The Open Biotechnology Journal




ISSN: 1874-0707 ― Volume 15, 2021
RESEARCH ARTICLE

A Comparative Proteomic Study of Thermobifida Cellulosilytica TB100T Secretome Grown on Carboxymethylcellulose and Rice Straw



Mai H. Elmahdy1, *, Ahmed F. Azmy1, Eman El-Gebaly1, 2, Amal Saafan3, Yasser Gaber1, 4
1 Department of Microbiology and Immunology, Faculty of Pharmacy, Beni-Suef University, 62511, Beni-Suef, Egypt
2 Department of Microbiology and Immunology, Faculty of Pharmacy, October 6 University, 12585, Cairo, Egypt
3 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Menoufia University, Shebeen El-kom, 51132, Egypt
4 Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Mutah University, 61710, Mutah, Jordan

Abstract

Background:

Cellulose, the major component of the plant cell wall, is the most abundant and cheap polymer on earth. It can be used by varieties of cellulolytic enzymes. Cellulases can hydrolyze cellulose to its glucose monomers, which can be fermented to many biotechnological products, such as biochemicals, bioplastics, and biofuels. Actinomycetes are potential sources of cellulases.

Objective:

The current study sheds light on the cellulolytic activity of Thermobifida cellulosilytica, a previously isolated thermophilic actinomycete, and the analysis of the lignocellulases produced in the secretome as a result of induction by different carbon sources.

Methods:

The cellulolytic activity was qualitatively confirmed by Congo red method showing a large halo zone around the colonies. The activity was also assayed using the 3,5-dinitrosalicylic acid (DNS) method. The secretome analysis was conducted by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) based proteomic approach.

Results:

The cellulolytic activity increased by two folds upon the growth of T. cellulosilytica on rice straw (RS) as a complex substrate comparatively to Carboxymethylcellulose (CMC) as a simple one. These results were highly assured by LC-MS/MS. Where more proteins (n=31) were produced in the RS secretome, CMC produced only six proteins, including only one cellulase. Different classes of proteins produced in the RS secretome were cellulases (26%), hemicellulases (16%), proteases (10%), and others (48%).

Conclusion:

Lignocellulases are inducible enzymes. RS as a complex substrate induced T. cellulosilytica for the expression of more lignocellulolytic enzymes than CMC.

Keywords: Carboxymethylcellulose , Carboxymethylcellulase , Thermobifida cellulosilytica , Rice straw , Secretome analysis , LC-MS/MS .


Article Information


Identifiers and Pagination:

Year: 2020
Volume: 14
First Page: 42
Last Page: 51
Publisher Id: TOBIOTJ-14-42
DOI: 10.2174/1874070702014010042

Article History:

Received Date: 26/11/2019
Revision Received Date: 22/01/2020
Acceptance Date: 27/02/2020
Electronic publication date: 01/06/2020
Collection year: 2020

© 2020 Elmahdy et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Microbiology and Immunology, Faculty of Pharmacy, Beni-Suef University, 62511, Beni-Suef, Egypt; Tel: +201096656500; Fax: +20 82 216 2136; E-mail: mai.elmahdy@pharm.bsu.edu.eg





1. INTRODUCTION

The lignocellulosic materials, left behind agricultural and forestry industries, are mainly composed of cellulose 40-50%, hemicellulose 20-40%, and lignin 20-30% of plant dry weight [1Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG. Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 2012; 5(1): 45.
[http://dx.doi.org/10.1186/1754-6834-5-45] [PMID: 22747961]
]. Their production was estimated by 1.5 x 10 12 tons per year [2Sukumaran RK, Singhania RR, Pandey A. Microbial cellulases-production, applications and challenges. JSIR 2005; 64: 832-44.]. Cellulose is a linear polymer ranging from 10,000-15,000 glucose units linked by β-1,4 glycosidic bonds [3Agbor VB, Cicek N, Sparling R, Berlin A, Levin DB. Biomass pretreatment: Fundamentals toward application. Biotechnol Adv 2011; 29(6): 675-85.
[http://dx.doi.org/10.1016/j.biotechadv.2011.05.005] [PMID: 21624451]
] and may reach up to 25,000 glucose units [4Juturu V, Wu JC. Microbial cellulases: Engineering, production and applications. Renew Sust Energ Rev 2014; 33: 188-203.
[http://dx.doi.org/10.1016/j.rser.2014.01.077]
]. Due to its highly ordered crystalline structure, water insolubility as well as the presence of a tough lignin layer around it, cellulose is slowly/hardly degraded and unusable, leading to its disposition and accumulation as waste in nature [5Yang W, Meng F, Peng J, et al. Isolation and identification of a cellulolytic bacterium from the Tibetan pig’s intestine and investigation of its cellulase production. Electron J Biotechnol 2014; 17: 262-7.
[http://dx.doi.org/10.1016/j.ejbt.2014.08.002]
]. This may lead to environmental pollution [6Bayer EA, Shoham Y, Lamed R. Cellulose-decomposing bacteria and their enzyme systems The prokaryotes 2006; 578-617.]. So, intervention to accelerate cellulose degradation and conversion to its fermentable sugars that can be used for biorefinery and industrial purposes is a must.

