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Article

Comparative Analysis of CAZymes from Trichoderma longibrachiatum LMBC 172 Cultured with Three Different Carbon Sources: Sugarcane Bagasse, Tamarind Seeds, and Hemicellulose Simulation

by
Alex Graça Contato
1,2,*,
Tiago Cabral Borelli
3,
Ana Karine Furtado de Carvalho
4,
Heitor Buzetti Simões Bento
5,
Marcos Silveira Buckeridge
6,
Janet Rogers
7,
Steven Hartson
7,
Rolf Alexander Prade
2 and
Maria de Lourdes Teixeira de Moraes Polizeli
1,8
1
Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto 14040-900, SP, Brazil
2
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA
3
Department of Cellular and Molecular Biology and Pathogenic Bioagents, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto 14040-900, SP, Brazil
4
Department of Basic and Environmental Sciences, Lorena School of Engineering, University of São Paulo, Lorena 12602-810, SP, Brazil
5
Department of Bioprocess Engineering and Biotechnology, School of Pharmaceutical Sciences, São Paulo State University, Araraquara 14800-903, SP, Brazil
6
Department of Botany, Institute of Biosciences, University of São Paulo, São Paulo 05508-090, SP, Brazil
7
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA
8
Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto 14040-900, SP, Brazil
*
Author to whom correspondence should be addressed.
Clean Technol. 2024, 6(3), 994-1010; https://doi.org/10.3390/cleantechnol6030050
Submission received: 6 June 2024 / Revised: 24 July 2024 / Accepted: 5 August 2024 / Published: 8 August 2024
Browse Figure
Figure 1
<p>CAZymes from secretome analysis of <span class="html-italic">T. longibrachiatum</span> LMBC 172 in culture condition: hemicellulose simulation, or sugarcane bagasse, or tamarind seeds. (<b>A</b>) Total CAZymes found. (<b>B</b>) Venn plot correlating the CAZymes found in each culture condition.</p> ">
Review Reports Versions Notes

Abstract

:
The examination of fungal secretomes has garnered attention for its potential to unveil the repertoire of secreted proteins, notably CAZymes (Carbohydrate-Active enzymes), across various microorganisms. This study presents findings on categorizing the secretome profile of CAZymes by their function and family, derived from the filamentous fungus Trichoderma longibrachiatum LMBC 172. The cultivation was performed through submerged fermentation with three distinct carbon sources: sugarcane bagasse, tamarind seeds, and a control simulating hemicellulose containing 0.5% beechwood xylan plus 0.5% oat spelt xylan. The secretome analysis revealed 206 distinct CAZymes. Each carbon source showed particularities and differences. Of these, 89 proteins were produced simultaneously with all the carbon sources; specifically, 41 proteins using only the hemicellulose simulation, 29 proteins when sugarcane bagasse was used as a carbon source, and only 3 when tamarind seeds were used. However, in this last condition, there was a high intensity of xyloglucanase GH74 production, thus reaffirming the richness of xyloglucan in the constitution of these seeds. When evaluating the proteins found in two conditions, 18 proteins were shown between the simulation of hemicellulose and sugarcane bagasse, 11 proteins between the simulation of hemicellulose and tamarind seeds, and 15 proteins between sugarcane bagasse and tamarind seeds. Among the proteins found, there are representatives of different families such as glycosyl hydrolases (GHs) that cleave cellulose, hemicellulose, pectin, or other components; carbohydrate esterases (CEs); polysaccharide lyases (PLs); carbohydrate-binding modules (CBMs); and auxiliary activity enzymes (AAs). These results demonstrate the importance of analyzing CAZymes secreted by microorganisms under different culture conditions.

1. Introduction

The rise in agro-industrial operations has resulted in the accumulation of substantial quantities of lignocellulosic residues sourced from diverse origins, including wood and various agricultural byproducts globally [1,2,3]. Annually, approximately 146 billion tons of waste are generated worldwide [4]. There has been a notable surge in economic interest in these residues in recent years due to their renewability and cost-effectiveness, offering substantial potential for chemical and bioenergy production, such as bioethanol [5,6,7].
Plant biomass is composed of intricately structured polymeric materials, including proteins, lignin, holocellulose (a composite of cellulose fibers enveloped in hemicellulose-pectin), ash, salts, and minerals [8,9]. The polysaccharide framework within the plant cell wall represents one of the most intricate structures, with its lignocellulosic constitution varying depending on its source [10,11]. These cell wall polysaccharides serve as energy reservoirs, which, upon efficient extraction, can be utilized to produce second-generation ethanol, particularly through the hydrolysis of sugarcane bagasse [12].
The process of converting lignocellulosic biomass into ethanol and other chemical compounds often relies on a multienzyme system that operates synergistically [5,13,14]. It is crucial to investigate various microorganisms and comprehend how they secrete enzymes relevant to these processes [15]. Consequently, examining the secretomes of various fungi has garnered attention. These studies offer insights into the secreted proteins, notably the CAZymes (Carbohydrate-Active enzymes), released by diverse microorganisms cultivated under different conditions [16,17,18,19]. CAZymes encompass numerous enzyme protein families, each classified based on protein sequence similarities and distinctive three-dimensional folding structures [20]. They are classified between glycosyl hydrolases (GHs) that cleave cellulose, hemicellulose, pectin, or other components; carbohydrate esterases (CEs); polysaccharide lyases (PLs); carbohydrate-binding modules (CBMs); and auxiliary activity enzymes (AAs). These analyses allow a better understanding of the ideal way to obtain proteins of industrial interest, in addition to enabling the discovery of proteins not yet described in the literature for the studied microorganisms [21].
One particularly intriguing microorganism is Trichoderma longibrachiatum, especially in the context of biotechnology and biomass bioconversion. T. longibrachiatum is distributed globally, with a predominant presence in warmer climates. Its colonies usually exhibit an initial off-white colony, which later transitions to a shade of greyish green with age [22]. The members of this clade have gained significant attention across different sectors due to their remarkable capacity to excrete substantial quantities of proteins and metabolites [23,24]. The enzymes produced by T. longibrachiatum are used in various industries, including food, beverages, textiles, and paper, due to their ability to degrade complex plant polysaccharides [25,26]. Additionally, some strains of Trichoderma, including T. longibrachiatum, are used as biocontrol agents, antagonizing plant pathogens and offering an eco-friendly alternative to chemical pesticides [27,28]. The study of the CAZymes from the secretome of T. longibrachiatum provides valuable insights into the molecular mechanisms of enzyme production and adaptation to different carbon sources, essential for engineering more efficient strains and understanding their ecological interactions. T. longibrachiatum can grow on a variety of substrates, including agro-industrial residues like sugarcane bagasse and tamarind seeds, making it an ideal model for biomass bioconversion studies and the development of sustainable biotechnological processes [23,26,29,30]. Moreover, the use of T. longibrachiatum in the degradation of agricultural residues not only adds value to these residues but also helps reduce the environmental impact associated with their improper disposal.
Sugarcane (Saccharum sp.) is classified as a monocotyledonous grass [31,32], with Brazil currently holding the title of the world’s largest sugarcane producer, primarily concentrated in the central-southern region of the country [33]. Following Brazil, other significant sugarcane-producing countries include India, China, Thailand, and Pakistan. In 2022, global sugarcane production reached a total of 1.92 billion tons, with Brazil producing 38% of the world total, India with 23%, and China producing 5%. These values make it the third-most produced commodity worldwide [34]. Therefore, given that each ton of sugarcane results in approximately 270 kg of bagasse, the global sugarcane crop in 2022 generated more than 500 million tons of bagasse. This significant quantity highlights the necessity of utilizing this abundant byproduct in countries with extensive sugarcane production [35]. Characterized by a secondary cell wall, sugarcane bagasse typically comprises approximately 32–45% cellulose, 20–32% hemicellulose, 17–32% lignin, and 1–9% ash, along with other constituents [36,37,38].
On the other hand, tamarind (Tamarindus indica L.) is a tropical fruit tree indigenous to equatorial Africa, India, and Southeast Asia, featuring both pulp and seeds encased in a tough shell [39]. The biggest tamarind producers in the world are countries such as India, Malaysia, Myanmar, Bangladesh, Sri Lanka, Thailand, the United Arab Emirates, and South American countries. When processing 1 kg of fresh tamarind, it will give 55% pulp, 30–40% seed, 6% peel, and 5% fiber. The seed is the main and underutilized byproduct of the tamarind pulp industry and contains approximately (70%) kernel and (30%) hard brown testa [40,41,42]. The processing techniques, particularly the removal of pulp from the pod or seeds from the pulp, as well as the handling and storage of the seed and pulp, are traditionally practiced in the growing region or country. However, the most common processing method involves completely removing the testa from the kernel. The testa is separated from the kernels either by roasting or by soaking the seeds in water. Given that the mineral content of the seed coat is higher than that of the cotyledon, it is expected that their thermal properties and behaviors differ, resulting in varying degrees of expansion and contraction. This difference aids in detaching the seed coat from the seed [42]. According to Gonçalves et al. [43], tamarind seed composition includes approximately 1.82 ± 0.01% ash, 33.07 ± 1.40% lignin, 33.31 ± 3.56% cellulose, and 10.45 ± 1.45% hemicellulose. Additionally, these seeds boast a significant xyloglucan content, accounting for roughly 40% of their dry mass [44], making them a promising resource for CAZymes exploration.
Due to their composition, sugarcane bagasse and tamarind seeds have been employed in cultivating microorganisms to generate microbial enzymes capable of breaking down lignocellulosic biomass [2,19,24,26,45]. In this context, the present study unveils the categorization of CAZymes by function and family within the secretome profile of the filamentous fungus T. longibrachiatum LMBC 172. The fungus was cultured via submerged fermentation with three distinct carbon sources: sugarcane bagasse, or tamarind seeds, or a control simulating hemicellulose. The hemicellulose simulation was chosen to be used because hemicellulose is the part of lignocellulose that needs a larger framework of enzymes for degradation [46]. The global annual production of hemicellulose is approximately 60 billion tons, making it the second-most abundant renewable component of lignocellulosic biomass, after cellulose [47].

