A Holistic Approach to Circular Bioeconomy Through the Sustainable Utilization of Microalgal Biomass for Biofuel and Other Value-Added Products.

Emissions from transportation and industry primarily cause global warming, leading to floods, glacier melt, and rising seas. Widespread greenhouse gas emissions and resulting global warming pose significant risks to the environment, economy, and society. The need for alternative fuels drives the development of third-generation feedstocks: microalgae, seaweed, and cyanobacteria. These microalgae offer traits like rapid growth, high lipid content, non-competition with human food, and growth on non-arable land using brackish or waste water, making them promising for biofuel. These unique phototrophic organisms use sunlight, water, and carbon dioxide (CO2) to produce biofuels, biochemicals, and more. This review delves into the realm of microalgal biofuels, exploring contemporary methodologies employed for lipid extraction, significant value-added products, and the challenges inherent in their commercial-scale production. While the cost of microalgae bioproducts remains high, utilizing wastewater nutrients for cultivation could substantially cut production costs. Furthermore, this review summarizes the significance of biocircular economy approaches, which encompass the utilization of microalgal biomass as a feed supplement and biofertilizer, and biosorption of heavy metals and dyes. Besides, the discussion extends to the in-depth analysis and future prospects on the commercial potential of biofuel within the context of sustainable development. An economically efficient microalgae biorefinery should prioritize affordable nutrient inputs, efficient harvesting techniques, and the generation of valuable by-products.

Galactosylceramidase deficiency and pathological abnormalities in cerebral white matter of Krabbe disease.

Krabbe Disease (KD) is an autosomal recessive disorder that results from loss-of-function mutations in the GALC gene, which encodes lysosomal enzyme galactosylceramidase (GALC). Functional deficiency of GALC is toxic to myelin-producing cells, which leads to progressive demyelination in both the central and peripheral nervous systems. It is hypothesized that accumulation of psychosine, which can only be degraded by GALC, is a primary initiator of pathologic cascades. Despite the central role of GALC in KD pathomechanism, investigations of GALC deficiency at a protein level are largely absent, due in part, to the lack of sensitive antibodies in the field. Leveraging two custom antibodies that can detect GALC at endogenous levels, we demonstrated that GALC protein is predominantly localized to oligodendrocytes in cerebral white matter of an infant brain, consistent with its functional role in myelination. Mature GALC could also be quantitatively detected as a 26 kDa band by western blotting and correlated to enzyme activity in brain tissues. The p.Ile562Thr polymorphic variant, which is over-represented in the KD population, was associated with reduced mature GALC protein and activity. In three infantile KD cases, homozygous null mutations in GALC lead to deficiency in total GALC protein and activity. Interestingly, although GALC activity was absent, normal levels of total GALC protein were detected by a sandwich ELISA using our custom antibodies in a later-onset KD brain, which suggests that the assay has the potential to differentiate infantile- and later-onset KD cases. Among the infantile KD cases, we quantified a 5-fold increase in psychosine levels, and observed increased levels of acid ceramidase, a key enzyme for psychosine production, and hyperglycosylated lysosomal-associated membrane protein 1, a marker for lysosomal activation, in periventricular white matter, a major pathological brain region, when compared with age-matched normal controls. While near complete demyelination was observed in these cases, we quantified that an early-infantile case (age of death at 10 months) had about 3-fold increases in both globoid cells, a pathological hallmark for KD, and CD8-positive T lymphocytes, a pathological marker for multiple sclerosis, in the white matter when compared with a slower progressing infantile case (age of death at 21 months), which suggests a positive correlation between clinical severity and neuropathology. Taken together, our findings have advanced the understanding of GALC protein biology in the context of normal and KD brain white matter. We also revealed new neuropathological changes that may provide insights to understand KD pathogenesis.

Identification of a Novel Thermostable Alkaline Protease from Bacillus megaterium-TK1 for the Detergent and Leather Industry.

