron microscopy and gel electrophoresis confirmed a selective adsorption of lipases on Diaion, thus endorsing simultaneous immobilization and purification. Gibbs free energy (∆G) evinced the spontaneity of the process (-17.9 kJ/mol ≤ ∆G ≤ -5.1 kJ/mol). Maximum immobilized protein concentration of 30 mg/g support by CRL. This biocatalyst was the most active in olive oil hydrolysis (hydrolytic activity of 126.0 ± 2.0 U/g) and in the synthesis of aromatic esters. Maximum conversion yield of 89.1% was attained after 150 Min for the synthesis of hexyl butyrate, followed by the synthesis of geranyl butyrate (87.3% after 240 Min) and propyl butyrate (80.0% after 150 Min). CRL immobilized on Diaion retained around 93% of its original activity after six consecutive cycles of 150 Min for the synthesis of hexyl butyrate.

Penicillin G acylase production by Mucor griseocyanus and the partial genetic analysis of its pga gene.

Penicillin acylases (penicillin amidohydrolase, EC 3.5.1.11) are a group of enzymes with many applications within the pharmaceutical industry, and one of them is the production of semi-synthetic beta-lactam antibiotics. This enzyme is mainly produced by bacteria but also by some fungi. In the present study, the filamentous fungus Mucor griseocyanus was used to produce penicillin acylase enzyme (PGA). Its ability to express PGA enzyme in submerged fermentation process was assessed, finding that this fungal strain produces the biocatalyst of interest in an extracellular way at a level of 570 IU/L at 72 h of fermentation; in this case, a saline media using lactose as carbon source and penicillin G as inducer was employed. In addition, a DNA fragment (859 bp) of the pga from a pure Mucor griseocyanus strain was amplified, sequenced, and analyzed in silico. The partial sequence of pga identified in the fungi showed high identity percentage with penicillin G acylase sequences deposited in NCBI through BLAST, especially with the ß subunit of PGA from the Alcaligenes faecalis bacterium¸ which is a region involved in the catalytic function of this protein. Besides, the identification of domains in the penicillin G acylase sequence of Mucor griseocyanus showed three conserved regions of this protein. The bioinformatic results support the identity of the gen as penicillin G acylase. This is the first report that involves sequencing and in silico analysis of Mucor griseocyanus strain gene encoding PGA.

Annotation of AMP-activated protein kinase genes and its comparative transcriptional analysis between high and low lipid producing strains of Mucor circinelloides.

BACKGROUND: AMP-activated protein kinase (AMPK) is an important regulator for lipid accumulation, potentially known to have an inhibitory role in lipid synthesis. It inactivates acetyl-CoA carboxylase (ACC), an important regulatory enzyme required for lipid synthesis. However, in Mucor circinelloides, AMPK and its association with lipid accumulation has not been studied yet. OBJECTIVES: To identify AMPK genes in M. circinelloides and to compare their expression levels in high and low lipid-producing strains of M. circinelloides to predict the possible roles of AMPK in lipid metabolism and to select candidate genes for further studies to enhance lipid accumulation. RESULTS: Two genes for α-subunit, one for ß-subunit and six for γ-subunit were identified and annotated. Bioinformatic analysis confirmed the presence of typical conserved domains in these genes. Furthermore, transcriptional profiling displayed marked differences in expression kinetics of subunits among the selected strains. The expression of AMPK genes decreased rapidly in WJ11, high lipid producer strain during the lipid accumulation phase while contrasting profile of expression was observed in CBS 277.49, low lipid producer strain. CONCLUSION: The present study has shown the association of AMPK genes with lipid metabolism at the transcriptional level. The involvement of Snf-α1, Snf-α2, Snf-ß, Snf-γ1, Snf-γ4, Snf-γ5 subunits were shown to be more pronounced and could potentially be further explored in future studies.

The DASH-type Cryptochrome from the Fungus Mucor circinelloides Is a Canonical CPD-Photolyase.

