A Neutral Thermostable β-1,4-Glucanase from Humicola insolens Y1 with Potential for Applications in Various Industries

We cloned a new glycoside hydrolase family 6 gene, Hicel6C, from the thermophilic fungus Humicola insolens Y1 and expressed it in Pichia pastoris. Using barley β-glucan as a substrate, recombinant HiCel6C protein exhibited neutral pH (6.5) and high temperature (70°C) optima. Distinct from most reported acidic fungal endo-β-1,4-glucanases, HiCel6C was alkali-tolerant, retaining greater than 98.0, 61.2, and 27.6% of peak activity at pH 8.0, 9.0, and 10.0, respectively, and exhibited good stability over a wide pH range (pH 5.0−11.0) and at temperatures up to 60°C. The K m and V max values of HiCel6C for barley β-glucan were 1.29 mg/mL and 752 μmol/min·mg, respectively. HiCel6C was strictly specific for the β-1,4-glucoside linkage exhibiting activity toward barley β-glucan, lichenan, and carboxy methylcellulose sodium salt (CMC-Na), but not toward laminarin (1,3-β-glucan). HiCel6C cleaved the internal glycosidic linkages of cellooligosaccharides randomly and thus represents an endo-cleaving enzyme. The predominant product of polysaccharide hydrolysis by HiCel6C was cellobiose, suggesting that it functions by an endo-processive mechanism. The favorable properties of HiCel6C make it a good candidate for basic research and for applications in the textile and brewing industries.

Neutral endo-glucanases are favored by the textile industry for biostoning of indigo-dyed denim fabric because they have less aggressive effects on the fabric and have superior low backstaining properties [7]. Because of their high reaction rate and excellent stability at high temperatures, thermophilic endo-glucanases present an outstanding option for the brewing industry to improve the brewing process at elevated temperatures. However, most commercially available cellulases are derived from mesophilic filamentous fungi such as Trichoderma spp., Penicillium spp., and Aspergillus spp. and have acidophilic properties [8]. Therefore, novel endo-glucanases with high efficiency, neutral activity, and good thermostability are in great demand.
Filamentous fungi of the Humicola spp. are excellent producers of neutral thermostable cellulases for industrial applications [9][10][11][12]. Thermophilic Humicola insolens Y1 can produce a variety of GH enzymes with neutral pH and high temperature optima [13][14][15]. In this study, we cloned a novel GH6 gene (Hicel6C) from H. insolens Y1, expressed it in Pichia pastoris, and determined the biochemical properties of the recombinant HiCel6C protein. Compared to other reported fungal endo-glucanases, HiCel6C is thermophilic and alkali-tolerant and has potential for applications in the textile and brewing industries.
Minimal dextrose (MD) medium, buffered glycerol complex (BMGY) medium, and buffered methanol complex (BMMY) medium were prepared according to the manual of the Pichia Expression Kit (Invitrogen).

Cloning of an Hicel6C cDNA
Total RNA was extracted from H. insolens Y1 using TRIzol Reagent (Invitrogen) after 48 h of cultivation in wheat bran medium. First-strand cDNA was synthesized using a PrimeScript RT reagent kit (TaKaRa). The glucanase encoding gene, Hicel6C, was identified in the genome sequence of H. insolens Y1 (whole genome sequencing in progress). An Hicel6C cDNA incorporating a C-terminal His 6 -tag coding sequence was generated using the specific primers Hicel6CF 5 0 -GGGAATTCGCTCCCAGCCCCAAGAGC-3 0 (EcoRI site underlined) and Hicel6CR 5 0 -GCGGCCGCTTAGTGGTGGTGGTGGTGGTGCCAGAACTTGAAGATGG-3 0 (NotI site underlined) and cloned into the vector pEASY-Blunt for sequencing.