The efficient hydrolysis of cellulosic waste materials, has been employed either chemically or enzymatically [4Juturu V, Wu JC. Microbial cellulases: Engineering, production and applications. Renew Sust Energ Rev 2014; 33: 188-203.
[http://dx.doi.org/10.1016/j.rser.2014.01.077]
, 7Mohanta YK. Isolation of cellulose-degrading actinomycetes and evaluation of their cellulolytic potential. Bioeng Biosci 2014; 2: 1-5.]. Chemical hydrolysis methods of cellulose degradation include incineration (the routine method), pyrolysis, steam explosion, and acidic or alkaline treatment [8Kulcu R, Yaldiz O. Determination of aeration rate and kinetics of composting some agricultural wastes. Bioresour Technol 2004; 93(1): 49-57.
[http://dx.doi.org/10.1016/j.biortech.2003.10.007] [PMID: 14987720]
]. Nowadays, application of these chemical methods has been dramatically limited and restricted only to be a preliminary/pretreatment step before enzymatic hydrolysis [9Hendriks AT, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 2009; 100(1): 10-8.
[http://dx.doi.org/10.1016/j.biortech.2008.05.027] [PMID: 18599291]
] due to their many disadvantages including environmental pollution, health problems, requirement of large equipment, and production of toxic chemicals or inhibitors as furfural or even undesirable non-sugar by-product [4Juturu V, Wu JC. Microbial cellulases: Engineering, production and applications. Renew Sust Energ Rev 2014; 33: 188-203.
[http://dx.doi.org/10.1016/j.rser.2014.01.077]
, 5Yang W, Meng F, Peng J, et al. Isolation and identification of a cellulolytic bacterium from the Tibetan pig’s intestine and investigation of its cellulase production. Electron J Biotechnol 2014; 17: 262-7.
[http://dx.doi.org/10.1016/j.ejbt.2014.08.002]
, 7Mohanta YK. Isolation of cellulose-degrading actinomycetes and evaluation of their cellulolytic potential. Bioeng Biosci 2014; 2: 1-5.].

On the other hand, the enzymatic degradation of cellulose can overcome all these drawbacks being clean, highly specific, and eco-friendly [7Mohanta YK. Isolation of cellulose-degrading actinomycetes and evaluation of their cellulolytic potential. Bioeng Biosci 2014; 2: 1-5., 10Lekshmi M, Jayadev A, Navami S. Isolation and screening of actinomycetes from marine samples for enzyme production. Int J Sci Eng Res 2014; 5: 199-204.]. The bioconversion of cellulosic wastes into its simple sugars has been employed by different hydrolytic and oxidative enzymes such as cellulases, hemicellulases (xylanases), lytic polysaccharide monooxygenases (LPMO) and peroxidases [1Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG. Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 2012; 5(1): 45.
[http://dx.doi.org/10.1186/1754-6834-5-45] [PMID: 22747961]
, 11Trigo C, Ball A. Production of extracellular enzymes during the solubilisation of straw by Thermomonospora fusca BD25. Appl Microbiol Biotechnol 1994; 41: 366-72.
[http://dx.doi.org/10.1007/BF00221233]
]. Cellulases are a group of three enzymes; endoglucanases or CMCase (carboxymethylcellulases) (E.C. 3.2.1.4), exoglucanases or CBHs (cellobiohydrolases) (E.C. 3.2.1.91) and β-glucosidases (BGLs) or cellobiases (E.C. 3.2.1.21), acting synergistically for complete hydrolysis of cellulose [4Juturu V, Wu JC. Microbial cellulases: Engineering, production and applications. Renew Sust Energ Rev 2014; 33: 188-203.
[http://dx.doi.org/10.1016/j.rser.2014.01.077]
]. Microbial lignocellulolytic enzymes are mainly produced from bacteria, fungi, and actinomycetes, the most effective secretors of lignocelluloses [12Budihal SR, Agsar D, Patil SR. Enhanced production and application of acidothermophilic Streptomyces cellulase. Bioresour Technol 2016; 200: 706-12.
[http://dx.doi.org/10.1016/j.biortech.2015.10.098] [PMID: 26556405]
].

Thermobifida cellulosilytica TB100T strain is an actinomycete that has been isolated from manure compost region [13Kukolya J, Nagy I, Láday M, et al. Thermobifida cellulolytica sp. nov., a novel lignocellulose-decomposing actinomycete. Int J Syst Evol Microbiol 2002; 52(Pt 4): 1193-9.
[PMID: 12148627]
]. This strain showed 97.4% similarity to Thermobifida fusca, one of the well-characterized cellulolytic actinomycetes [13Kukolya J, Nagy I, Láday M, et al. Thermobifida cellulolytica sp. nov., a novel lignocellulose-decomposing actinomycete. Int J Syst Evol Microbiol 2002; 52(Pt 4): 1193-9.
[PMID: 12148627]
]. T. cellulosilytica becomes the subject of many recent researches due to its production of many important industrial enzymes such as cutinases and lignocellulases. Cutinases are efficiently used in plastic biodegradation, especially hydrolysis of PET (polyethylene terephthalate) [14Kawai F, Kawabata T, Oda M. Current knowledge on enzymatic PET degradation and its possible application to waste stream management and other fields. Appl Microbiol Biotechnol 2019; 103(11): 4253-68.
[http://dx.doi.org/10.1007/s00253-019-09717-y] [PMID: 30957199]
]. Lignocellulases composed of cellulases, hemicellulases, and ligases are responsible for effective hydrolysis of lignocellulosic substrates [15Obeng EM, Adam SNN, Budiman C, Ongkudon CM, Maas R, Jose J. Lignocellulases: A review of emerging and developing enzymes, systems, and practices. Bioresour Bioprocess 2017; 4: 1-22.
[http://dx.doi.org/10.1186/s40643-017-0146-8]
].