2. Material and Methods

2.1. Maintenance of the Fungus and Culture Medium

The fungi T. longibrachiatum LMBC 172 used in this work were isolated from tree trunks in Ribeirão Preto, SP, Brazil. The identification and deposition with the GenBank accession code OQ255882.1 were detailed by Contato et al. [45]. Microorganism maintenance involved spore inoculation on potato dextrose agar medium (PDA) (Sigma-Aldrich, Saint Louis, MO, USA), with subsequent transfers performed in glass tubes containing the same medium. Incubation occurred at a temperature of 30 °C. Afterwards, the tubes were kept under refrigeration for up to 30 days. In addition, they were cryopreserved at −80 °C to maintain the strain for long periods of time.

2.2. Plant Material

The sugarcane bagasse originated from Pedra Agroindustrial S/A sugarcane mill (Serrana, SP, Brazil) and comprised a blend of straw, leaves, and culms from various sugarcane varieties (CTC-4, CTC-7, CTC-20, IAC95500, RB867515, and RB966928). To sanitize the material, it was immersed in 92 °GL ethanol for 1 h, followed by rinsing with distilled water. Subsequently, the material underwent drying in an oven at 50 °C for 3 days and was then milled using a 25-mesh sieve knife mill (SL-32-SOLAB).
Tamarind (Tamarindus indica, Fabaceae) seeds were sourced from the campus of the University of Sao Paulo, Ribeirão Preto, SP, Brazil. The seeds underwent boiling in water for 1 h, followed by drying in an oven at 50 °C for 3 days to ensure sanitary quality and prevent the growth of other fungi. The seeds were milled using a 20-mesh sieve knife mill (SL-32-SOLAB).

2.3. Submerged Culture of T. longibrachiatum LMBC 172 for Protein Secretion Induction

The submerged culture procedure followed the methodology outlined by Contato et al. [45]. A spore solution containing 106–107 spores/mL was prepared from the fungus. The fungus was cultured in test tubes, suspended in sterile distilled water, and spore counts were conducted using a microscope and a Neubauer chamber. This suspension was then inoculated into 125 mL Erlenmeyer flasks containing 25 mL of Khanna medium (comprising Khanna’s salt solution [20×]: NH4NO3 (2.0 g), KH2PO4 (1.3 g), MgSO4·7H2O (0.362 g), KCl (0.098 g), ZnSO4·H2O (0.007 g), MnSO4·H2O (0.0138 g), Fe2(SO4)3·6H2O (0.0066 g), CuSO4·5H2O (0.0062 g), with distilled water q.s. (100 mL) (5.0 mL); yeast extract (0.1 g); carbon source (1.0 g); distilled water q.s. to 100 mL) [48]. The media were supplemented, individually, with 1% (w/v) of two different lignocellulosic residues: sugarcane bagasse and tamarind seeds. Additionally, a control simulating hemicellulose was performed, individually, using a mixture containing 0.5% beechwood xylan and 0.5% oat spelt xylan (Sigma-Aldrich, Saint Louis, MO, USA). The 1% (w/v) ratio of lignocellulosic residues and hemicellulose simulation was chosen to evaluate the impact on CAZymes because this concentration was usually reported in studies that successfully evaluated the secretome profile of filamentous fungi [19,49,50]. After, the Erlenmeyer flasks were incubated at 30 °C under static conditions for up to 72 h, as optimal conditions for protein induction as described by Contato et al. [45].

2.4. Sample Processing

The culture supernatant of T. longibrachiatum cultivated in the residues under submerged conditions was harvested through filtration with Whatman filter paper Grade 1 in a vacuum pump following a 72 h period. Subsequently, it was concentrated using ultrafiltration (10,000 MWCO, PES membrane, Vivaspin, Littleton, CO, USA), then washed twice with 5 mL of 50 mM sodium acetate buffer at pH 5.0. The proteins were then subjected to separation via SDS-PAGE electrophoresis [51].