Heterologous expression in P. pastoris
The Hicel6C cDNA sequence lacking the signal peptide coding sequence was digested with EcoRI and NotI and ligated into the corresponding sites of the pPIC9 vector to create an inframe fusion with the α-factor signal peptide yielding pPIC9-Hicel6C. The recombinant plasmid was linearized using BglII and transformed into P. pastoris GS115 competent cells by electroporation using a Gene PulserX cell Electroporation System (Bio-Rad, USA). Positive transformants were cultured on MD plates and grown for 1-2 days at 30°C until single colonies appeared. These colonies were then transferred to 5 mL of BMGY medium and grown at 30°C for 2 d. The cells were collected by centrifugation and resuspended in 1 mL of BMMY medium containing 0.5% methanol for induction. Following induction, the culture supernatant was collected by centrifugation (12,000 × g, 4°C, 10 min) for use in a glucanase activity assay with barley β-glucan as the substrate. The positive transformant exhibiting the highest endo-glucanase activity was selected for fed-batch-mode fermentation in a 3-L fermenter containing 2 L of growth medium. The entire procedure was carried out according to the Invitrogen Pichia Expression Kit manual with some modifications. The positive transformant was first grown in a 250-mL flask containing 50 mL of yeast peptone dextrose medium at 30°C with agitation at 220 rpm for 48 h followed by overnight growth in a 1-L flask containing 200 mL of the same medium at 30°C and 220 rpm. Then the entire culture was transferred into a 3-L fermenter containing 2 L of basal salt medium with PMT1 trace salts solution and grown at 30°C and pH 6 with agitation at 1000 rpm and an aeration rate of 1.5 vvm. A glucose-fed batch phase was activated by addition of 25% glucose in PMT1 solution at 36 mL/h/L for 4 h when the glucose in the medium was completely consumed. A mixture of glucose and methanol (8:1) was then added at a rate of 9 mL/h/L to acclimatize the cells to methanol. The final methanol-fed batch phase was initiated for induction and expression of the recombinant protein for 108 h. The glucose was consumed completely and the dissolved oxygen level was maintained above 20% during the process. During induction/expression, the glucanase activity in the culture supernatant was assayed at multiple time intervals.

Purification and identification of recombinant HiCel6C
The culture supernatant was collected by centrifugation (6000 × g, 4°C, 20 min) to remove cell debris and undissolved materials followed by concentration through a Vivaflow 200 ultrafiltration membrane with a 10-kDa molecular weight cut-off (Vivascience, Germany). The crude enzyme was loaded onto a His-Trap Sepharose XL FPLC column (Amersham Pharmacia, Sweden) pre-equilibrated with NTA0 buffer (20 mM Tris-HCl, pH 7.6, 0.5 M NaCl, 10% glycerol) and eluted using a linear gradient of imidazole (0.0-0.5 M) in NTA0 buffer at a flow rate of 3.0 mL/min. Fractions with high enzymatic activity were collected. The purity of the protein was determined by SDS-PAGE and staining with Coomassie Brilliant Blue G-250. For identification, the protein band was excised from the gel, digested with trypsin, and sequenced using liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) at the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences. To determine the protein concentration, a Bradford assay was used with bovine serine albumin as the standard. To remove N-glycosylation, purified recombinant HiCel6C (2 μg) was treated with 500 U of endo-β-N-acetylglucosaminidase H (Endo H) for 2 h at 37°C according to the supplier's instructions (New England Biolabs, USA) and analyzed by SDS-PAGE. The native molecular weight (MW) of HiCel6C was determined by size exclusion chromatography using a 3-mL elution volume and a Superdex 200 exclusion column (Amersham Pharmacia, Sweden). The column was equilibrated and eluted with appropriate buffer (20 mM Tris-HCl, 300 mM NaCl, pH 7.2) at a flow rate of 0.2 mL/min and calibrated with lysozyme from chicken egg white (MW = 14,300), α-chymotrypsinogen A from bovine pancreas (MW = 24,500), albumin egg (MW = 45,000), and albumin bovine V (MW = 67,000). The apparent MW of HiCel6C was calculated from the calibration curve of log (MW) vs. elution volume.

Glucanase activity assay
Glucanase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method [16]. The standard assay system consisted of 100 μL of appropriately diluted enzyme and 900 μL of 100 mM Na 2 HPO 4 -citric acid (pH 6.5) containing 1.0% (w/v) barley β-glucan at 70°C for 10 min. The reaction was terminated by the addition of 1.5 mL of DNS reagent and then boiled for 5 min. When the reaction mixture cooled to room temperature, the absorbance at 540 nm was determined. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under the above conditions. Glucose was used as the standard.