Previous studies are focused on the identification of culture conditions, phenotypic, genotypic and taxonomic data of T. cellulosilytica TB100 T [ 13Kukolya J, Nagy I, Láday M, et al. Thermobifida cellulolytica sp. nov., a novel lignocellulose-decomposing actinomycete. Int J Syst Evol Microbiol 2002; 52(Pt 4): 1193-9.
[PMID: 12148627]
]. Little studies assessed the cellulolytic activity of this thermophilic strain [ 16Kukolya J, Dobolyi C, Hornok L. Isolation and identification of thermophilic cellulolytic actinomycetes. Acta Phytopathol Entomol Hung 1997; 32: 97-107. ]. Our research aimed to evaluate the cellulolytic potential of T. cellulosilytica grown on different carbon sources. This is the first work concerned with studying the effect of substrate type on the expression of different cellulases in the secretome collected from T. cellulosilytica using LC-MS/MS based proteomic analysis. This study can help the researchers interested in the production of thermophilic cellulases for industrial use.

2. MATERIALS AND METHODS

2.1. Bacteria and Culture Conditions

Thermobifida cellulosilytica TB100T (DSM 44535) used in the present study was ordered from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), Germany. The lyophilized strain was aseptically resuspended in 1 ml of Czapek peptone broth containing: sucrose 30 g/L, NaNO3 3 g/L, K2HPO4 1 g/L, MgSO4.7H2O 0.5 g/L, KCl 0.5 g/L, FeSO4.7H2O 0.01 g/L, yeast extract 2 g/L, peptone 5 g/L and water to 1L. PH was adjusted to 7.3. A volume of 100 μL was added to 5 mL Czapek peptone broth for refreshment and was incubated at 45οC for 3-5 days. A glycerol stock was prepared and preserved at -80οC.

2.2. Qualitative Detection of The Cellulolytic Activity of T. cellulosilytica

The selected strain was cultivated on CMC agar media containing: CMC 0.5 g/L, NaNO3 0.1 g/L, K2HPO4 0.1 g/L, MgSO4 0.05 g/L, yeast extract 0.05 g/L, agar 20 g/L and water to 1L by spot inoculation method. The plates were incubated at 50οC for 72 h then flooded with 0.1% Congo red dye, gently shaken for 20 min, and excess dye was poured off. Finally, the plates were washed with 1M NaCl, gently shaken for 15 min and excess NaCl was poured off. The formation of a clear halo zone around the colony, that can be visualized as a purple color when flooded with 1 M HCl is considered as a positive result [17Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 1982; 43(4): 777-80.
[http://dx.doi.org/10.1128/AEM.43.4.777-780.1982] [PMID: 7081984]
].

2.3. Effect of Different Substrates on Cellulase Production

Cellulase production was determined using CMC and RS, individually, as simple and complex substrate in the production medium containing the following constituents: CMC 5 g/L or pre-treated RS 1% (w/v), MgSO4.7H2O 0.2 g/L, KNO3 0.75 g/L, K2HPO4 0.5 g/L, FeSO4.7H2O 0.02 g/L, CaCl2 0.04 g/L, yeast extract 2 g/L and water to 1 L, pH was adjusted to 7.5. The RS was thermally and chemically pre-treated by NaOH. RS treatment was done as described by Taniguchi [18Taniguchi M, Tanaka M, Matsuno R, et al. Evaluation of chemical pretreatment for enzymatic solubilization of rice straw. Eur J Appl Microbiol Biotechnol 1982; 14: 35-9.
[http://dx.doi.org/10.1007/BF00508001]
] as following: RS was cut into small pieces, washed with tap water for removal of surface dust, and soaked in 2.5% NaOH for 1h with a proportion of 1:10 (w/v). The mixture was autoclaved at 121οC for 30 min and then filtered. The residue was washed with distilled water several times till the supernatant pH became neutral. Finally, the residue was filtered again and dried in the oven at 80οC for 10 min. The microorganism was added to the flasks containing the previous production media with the corresponding substrates. The flasks were incubated at 50οC, shaken at 150 rpm for 11 days. A sample of one mL was periodically withdrawn every 24 h interval for assay of cellulase activity of T. cellulosilytica.

2.4. Cellulase Assay

After the specified incubation time, one milliliter of the production medium was withdrawn and centrifuged at 7000 xg for 10 min. Two hundred and fifty microliters of CFE (Cell Free Extract) was added to a reaction mixture composed of: 500 µL of CMC and 250 µL of 50 mM phosphate buffer, pH was adjusted to 7. The reaction mixture was incubated at 50οC for 2 h, and the resulted reducing sugars were quantified by the DNS method [19Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959; 31: 426-8.
[http://dx.doi.org/10.1021/ac60147a030]
] using a microtiter plate reader (Tecan Sunrise, Switzerland). A standard curve was constructed based on at least six different glucose concentrations to estimate the reducing sugars released. One international unit of enzymatic activity was defined as the amount of enzyme that released 1 μmol of reducing sugar per min.