An increased need by the green industry for enzymes that can be exploited for eco-friendly industrial applications led us to isolate and identify a unique protease obtained from a proteolytic Bacillus megaterium-TK1 strain from a seawater source. The extracellular thermostable serine protease was processed by multiple chromatography steps. The isolated protease displayed a relative molecular weight (MW) of 33 kDa (confirmed by zymography), optimal enzyme performance at pH 8.0, and maximum enzyme performance at 70 °C with 100% substrate specificity towards casein. The proteolytic action was blocked by phenylmethylsulfonyl fluoride (PMSF), a serine hydrolase inactivator. Protease performance was augmented by several bivalent metal cations. The protease tolerance was studied under stringent conditions with different industrial dispersants and found to be stable with Surf Excel, Tide, or Rin detergents. Moreover, this protease could clean blood-stained fabrics and showed dehairing activity for cow skin with significantly reduced pollution loads. Our results suggest that this serine protease is a promising additive for various eco-friendly usages in both the detergent and leather industries.

Identification of Anti-Inflammatory Compounds from Hawaiian Noni (Morinda citrifolia L.) Fruit Juice.

Noni (Morinda citrifolia L.) fruit juice has been used in Polynesia as a traditional folk medicine and is very popular worldwide as a functional food supplement. In this study, compounds present in Hawaiian Noni fruit juice, with anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were identified. Five compounds were isolated using a bioassay-driven technique and phytochemical analysis of noni fruit juice: asperulosidic acid (1), rutin (2), nonioside A (3), (2E,4E,7Z)-deca-2,4,7-trienoate-2-O-ß-d-glucopyranosyl-ß-d-glucopyranoside (4), and tricetin (5). The structures of these five compounds were determined via NMR spectroscopy and LC/MS. In an anti-inflammatory assay, compounds 1-5 inhibited the production of nitric oxide (NO), which is a proinflammatory mediator, in LPS-stimulated macrophages. Moreover, the mechanisms underlying the anti-inflammatory effects of compounds 1-5 were investigated. Parallel to the inhibition of NO production, treatment with compounds 1-5 downregulated the expression of IKKα/ß, I-κBα, and NF-κB p65 in LPS-stimulated macrophages. Furthermore, treatment with compounds 1-5 downregulated the expression of nitric oxide synthase and cyclooxygenase-2. Thus, these data demonstrated that compounds 1-5 present in noni fruit juice, exhibited potential anti-inflammatory activity; these active compounds may contribute preventively and therapeutically against inflammatory diseases.

Hidden Twins: SorCS Neuroreceptors Form Stable Dimers.

SorCS1, SorCS2 and SorCS3 belong to the Vps10p-domain family of multiligand receptors. Genetic and functional studies have linked SorCS receptors to psychiatric disorders, Alzheimer's disease and type 2 diabetes, demonstrating critical roles in neuronal functionality and metabolic control. Surprisingly, their structural composition has so far not been studied. Here we have characterized SorCS1, SorCS2 and SorCS3 using biochemical methods and electron microscopy. We found that their purified extracellular domains co-exist in stable dimeric and monomeric populations. This was supported by co-immunoprecipitation experiments, where membrane-bound dimers were successfully pulled down from cell lysate. While dimers were virtually unbreakable, dimerization of the monomeric population was promoted through enzymatic deglycosylation. We conclude that post-translational modifications, specifically the degree and pattern of glycosylation, regulate the oligomeric state of the protein. Hence, cells may dictate ligand specificity by controlling the ratio between monomers and dimers and, therefore, regulate the multiple functions of SorCS receptors.

Characterization of a solvent, surfactant and temperature-tolerant laccase from Pleurotus sp. MAK-II and its dye decolorizing property.

OBJECTIVE: Laccase is industrially important but a major challenge is the production of an ideal laccase with suitable physicochemical properties to tolerate temperature, surfactants, metal ions and solvents towards its potential application in bioremediation. RESULTS: A laccase with a molecular mass of 43 kDa was purified from Pleurotus sp. MAK-II. It was optimally active at pH 4.5 and 60 °C using ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) as substrate. The laccase was susceptible to NaN3 and NaCN. Activity was strongly enhanced by Cu(2+), Mg(2+) and Ca(2+). The purified laccase showed stability towards various surfactants and solvents and decolorized, in the presence of violuric acid as redox mediator, the diazo dye Congo Red and the anthraquinone dye Remazol Brilliant Blue R to the extent of 96 and 72 %, respectively. CONCLUSION: The ideal physicochemical properties of Pleurotus sp. MAK-II-derived laccase suggest that it could be effectively used in the textile dye industry.