2.5. Characterization of the T. longibrachiatum LMBC 172 by Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)

For secretome LC–MS/MS analysis, 15–20 μg of total secretome proteins was loaded onto an SDS-PAGE on 12% separation gel. The gel electrophoresis employed preparative PAGE to isolate the protein secretomes from complex carbohydrate and phenolic species present in the supernatant. Electrophoresis ceased once the bromophenol blue tracking dye had migrated 2–3 cm into the separating gel. Subsequently, the gel was stained with Coomassie blue, and the entire protein banding profile was excised for processing via LC–MS/MS [52].
Isolated gel bands underwent reduction with Tris (2-carboxyethyl) phosphine, followed by alkylation using 2-iodoacetamide, and overnight digestion with 8 μg/mL trypsin in ammonium bicarbonate buffer. Peptides were extracted from the gel segments and desalted using C18 pipet tips following the manufacturer’s guidelines (Agilent P/N A57003100, Agilent Technologies, Santa Clara, CA, USA). The desalted peptides were then dissolved in 0.1% aqueous formic acid and injected onto a 75-micron × 50 cm capillary HPLC column packed with 2-micron C18 particles (Thermo P/N 164942, ThermoFisher Scientific, Waltham, MA, USA) with a vented trap column setup. Peptide separation was achieved using a 60 min gradient of formic acid/acetonitrile and ionization was performed in a Nanospray Flex ion source equipped with stainless-steel emitters linked to a quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA).
Peptide ions were subjected to analysis using a “high-low” “top-speed” data-dependent MS/MS strategy. Precursors were initially analyzed at high resolution in the Orbitrap sector, followed by MS/MS selection in the quadrupole sector, fragmentation by HCD in the ion routing multipole, and subsequent analysis of fragment ions in the ion trap sector.
Each sample underwent LC-MS/MS analysis twice, with the two RAW data files merged into a single sample for database searching using MaxQuant (version v2.0.1.0, Max-Planck-Institute of Biochemistry, Planegg, Germany) [53]. Spectra were searched against a database containing 402,135 protein sequences obtained from the NCBI on 27 May 2022, using “Trichoderma” as the genus search term. Searches were annotated using Python version v3.11 (Python Software Foundation, Wilmington, DE, USA) to annotate NCBI T. longibrachiatum IDs by transferring annotations from related curated proteins at Uniprot (https://www.uniprot.org/ accessed on 18 December 2022). Sequences with a false discovery rate (FDR or q-value) greater than 0.00 were removed from the analysis. Finally, we identified conserved CAZy domains using Hidden Markov Model (HMM) profiles available on the dbCAN2 web platform (https://bcb.unl.edu/dbCAN2/index.php accessed on 18 December 2022). Only domains with e-values > 10−17 and coverage > 0.35 were considered.