2.5. Partial Characterization of Crude Extract Produced from T. cellulosilytica

The CFE resulted from T. cellulosilytica growing on CMC as sole carbon source was used for characterization as following:

2.5.1. Optimum PH

Different buffer systems with different pH were prepared. One hundred mM Citrate buffer with pH 4.5, 5 and 5.5 and 100 mM phosphate buffer with pH 6, 6.5, 7, 7.5, 8 and 8.5 were used. The reaction mix was made with each prepared buffer and incubated at 50οC for 2 h. The enzyme activity was assayed by the DNS method.

2.5.2. Optimum Temperature

Five hundred microliters of CMC 0.5%, 250 μL buffer pH 8 and 250 μL supernatant were mixed in Eppendorf tubes. The tubes were incubated at different temperatures (4, 25, 30, 37, 50, and 70οC) for 2 h. The enzyme activity was assayed by the DNS method.

2.5.3. Optimum Substrate Concentration

A typical reaction was prepared as following: in Eppendorf tubes, 500 μL of different concentrations of CMC (0.1%, 0.25%, 0.5%, 0.75%, and 1%), 250 μL buffer pH 8 and 250 μL supernatant were mixed. The tubes were then incubated at 50οC for 2 h. A negative control tube containing distilled water instead of supernatant was incubated under the same conditions. The reducing sugar content was measured using the DNS method.

2.6. Secretome Analysis of T. cellulosilytica Grown on Different Cellulose Substrates

2.6.1. Strain Cultivation and Secretomic Protein Extraction

T. cellulosilytica TB100T strain was cultivated on the production medium containing the corresponding substrates (0.5% CMC and 1% RS, separately). The flasks were incubated at 50οC with shaking at 150 rpm for 7 days. The secretome from each substrate was harvested by centrifugation at 7000 xg at 4οC for 15 min twice to remove the bacterial pellets completely. The supernatant was concentrated and diafiltered through Vivaspin dialysis membrane with 5 kDa MWCO (GE Healthcare Life Sciences, Uppsala, Sweden) with 10 mM PO4-3 buffer pH 7, to one-tenth its starting volume. The protein concentration of each sample was determined using the bicinchoninic acid assay method (BCA). The resultant volume aliquots were preserved at -80οC.

2.6.2. Protein Digestion and LC-MS/MS Analysis

The secretory proteins from each sample were chemically denaturated by 8 M urea, reduced with 200 mM DTT (dithiothreitol) for 30 min at room temperature, alkylated with 1 M IAA (iodoacetamide) for 45 min to 1 h at room temperature in the dark and then enzymatically digested by trypsin containing procaine. The tryptic digestion was performed at 37 οC for 18 h (overnight) and then terminated by acidification with 100% FA (formic acid) to pH range 2-3 [20Adav SS, Cheow ESH, Ravindran A, Dutta B, Sze SK. Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass. J Proteomics 2012; 75(12): 3694-706.
[http://dx.doi.org/10.1016/j.jprot.2012.04.031] [PMID: 22575269]
]. The resulting peptides were analyzed by Ekspert NanoLC425 HPLC system (Eksigent, USA) for chromatographic separation. Peptides (with 2 μL injection volume) from each sample were loaded onto trapping cartridge (10 x 0.5 mm) packed with CHROMXP C18CL 5μm particles, washed using mobile phase A (0.1% FA in DI-Water) at a flow rate of 10 μL/min for 3 min. Subsequently, peptides were fractionated on the analytical column (150 x 0.3 mm) packed with CHROMXP C18CL, 120 Aο, 3 μm particles. The mobile phase A and B (0.1% FA in acetonitrile) were run at a flow rate of 2 μl/min over 55 min to establish gradient elution that was 38 min 3-30% B; followed by 5 min 30-40% B and 2 min 40-80% B; maintained at 80% B for 3 min and finally equilibrated at 3% B.

Peptide fractions eluted from the HPLC system were directly injected into the mass spectrometer with nanospray® Ion Source. Data acquisition was set with a Triple TOF 5600+ system (AB SCIEX software), and the MS data were acquired in the positive ion mode, with a selected mass range of 400-1250 m/z. Smart IDA (Information Dependent Acquisition) was obtained through high-resolution TOF/MS survey scan followed by a product ion scan resulted from MS/MS analysis for the most abundant 40 ions whose threshold was over 150 counts, and charge ranged from +2 to +5 with cycle speed of 1.5 s.

2.6.3. Mass Spectrometric Data Analysis

Data acquisition was performed with Analyst® TF 1.7.1 (AB SCIEX software). MS raw data files from the Triple TOF 5600+ system were converted into MGF (Mascot Generic Format) files, which were then analyzed by Peptide Shaker (v1.16.27) for protein identification. The databases used for searching T. cellulosilytica TB100 were Swiss-prot and TrEMBL database containing 3,587 proteins [21Bairoch A, Apweiler R. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res 2000; 28(1): 45-8.
[http://dx.doi.org/10.1093/nar/28.1.45] [PMID: 10592178]
]. The X!Tandem Algorithm was used for peptide identification. The data search parameters were set up as follows: Trypsin digestion with a maximum of two missed cleavage sites was considered along with fixed modification of cysteines by carbamidomethylation. Acetylation of lysine residues, peptide N termini, deamidation of asparagine and glutamine residues; and methionine oxidation were set as variable modifications. The presence of signal peptide sequences was determined using the ProtParam database [22Walker JM. The proteomics protocols handbook 2005; 969.
[http://dx.doi.org/10.1385/1592598900]
].