Characterization of a novel endoglucanase from Ganoderma lucidum.

We evaluated the production and characterization of endoglucanase from Ganoderma lucidum using different lignocellulose biomasses. We purified a novel carboxymethyl cellulose (CMC) hydrolyzing endoglucanase from the white-rot fungus G. lucidum when the medium was supplemented with 1% (w/v) wheat bran. Endoglucanase was purified 12.5-fold via ammonium sulfate fractionation, Sephadex G-100, and Q-Sepharose column chromatography with a final yield of 15%. SDS-PAGE analysis revealed that the endoglucanase had a molecular mass of 64.0 kDa. The optimal activity of purified endoglucanase was at pH 5.0 and 35 °C, though it was stable between pH 4.0-7.0 and temperatures of 30-60 °C. The purified enzyme was specific to CMC as a suitable substrate. The metal ions Hg(2+), Fe(2+), and Cr(2+) inhibited enzyme activity, while Ca(2+), Mg(2+), and Mn(2+) enhanced enzyme activity. The endoglucanase showed high activity and stability in the presence of different surfactants and non-polar hydrophobic organic solvents. This endoglucanase is tolerant to high temperature, metal ions, surfactants, and solvents, suggesting that it is appropriate for use in biomass conversion for biofuel production under harsh environmental conditions.

Characterization of lignocellulolytic enzymes from white-rot fungi.

The development of alternative energy sources by applying lignocellulose-based biofuel technology is critically important because of the depletion of fossil fuel resources, rising fossil fuel prices, security issues regarding the fossil fuel supply, and environmental issues. White-rot fungi have received much attention in recent years for their valuable enzyme systems that effectively degrade lignocellulosic biomasses. These fungi have powerful extracellular oxidative and hydrolytic enzymes that degrade lignin and cellulose biopolymers, respectively. Lignocellulosic biomasses from either agricultural or forestry wastes are abundant, low-cost feedstock alternatives in nature but require hydrolysis into simple sugars for biofuel production. This review provides a complete overview of the different lignocellulose biomasses and their chemical compositions. In addition, a complete list of the white-rot fungi-derived lignocellulolytic enzymes that have been identified and their molecular structures, mechanism of action in lignocellulose hydrolysis, and biochemical properties is summarized in detail. These enzymes include ligninolytic enzymes (laccase, manganese peroxidase, lignin peroxidase, and versatile peroxidase) and cellulolytic enzymes (endo-glucanase, cellobiohydrolase, and beta-glucosidase). The use of these fungi for low-cost lignocellulolytic enzyme production might be attractive for biofuel production.

Strep-tag II and Twin-Strep based cassettes for protein tagging by homologous recombination and characterization of endogenous macromolecular assemblies in Saccharomyces cerevisiae.

Peptide sequences fused to a gene of interest facilitate the isolation of proteins or protein complexes from cell extracts. In the case of fluorescent protein tags, the tagged protein can be visually localized in living cells. To tag endogenous genes, PCR-based homologous recombination is a powerful approach used in the yeast Saccharomyces cerevisiae. This approach uses short, homologous DNA sequences that flank the tagging cassette to direct recombination. Here, we constructed a set of plasmids, whose sequences were optimized for codon usage in yeast, for Strep-tag II and Twin-Strep tagging in S. cerevisiae. Some plasmids also contain sequences encoding for a fluorescent protein followed by the purification tag. We demonstrate using the yeast pyruvate dehydrogenase (PDH) complex that these plasmids can be used to purify large protein complexes efficiently. We furthermore demonstrate that purification from the endogenous pool using the Strep-tag system results in functionally active complexes. Finally, using the fluorescent tags, we show that a kinase and a phosphatase involved in regulating the activity of the PDH complex localize in the cells' mitochondria. In conclusion, our cassettes can be used as tools for biochemical, functional, and structural analyses of endogenous multi-protein assemblies in yeast.

Brain site-specific proteome changes in aging-related dementia.

