TITLE: Characterization of two thermophilic cellulases from Talaromyces leycettanus JCM12802 and their synergistic action on cellulose hydrolysis
AUTHORS: Yuan Gu, Fei Zheng, Yuan Wang, Xiaoyun Su, Yingguo Bai, Bin Yao, Huoqing Huang, Huiying Luo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The genes of two Thermophilic cellulases (</span><span style = "font-style:italic;">Tl</span><span>Cel5A and </span><span style = "font-style:italic;">Tl</span><span>Cel6A) belonging to GH5 and GH6 respectively were obtained from</span><span style = "font-style:italic;">Talaromyces leycettanus</span><span> JCM12802 and proteins of them were expressed in </span><span style = "font-style:italic;">Pichia pastoris</span><span>. The properties of the cellulases were investigated by determining their optimal temperature, optimal pH, temperature stability, pH stability, and kinetic parameters. Through the hydrolysis of oligosaccharides analysis, the effect of two cellulases (</span><span style = "font-style:italic;">Tl</span><span>Cel5A and </span><span style = "font-style:italic;">Tl</span><span>Cel6A) on oligosaccharides can be known. Then, under the optimal conditions, the synergistic effect between two cellulases (</span><span style = "font-style:italic;">Tl</span><span>Cel5A and </span><span style = "font-style:italic;">Tl</span><span>Cel6A) mixed in different ratios was determined.</span></div></div>

[STEPS]
?. Cloning of the cellulase-encoding genes
?. The full length cellulase-encoding genes,Tlcel5A and Tlcel6A, were identified in the genome sequence of T. leycettanusJCM12802, and obtained by PCR amplification with specific primers GH5F/GH5R and GH6F/GH6R (Table 1).
?. The PCR conditions were as follows: 95°C for 3min, followed by 30 cycles of 95°C for 30 s, 60°C for 60 s, and 72 ◦C for 1min.
?. cDNAs were then synthesized in vitro using the ReverTra Ace-a-™ kit. The cDNAs coding for the mature TlCel5A and TlCel6A were amplified with primers GH5F1/GH5R1 and GH6F1/GH6R1 harboring restriction sites, respectively. (Table 1).
?. The PCR products were inserted into plasmid pGEM-T Easy and then transformed into E. coli Trans1-T1.
?. Sequence and structure analysis
?. The nucleotide and amino acid sequences were used for BLAST analysis at NCBI (http:// www.ncbi.nlm.nih.gov/BLAST/).
?. Simplified sequence alignment and prediction of the isoelectric point and molecular weight were conducted by using the Vector NTI Suite 10.0 software.
?. Signal peptide prediction was carried out with SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/).
?. Multiple alignment of protein sequences was performed with the ClustalX 2.1 (http://www.clustal.org, [1], and the results were demonstrated by ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi, [2].
?. The NetNglyc server was used to predict the putative N-glycosylation sites (http://www.cbs.dtu.dk/services/NetNGlyc/).
?. And the modeled structures were predicted and visualized by using the SWISS-MODEL [3] and Pymol [4].
?. Heterogeneous expression in P. pastoris
?. The cDNA fragments coding for the mature proteins of TlCel5A and TlCel6A were digested with restriction enzymes SnabI or EcoRI and NotI and subcloned into plasmid pPIC9 to yield expression vectors pPIC9-Tlcel5A and pPIC9-Tlcel6A, respectively.
?. The recombinant plasmids were then linearized with Bgl II and individually transformed into P. pastoris GS115 competent cells by electroporation.
?. MD plates can be used to select the transformants and pick some positive colonies into 3 mL BMGY with 10 mL tubes for two days and change 1 mL BMMY for another two days at 30 °C and 220 rpm.
?. The culture supernatants were collected by centrifugation at 5,000 rpm for 10 min, followed by cellulase activity assay as described below.
?. The most active transformants of TlCel5A and TlCel6A were grown in 1 L shake-flasks containing 400 mL BMGY at 220 rpm and 30 °C for 2 days, respectively.
?. The cultures were pelleted by centrifugation and resuspended in 200 mL of BMMY for 3-day-growth at 30 °C and 200 rpm. Methanol was then added at the final concentration of 0.5 % every 24 h for continuous induction of 3 days.
?. The culture supernatants were collected by centrifugation at 4500× g for further analysis.
?. Purification of recombinant TlCel5A and TlCel6A
?. The viva flow 200 ultrafiltration membrane system (Sartorius, Germany) with 5 kDa cut-off was used for the concentration and buffer exchange (to 0.1 M Tris-HCl, pH 8.0) of the crude enzymes.
?. The HiTrapTM Desalting column was used to desalt and the HiTrap Q Sepharose XL FPLC column (GE Healthcare) pre-equilibrated with 0.1 M Tris-HCl (pH 8.0) was used to purify for the recombinant proteins.
?. The gradient NaCl of 0‒0.7 M at the flow rate of 3 mL/min was used to elute the proteins.
?. Fractions showing cellulase activities were pooled and further desalted with a 5 kDa molecular cut-off concentration tube (Millipore) using 0.1 M McIlvaine buffer (pH 3.5 or 4.5).
?. The purified proteins were separated on 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity and molecular mass analysis.
?. Protein concentration was determined by using the Bradford method.
?. Deglycosylation of the purified recombinant TlCel5A and TlCel6ATo remove N-glycosylation, the purified recombinant TlCel5A and TlCel6A were treated with Endo H for 2 h at 37 °C following the manufacturer’s instructions (New England Biolabs), and checked by SDS-PAGE.