2.6.4. Statistical Analysis

All experiments were done in at least two independent replicates, and the presented data are the mean values of replicates ± standard deviation using Microsoft Excel 2010 (Microsoft, USA). Differences between means were examined by one-way ANOVA (analysis of variance) followed by the Student’s t test using SPSS 22.0 software (SPSS Inc., Chicago, USA). The significance of differences was considered at a probability value less than 0.05.

3. RESULTS

3.2. Cellulolytic Activity Assay Using Different Carbon Sources

The cellulolytic activity of T. cellulosilytica TB100T was estimated upon its growth on either CMC or RS. The cellulase production was gradually increased till reaching its maximum activity that was estimated at 0.132± 0.080 IU/mL and 0.298±0.161 IU/mL on the 8th day and 7th day of incubation for both CMC and RS substrates, respectively. Subsequently, the cellulolytic activity decreased with longer incubation (Fig. 2).

3.3. Partial Characterization of Crude Extract

The cellulolytic activity of the CFE was optimal at pH 4.5 and 7.5 evaluated by 0.137 and 0.132 IU/mL, respectively, (Fig. 3A). The optimum temperature of cellulase was 50οC with a maximum activity of 0.136 IU/mL. Cellulase enzyme was able to tolerate temperature up to 70οC (Fig. 3B). The concentration of 0.75% of CMC was found to induce the highest amount of cellulase estimated by 0.132 IU/mL (Fig. 3C).

3.4. Secretome Analysis

The extracellular proteins of T. cellulosilytica TB100T, secreted in the presence of different substrates, were confidently identified by LC-MS/MS based proteomic analysis. After the enzymatic digestion of the secretome proteins by trypsin, a total of 9 peptides and 127 peptides were detected in the MS/MS spectra of CMC and RS, (Supplementary files 2 and 3). The resulted peptides were then searched against different databases giving 6 proteins and 31 proteins were produced in the CMC and RS, respectively, (Supplementary files 4 and 5).

The molecular weight and isoelectric point (PI) of all identified proteins were ranged from 15-106.3 kDa and 3.52-6.43 as shown in Tables 1 and 2. Most produced proteins had an acidic pH and extracellularly secreted by the action of signal peptides.

The secretomic produced proteins were functionally categorized and pie charted according to their roles in the deconstruction of lignocellulosic biomass (Fig. 4). Regarding CMC, the only secreted cellulolytic enzyme was glucanase (7%). On the other hand, the proteins detected in the RS secretome were grouped as cellulases (26%), hemicellulases (16%), proteases (10%), and other proteins (48%) as shown in (Fig. 4). Out of 31 proteins, 9 proteins (8.43) belonged to glycoside hydrolases (GHs) which were classified into different families according to (Carbohydrate Active enZYmes) CAZY database (http://www.cazy.org) [23Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 2014; 42: D490-5.
[http://dx.doi.org/10.1093/nar/gkt1178] [PMID: 24270786]
]. The GH families included were GH6 (2), GH9 (2), GH10 (2), GH43 (1), GH48 (1) and GH81 (1). In addition, ten enzymes were annotated as containing carbohydrate binding modules (CBMs).

4. DISCUSSION

Actinomycetes are considered as potential sources of bioactive secondary metabolites like antibiotics, enzymes and growth factors [12Budihal SR, Agsar D, Patil SR. Enhanced production and application of acidothermophilic Streptomyces cellulase. Bioresour Technol 2016; 200: 706-12.
[http://dx.doi.org/10.1016/j.biortech.2015.10.098] [PMID: 26556405]
]. Cellulose active enzymes, “cellulases” are considered as important enzymes certainly produced from actinobacteria, including T. cellulosilytica TB100T. An additional thermophilic property of T. cellulosilytica TB100T strain makes it a potential source for the production of thermostable cellulases, the cause of choice of the provided actinomycete strain in this study.

We confirmed the cellulolytic activity of T. cellulosilytica TB100T by Congo red which is the most widely used method for preliminary screening of cellulolytic actinomycetes, bacteria and fungi [5Yang W, Meng F, Peng J, et al. Isolation and identification of a cellulolytic bacterium from the Tibetan pig’s intestine and investigation of its cellulase production. Electron J Biotechnol 2014; 17: 262-7.
[http://dx.doi.org/10.1016/j.ejbt.2014.08.002]
]. Congo red binds strongly with β-1,4-D glycoside linkage in polysaccharides, forming red complex [24Santhi VS, Gupta A, Saranya S, Jebakumar SRD. A novel marine bacterium Isoptericola sp. JS-C42 with the ability to saccharifying the plant biomasses for the aid in cellulosic ethanol production. Biotechnol Rep (Amst) 2014; 1-2: 8-14.
[http://dx.doi.org/10.1016/j.btre.2014.05.002] [PMID: 28435797]
]. This strong complex does not form except with polymers [25Wood PJ, Erfle JD, Teather RM. Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases. Methods Enzymol 1988; 160: 59-74.
[http://dx.doi.org/10.1016/0076-6879(88)60107-8]
]. So, a large clear halo zone formed around colonies due to degradation of CMC polymer as a role of cellulase produced from T. cellulosilytica. Although the formation of a large clear zone on CMC agar plates, it doesn’t imply good productivity in liquid media [17Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 1982; 43(4): 777-80.
[http://dx.doi.org/10.1128/AEM.43.4.777-780.1982] [PMID: 7081984]
].