Cryptochromes and photolyases are blue-light photoreceptors and DNA-repair enzymes, respectively, with conserved domains and a common ancestry [1-3]. Photolyases use UV-A and blue light to repair lesions in DNA caused by UV radiation, photoreactivation, although cryptochromes have specialized roles ranging from the regulation of photomorphogenesis in plants, to clock function in animals [4-7]. A group of cryptochromes (cry-DASH) [8] from bacteria, plants, and animals has been shown to repair in vitro cyclobutane pyrimidine dimers (CPDs) in single-stranded DNA (ssDNA), but not in double-stranded DNA (dsDNA) [9]. Cry-DASH are evolutionary related to 6-4 photolyases and animal cryptochromes, but their biological role has remained elusive. The analysis of several crystal structures of members of the cryptochrome and photolyase family (CPF) allowed the identification of structural and functional similarities between photolyases and cryptochromes [8, 10-12] and led to the proposal that the absence of dsDNA repair activity in cry-DASH is due to the lack of an efficient flipping of the lesion into the catalytic pocket [13]. However, in the fungus Phycomyces blakesleeanus, cry-DASH has been shown to be capable of repairing CPD lesions in dsDNA as a bona fide photolyase [14]. Here, we show that cry-DASH of a related fungus, Mucor circinelloides, not only repairs CPDs in dsDNA in vitro but is the enzyme responsible for photoreactivation in vivo. A structural model of the M. circinelloides cry-DASH suggests that the capacity to repair lesions in dsDNA is an evolutionary adaptation from an ancestor that only had the capacity to repair lesions in ssDNA.

Strong inhibitory activities and action modes of lipopeptides on lipase.

Lipopeptides have been reported to exhibit anti-obesity effects. In this study, we obtained a Bacillus velezensis strain FJAT-52631 that could coproduce iturins, fengycins, and surfactins. Results showed that the FJAT-52631 crude lipopeptide, purified fengycin, iturin, and surfactin standards exhibited strong inhibition activities against lipase with dose-dependence manners (half maximal inhibitory concentration (IC50) = 0.011, 0.005, 0.056, and 0.005 mg/mL, respectively). Moreover, fengycin and surfactin had the comparable activities with orlistat, but iturin not. It was revealed that the inhibition mechanism and type of the lipopeptides were reversible and competitive. The quenching mechanism of lipase was static and only one binding site between lipase and lipopoeptide was inferred from the fluorescence analysis. The docking analysis displayed that fengycin and surfactin could directly interact with the active amino acid residues (Ser or Asp) of lipase, but not with iturin. Our work suggests that the B. velezensis lipopeptides would have great potential to act as lipase inhibitors.

Preparative scale application of Mucor circinelloides ene-reductase and alcohol dehydrogenase activity for the asymmetric bioreduction of α,ß-unsaturated γ-ketophosphonates.

The fungus Mucor circinelloides exhibits high potential for green chemistry and technological applications. Recently M. circinelloides, which so far was considered mainly as a platform for biodiesel production, was found to exhibit high ene-reductase activity. In our current research we applied this promising microorganism to the biotransformation of a series of α,ß-unsaturated γ-ketophosphonates. The biotransformations were conducted using cheap corn steep liquor or minimal media. The products were obtained with excellent enantiomeric purity (>99% ee in most cases) and in good isolated yields, highlighting the great potential of this microorganism for asymmetric synthesis. Moreover, the products obtained may be further applied as chiral building blocks for the synthesis of biologically active compounds, such as glutamic acid or fosmidomycin derivatives.

Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization.

FAD-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is an enzyme utilized industrially in glucose sensors. Previously, FAD-GDH isolated from Mucor prainii (MpGDH) was demonstrated to have high substrate specificity for glucose. However, MpGDH displays poor thermostability and is inactivated after incubation at 45 °C for only 15 min, which prevents its use in industrial applications, especially in continuous glucose monitoring (CGM) systems. Therefore, in this study, a chimeric MpGDH (Mr144-297) was engineered from the glucose-specific MpGDH and the highly thermostable FAD-GDH obtained from Mucor sp. RD056860 (MrdGDH). Mr144-297 demonstrated significantly higher heat resistance, with stability at even 55 °C. In addition, Mr144-297 maintained both high affinity and accurate substrate specificity for D-glucose. Furthermore, eight mutation sites that contributed to improved thermal stability and increased productivity in Escherichia coli were identified. Collectively, chimerization of FAD-GDHs can be an effective method for the construction of an FAD-GDH with greater stability, and the chimeric FAD-GDH described herein could be adapted for use in continuous glucose monitoring sensors.

Lipase-Catalyzed Chemoselective Ester Hydrolysis of Biomimetically Coupled Aryls for the Synthesis of Unsymmetric Biphenyl Esters.