This study is aimed at gaining insights into the brain site-specific proteomic senescence signature while comparing physiologically aged brains with aging-related dementia brains (for example, Alzheimer's disease (AD)). Our study of proteomic differences within the hippocampus (Hp), parietal cortex (pCx) and cerebellum (Cb) could provide conceptual insights into the molecular mechanisms involved in aging-related neurodegeneration. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) brain site-specific proteomic strategy, we identified 950 proteins in the Hp, pCx and Cb of AD brains. Of these proteins, 31 were significantly altered. Most of the differentially regulated proteins are involved in molecular transport, nervous system development, synaptic plasticity and apoptosis. Particularly, proteins such as Gelsolin (GSN), Tenascin-R (TNR) and AHNAK could potentially act as novel biomarkers of aging-related neurodegeneration. Importantly, our Ingenuity Pathway Analysis (IPA)-based network analysis further revealed ubiquitin C (UBC) as a pivotal protein to interact with diverse AD-associated pathophysiological molecular factors and suggests the reduced ubiquitin proteasome degradation system (UPS) as one of the causative factors of AD.

Brain-site-specific proteome changes induced by neuronal P60TRP expression.

p60 transcription regulator protein (p60TRP) facilitates the processing of the amyloid precursor protein towards the non-amyloidogenic pathway by inhibiting the ß-secretase action. This protein was initially identified to be downregulated in the temporal lobe of brains from Alzheimer's disease patients. p60TRP is one of the G-protein-coupled receptor (GPCR)-associated proteins which directly influences the signalling capacity of GPCRs. In the present study, we investigated the brain-region-specific proteome profile of transgenic p60TRP mice to gain an insight into the molecular events mediated by the long-term effect of neuronal p60TRP overexpression on brain proteome changes and its potential implication for neuronal functions in the central nervous system. Using a proteomics research approach based on isobaric tags for relative and absolute quantitation, we identified 2,025 proteins, whereby 1,735 proteins were quantified, out of which 56 were found to be significantly altered in the cortex and/or hippocampus of neuronal transgenic neuronal p60TRP mice. Our data suggests that in vivo overexpression of neuronal p60TRP significantly affects cognitive and neuroprotective capacities.

Secretome analysis of Ganoderma lucidum cultivated in sugarcane bagasse.

Harmful environmental issues of fossil-fuels and concerns about petroleum supplies have spurred the search for renewable alternative fuels such as biofuel. Agricultural crop residues represent an abundant renewable resource for future biofuel. To be a viable alternative, a biofuel should provide a net energy gain, have environmental benefits, be economically feasible, and should also be producible in large quantities without reducing food supplies. We used these criteria to evaluate the white rot basidiomycota-derived fungus Ganoderma lucidum that secretes substantial amounts of hydrolytic and oxidative enzymes useful for the degradation of lignocellulosic biomass that were not described hitherto. The current bottleneck of lignocellulosic biofuel production is the hydrolysis of biomass to sugar. To understand the enzymatic hydrolysis of complex biomasses, we cultured G. lucidum with sugarcane bagasse as substrate and qualitatively analyzed the entire secretome. The secreted lignocellulolytic enzymes were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and diverse enzymes were found, of which several were novel lignocellulosic biomass hydrolyzing enzymes. We further explored G. lucidum-derived cellulose, hemicellulose and lignin degrading enzymes as valuable enzymes for the second generation of biofuel obtained from a lignocellulose substrate such as sugarcane bagasse.

Tianma modulates blood vessel tonicity.

Tianma is a traditional Chinese medicine (TCM) often used for the treatment of hypertension and heart diseases. To elucidate the function of tianma at the molecular level, we investigated the effect of tianma on vascular functions and aortic protein metabolism. We found that long-term treatment with tianma (~2.5g/kg/day for three months) in one-year-old rats could enhance acetylcholine (ACh)-induced vasorelaxation in endothelium-intact thoracic aortic rings against both KCl (80 mM)- and phenylephrine (PE)-induced contraction. By using the iTRAQ (isobaric tag for relative and absolute quantification) technique, we confirmed from the functional data at the proteome level that tianma treatment down-regulated the expressions of contractile proteins (e.g. Acta2) and other related structural proteins (e.g. desmin), and up-regulated the expressions of extracellular matrix (ECM) glycoproteins (e.g. Fbln5) and anti-thrombotic proteins (e.g. Anxa2) in aortic tissue. By inductive reasoning, tianma could perform its vasodilatory effect not only by inhibiting vascular smooth muscle contraction, but also by enhancing blood vessel elasticity and stabilizing the arterial structure. Thus, tianma might become a novel therapeutic herbal medicine for cardiovascular diseases by regulating the aortic proteome metabolism.