It is also worth noting that the maximum CMCase activity of a thermophilic actinomycete, Thermomonospora fusca BD25 which is the closest one to the strain of our study, was also weak and estimated at 0.05 IU/mL, nearly half the CMCase activity from our strain of interest [26Tuncer Mr, Ball AS, Rob A, et al. Optimization of extracellular lignocellulolytic enzyme production by a thermophilic actinomycete Thermomonospora fusca BD25. Enzyme Microb Technol 1999; 25: 38-47.
[http://dx.doi.org/10.1016/S0141-0229(99)00012-5]
]. Similarly, lower weak cellulolytic activity was produced maximally from Geobacillus sp. DUSELR7 on day 7 and from Brevibacillus sp. DUSELG12 on day 10 as 0.058 IU/mL and 0.02 IU/mL respectively [27Rastogi G, Muppidi GL, Gurram RN, et al. Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine, Lead, South Dakota, USA. J Ind Microbiol Biotechnol 2009; 36(4): 585-98.
[http://dx.doi.org/10.1007/s10295-009-0528-9] [PMID: 19189143]
]. In another study, the cellulase activity of Bacillus subtilis AS3 was 0.07 IU/mL in basal control medium and increased 6 folds to be 0.43 IU/mL after cultivation in the optimized medium using Placket-Burman design [28Deka D, Bhargavi P, Sharma A, Goyal D, Jawed M, Goyal A. Enhancement of cellulase activity from a new strain of Bacillus subtilis by medium optimization and analysis with various cellulosic substrates. Enzyme Res 2011; 2011151656
[http://dx.doi.org/10.4061/2011/151656] [PMID: 21637325]
].

Fig. (2)
Effect of incubation period/fermentation time on the cellulolytic activity of T. cellulosilytica using different carbon sources, CMC and RS. The symbols (♦) with a red line representing 0.5% CMC and (■) with blue line representing 1% RS.


Fig. (3)
Partial characterization of cellulase including:
A. Effect of different pH () on cellulase production.
B. Effect of different incubation temperatures () on cellulase production.
C. Effect of different CMC concentration () on cellulase production.


Fig. (4)
Different classes of proteins identified in the T. cellulosilytica TB100 secretome using (A). CMC as a simple substrate and (B). rice straw as a complex substrate after analysis by LC-MS/MS data.


Table 1
Functional classification of proteins detected in the secretome of T. cellulosilytica TB100 using CMC as simple substrate.


Table 2
Functional classification of proteins detected in the secretome of T. cellulosilytica TB100 using RS as complex substrate.


On the other hand, there are some actinobacterial strains in other studies that produced CM Case with moderate and high maximum activity including actinomycete BAY 21 with 1.381±0.024 IU/mL [7Mohanta YK. Isolation of cellulose-degrading actinomycetes and evaluation of their cellulolytic potential. Bioeng Biosci 2014; 2: 1-5.] and Streptomyces DSK59 with 20 IU/mL [12Budihal SR, Agsar D, Patil SR. Enhanced production and application of acidothermophilic Streptomyces cellulase. Bioresour Technol 2016; 200: 706-12.
[http://dx.doi.org/10.1016/j.biortech.2015.10.098] [PMID: 26556405]
]. Another alkalothermophilic actinomycete produced CM Case with the activity of 23 IU/mL, 8.5 IU/mL and 12.5 IU/mL when cellulose paper powder, wheat bran and corncob were used as the substrate, respectively [29George SP, Ahmad A, Rao MB. Studies on carboxymethyl cellulase produced by an alkalothermophilic actinomycete. Bioresour Technol 2001; 77(2): 171-5.
[http://dx.doi.org/10.1016/S0960-8524(00)00150-4] [PMID: 11272024]
].

Regarding the cellulase, it is produced gradually till reaching maximum activity followed by a decline with longer incubation. Longer incubation period enhances the production of proteases. Proteases degrade subsequent enzymes resulting in diminished cellulolytic activity [30Singh S, Moholkar VS, Goyal A. Isolation, identification, and characterization of a cellulolytic Bacillus amyloliquefaciens strain SS35 from rhinoceros dung. ISRN Microbiol 2013; 2013728134
[http://dx.doi.org/10.1155/2013/728134] [PMID: 23762763]
]. Prasad reported (2013) other reasons related to feedback inhibition occurred by the accumulation of end products [31Prasad P, Singh T, Bedi S. Characterization of the cellulolytic enzyme produced by Streptomyces griseorubens [Accession No. AB184139] isolated from Indian soil. J King Saud University Sci 2013; 25: 245-50.
[http://dx.doi.org/10.1016/j.jksus.2013.03.003]
].

Unlike most researches interested in the isolation of cellulolytic strains, the first study directed toward secretomic analysis of T. cellulosilytica TB100T was conducted [32Liu D, Li J, Zhao S, et al. Secretome diversity and quantitative analysis of cellulolytic Aspergillus fumigatus Z5 in the presence of different carbon sources. Biotechnol Biofuels 2013; 6(1): 149.
[http://dx.doi.org/10.1186/1754-6834-6-149] [PMID: 24131596]
]. The secretory protein profiles of T. cellulosilytica grown on different carbon sources, including CMC and RS, were compared. The proteomic study revealed a significant up-regulation in the lignocellulolytic enzymes expression of in the case of RS than CMC.