3. Results and Discussion

Secretome Protein Composition

To elucidate the secretome of T. longibrachiatum LMBC 172, we gathered the culture supernatants and subjected them to LC-MS/MS analysis. Protein identifications were conducted by searching against a database of Trichoderma sequences obtained from the NCBI, and subsequent annotation was performed on these identified proteins. Our analyses identified 206 distinct proteins on the sum of the three different conditions (supplemented with sugarcane bagasse, or tamarind seeds, or the hemicellulose simulation) in the secretome of T. longibrachiatum LMBC 172 (all non-anchored extracellular proteins), of which 159 proteins were shown in the control simulating hemicellulose, 151 proteins when 1% sugarcane bagasse was used for the culture, and 118 proteins when the residue used was 1% tamarind seeds (Figure 1A). Although many proteins were coincident in the analyzed experimental conditions, there were differences in the number of proteins found, principally due to the use of beechwood xylan plus oat spelt xylan, simulating the constitution of hemicellulose, where there is a strong presence of xylan [54] (Figure 1B). The sugarcane bagasse, as demonstrated by Scarcella et al. [14], has a constitution of 159.44 ± 23.81 of xylose as the main sugar, 11-fold more than glucose (14.08 ± 4.07), the second-most abundant non-cellulosic monosaccharide. These values show the high content of hemicellulose in the structure of sugarcane bagasse. The tamarind seed is rich in xyloglucan, one of the most abundant hemicellulose polymers after xylan [44,55], thus justifying a smaller number of proteins found in the secretome when this lignocellulosic residue was used.
As can be seen in Figure 1B, of these 206 proteins found, 89 are found in all culture conditions (hemicellulose simulation, or sugarcane bagasse, or tamarind seeds). The 89 proteins found with the three different carbon sources and which biomass they degrade are shown in Table 1. Among the proteins found, there are representatives of different families such as glycosyl hydrolases (GHs) that cleave cellulose, hemicellulose, pectin, or other components; carbohydrate esterases (CEs); polysaccharide lyases (PLs); carbohydrate-binding modules (CBMs); and auxiliary activity enzymes (AAs).
Taking into analysis only the use of the hemicellulose simulation, the major proteins produced according to their iBAQ value (sum of all the peptide intensities divided by the number of observable peptides of a protein) were endo-1,4-β-xylanases, xylan 1,4-β-xylosidase, α-L-arabinofuranosidase, and α-galactosidase (Table 1). However, it was shown that 41 proteins were produced when only this carbon source was used (Figure 1B, Table 2). Most of these proteins are responsible for the degradation of xylan and its ramifications [56].
On the other hand, it was shown that T. longibrachiatum LMBC 172 secreted 29 unique proteins with the use of sugarcane bagasse as the carbon source (Figure 1B, Table 3), with emphasis on 2 glycosyl hydrolases of the GH92 family, a family recognized to belong to α-mannosidases [57], in addition to 2 more mannan endo-1,6-α-mannosidases from the GH76 family, in agreement with the study by Scarcella et al. [14], who showed a mannose composition in sugarcane bagasse. Another enzyme found that agrees with the study by Scarcella et al. [14] is the α-fucosidase A of the GH95 family, given the existence of fucose in the constitution of this residue. Another interesting finding when using sugarcane bagasse for the culture was the great iBAQ shown in the production of a glycosyl hydrolase from the GH93 family: the GH93 family hydrolyses linear α-1,5-L-arabinan [58].
A different analysis was shown when tamarind seeds were used for the culture, since only three proteins were produced exclusively with the use of this lignocellulosic residue (Figure 1B), which were a carbohydrate-binding module CBM1, a glycosyl hydrolase of the GH5 family, and curiously a laccase, an enzyme recognized for being a polyphenol oxidase [59] (Table 4). However, an interesting verification is the fact of the great iBAQ shown in the production of a xyloglucanase of the GH74 family when using tamarind seeds in comparison with the other conditions (Table 1), thus reaffirming the richness of xyloglucan in the constitution of these seeds.
Other important data to be analyzed are the proteins found in only two of the three conditions tested and the reasons for this. When using the simulation of hemicellulose and sugarcane bagasse for the culture, 18 proteins in common are shown, which are not found when the culture was made with tamarind seeds (Figure 1B). Among the most interesting are a lysozyme of the GH25 family, which promotes the hydrolysis of the β-1,4 glycosidic bonds between residues of N-acetylmuramic acid (Mur2Ac) and N-acetyl-D-glucosamine (GlcNAc) in a peptidoglycan [60]; and a β-glucuronidase of the GH79 family (Table S1—Supplementary Material). However, the great majority of these 18 proteins are enzymes that cleave xylan, a polysaccharide seen in smaller amounts in tamarind seeds.
The correlation of the CAZymes shown in the secretome of T. longibrachiatum LMBC 172 when cultivated with the hemicellulose simulation or with tamarind seeds was performed, and it was seen that 11 proteins are found (Figure 1B); specifically, α-L-arabinofuranosidases belonging to the GH54 family were investigated. These enzymes are responsible for hydrolyzing terminal α-1,5-glycosidic linkages to arabinofuranosides in arabinan, as well as α-1,2 and α-1,3-linkages to arabinofuranosides of arabinan, arabinoxylan, and arabinogalactan. They operate synergistically with other hemicellulolytic enzymes, effectively removing L-arabinose side chains that might otherwise impede the activity of backbone-degrading enzymes [61]. Other proteins found are the glycosyl hydrolases of the GH7 family that cleave β-1,4 glycosidic bonds in cellulose/β-1,4-glucans [62] (Table S2—Supplementary Material). It is agreement the presence, simultaneous between these conditions (hemicellulose simulation and tamarind seeds), of enzymes that cleave cellulose existing in the medium.
Analyzing the CAZymes shown simultaneously among the cultures performed with lignocellulosic residues, sugarcane bagasse, or tamarind seeds, 15 proteins are found in common (Figure 1B), especially a feruloyl esterase C, a mannosyl-oligosaccharide α-1,2-mannosidase of the GH47 family, an AA9 lytic polysaccharide monooxygenase, and a glycosyl hydrolase from the GH39 family, recognized to belong to β-xylosidase [63] (Table S3—Supplementary Material). However, the great majority are CAZymes from the GH3 family, which currently groups together exo-acting β-D-glucosidases, α-L-arabinofuranosidases, and β-D-xylopyranosidases [64], enzymes known to be hemicelluloses.
The secreted enzymes of T. longibrachiatum differ when using sugarcane bagasse, or tamarind seeds, or hemicellulose simulation due to the distinct composition of the polysaccharides present in these carbon sources. Sugarcane bagasse primarily consists of cellulose, hemicellulose, and lignin, with cellulose being the most abundant polysaccharide [36,37,38]. As a result, the secretome of T. longibrachiatum grown on sugarcane bagasse is rich in cellulases, such as GH3, GH5, GH6, and GH7, which are crucial for breaking down cellulose into glucose monomers (Table 1 and Table 3). Additionally, the presence of hemicellulose and lignin in sugarcane bagasse stimulates the production of hemicellulases (e.g., xylanases, GH10) and lignin-degrading auxiliary activity enzymes (AAs) like AA3 and AA9 (Table 1 and Table S3—Supplementary Material), facilitating the comprehensive degradation of this complex biomass.
In contrast, tamarind seeds contain high levels of xyloglucans, which are polysaccharides composed of a cellulose backbone with xylose, galactose, and fucose side chains [43,44]. The unique structure of xyloglucans in tamarind seeds necessitates the production of specific enzymes, such as xyloglucanases (GH74), to effectively hydrolyze these complex sugars [19]. Consequently, the secretome of T. longibrachiatum cultured with tamarind seeds is particularly enriched in enzymes that target xyloglucans (Table 1), reflecting the adaptation of the fungus to the predominant polysaccharides in this carbon source.
Hemicellulose simulation, which likely includes a mixture of various hemicellulosic sugars such as xylans, mannans, and arabinogalactans, prompts the secretion of a diverse array of hemicellulases tailored to these components. Enzymes such as xylanases (GH11), mannosidases (GH76), and arabinofuranosidases (GH54) are produced to degrade the heterogeneous polysaccharide structure of hemicellulose into fermentable sugars (Table 1 and Table 2; and Tables S1 and S2—Supplementary Material) [46,47].
The differences in secreted enzymes when using sugarcane bagasse, or tamarind seeds, or hemicellulose are thus directly influenced by the specific polysaccharide compositions of these substrates. The fungus adapts its enzymatic machinery to efficiently break down the available sugars, producing a tailored set of CAZymes that correspond to the structural complexity and specificities of the given carbon source. This adaptive enzyme production ensures the optimal utilization of the provided biomass, demonstrating the metabolic versatility of T. longibrachiatum.
These results demonstrate the importance of analysis studies of CAZymes secreted by microorganisms in different culture conditions, since their abundance in relation to protein intensity can present different results [65]. In addition, they agree with previous works published by our group, such as that of Contato et al. [45], which provides, under the same culture conditions, the catalytic activity of the enzymes found in the T. longibrachiatum LMBC 172 secretome shown in this study. The research identified a total of 206 distinct CAZymes in the secretome of T. longibrachiatum LMBC 172, with 89 proteins consistently produced across all three conditions (sugarcane bagasse, or tamarind seeds, or hemicellulose simulation). Notably, specific proteins were uniquely produced depending on the carbon source, including 41 proteins for hemicellulose simulation, 29 for sugarcane bagasse, and 3 for tamarind seeds. Tamarind seeds, specifically, induced a high production of xyloglucanase GH74, reflecting their high xyloglucan content [44].
The identified CAZymes belong to various families, such as glycosyl hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), carbohydrate-binding modules (CBMs), and auxiliary activity enzymes (AAs). The study underscores the adaptive mechanisms of T. longibrachiatum in secreting differential enzymes based on the carbon source, which is crucial for the degradation of specific components of lignocellulosic biomass. This differential enzyme secretion highlights the fungus’s ability to utilize diverse carbon sources effectively [23,24,26,29,66,67].
From a biotechnological perspective, the detailed secretome analysis of T. longibrachiatum can drive the development of efficient enzyme cocktails for biomass conversion processes, thereby enhancing the production of biofuels and biochemicals [24,68,69]. The specific enzymes identified in this study hold potential for further engineering or optimization for various industrial applications, including the food, beverage, textile, and paper industries [25,68,70,71]. Additionally, utilizing agricultural residues like sugarcane bagasse and tamarind seeds as carbon sources for enzyme production promotes sustainable biomass utilization, reducing environmental waste and supporting circular bioeconomy initiatives [14,26,45,72]. The ability of T. longibrachiatum to produce different enzymes tailored to specific substrates suggests potential for customized enzyme production to meet specific industrial needs.
Environmentally, this study supports efforts to mitigate the impact of waste disposal by leveraging agro-industrial residues, thus promoting the efficient use of renewable resources. The findings advocate for eco-friendly alternatives to traditional chemical processing methods, reducing reliance on non-renewable resources and minimizing environmental pollution. Furthermore, this research advances the understanding of molecular mechanisms behind enzyme production and secretion in fungi, contributing significantly to the field of fungal biotechnology. Insights gained from this study can inform future research on other fungal species and their potential applications in various biotechnological processes.
In summary, this study provides a comprehensive analysis of the CAZyme secretome of T. longibrachiatum under different conditions, highlighting significant biotechnological advancements and sustainable industrial applications. The research emphasizes the potential for developing efficient and tailored enzyme solutions to meet the growing demand for sustainable biomass conversion and industrial bioprocessing.

