Expression analyses of endoglucanase gene in Penicillium oxalicum and Trichoderma viride

INTRODUCTION

Cellulose is a water insoluble polysaccharide and the most abundant degradable hydrocarbons in nature, which is composed of D-glucopyranosyl units joined by 1,4-glycosidic bonds. Cellulose plays an important role in changing stabilized carbon to CO₂, as well as producing sustainable biological and bio-energy products to replace fossil fuels (Chojoghi et al. 2005). Biological degradation of cellulose in soil could be performed by cellulase enzyme produced endogen-
ously by many microorganisms such as fungi, bacteria and Protista (Li et al. 2009). A number of enzymes with cellulose activity classified in family 9 from various organisms from which cellulases have been reported (Arai et al. 2003).

Cellulolytic enzymes should be either secreted in the environment or connected to outer surface ofcellulolytic microorganisms. These enzymes usually act together synergistically. They are able to hydrolyze β-1, 4 glycosidic bands in cellulose and change it to CO₂ and H₂O (in aerobic condition), and CO₂, H₂O, and CH₄ (in anaerobic condition). These enzymes are; 1-4-β-D- glucanase or endoglucanases (EG, EC 3.2.1.4), which hydrolyze internal glycosidic bands and 1-4-β-D-glucancellobiosehyrolase or exoglucanase (CBH, EC 3.2.1.9.1), which releases cellobiose units from the fungi can grow on cellulose as the sole source of carbon. Cellulase enzyme is one of the most important ends of the cellulose chain; 1-4-β-D-glucosidases (βGLC, 3.2.1.21) which separates glucose units from linear cellulose (cello-oligosaccharides) (Coughlan, 1990). Some microorganisms such as fungi are able to degrade cellulose. Fungi play an important role in degrading the cellulosic compounds due to their strong enzymatic systems (Jorgensen & Olsson 2006).

Detection and manipulation of some fungal species as cellulose producers has been interested many research centers, because of their application in starch processing, grain alcohol fermentation, extraction of fruit juices, pulp and paper industry and textile industry (Gao et al. 2008). Among hundreds of fungal species which are capable of developing on materials containing cellulose as the source of carbon, some of them could produce cellulose enzyme to degrade cellulose (Suurnakki et al. 2000). Previously, we have reported that some fungal species of different genera (Fusarium solani,Trichoderma viride,
T. reesei, T. koningii and Aspergillus terreus) can produce cellulolytic enzymes with high activity rate (Leylai et al. 2011a). Saprophytic fungi could be regarded as one of the most beneficial sources of cellulolytic enzymes. More than one hundred industrial enzymes have been introduced in agricultural, food and paper industries (Bhat 2000).

Withregard to the important role of these enzymes in industry, in this study, we attempted to determine the profile of endogluconase gene expression and its related proteins in two selected isolates of fungi with high cellulase activity.

MATERIALS AND METHODS

Fungal culture and media

Sampling was performed from soil of agricultural areas in north of Iran during 2009-2010. Soil suspension was prepared and cultured on water-agar medium. After growing fungi for five days, a six-mm disc of fungal colony was transferred to Potato Dextrose Agar (PDA, Merk, Germany) medium. A total of 300 isolates were used for celluase activity assay. Cellulose degradation in vitro was evaluated by providing minimal medium containing nitrogen sources, micro-elements, and carboxymethyl cellulose as the only carbon source (Leylai et al. 2011b). Induction of isolates was performed by carboxymethyl cellulose (CMC) to release cellulase in culture medium inside 100-ml conical flask containing fresh hypha of fungi. Experiments were done at 25 ºC in three independent replications.