Biochemical characterization of purified recombinant glucanase
Barley β-glucan was used as the substrate for biochemical characterization of purified recombinant HiCel6C. The optimal pH of the purified recombinant HiCel6C was determined in various buffers with pH values ranging from 3.0 to 11.0 at 70°C for 10 min. The pH stability of HiCel6C was determined by measuring the residual enzymatic activity under standard conditions (pH 6.5, 70°C, and 10 min) after incubation at 37°C for 1 h at various pH values of 2.0-12.0. The buffers used were 100 mM Na 2 HPO 4 -citric acid (pH 2.0-8.0), 100 mM Tris-HCl (pH 8.0-9.0), and 100 mM glycine-NaOH (pH 9.0-12.0).
The optimal temperature for HiCel6C activity in the temperature range 40-90°C was determined by measuring activity in 100 mM Na 2 HPO 4 -citric acid (pH 6.5) for 10 min. The thermostability of the enzyme was determined by measuring the residual activity under standard conditions after incubation at 60, 65, and 70°C for various time periods.
To study the effects of metal ions and chemical reagents on the activity of purified recombinant HiCel6C, various metal ions (KCl, NaCl, CaCl 2 , CoCl 2 , NiCl 2 , CuSO 4 , MgSO 4 , MnSO 4 , ZnSO 4 , FeCl 3 , Pb(CH 3 COO) 2 , or AgNO 3 ) or reagents (SDS, β-mercaptoethanol, CTAB, or EDTA) were added to the reaction system to a final concentration of 1 or 10 mM. The residual enzyme activity was determined under the standard assay conditions. An experiment without any added ion or chemical reagent was carried out as a control.

Analysis of hydrolysis products
The reaction system containing 3 U of purified HiCel6C, 0.6 mg of cellooligosaccharide (cellobiose, cellotriose, cellotetraose, cellopentaose, or cellohexaose), and 10 mg of CMC-Na or 2 mg of barley β-glucan in 1 mL of 200 mM Na 2 HPO 4 -citric acid (pH 6.5) was incubated at 50°C for 20 h. For analysis of initial products, the reaction system containing 0.3 U of purified HiCel6C and 0.2 mg of cellohexaose in 1 mL of 200 mM Na 2 HPO 4 -citric acid (pH 6.5) was incubated at 70°C for 10 min. The excess enzyme was removed from the reaction system using a Nanosep centrifugal 3K device (Pall, USA). The hydrolysis products were analyzed using high-performance anion exchange chromatography (HPAEC) with a model 2500 system from Dionex (USA). Glucose and cellooligosacchardies were used as standards.

Nucleotide sequence accession number
The nucleotide sequence of the endo-1,4-β-glucanase gene (Hicel6C) from H. insolens Y1 was deposited in the GenBank database under accession no. KM588315.

Results and Discussion
Cloning and sequence analysis of an Hicel6C cDNA from H. insolens Y1 We isolated a 1149-bp, full-length Hicel6C cDNA from H. insolens Y1 that encoded a polypeptide 382 amino acids in length. The deduced HiCel6C protein contained a putative signal peptide at the N-terminus (residues 1-18) and a GH6 catalytic domain. The calculated molecular mass and pI value were estimated to be 41.7 kDa and 7.26, respectively. Further sequence analysis using the NetNGlyc Server identified three potential N-glycosylation sites (Asn-Xaa-Thr/ Ser, where Xaa is not Pro). The deduced HiCel6C amino acid sequence shared the highest identity of 83% with a putative GH6 protein from Chaetomium atrobrunneum (AGV05123.1) and sequence identities of 57 and 38% with endo-β-1,4-glucanase (Cel6B, Q7SIG5.1) and cellobiohydrolase II (Cel6A, Q9C1S9.1), respectively, from H. insolens (Fig 1). Based on sequence alignment and homology modeling, HiCel6C has greater sequence and structural similarity to H. insolens Cel6B [17]. Both enzymes have an open N-terminal loop which is conserved among GH6 family members, but lack the C-terminal active-site-enclosing loop which is present in their cellobiohydrolase counterparts (S1 Fig). The two putative conserved catalytic residues of HiCel6C were predicted to be Asp152 and Asp330 corresponding to Asp139 and Asp316 of Cel6B.