?. Enzyme activity measurementsThe cellulase activities were determined using 1 % (w/v) CMC-Na, barley β-glucan or lichenan as the substrate.
?. The reaction mixtures containing 100 μL of properly diluted enzyme solution and 900 μL substrate solution in 0.1 M McIlvaine buffer (pH 3.5 and 4.5) were incubated at 75 °C or 80 °C (optimum temperature) for 10 min. (When using 5 mg/mL Avicel as the substrate, the reaction period was lengthened to 60 min)
?. The amounts of reducing sugars were determined via the 3,5-dinitrosalicylic acid (DNS) method. [5] One unit (U) of enzyme activity was defined as the amount of enzyme producing 1 μmol of reducing    sugar per minute under the assay conditions.
?. Biochemical characterization of TlCel5A and TlCel6ACMC-Na, barley-glucan and PASC were used as the substrates for enzyme characterization.
?. The optimal pH was determined at 75 °C for TlCel5A and 80 °C for TlCel6A, respectively, in 0.1 M McIlvaine buffer with pH ranging from 2.0 to 8.0.
?. To test the pH stability, the purified enzymes were pre-incubated at 37 °C for 60 min in buffers with pH ranging from 1.0 to 12.0 (0.1 M KCl-HCl for pH 1.0‒2.0, 0.1 M McIlvaine buffer for 2.0 to 8.0, and 0.1 M glycine-NaOH for pH 9.0‒12.0).
?. The residual enzyme activities were determined at 75 °C and pH 3.5 for TlCel5A and 80 °C (barley β-glucan) or 70 °C (PASC) and pH 4.5 for TlCel6A, respectively.
?. To determine the optimal temperature, the cellulase activities were determined at temperatures ranging from 30 °C to 90 °C and pH 3.5 for TlCel5A or pH 4.5 for TlCel6A for 10 min, respectively.
?. For thermal stability assays, TlCel5A and TlCel6A were incubated at temperatures of 70 °C, 75 °C or 80 °C and pH 3.5 or 4.5 for different periods of time.
?. For half-life determination, 0.05 mg of TlCel5A or 0.2 mg of TlCel6A was incubated at 60 °C, 65 °C or 70 °C for 0.5‒24 h
?. Residual activities were determined as described above.
?. Substrate specificity and kinetic parametersSubstrate specificity of TlCel5A and TlCel6A were determined by using 1 % (w/v) CMC-Na, barley β-glucan, Avicel, lichenan, laminarin, xyloglucan, birchwood xylan, konjac flour, locus bean gum, glucomannan, or 0.2 mM of pNPGlu, pNPGal, pNPC as the substrate.
?. The kinetic parameters (Km, Vmax and kcat/Km) of TlCel5A were determined by incubating the enzyme with 0.25 to 10 mg/mL CMC-Na at pH 3.5 and 75 °C for 5 min.
?. For TlCel6A, the kinetic parameters were determined at pH 4.5 and 80 °C for 5 min with 0.25‒10.0 mg/mL barley β-glucan as the substrate.
?. The enzyme activities were determined by using the DNS method. The kinetic constants were calculated using the Lineweaver-Burk plots by GraphPad Prism 6.0 (http://www.graphpad.com/scientific-software/prism/).
?. Analysis of the cellooligosaccharides hydrolysis productsThe hydrolysis products of cellooligosaccharides by TlCel5A and TlCel6A were detected by high-performance anion exchange chromatography (HPAEC, model 2500, Dionex, Sunnyvale, CA).
?. Purified TlCel5A (0.05 U) or TlCel6A (0.02 U) was added into 1 mL of cellooligosaccharide solution containing 200 μg of cellotetraose, cellopentaose, or cellohexaose and incubated at 60 °C for 0 min, 1 min, 5 min, 30 min, 1 h, 4 h or 5 h.
?. After enzyme inactivation by boiling water bath, the hydrolysates were diluted 100 times with ddH2O, and 100 µL of each sample was injected into the column of HPAEC.
?. The oligosaccharides were eluted by 100 mM NaOH. And the standards of oligosaccharides consisted of glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose.
?. The amount of each hydrolysate was calculated based on the peak area. One enzyme unit (U) was defined as the amount of enzyme to hydrolyze 1 µmol of substrate per minute under the assay conditions. The catalytic efficiency values (kcat/Km) of TlCel6A against cellooligosaccharides were calculated according to Xia et al. (2016).
?. Synergistic of TlCel5A and TlCel6A on cellulose hydrolysis
?. When using Avicel as the substrate, TlCel5A and TlCel6A were combined at the ratios of 0.4:3.6, 0.8:3.2, 1.2:2.8, 1.6:2.4, 2.0:2.0, 2.4:1.6, 2.8:1.2, 3.2:0.8, 3.6:0.4 in the total amount of 4.0 mM and incubated with 5 mg/mL of Avicel in 50 mM McIlvaine buffer, pH 4.0 at 60 °C for 24 h. The amounts of reducing sugars released were determined using the DNS method.
?. When using SECS or Avicel as the substrate, 8 μM of TlCel5A or TlCel6A alone, or 4 μM TlCel5A and 4 μM TlCel6A was incubated with 5 mg/mL substrate at pH 4.0 and 60 °C for 12 or 18 h.
?. With 2.5 mg/mL PASC as the substrate, 2 μM of TlCel5A or TlCel6A alone or 1 μM TlCel5A and 1 μM TlCel6A was added.
?. The amounts of reducing sugars released were determined by using the DNS method.
?. The amounts of glucose released were analyzed by a one-factor ANOVA of SPSS19.0 to assess the synergistic effects of TlCel5A and TlCel6A. Statistical differences were considered to be significant at P