Lignocellulases are inducible enzymes [20Adav SS, Cheow ESH, Ravindran A, Dutta B, Sze SK. Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass. J Proteomics 2012; 75(12): 3694-706.
[http://dx.doi.org/10.1016/j.jprot.2012.04.031] [PMID: 22575269]
]. Their production is dependent on the type or nature of the substrate used the culture media, which is known as substrate-induced phenomena [33Juhasz T, Szengyel Z, Reczey K, et al. Characterization of cellulases and hemicellulases produced by Trichodermareesei on various carbon sources. Process Biochem 2005; 40: 3519-25.
[http://dx.doi.org/10.1016/j.procbio.2005.03.057]
, 34Manavalan A, Adav SS, Sze SK. iTRAQ-based quantitative secretome analysis of Phanerochaete chrysosporium. J Proteomics 2011; 75(2): 642-54.
[http://dx.doi.org/10.1016/j.jprot.2011.09.001] [PMID: 21945728]
]. Do Vale and his colleagues (2012) showed that the complexity of the substrate affected the variety of the produced enzymes [35Do Vale LH, Gómez-Mendoza DP, Kim MS, et al. Secretome analysis of the fungus Trichoderma harzianum grown on cellulose. Proteomics 2012; 12(17): 2716-28.
[http://dx.doi.org/10.1002/pmic.201200063] [PMID: 22745025]
]. The CMC secretome contained six secretory proteins involving only one cellulase (glucanase). This low expression level of cellulases produced in the CMC secretome has an advantage of better isolation, purification, and studying the kinetic parameters of this cellulase, leading to decrease production cost on the large industrial scale.

On the contrary, more lignocelluloses (31 proteins) were identified in the RS secretome. The different classes of proteins produced were cellulases, hemicellulases, proteases, and other proteins. These varieties of enzymes were produced in the RS secretome due to its complex composition of cellulose (41%), hemicellulose (20%) tightly packed with lignin (12%) [36Narra M, Dixit G, Divecha J, Madamwar D, Shah AR. Production of cellulases by solid state fermentation with Aspergillus terreus and enzymatic hydrolysis of mild alkali-treated rice straw. Bioresour Technol 2012; 121: 355-61.
[http://dx.doi.org/10.1016/j.biortech.2012.05.140] [PMID: 22864171]
].

The lignin content of RS hinders the accessibility of the hydrolytic enzymes to their substrates [20Adav SS, Cheow ESH, Ravindran A, Dutta B, Sze SK. Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass. J Proteomics 2012; 75(12): 3694-706.
[http://dx.doi.org/10.1016/j.jprot.2012.04.031] [PMID: 22575269]
]. Therefore, thermal and chemical pretreatment of RS is an essential process to make their cellulosic content more accessible to cellulases [37Binod P, Sindhu R, Singhania RR, et al. Bioethanol production from rice straw: An overview. Bioresour Technol 2010; 101(13): 4767-74.
[http://dx.doi.org/10.1016/j.biortech.2009.10.079] [PMID: 19944601]
]. However, RS pretreatment is two-sided coin having drawbacks, including the formation of inhibitory products such as phenolics, aliphatic acids, and furan derivatives making feedback inhibition on the hydrolytic enzymes and further the fermentation process [38Chandel AK, Singh OV. Weedy lignocellulosic feedstock and microbial metabolic engineering: Advancing the generation of ‘Biofuel’. Appl Microbiol Biotechnol 2011; 89(5): 1289-303.
[http://dx.doi.org/10.1007/s00253-010-3057-6] [PMID: 21181146]
].

Most of the produced proteins contain carbohydrate-binding modules (CBMs) previously known as cellulose then carbohydrate binding domains (CBDs). CBMs are non-catalytic domains covalently bonded to the hydrolytic enzymes of different GH families. Like GHs, CBMs were classified into families ranged from family1 to 49 according to amino acid sequence similarities [39Moser F, Irwin D, Chen S, Wilson DB. Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8. Biotechnol Bioeng 2008; 100(6): 1066-77.
[http://dx.doi.org/10.1002/bit.21856] [PMID: 18553392]
]. CBMs are responsible for protein-polysaccharide recognition [40Boraston AB, Bolam DN, Gilbert HJ, Davies GJ. Carbohydrate-binding modules: Fine-tuning polysaccharide recognition. Biochem J 2004; 382(Pt 3): 769-81.
[http://dx.doi.org/10.1042/BJ20040892] [PMID: 15214846]
]. They can target (hemi) cellulases to their carbohydrate polymers by increasing their concentrations at the substrates sites. Then, they change the carbohydrate structure, promoting their degradation by the effect of the hydrolytic enzymes [39Moser F, Irwin D, Chen S, Wilson DB. Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8. Biotechnol Bioeng 2008; 100(6): 1066-77.
[http://dx.doi.org/10.1002/bit.21856] [PMID: 18553392]
, 41Shoseyov O, Shani Z, Levy I. Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev 2006; 70(2): 283-95.
[http://dx.doi.org/10.1128/MMBR.00028-05] [PMID: 16760304]
]. So, CBMs have a positive effect on increasing the rate and yield of the lignocellulolytic enzymes.