Sugar and protein assay

Sugar assay was done four days after induction and continued for one month. Released glucose in culture media was detected by using ammonium molybdate reagent. Absorption was read by spectrophotometer at 575 nm wave length (Kossem & Nannipieri 1995). Endoglucanase activity was determined in a duplicate reaction mixture containing 200 μl 1% CMC-Na (w/v, dissolved in 100 mM sodium acetate buffer, pH 6) and 100 mM NaCl. The reactions were allowed to proceed for 10 min at 50 °C and terminated by adding 0.5 ml dinitrosalicylic acid (DNS) and then heating in a boiling water bath for 10 min. The absorbance was measured at 540 nm, using Novospect II spectrophotometer (Pharmacia). One unit of enzyme is defined as the amount of enzyme that catalyzes the formation of 1 μmol glucose/min under defined assay conditions (Li et al. 2009). Protein purification procedures were carried out at 4°C with chromatographic monitored at 280 nm wave length. Protein concentrations were determined by the method of Bradford (Bradford 1976), using serum albumin as standard treatment. SDS–PAGE was performed in 10% SDS–PAGE and proteins were visualized by silver staining (Morrissey 1981). A band of about 66 kDa was cut out and kept in CMC medium for 24 h at 25°C. Protein extraction and purification from gel was performed using AllPrep Mini kit (Qiagen) according to manufacturer’s protocol. The sequence of N-terminal amino acid of the intact enzyme was determined by using an automated sequencer (Applied Biosystem, ABI, USA).

RNA isolation and reverse transcription

Total RNAs from induced hypha in culture medium was performed using a standard phenol-chloroform procedure and purified using RNA purification Kit (Qiagene). Total RNA was quantified using a Scandrop spectrophotometer (Analytika, Germany) and RNA quality was assessed by 1% agarose gel electrophoresis stained by Ethidium bromide. The integrity of RNA was evaluated with an Agilent 2100 bioanalyzer (Agilent Technologies). First-strand cDNA was synthesized from total RNA using Roch extraction solution (Roch, Germany). 50-100 ng of the total RNA from each CMC stimulated cell was used to prepare double-strand cDNA using Ominiscrip kit (Qiagene, Germany) according to manufacturer’s protocol.

Primer design and polymerase chain reaction

Primers used in this study were designed by using NTI software and Primer 3 site (http://frodo.wi.mit.
edu). The primers were checked for secondary structures with oligoanalyser software (http://www.
idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx). The primers used in this study are summarized in Table 1.

Table 1. The primers used in this study.

*PO: Penicillium oxalicum 1SMS and TV: Trichoderma viride 156M

PCR assay

To identify β-1,4-endoclocanase sequence, the PCR assay was performed by using related specific primers (Table 1). The reaction mixtures were prepared in a total volume of 25 μl with a final concentration of 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.2 mM of each dNTP and 1.5 mM MgCl₂. For each reaction, 1.5 U of Taq DNA polymerase (Fermentas, France), 15 pmol of each primer and 25 ng of fungal template DNA were added. The PCR reaction was performed in a thermocycler (Eppendorf Mastercycler Gradient, Hamburg, Germany) using the following program: an initial denaturation step at 94° for 2 min, 40 cycles of 94 °C (30 s), 60 °C - 66 °C (30 s), 72 °C (60 s), and a final extension step at 72 °C for 10 min. A negative control, deleting DNA template, was used in every set of reactions. PCR products were separated by electrophoresis on 1.5% agarose gels stained with Ethidium bromide (0.5 µg/ml) in 40 mM Tris–acetate and 1.0 mM EDTA and then photographed under UV light. Extraction of purified PCR fragment was done using a Silica Bead DNA Gel Extraction kit (Fermentase, USA), according to the manufacturer’s protocol.

DNA Sequencing

The 5’-end DNA sequencing was conducted with an ABI 3700 sequencer at Macrogen Company in South Korea. The cDNA sequences were screened for quality and then queried using the National Center for Biotechnology Information (NCBI) standalone BlastAll program against the BLASTN (nucleic acid), TBLASTX (translated protein query search), Swissprot, version 6.0 UniProt and UniRef100. PROSITEs were specified using NPSA server (http://npsapbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_server.html). Sequence similarities above 98% and E values less than 1E^-10 were considered as statistically significant positive matches (data not presented). Sequences that matched to hypothetical proteins or had low similarity to a known protein were considered as unknown genes.