4. Conclusions

The analysis of the secretome of T. longibrachiatum LMBC 172 cultured under submerged fermentation in two different lignocellulosic residues, sugarcane bagasse or tamarind seeds, in addition to a hemicellulose simulation as the control, revealed a total of 206 CAZymes. Each carbon source showed particularities and differences. Of these, 89 proteins were produced simultaneously with all the carbon sources, 41 proteins using only the hemicellulose simulation, 29 proteins when sugarcane bagasse was used as a carbon source, and only 3 when tamarind seeds were used. Among the proteins found, there are representatives of different families such as glycosyl hydrolases (GHs) that cleave cellulose, hemicellulose, pectin, or other components; carbohydrate esterases (CEs); polysaccharide lyases (PLs); carbohydrate-binding modules (CBMs); and auxiliary activity enzymes (AAs). These results demonstrate the importance of analysis studies of CAZymes secreted by microorganisms in different culture conditions, since their abundance in relation to protein intensity can present different results. However, it has limitations that need addressing. The secretome analysis, though detailed, was based on a limited number of carbon sources, and expanding the range of substrates could provide a broader understanding of the enzyme production capabilities of T. longibrachiatum. Future research should explore the genetic and metabolic pathways involved in enzyme regulation and secretion, as well as investigate the synergistic effects of mixed carbon sources on enzyme profiles.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cleantechnol6030050/s1, Table S1: Comprehensive LC-MS/MS secretome analysis for 18 proteins found using the simulation of hemicellulose and sugarcane bagasse classified according to which biomass they degrade; Table S2: Comprehensive LC-MS/MS secretome analysis for 11 proteins found using the simulation of hemicellulose and tamarind seeds classified according to which biomass they degrade; Table S3: Comprehensive LC-MS/MS secretome analysis for 15 proteins found using sugarcane bagasse and tamarind seeds classified according to which biomass they degrade.

Author Contributions

A.G.C.: methodology, investigation, data curation, formal analysis, writing—original draft. T.C.B.: methodology, investigation, data curation, formal analysis. A.K.F.d.C.: writing—review and editing. H.B.S.B.: writing—review and editing. M.S.B.: funding acquisition, conceptualization. J.R.: methodology, investigation, data curation, formal analysis, writing—review and editing. S.H.: methodology, investigation, data curation, formal analysis, writing—review and editing. R.A.P.: methodology, investigation, data curation, formal analysis, funding acquisition, supervision, writing—review and editing. M.d.L.T.d.M.P.: funding acquisition, conceptualization, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the doctorate grants awarded to A.G. Contato (Processes nº 2017/25862-6 and 2021/07066-3), and to T.C. Borelli (Process nº 2021/08235-3); and financial support from the National Institute of Science and Technology of Bioethanol (Process FAPESP 2014/50884-5), and Process FAPESP 2018/07522-6. Conselho Nacional de Desenvolvimento Científico (CNPq), processes 465319/2014-9. M.L.T.M. Polizeli (process 310340/2021-7) and M.S. Buckeridge are Research Fellows of CNPq.

Institutional Review Board Statement

The study adheres to all ethical guidelines, including compliance with the legal regulations of the countries where the research was conducted.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets produced and analyzed during this study are accessible upon reasonable request from the corresponding author.

Acknowledgments

Mass spectrometry analyses and primary database searches were performed at the Center for Genomics and Proteomics, situated at Oklahoma State University.

Conflicts of Interest

The authors assert that there are no apparent competing financial interests or personal relationships that might have influenced the findings presented in this paper.