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions. The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time, temperature, pH, organic solvent, and water-organic solvent ratios, to improve the chemoselectivity, and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize the observed differences in efficacies of the lipases to convert biphenyl esters. The optimized reaction conditions were transferred to the preparative scale with high yields, underlining the potential of the presented biomimetic approach as an alternative strategy to the commonly used transition metal-based strategies for the synthesis of diverse biphenyl esters.

Novel Dual-Functional Enzyme Lip10 Catalyzes Lipase and Acyltransferase Activities in the Oleaginous Fungus Mucor circinelloides.

Lipases or triacylglycerol (TAG) lipases belong to the α/ß-hydrolases superfamily, which are enzymes capable of catalyzing the hydrolysis of the ester bond between fatty acids and glycerol. Interestingly, some lipases have been found to not only possess hydrolysis activity but also acyltransferase activity in yeasts and microalgae. Our present study reported a novel dual-functional Mucor circinelloides lipase Lip10 with a slight lipolysis activity but a noteworthy phospholipid/diacylglycerol acyltransferase (PDAT) activity. The purified Lip10 mutants prefer to utilize phosphatidyl serine to form TAG over phosphatidyl ethanolamine and phosphatidylcholine. Site-directed mutagenesis indicated that the histidine residue in the acyltransferase motif H-(X)4-D is indispensable for the PDAT activity of Lip10. Overexpression of the acyltransferase motif of Lip10 promoted cell growth by 12% and increased lipid production by 14% compared to the control, whilst overexpression of the lipase motif induced lipid degradation in M. circinelloides.

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

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

Thiographene synthesized from fluorographene via xanthogenate with immobilized enzymes for environmental remediation.

Graphene, graphene oxide and their related thiographene-, hydroxygraphene- or fluorographene-based materials have broad applications. We report on the thiol-functionalization of fluorographene via xanthogenate. Such thiographene contains 5.1 at% of sulphur in the form of thiol groups, which is the highest thiol content reported to date. Such tailored thiographene allows the immobilization of two types of enzymes. Here, we explore the functionalization of highly thiolated graphene with enzymes via physisorption or covalent linkage producing an important heterogeneous biocatalyst platform for wastewater treatment applications. Thiographene modified with a lipase from Mucor miehei can find utilization in lipid-rich wastewater treatment whereas the catalase-modified thiographene is intended for bioremediation applications. Upon increasing concentration of the thiol groups on graphene, protein loading of the catalase was increased by 16% and the ester bond cleavage activity of the thiographene-immobilized lipase was 129% that of the free lipase. We expect that such a highly active heterogeneous thiographene-based biocatalyst will find a use in water remediation applications.

Alcohol dehydrogenase 1 participates in the Crabtree effect and connects fermentative and oxidative metabolism in the Zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of adh mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh+ strain R7B, the ADH-defective (Adh-) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD+-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the adhl gene. Complementation with the wild-type allele adh1+ by transformation of M5 remedied all the defects caused by the adh1 mutation. These findings indicate that in M. circinelloides, the product of the adh1 gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of M. circinelloides possess low mRNA levels of the ethanol assimilation genes (adl2 and acs2), which could explain their inability to grow in the alcohol.

CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes.

Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the ß-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.

Produção e purificação integrada de protease fibrinolítica de Mucor subtilissimus UCP 1262 / Integrated production and purification of fibrinolytic protease from Mucor subtilissimus UCP 1262

As enzimas fibrinolíticas podem ser obtidas de micro-organismos por meio de processos fermentativos. O presente trabalho teve como objetivo avaliar a produção e extração integrada da protease fibrinolítica de Mucor subtilissimus UCP 1262 usando sistema de duas fases aquosas (SDFA). O processo integrado foi realizado para avaliar a produção, partição e recuperação da protease fibrinolítica, segundo planejamento experimental 23, utilizando como variáveis independentes a massa molar do polietileno glicol (PEG), a concentração do PEG e a concentração do sulfato de sódio. A maior atividade fibrinolítica (15,40U/mL) foi obtida na fase rica em sulfato de sódio no ensaio composto por 10% de sal e 18% de PEG 8000 (g/mol). Recuperações superiores a 80% foram obtidas. A protease fibrinolítica apresentou pH ótimo 7,0, estabilidade entre os pH 6,0 e 8,5, temperatura ótima 50°C, sendo estável de 10°C a 50°C. A enzima foi classificada como uma serino protease, com massa molecular de 52kDa. Como resultado, o processo é notavelmente eficaz para pré-purificar a protease fibrinolítica com baixo custo e rapidez significativa. Quando comparada a outras técnicas de produção e purificação isoladas, a fermentação extrativa é um processo digno a ser substituto das etapas iniciais de separação convencionais.(AU)