Gastrodia elata Blume (tianma) mobilizes neuro-protective capacities.

Tianma (Gastrodia elata Blume) is a traditional Chinese medicine (TCM) often used for the treatment of headache, convulsions, hypertension and neurodegenerative diseases. Tianma also modulates the cleavage of the amyloid precursor protein App and cognitive functions in mice. The neuronal actions of tianma thus led us to investigate its specific effects on neuronal signalling. Accordingly, this pilot study was designed to examine the effects of tianma on the proteome metabolism in differentiated mouse neuronal N2a cells using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach. We identified 2178 proteins, out of which 74 were found to be altered upon tianma treatment in differentiated mouse neuronal N2a cells. Based on the observed data obtained, we hypothesize that tianma could promote neuro-regenerative processes by inhibiting stress-related proteins and mobilizing neuroprotective genes such as Nxn, Dbnl, Mobkl3, Clic4, Mki67 and Bax with various regenerative modalities and capacities related to neuro-synaptic plasticity.

Tianma modulates proteins with various neuro-regenerative modalities in differentiated human neuronal SH-SY5Y cells.

Tianma (Rhizoma gastrodiae) is the dried rhizome of the plant Gastrodia elata Blume (Orchidaceae family). As a medicinal herb in traditional Chinese medicine (TCM) its functions are to control convulsions, pain, headache, dizziness, vertigo, seizure, epilepsy and others. In addition, tianma is frequently used for the treatment of neurodegenerative disorders though the mechanism of action is widely unknown. Accordingly, this study was designed to examine the effects of tianma on the proteome metabolism in differentiated human neuronal SH-SY5Y cells to explore its specific effects on neuronal signaling pathways. Using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach, we identified 2390 modulated proteins, out of which 406 were found to be altered by tianma in differentiated human neuronal SH-SY5Y cells. Based on the observed data, we hypothesize that tianma promotes neuro-regenerative signaling cascades by controlling chaperone/proteasomal degradation pathways (e.g. CALR, FKBP3/4, HSP70/90) and mobilizing neuro-protective genes (such as AIP5) as well as modulating other proteins (RTN1/4, NCAM, PACSIN2, and PDLIM1/5) with various regenerative modalities and capacities related to neuro-synaptic plasticity.

New insights into the brain protein metabolism of Gastrodia elata-treated rats by quantitative proteomics.

Gastrodia elata (tianma) is a traditional Chinese herbal medicine (TCM) often used for the treatment of cerebrovascular diseases. In this study, we investigated the effects of tianma on the brain protein metabolism by quantitative proteomics to gain evidence for a direct relationship between tianma treatment and brain functions. One-year-old rats were treated with tianma (~2.5 g/kg/day) for 3months and the brain tissue proteome was analyzed by using the iTRAQ (isobaric tag for relative and absolute quantification) technology. According to our results, the long-term treatment with tianma could modulate the brain protein metabolism at the proteome level by down-regulating the expressions of various proteins, such as Gnao1 and Dctn2, which are related to neuronal growth cone control and synaptic activities. In addition, tianma treatment also induced the up-regulation of molecular chaperons and proteins related to the misfolded protein response, like Anxa5, and also other proteins involved in Huntington's disease (HD) (e.g. Pacsin1 and Arf3). Concluding, tianma could eventually contribute to activities related to synaptic plasticity and neuro-restorative processes and thus might be a novel candidate agent for the treatment of neurodegenerative diseases by regulating the brain proteome.

Neuronal p60TRP expression modulates cardiac capacity.

Heart failure, including myocardial infarction, is the leading cause for death and the incidence of cardiovascular diseases is predicted to continue to rise worldwide. In the present study we investigated the whole heart proteome profile of transgenic p60-Transcription Regulator Protein (p60TRP) mice to gain an insight into the molecular events caused by the long-term effect of neural p60TRP over-expression on cardiac proteome changes and its potential implication for cardiovascular functions. Using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach, we identified 1148 proteins, out of which 116 were found to be significantly altered in the heart of neural transgenic p60TRP mice. Based on the observed data, we conclude that in vivo neural over-expression of transgenic p60TRP with its neuroprotective therapeutic potential significantly affects cardiovascular capacities.