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Cleantechnol 06 00050 g001
Figure 1. CAZymes from secretome analysis of T. longibrachiatum LMBC 172 in culture condition: hemicellulose simulation, or sugarcane bagasse, or tamarind seeds. (A) Total CAZymes found. (B) Venn plot correlating the CAZymes found in each culture condition.
Figure 1. CAZymes from secretome analysis of T. longibrachiatum LMBC 172 in culture condition: hemicellulose simulation, or sugarcane bagasse, or tamarind seeds. (A) Total CAZymes found. (B) Venn plot correlating the CAZymes found in each culture condition.
Cleantechnol 06 00050 g001
Table 1. Comprehensive LC-MS/MS secretome analysis for 89 proteins found in cultures with hemicellulose simulation, sugarcane bagasse, and tamarind seeds, classified according to which substrates they degrade.
Table 1. Comprehensive LC-MS/MS secretome analysis for 89 proteins found in cultures with hemicellulose simulation, sugarcane bagasse, and tamarind seeds, classified according to which substrates they degrade.
Degraded BiomassiBAQ (a) Hemicellulose SimulationiBAQ (a) Sugarcane BagasseiBAQ (a) Tamarind SeedsProtein IDs
(b)		FamilyMS/MS View: Identified ProteinsMolecular Weight (kDa) (c)
amido4.13 × 10108.41 × 108 9.24 × 108 A0A2T3YUD7GH15glucoamylase8
amido1.78 × 1010 7.88 × 108 2.24 × 108 A0A2T3ZF22GH15glucoamylase33
amido1.62 × 108 1.18 × 107 1.29 × 107 A0A2T3YUB0GH13α-amylase112
carboxylic ester1.69 × 107 6.71 × 107 1.76 × 106 A0A6V8QQW9CE1carboxylic ester hydrolase36
cellulose5.20 × 1010 3.98 × 109 1.50 × 1010Q6QTF2GH12endoglucanase I36
cellulose2.32 × 1010 5.84 × 108 7.30 × 109 A0A6V8R3W7GH6exoglucanase 264
cellulose1.48 × 1010 2.46 × 109 2.25 × 1010 A0A6V8QY83GH7exoglucanase 128
cellulose5.55 × 109 3.75 × 107 6.87 × 107 A0A6V8R5D3GH3β-glucosidase A26
cellulose3.06 × 109 3.40 × 108 4.86 × 109 A0A142C169GH71,4-β-D-glucan cellobiohydrolase33
cellulose2.10 × 109 1.57 × 108 3.46 × 108 A0A2T3YQZ3GH5glycoside hydrolase45
cellulose1.04 × 1097.80 × 108 8.69 × 107KAH8124777.1CBM35carbohydrate-binding module44
cellulose4.81 × 1082.78 × 1081.20 × 109A0A2T3ZAP7CBM1carbohydrate-binding module88
cellulose1.69 × 1089.14 × 1064.78 × 107A0A2T3ZMC2CBM1carbohydrate-binding module33
cellulose2.96 × 1071.90 × 1082.20 × 108A0A6V8R7F2GH5endoglucanase II56
cellulose2.16 × 1074.65 × 1067.80 × 106A0A0W7VDH7GH6exoglucanase45
cellulose3.16 × 1061.85 × 1093.86 × 108A0A6V8R5M5GH3β-glucosidase celA57
chitin2.00 × 1081.74 × 1083.72 × 106XP_024756832.1GH18glycoside hydrolase56
chitin7.88 × 1071.66 × 1073.31 × 105A0A2T3YRL6GH18glycoside hydrolase46
chitin6.24 × 1075.66 × 1076.51 × 106A0A2K0U0B3GH18chitinase26
chitin2.28 × 1061.78 × 1082.20 × 106A0A0B5AH01GH18chitinase98
cutin1.59 × 1092.00 × 1072.33 × 107A0A2T3ZC81CE5cutinase42
ester carboxylic1.05 × 1081.65 × 1081.42 × 108A0A6V8R506CE1carboxylic ester hydrolase55
GMC2.17 × 1071.33 × 1082.46 × 107A0A6V8R691AA3glucose–methanol–choline GMC oxidoreductase57
hemicellulose2.38 × 10119.25 × 1093.25 × 108A0A6V8RCI3GH3xylan 1,4-β-xylosidase32
hemicellulose1.93 × 10111.27 × 10103.18 × 1010A0A6V8R417GH54α-L-arabinofuranosidase85
hemicellulose1.72 × 10111.88 × 1096.96 × 109A0A6V8R4W9GH11endo-1,4-β-xylanase34
hemicellulose1.35 × 10116.71 × 1084.40 × 108A0A6V8QYX8GH27α-galactosidase57
hemicellulose1.27 × 10112.83 × 10106.35 × 109A0A088MAZ4GH11endo-1,4-β-xylanase38
hemicellulose3.10 × 10101.02 × 1083.73 × 109A0A2T3YZH0GH11endo-1,4-β-xylanase55
hemicellulose1.27 × 10104.52 × 1084.59 × 108A0A6V8R480GH721,3-β-glucanosyltransferase105
hemicellulose1.03 × 10101.33 × 1081.20 × 107G9NNL4GH12glycoside hydrolase55
hemicellulose6.63 × 1096.27 × 1081.97 × 108A0A6V8QQS5GH54α-L-arabinofuranosidase88
hemicellulose5.49 × 1092.57 × 1083.19 × 106A0A6V8QVE6GH16endo-1,3(4)-β-glucanase31
hemicellulose4.93 × 1092.29 × 1083.19 × 109A0A2T3Z959GH62α-L-arabinofuranosidase66
hemicellulose4.40 × 1093.00 × 1071.36 × 108A0A6V8R688GH721,3-β-glucanosyltransferase33
hemicellulose2.17 × 1094.19 × 1065.16 × 105G9N9X8GH11glycoside hydrolase24
hemicellulose1.71 × 1091.60 × 1091.86 × 109A0A6V8QV79GH43arabinoxylan arabinofuranohydrolase26
hemicellulose1.28 × 1097.97 × 1071.97 × 106A0A6V8R523GH2β-mannosidase A88
hemicellulose1.24 × 1092.84 × 1078.89 × 107A0A2P4ZDF2GH27α-galactosidase67
hemicellulose1.09 × 1094.23 × 1088.82 × 106A0A6V8R4Z6GH30xylanase52
hemicellulose9.44 × 1086.64 × 1084.68 × 108A0A6V8QNB0GH16glycoside hydrolase50
hemicellulose8.42 × 1084.51 × 1085.59 × 109UKZ86534.1GH74xyloglucanase78
hemicellulose8.41 × 1082.62 × 1073.68 × 107A0A6V8QP12CE5acetylxylan esterase 253
hemicellulose7.38 × 1082.48 × 1081.65 × 108A0A6V8QM46GH17glucan endo-1,3-β-glucosidase eglC88
hemicellulose7.33 × 1081.03 × 1074.09 × 107G9NGV2GH27α-galactosidase60
hemicellulose3.32 × 1083.81 × 1081.23 × 107A0A2T3ZAU0GH30glycoside hydrolase56
hemicellulose2.74 × 1081.65 × 1081.44 × 106A0A6V8R899GH64glucan endo-1,3-β-glucosidase56
hemicellulose2.48 × 1082.79 × 1089.46 × 106A0A6V8QQA1GH721,3-β-glucanosyltransferase9
hemicellulose1.38 × 1085.95 × 1063.06 × 106A0A2N1L3Y3CE5acetylxylan esterase46
hemicellulose1.32 × 1085.86 × 1063.99 × 106A0A0F9XN15GH27α-galactosidase57
hemicellulose1.11 × 1083.25 × 1053.10 × 107A0A6V8R5J5GH55glucan 1,3-β-glucosidase57
hemicellulose1.07 × 1085.67 × 1065.74 × 106A0A6V8RC59GH27α-galactosidase36
hemicellulose7.91 × 1071.10 × 1074.34 × 107A0A2T4B0H5GH54α-L-arabinofuranosidase102
hemicellulose7.85 × 1073.88 × 1061.98 × 105Q6QNU8GH11endo-1,4-β-xylanase88
hemicellulose6.42 × 1075.76 × 1081.14 × 108A0A6V8QJT1GH55glucan 1,3-β-glucosidase79
hemicellulose5.74 × 1072.15 × 1073.48 × 106A0A6V8QPF0GH2β-mannosidase A125
hemicellulose5.66 × 1076.36 × 1074.29 × 106A0A2T3YZN9GH78glycoside hydrolase40
hemicellulose4.26 × 1072.35 × 1071.44 × 107A0A2T3YYK7GH71glycoside hydrolase27
hemicellulose2.47 × 1071.06 × 10105.93 × 108A0A6V8QIP8GH10endo-1,4-β-xylanase C49
hemicellulose2.12 × 1073.21 × 1071.65 × 106A0A2T4C2Y0GH721,3-β-glucanosyltransferase51
hemicellulose1.92 × 1071.59 × 1067.44 × 105A0A2T4AVU1GH17glycoside hydrolase88
hemicellulose1.85 × 1075.82 × 1081.93 × 107A0A2T3YYG3GH30glycoside hydrolase63
hemicellulose1.13 × 1078.23 × 1063.68 × 104G0RWY3GH18endo-1,4-β-xylanase77
hemicellulose8.92 × 1066.19 × 1083.87 × 107A0A6V8R5H6GH35β-galactosidase60
hemicellulose8.60 × 1061.64 × 1061.88 × 105A0A2T3Z3S2GH27α-galactosidase87
hemicellulose3.74 × 1062.33 × 1082.02 × 106A0A2T3ZG69GH31glycoside hydrolase89
hemicellulose3.40 × 1066.90 × 1092.42 × 108XP_024755433.1GH10glycoside hydrolase57
hemicellulose1.49 × 1063.32 × 1072.49 × 107A0A6V8R3D3GH3xylan 1,4-β-xylosidase65
hemicellulose1.39 × 1061.87 × 1082.57 × 106A0A6V8QXM8GH67α-glucuronidase55
hemicellulose6.91 × 1051.42 × 1074.53 × 106A0A6V8QX14GH76mannan endo-1,6-α-mannosidase44
hemicellulose6.50 × 1058.42 × 1062.19 × 105A0A2T3YZP2GH79glycoside hydrolase97
lignin1.69 × 1091.44 × 1091.83 × 106A0A6V8QZK1AA3laccase70
oxygen1.25 × 1084.76 × 1078.71 × 105A0A6V8R4F9AA3FAD-dependent monooxygenase54
pectin1.26 × 10116.11 × 1091.58 × 109A0A2T3YXQ4CE5carbohydrate esterase56
pectin3.20 × 10103.13 × 1071.34 × 109A0A6V8R602GH28endopolygalacturonase56
pectin1.52 × 10101.12 × 1092.57 × 108XP_024766320.1CE5carbohydrate esterase49
pectin5.45 × 1094.68 × 1086.53 × 108A0A2K0T4R7CE5cutinase48
pectin3.53 × 1091.35 × 1061.05 × 107A0A6V8QQH8PL7alginate lyase88
pectin2.31 × 1091.04 × 1073.93 × 107A0A6V8R557PL1polysaccharide lyase42
pectin1.53 × 1094.76 × 1075.34 × 108A0A2T3YYD8CE8pectinesterase36
pectin5.65 × 1084.01 × 1084.44 × 107A0A2T3YTH0GH28glycoside hydrolase65