RESULTS

Sugar and protein assay

Sugar and protein released in inducer medium were measured at different time intervals. There was fluctuation in the amount of released glucose during sugar measurement in studied isolates.

 Presence of glucose in fungal culture medium showed cellulose decomposition. According to these results, variation in amount of glucose are significantly observed during the measurement which is due to fungal activity to provide required conditions for growing and producing the maximum amount of enzyme. Regarding the amount of the released sugar and protein, isolates were graded; two isolates with high activity were selected for future experiments (Table 2). The maximum amount of the enzyme was obtained 13 days after inoculation for different isolates in culture media. Results showed that the amount of enzyme was decreased. This might be due to either aging of fungi and presence of secondary metabolites or reduction of materials in culture media. The resulted data indicated that two isolates, Penicillium oxalicum 1SMS and Trichoderma viride 156MS possessed the maximum enzyme production rates which were 0.078 mg/L and 0.075 mg/L, respectively.

Endoglucanase enzyme evaluation showed that the two isolates could produce 0.06578 and 0.05989 mg/L protein, respectively. SDS-PAGE analysis also indicated a protein with molecular weight of 45-66.2 kDa, which is able to release glucose according to enzyme verification test (Fig 1).

Table 2. Bioinformatics analysis of N-glycosylation site related to endoglucanase gene in two studied fungal species

Randomized probability: 5.138e-03

Site: 78 to 81 NTTL. Identity

Site: 164 to 167 NGSL. Identity

Site: 204 to 207 NGTL. Identity

Site: 208 to 211 NTSH. Identity

Site: 394 to 397 NSTA. Identity

Fig. 1. SDS–PAGE analysis of purified enzyme stained with coomassie R-250 brilliant blue. (A): protein profile produced by Trichoderma viride (lan 1) and (B):.Peninicilium oxalicum at inducer CMC medium (lane 2). M: protein size marker.

Trichoderma viride 156MS

Amplified product of endogluconase gene in Trichoderma viride 156MS isolates, which was provided by using specific primer, showed a yielded fragment ranging from 1400 to 1600 bp (Fig 2A). This sequence showed 98% similarity with the same gene in Trichoderma isolates having a fragment of 1700 bp in database. The sequence of T. viride156MS which was characterized in this part has been recorded in GenBank under accession number of JN247430. Bioinformatic analyses of queried sequence showed an open reading frame (Fig 2B).

Penicillium oxalicum 1SMS

Amplified fragments produced in PCR reaction by usingspecific primer showed a ranging in size from 1400 to 1700 bp on electrophoresisgel (Fig 3A). Nucleotide sequence related to coding region of eg1 in Penicillium oxalicum cited in NCBI database, has the size of 1437 bp. Comparison of amplified sequences with those of 5-P flanking regions of cellulase genes in other fungi revealed the large areas of homology about 98%. The sequence related to coding region of β-1,4 endoglucanase (CDs) showed a high degree of nucleotide sequence similarity to genes assigned in the genomes of Penicillium isolates. Protein sequence also showed a strong sequence similarity to endoglucanase genes in Penicillium isolates. The sequence determinationof this isolate has been deposited in GenBank under accession number of JF309107. Bioinformatics analysis of queried sequence showed an open reading frame (Fig 3B).

Fig. 2. (A) Related fragments of endoglucanase gene (Line 3) and cDNA of expressed eg1 gene (Lines 1-2) amplified by using specific primers for Trichoderma viride 156MS. (B) ORF of coding sequence region (cDNA) related to Endoglucanase gene in T. viride 156MS.

Fig. 3. (A) Related fragments of endoglucanase gene (Line 1) and cDNA of expressed eg1 gene (Lines 2-3) amplified

by using specific primers for Penicillium oxalicum 1SMS.(B) ORF of coding sequence region related to

endoglucanase gene in P. oxalicum 1SMS.