Phenotyping of tianma-stimulated differentiated rat neuronal b104 cells by quantitative proteomics.

Gastrodia elata blume (tianma) is a traditional Chinese herb often used in the treatment of convulsions, headaches, and hypertension. Although interest in neuronal-related actions of tianma is increasing, minimal studies have been conducted to determine its specific effects on neuronal cells. This study was designed to examine the effects of tianma on the metabolism in differentiated neuroblastoma cells using the isobaric tag for relative and absolute quantitation (iTRAQ) technology. Stimulation of these cells with tianma caused changes in the expression of 38 proteins that were subsequently classified according to their physiological functions and association with neurodegenerative diseases. We identified six proteins with altered functional activities in neurodegenerative disease states that were modulated by tianma: triosephosphate isomerase (Tpi1), peptidyl-prolyl cis-trans isomerase A (Ppia), neural cell adhesion molecule 1 (Ncam1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (Uchl1), septin-2 (Sept2) and heat shock protein 90 (Hsp90aa1). We postulate that tianma mediates its neuroprotective effects via upregulation of Ncam1, Hsp90aa1, Tpi1 and Ppia while downregulating Sept2 and Uchl1. These changes in protein expression aid in the restoration of the intracellular environment to a metabolically balanced state, promoting cell survival. Based on these observed data, we conclude that tianma has therapeutic potential, especially for neurodegenerative diseases.

iTRAQ-based quantitative secretome analysis of Phanerochaete chrysosporium.

The basidiomycete fungi such as Phanerochaete chrysosporium secrete large amount of hydrolytic and oxidative enzymes and degrade lignocellulosic biomass. The lignin depolymerizing proteins were extensively studied, but cellulose, hemicellulose and pectin hydrolyzing enzymes were poorly explored. In this study P. chrysosporium was grown in cellulose, lignin and mixture of cellulose and lignin, and secretory proteins were quantified by isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS). An iTRAQ quantified 117 enzymes comprising cellulose hydrolyzing endoglucanases, exoglucanases, beta-glucosidases; hemicelluloses hydrolyzing xylanases, acetylxylan esterases, mannosidases, mannanases; pectin-degrading enzymes polygalacturonase, rhamnogalacturonase, arabinose and lignin degrading protein belonging to oxidoreductase family. Under cellulose and cellulose with lignin culture conditions, enzymes such as endoglucanases, exoglucanases, ß-glucosidases and cellobiose dehydrogenase were significantly upregulated and iTRAQ data suggested hydrolytic and oxidative cellulose degradation. When lignin was used as a major carbon source, enzymes such as copper radical oxidase, isoamyl oxidase, glutathione S-transferase, thioredoxin peroxidase, quinone oxidoreductase, aryl alcohol oxidase, pyranose 2-oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase were expressed and significantly regulated. This study explored cellulose, hemicellulose, pectin and lignin degrading enzymes of P. chrysosporium that are valuable for lignocellulosic bioenergy.

Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes.

The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study characterized 102 highly confident unique proteins in the secretome with zero false discovery rate based on decoy strategy. The iTRAQ technique identified and relatively quantified many hydrolyzing enzymes such as cellulases, hemicellulases, glycoside hydrolases, proteases, peroxidases, and protein translocating transporter proteins during fermentation. The enzymes have potential application in lignocellulosic biomass hydrolysis for biofuel production, for example, the cellulolytic and hemicellulolytic enzymes glucan 1,4-alpha-glucosidase, alpha-glucosidase C, endoglucanase, alpha l-arabinofuranosidase, beta-mannosidase, glycosyl hydrolase; proteases such as tripeptidyl-peptidase, aspergillopepsin, and other enzymes including cytochrome c oxidase, cytochrome c oxidase, glucose oxidase were highly expressed in A. niger and its mutant secretion. In addition, specific enzyme production can be stimulated by controlling pH of the culture medium. Our results showed comprehensive unique secretory protein profile of A. niger, its regulation at different pH, and the potential application of iTRAQ-based quantitative proteomics for the microbial secretome analysis.