pectin2.87 × 1084.01 × 1051.85 × 108A0A2T3YUA1GH28glycoside hydrolase34
pectin1.79 × 1081.61 × 1077.61 × 106A0A2T3ZG56GH28glycoside hydrolase56
pectin1.13 × 1083.68 × 1071.33 × 108A0A395NND2GH28glycoside hydrolase50
pectin8.22 × 1076.60 × 1071.40 × 107A0A2T3ZCA4CE16carbohydrate esterase26
pectin1.94 × 1073.74 × 1051.17 × 105A0A2T3YZM0GH18glycoside hydrolase39
pectin2.24 × 1061.43 × 1069.20 × 107A0A6V8RBF6PL1pectate lyase C68
pectin1.61 × 1055.72 × 1065.28 × 106A0A2T3YUJ2GH28glycoside hydrolase71
phosphate3.60 × 1086.77 × 1076.38 × 105A0A6V8QHF9CBM21acid phosphatase44
(a) The iBAQ corresponds to the sum of all the peptide intensities divided by the number of observable peptides of a protein. (b) Accession number with protein information and family information were obtained from UniProt/Swiss-Prot database or NCBI database. (c) Hypothetical molecular weight of the proteins.
Table 2. Comprehensive LC-MS/MS secretome analysis for 41 proteins found only in the hemicellulose simulation conditions classified according to which biomass they degrade.
Table 2. Comprehensive LC-MS/MS secretome analysis for 41 proteins found only in the hemicellulose simulation conditions classified according to which biomass they degrade.
Degraded BiomassiBAQ (a)
Hemicellulose Simulation		Protein IDs
(b)		FamilyMS/MS View:
Identified Proteins		Molecular Weight (kDa) (c)
amido6.46 × 107A0A2T4B8C2CE50amidase49
cellulose1.05 × 108A0A2T3Z508CBM1carbohydrate-binding module28
cellulose4.09 × 107G9P6M2CE5carbohydrate esterase53
cellulose3.37 × 107A0A2K0SW07CBM1carbohydrate-binding module78
cellulose2.07 × 107A0A2T3YUC4GH3β-glucosidase55
cellulose1.41 × 107A0A2T3YX19CBM1carbohydrate-binding module52
cellulose1.37 × 107A0A2T4BD17CBM1carbohydrate-binding module42
cellulose1.09 × 107A0A2T3Z5Y5CBM18carbohydrate-binding module55
cellulose9.31 × 106A0A6V8R0S9AA9lytic polysaccharide monooxygenase40
cellulose3.65 × 106G9NFW5GH5glycoside hydrolase50
cellulose2.95 × 106A0A395NJN7GH7cellobiohydrolase25
cellulose2.28 × 106A0A2K0TAX7AA9copper radical oxidase43
cellulose1.96 × 106G9N4X9GH5glycoside hydrolase115
chitin2.27 × 108A0A6V8R2F4GH18chitinase44
chitin7.82 × 106A0A2K0T4Z2GH18chitinase49
hemicellulose3.57 × 108A0A6V8QJU6CE6acetylxylan esterase46
hemicellulose8.52 × 107A0A2H2ZRV5CE5acetylxylan esterase36
hemicellulose7.56 × 107A0A0W7VKU2GH54α-L-arabinofuranosidase102
hemicellulose6.34 × 107G9N626GH27α-galactosidase32
hemicellulose4.66 × 107A0A395NVC4GH3xylan 1,4-β-xylosidase58
hemicellulose4.39 × 107A0A6V8QTS4GH16glucan endo-1,3-β-glucosidase44
hemicellulose3.29 × 107A0A6V8QPH8GH76mannan endo-1,6-α-mannosidase34
hemicellulose3.03 × 107KAH6604482.1GH11glycoside hydrolase56
hemicellulose3.01 × 107A0A2K0TP30GH16glycoside hydrolase49
hemicellulose2.69 × 107G9NUB8GH62α-L-arabinofuranosidase51
hemicellulose2.44 × 107A0A2T3YUG9GH3xylan 1,4-β-xylosidase25
hemicellulose1.82 × 107A0A2T4ASM6GH3xylan 1,4-β-xylosidase31
hemicellulose1.54 × 107G9MSH9GH3xylan 1,4-β-xylosidase15
hemicellulose1.40 × 107A0A2T3YT55GH54α-L-arabinofuranosidase40
hemicellulose1.24 × 107A0A395NS24GH54α-L-arabinofuranosidase27
hemicellulose9.94 × 106A0A2T4BTG8GH54α-L-arabinofuranosidase46
hemicellulose7.48 × 106G9MZ65GH54α-L-arabinofuranosidase28
hemicellulose6.24 × 106G9MV41GH27α-galactosidase69
hemicellulose5.24 × 106A0A6V8R9B0GH27α-galactosidase42
hemicellulose4.48 × 106A0A2K0UKQ2GH3xylan 1,4-β-xylosidase26
hemicellulose3.55 × 106G9P179GH3xylan 1,4-β-xylosidase55
hemicellulose2.72 × 106A0A395NKK0GH12glycoside hydrolase120
hemicellulose1.11 × 106G9NPZ0GH64glycoside hydrolase45
pectin1.20 × 108G9NBD3CE5carbohydrate esterase65
pectin4.64 × 107G9NXF6CE5carbohydrate esterase52
pectin1.60 × 106G9NPZ7CE5carbohydrate esterase49
(a) The iBAQ corresponds to the sum of all the peptide intensities divided by the number of observable peptides of a protein. (b) Accession number with protein information and family information were obtained from UniProt/Swiss-Prot database or NCBI database. (c) Hypothetical molecular weight of the proteins.
Table 3. Comprehensive LC-MS/MS secretome analysis for 29 proteins enzymes found only in the sugarcane bagasse culture classified according to which biomass they degrade.
Table 3. Comprehensive LC-MS/MS secretome analysis for 29 proteins enzymes found only in the sugarcane bagasse culture classified according to which biomass they degrade.
Degraded BiomassiBAQ (a)
Sugarcane Bagasse		Protein IDs
(b)		FamilyMS/MS View:
Identified Proteins		Molecular Weight
(kDa) (c)
amido4.51 × 106B5BQC3GH13α-amylase57
carboxylic ester1.37 × 108A0A6V8QU70CE1carboxylic ester hydrolase56
carboxylic ester3.42 × 107A0A2T3ZJ05CE1carboxylic ester hydrolase26
carboxylic ester1.03 × 107A0A6V8R5C2CE1carboxylic ester hydrolase70
carboxylic ester8.95 × 106A0A2K0U229CE1carboxylic ester hydrolase60
cellulose9.08 × 107A0A6V8QSJ6GH3β-glucosidase F46
cellulose3.84 × 107A0A395N8R8GH2glycoside hydrolase25
cellulose1.99 × 107A0A6V8QPG8GH31α-glucosidase31
cellulose9.93 × 106G9P291GH3glycoside hydrolase25
cellulose4.39 × 106A0A2T4CHF3GH3β-glucosidase96
cellulose2.32 × 106A0A2T3YT78GH3β-glucosidase28
chitin2.29 × 107V9I0I2GH18chitinase42
chitin4.68 × 106A0A395NWN8GH75endo-chitanase88
fucose1.33 × 107A0A6V8QZS6GH95α-fucosidase A26
hemicellulose2.46 × 108A0A2T3ZFW9GH92glycoside hydrolase88
hemicellulose9.26 × 107A0A6V8QLJ6GH76mannan endo-1,6-α-mannosidase25
hemicellulose3.34 × 107A0A6V8QYK7GH43arabinoxylan arabinofuranohydrolase56
hemicellulose3.21 × 107A0A2T3ZL91GH16glycoside hydrolase72
hemicellulose1.86 × 107A0A6V8R1B3GH51α-L-arabinofuranosidase36
hemicellulose9.40 × 106A0A0F9XAZ2GH43glycoside hydrolase47
hemicellulose5.84 × 106A0A6V8R596GH5glucan endo-1,6-β-glucosidase B26
hemicellulose4.36 × 106A0A2T3YUM4GH55glycoside hydrolase80
hemicellulose4.34 × 106A0A2P4ZLF1GH6α-galactosidase27
hemicellulose2.56 × 106A0A395NYK8GH71glycoside hydrolase42
hemicellulose2.42 × 106A0A1T3CS19GH76mannan endo-1,6-α-mannosidase58
hemicellulose2.13 × 106G9PBD3GH721,3-β-glucanosyltransferase49
hemicellulose1.74 × 106G9NFR0GH93glycoside hydrolase49
hemicellulose1.67 × 106A0A2T3YT16GH17glycoside hydrolase23
hemicellulose1.49 × 106A0A2T3YQY4GH92glycoside hydrolase25
(a) The iBAQ corresponds to the sum of all the peptide intensities divided by the number of observable peptides of a protein. (b) Accession number with protein information and family information were obtained from UniProt/Swiss-Prot database or NCBI database. (c) Hypothetical molecular weight of the proteins.
Table 4. Comprehensive LC-MS/MS secretome analysis for three proteins found only in the tamarind seeds condition classified according to which biomass they degrade.
Table 4. Comprehensive LC-MS/MS secretome analysis for three proteins found only in the tamarind seeds condition classified according to which biomass they degrade.
Degraded
Biomass		iBAQ (a)
Tamarind Seeds		Protein IDs
(b)		FamilyMS/MS View:
Identified Proteins		Molecular Weight
(kDa) (c)
cellulose3.03 × 108G9PBZ8GH5glycoside hydrolase88
cellulose3.01 × 106A0A2T3YZD8CBM1carbohydrate-binding module34
lignin1.07 × 108A0A2T3YR43AA3laccase45
(a) The iBAQ corresponds to the sum of all the peptide intensities divided by the number of observable peptides of a protein. (b) Accession number with protein information and family information were obtained from UniProt/Swiss-Prot database or NCBI database. (c) Hypothetical molecular weight of the proteins.
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MDPI and ACS Style