The absence of BGLs was noticed in the secretome of T. cellulosilytica grown on the corresponding substrates. This result was assured by quantitative assay for BGLs activity using pNPG (para nitro phenyl beta- D-glucopyranoside) as a substrate showing its negligible activity (data not shown). Our similar result was reported by other researches referring to the cause of the intracellular formation of this enzyme [42Singh S, Tiwari R, Renuse S, Pranaw K, Nain L. Proteomic analysis of Streptomyces sp. ssr-198 grown on paddy straw. J Basic Microbiol 2015; 55(6): 790-7.
[http://dx.doi.org/10.1002/jobm.201400639] [PMID: 25639595]
]. So, commercial cellulase preparations should be amended with BGLs from other sources, especially fungal ones, which are the highest BGL producers for efficient lignocellulolytic biomass hydrolysis [43Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A. Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresour Technol 2013; 127: 500-7.
[http://dx.doi.org/10.1016/j.biortech.2012.09.012] [PMID: 23069613]
].

The production, activity, and stability of actinomycetes, cellulases are greatly affected by pH and temperature [44McCarthy A. Lignocellulose-degrading actinomycetes. FEMS Microbiol Lett 1987; 46: 145-63.
[http://dx.doi.org/10.1111/j.1574-6968.1987.tb02456.x]
]. Thermophilic cellulase system showed stability and activity at a wide pH range from 4.5-8.5 with a slight non-significant increase at pH 4.5 and 7.5 (p< 0.05). Other researches showed similar results in which the optimum pH range of cellulases was generally reported from 4.2-5.2 [45Zinchenko G. Optimization of enzymatic hydrolysis of fibre sludge from pulp mill. Central Ostrobothnia University of Applied Sciences 2012.]. However, Prasad and co-workers (2013) assured that maximum cellulase activity from Streptomyces griseorubens (Accession No. AB184139) was at pH 7 [31Prasad P, Singh T, Bedi S. Characterization of the cellulolytic enzyme produced by Streptomyces griseorubens [Accession No. AB184139] isolated from Indian soil. J King Saud University Sci 2013; 25: 245-50.
[http://dx.doi.org/10.1016/j.jksus.2013.03.003]
]. The activity of CM Case from Bacillus sp. SMIA-2 was found to be optimum at pH 8 [46Ladeira SA, Cruz E, Delatorre AB, et al. Cellulase production by thermophilic Bacillus sp: SMIA-2 and its detergent compatibility. Electron J Biotechnol 2015; 18: 110-5.
[http://dx.doi.org/10.1016/j.ejbt.2014.12.008]
]. The main reason may be attributed to the complex cellulase system required for complete cellulose degradation. This complex system is composed of 3 different enzymes acting synergistically [47Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS. Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol Mol Biol Rev 2002; 66(3): 506-77.
[http://dx.doi.org/10.1128/MMBR.66.3.506-577.2002] [PMID: 12209002]
]. Each enzyme has its optimum pH that ensures proper working.

Cellulase isolated from T. cellulosilytica showed the maximum activity at 50οC with 94% of retained activity at 70οC. Other researchers showed similar results from Streptomyces lividans66, Bacillus sp., MSL2 strain, Bacillus sp DUSELR13, and Geobacillus sp WSUCF1 [48Théberge M, Lacaze P, Shareck F, Morosoli R, Kluepfel D. Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene. Appl Environ Microbiol 1992; 58(3): 815-20.
[http://dx.doi.org/10.1128/AEM.58.3.815-820.1992] [PMID: 1575483]
, 49Sriariyanun M, Tantayotai P, Yasurin P, et al. Production, purification and characterization of an ionic liquid tolerant cellulase from Bacillus sp. isolated from rice paddy field soil. Electron J Biotechnol 2016; 19: 23-8.
[http://dx.doi.org/10.1016/j.ejbt.2015.11.002]
]. This higher temperature reaction leads to faster reaction rates and better degradation of cellulosic materials [46Ladeira SA, Cruz E, Delatorre AB, et al. Cellulase production by thermophilic Bacillus sp: SMIA-2 and its detergent compatibility. Electron J Biotechnol 2015; 18: 110-5.
[http://dx.doi.org/10.1016/j.ejbt.2014.12.008]
]. The maximum cellulolytic activity achieved at 0.75% concentration of CMC. Higher concentrations were too viscous that hindered the cellulolytic assay.

CONCLUSION

T. cellulosilytic a is a promising source of thermostable cellulases that are active over a wide range of pH and temperature. Also, LC-MS/MS based proteomic analysis revealed that T. cellulosilytic a has variable cellulases that are efficient for the degradation of simple/complex lignocellulosic materials. T. cellulosilytic a is an interesting actinomycete for more studies in the production of thermostable cellulases for successful applications, such as biofuel production.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No animals/humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

The authors are thankful for the proteomics unit in the children’s cancer hospital, especially Dr. Sameh Magdeldin, Dr. Hassan Shikshaky, and Dr. Aya Osama for the proteomic analysis. We would like to thank Dr. Haitham Saeed for supporting this study.

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher’s web site along with the published article.

Supplementary file 1: Pellets of T. cellulosilytica grown on 0.5% CMC and 1% RS after 2 days and 7 days of incubation.

Supplementary file 2: Peptides identified in the CMC secretome by LC-MS/MS technique.

Supplementary file 3: Proteins identified in the CMC secretome by LC-MS/MS technique.

Supplementary file 4: Peptides identified in the RS secretome by LC-MS/MS technique.

Supplementary file 5: Proteins identified in the RS secretome by LC-MS/MS technique.

Download File


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Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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