Expression, purification, and identification of HiCel6C
Using cDNA generated from strain Y1 as template and Hicel6CF and Hicel6CR primers, we amplified a fragment of the Hicel6C gene lacking the signal peptide coding sequence. The resulting sequence was digested with EcoRI and NotI and ligated into the vector pPIC9. The recombinant plasmid pPIC9-Hicel6C was integrated into the chromosome of P. pastoris GS115. The transformant exhibiting the highest endo-glucanase activity was selected for high-cell-density fermentation in a 3-L fermenter. Recombinant HiCel6C protein was purified to electrophoretic homogeneity by Ni-NTA affinity chromatography (S2 Fig). The yield of HiCel6C was approximately 200.2 mg/L. The purified fractions from the fifth and sixth collection tubes, which had the highest protein concentrations (S2 Fig), were combined for further activity analysis. Size exclusion chromatography revealed that HiCel6C eluted as a single peak with a MW of approximately 36.3 kDa, suggesting that HiCel6C was monomeric in solution (S3 Fig). The lower apparent MW after size exclusion chromatography than the calculated molecular weight of 41.7 kDa may be due to nonglobular folding of the protein. Purified HiCel6C migrated as a single band of~50.0 kDa on SDS-PAGE which was greater than its calculated molecular weight. After deglycosylation with Endo H, the molecular mass decreased by~8 kDa (Fig 2). N-glycosylation often occurs when proteins are expressed heterologously in P. pastoris [18]. The observed variation in apparent molecular mass of HiCel6C can be ascribed to glycosylation of its three putative N-glycosylation sites.
To identify the purified protein, seven peptides, KSPKPSTPTGDVNPFEGK, KQIVGLV LYNLPDR, DGLQLYKDTFVKPYAEK, PNLPLAAKEFATVLK, GFATNVSNYNPFNALVR, VHLPGAR, and AGEWFDEYAQMLVKNADKSIFK, obtained by LC-ESI-MS/MS were compared to the deduced HiCel6C amino acid sequence. The complete match of these sequences indicated that the purified protein was in fact recombinant HiCel6C.

Characterization of purified recombinant HiCel6C
Most fungal endo-β-1,4-glucanases-such as endoglucanase II from Trichoderma reesei [19], EglA from Aspergillus niger VTCC-F021 [20], and EgG5 from Phialophora sp. G5 [21]-have an acidic pH optimum (pH 4.0-6.0). However, the cellulases and hemicellulases from Humicola sp. such as rCBH1.2 from H. grisea var. thermoidea (pH optimum of 8.0) [22] and XynA and Man5A from H. insolens Y1 [13,14] are alkali-tolerant. HiCel6C exhibited a similar preference for alkaline conditions with a pH optimum of 6.5 ( Fig 3A) and it retained greater than 98% of its peak activity at pH 8.0 and 61.2 and 27.6% of peak activity at pH 9.0 and 10.0, respectively. The enzyme was also stable over a wide pH range of 5.0-11.0 at 37°C with retention of greater than 80% of activity after 1 h of incubation. When the temperature was increased to 60°C, HiCel6C activity was stable only in the pH range 5.0-11.0 (Fig 3B). The profile splitting in Fig 3A and 3B was caused by buffer changes required for the assay. Due to the strength and nature of ions, enzyme activity may vary considerably in different buffer systems even at identical pH. Electrostatic potential analysis indicated that the number of basic residues located on the HiCel6C protein surface was greater than for its acidophilic counterparts (data not shown). This characteristic may be an important factor in the high adaptability and stability of HiCel6C under alkaline conditions. Previous studies demonstrated that neutral cellulases tend to cause much less indigo backstaining and higher abrasion than acid enzymes during biostoning of denim garments [23,24]. The excellent alkali-tolerance of neutral HiCel6C makes it particularly well suited for applications in the textile industry.
The optimal temperature for HiCel6C activity was estimated to be 70°C (Fig 3C) which is greater than generally known fungal endo-β-1,4-glucanases which have temperature optima of 50-60°C and are stable up to 50-55°C. Under high temperature conditions, β-1,4-glucanases from thermophilic fungi are more active and more stable than those from mesophiles [25][26][27].
To date, only a few fungal endo-β-1,4-glucanases, such as those from Talaromyces emersonii CBS394.64 [28], Penicillium pinophilum [2], and Phialophora sp. G5 [29], have been shown to have temperature optima similar to or higher than that of HiCel6C. We also investigated the thermal stability of purified HiCel6C (Fig 3D). After incubation at 60°C for 1 h, the enzyme retained greater than 90% of its initial activity. Thermostable endoglucanases are of great interest due to their potential applications in various industrial processes such as bioconversion of biomass into fermentative products, improvement of barley malting in brewing, and modification of the coarse mechanical pulp and reduction of the amount of chlorine used for bleaching in the pulp and paper industry.
The effects of metal ions and chemical reagents on HiCel6C activity were determined at concentrations of 1 and 10 mM (Table 1). Most metal ions and chemicals had no significant effect on HiCel6C activity with the exceptions of CTAB and Cu 2+ at 10 mM and Mn 2+ at 1 and 10 mM. HiCel6C exhibited significant resistance to SDS at concentrations of 1 and 10 mM, retaining 76.3 and 31.6% of activity, respectively. Thus HiCel6C has properties that are advantageous for applications in laundry detergents and the textile industry.