Contato, A.G.; Borelli, T.C.; de Carvalho, A.K.F.; Bento, H.B.S.; Buckeridge, M.S.; Rogers, J.; Hartson, S.; Prade, R.A.; Polizeli, M.d.L.T.d.M. Comparative Analysis of CAZymes from Trichoderma longibrachiatum LMBC 172 Cultured with Three Different Carbon Sources: Sugarcane Bagasse, Tamarind Seeds, and Hemicellulose Simulation. Clean Technol. 2024, 6, 994-1010. https://doi.org/10.3390/cleantechnol6030050

AMA Style

Contato AG, Borelli TC, de Carvalho AKF, Bento HBS, Buckeridge MS, Rogers J, Hartson S, Prade RA, Polizeli MdLTdM. Comparative Analysis of CAZymes from Trichoderma longibrachiatum LMBC 172 Cultured with Three Different Carbon Sources: Sugarcane Bagasse, Tamarind Seeds, and Hemicellulose Simulation. Clean Technologies. 2024; 6(3):994-1010. https://doi.org/10.3390/cleantechnol6030050

Chicago/Turabian Style

Contato, Alex Graça, Tiago Cabral Borelli, Ana Karine Furtado de Carvalho, Heitor Buzetti Simões Bento, Marcos Silveira Buckeridge, Janet Rogers, Steven Hartson, Rolf Alexander Prade, and Maria de Lourdes Teixeira de Moraes Polizeli. 2024. "Comparative Analysis of CAZymes from Trichoderma longibrachiatum LMBC 172 Cultured with Three Different Carbon Sources: Sugarcane Bagasse, Tamarind Seeds, and Hemicellulose Simulation" Clean Technologies 6, no. 3: 994-1010. https://doi.org/10.3390/cleantechnol6030050

APA Style

Contato, A. G., Borelli, T. C., de Carvalho, A. K. F., Bento, H. B. S., Buckeridge, M. S., Rogers, J., Hartson, S., Prade, R. A., & Polizeli, M. d. L. T. d. M. (2024). Comparative Analysis of CAZymes from Trichoderma longibrachiatum LMBC 172 Cultured with Three Different Carbon Sources: Sugarcane Bagasse, Tamarind Seeds, and Hemicellulose Simulation. Clean Technologies, 6(3), 994-1010. https://doi.org/10.3390/cleantechnol6030050

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