Fibrinolytic enzymes can be obtained from microorganisms through fermentative processes. The study aimed to evaluate the fibrinolytic protease production and integrated extraction from Mucor subtilissimus UCP 1262 by extractive fermentation using Aqueous Two-Phase Systems (ATPS). The integrated process was carried out to assess the production, partition and fibrinolytic enzyme recovery, according to a 2 3 -experimental design, using as independent variables Polyethylene glycol (PEG) molar mass, PEG and sodium sulphate concentration, concentration. The highest fibrinolytic activity (15.40U/mL) was obtained in sodium sulfate rich phase in the assay comprising of 10% of salt and 18% of PEG 8000 (g/mol). Yield greater than 80% was obtained. The fibrinolytic protease presented optimum pH 7.0 and stability between pH 6.0 and 8.5, and optimum temperature 50°C, stable between 10°C to 50°C. The enzyme was classified as a serine-protease with 52kDa of molecular weight. As a result, the process is remarkably effective to pre-purify the fibrinolytic protease with a low cost and significantly faster processing time. When compared to other isolated production and purification techniques the extractive fermentation is worthy of being a candidate to replace the initial stages of conventional separation processes.(AU)

Apocarotenoids produced from ß-carotene by dioxygenases from Mucor circinelloides.

Mucor circinelloides exhibits the complex sexual behaviour that is induced in other Mucoromycotina by a family of apocarotenoids called trisporoids. The genome of M. circinelloides contains four genes encoding putative carotenoid cleavage dioxygenases. The gene products of two of them were sufficient to convert ß-carotene into the precursors of three families of apocarotenoids, both in vitro and in the Escherichia coli heterologous in vivo system. The first of these products, CarS, cleaved the C40 ß-carotene into the C15 precursor of cyclofarnesoids and a C25 apocarotenal that was converted by the second enzyme, AcaA, into the C18 precursor of trisporoids and the C7 precursor of methylhexanoids. Apocarotenoids were not found in single or mixed cultures of the two strains of opposite sex, whose interaction readily produced zygospores, the sexual fusion cells.

Effect of acute exposure in swiss mice (Mus musculus) to a fibrinolytic protease produced by Mucor subtilissimus UCP 1262: An histomorphometric, genotoxic and cytological approach.

The fibrinolytic enzyme produced by Mucor subtilissimus UCP 1262 was obtained by solid fermentation and purified by ion exchange chromatography using DEAE-Sephadex A50. The enzyme toxicity was evaluated using mammalian cell lineages: HEK-293, J774.A1, Sarcoma-180 and PBMCs which appeared to be viable at a level of 80%. The biochemical parameters of the mice treated with an acute dose of enzyme (2000 mg/mL) identified alterations of AST and ALT and the histomorphometric analysis of the liver showed a loss of endothelial cells (P < 0.001). However, these changes are considered minimal to affirm that there was a significant degree of hepatotoxicity. The comet assay and the micronucleus test did not identify damage in the DNA of the erythrocytes of the animals treated. The protease did not degrade the Aα and Bß chains of human and bovine fibrinogens, thus indicating that it does not act as anticoagulant, but rather as a fibrinolytic agent. The assay performed to assess blood biocompatibility shows that at dose of 0.3-5 mg/mL the hemolytic grade is considered insignificant. Moreover, the enzyme did not prolong bleeding time in mice when dosed with 1 mg/kg. These results indicate that this enzyme produced is a potential competitor for developing novel antithrombotic drugs.

Sustainable enzymatic approaches in a fungal lipid biorefinery based in sugarcane bagasse hydrolysate as carbon source.

Single cell oil (SCO) was produced from enzymatically hydrolysed sugarcane bagasse by Mucor circinelloides. The fungus was cultured in the hydrolysate medium rich in glucose and xylose being able to assimilate both sugars simultaneously, attaining satisfactory values of lipid accumulation (25 wt%). The main concepts addressed herein were the utilization of these lipids for the production of (i) ethyl esters of fuel grade, and (ii) concentrate of polyunsaturated fatty acids for nutraceutical applications. It was noticed that the fungal lipids also contained carotenoids and that the fungal biomass presented lipolytic activity. The concept of integrating an M. circinelloides-based biorefinery into the sugarcane energy matrix may, thus, present a relevant alternative for the production of high value-added products.