Analysis of hydrolysis products
To explore the mode of action of HiCel6C, we analyzed the products of cellooligosaccharide hydrolysis by HiCel6C using high-performance anion exchange chromatography (HPAEC). HiCel6C did not degrade cellobiose, but had low activity against cellotriose to produce glucose and cellobiose (S4 Fig). In the presence of HiCel6C, cellotetraose was degraded completely with cellobiose as the main product. Under the same conditions, cellopentaose was hydrolyzed to cellobiose, cellotriose, and small amounts of glucose. The products of cellohexaose hydrolysis during the initial 10 min included cellobiose, cellotriose, cellotetraose, and a trace amount of glucose (S5 Fig). Upon further incubation, cellohexaose was hydrolyzed completely to cellobiose, cellotriose, and a small amount of glucose (S4 Fig). These results indicated that HiCel6C cuts cellooligosaccharides randomly and thus represents a typical endo-cleaving enzyme. HiCel6C provides another example of GH6 endoglucanases that lack the C-terminal activesite-enclosing loop. The C-terminal active-site-enclosing loop may be the crucial element that differentiates GH6 endo-and exo-glucanases. This hypothesis was verified by site-directed mutation of the C-terminus-proximal loop in cellobiohydrolase A of Cellulomonas fimi, which enhanced its endoglucanase activity [31].
We also examined the reaction products generated hydrolysis of barley β -glucan and CMC-Na by HiCel6C (Fig 4). Cellobiose was the predominant hydrolysis product of both barley β-glucan and CMC-Na followed by small amounts of glucose and cellotriose. This result provided evidence for a potential processive mechanism of HiCel6C activity similar to that of Cel5 from Hahella chejuensis [32], Cel5H from S. degradans [33], and EG1 from V. volvacea [34] all of which have been reported to be processive endoglucanases of the GH5 family.

Conclusions
We identified a novel GH6 endo-β-1,4-glucanase gene from H. insolens Y1 and overexpressed it in P. pastoris GS115. Recombinant HiCel6C protein exhibited optimal activity at pH 6.5 and 70°C when barley β-glucan was used as substrate. HiCel6C was alkali-tolerant and remained stable over a pH range of 5.0-11.0 and at temperatures up to 60°C. Substrate specificity and hydrolysis product analysis indicated that HiCel6C is a typical β-1,4-glucanase with an endocleaving mode. These properties make HiCel6C of considerable interest for basic research and for various industrial applications, especially in the textile and brewing industries.