Full Text - Effect of laundry detergent formulation on the performance of alkaline phytoproteases

Production of cold-adapted cellulase by Verticillium sp. isolated from Antarctic soils

Nengfei Wang*¹ · Jiaye Zang¹ · Kaili Ming¹ · Yu Liu¹ · Zuohao Wu¹ · Hui Ding¹

¹The First Institute of Oceanography, State Oceanic Administration, Key Laboratory of Marine Bioactive Substance, Qingdao Shandong, PR China

Financial support: This work was supported by the National Science and Technology projects of China research grant (Grant No. 2011BAD14B04), Basic Scientific Fund for National Public Research Institutes of China, Nonprofit scientific research of Marine (Grant No. 201005031, 200905022-2, 201005020, and 201105028-02) and International cooperation subject of Ministry of Science and Technology (Grant No. 2010DFA24340).

Keywords: Antarctic soil, cellulose, filter paper cellulase assay, psychrotrophic fungi, Verticillium sp.

Background: Cellulose can be converted to ethanol by simultaneous saccharification and fermentation (SSF). The difference between the optimal temperature of cellulase and microbial fermentation, however, has been identified as the critical problem with SSF. In this study, one fungal strain (AnsX1) with high cellulase activity at low temperature was isolated from Antarctic soils and identified as Verticillium sp. by morphological and molecular analyses.

Results: The biochemical properties of crude AnsX1 cellulase samples were studied by filter paper cellulase assay. The maximum cellulase activity was achieved at low temperature in an acidic environment with addition of metal ions. Furthermore, AnsX1 cellulase demonstrated 54-63% enzymatic activity at ethanol concentrations of 5-10%. AnsX1 cellulase production was influenced by inoculum size, carbon and nitrogen sources, and elicitors. The optimal culture conditions for AnsX1 cellulase production were 5% inoculum, wheat bran as carbon source, (NH₄)₂SO₄as nitrogen source, and sorbitol added in the medium.

Conclusions: Our present work has potential to enable the development of an economic and efficient cold-adapted cellulase system for bioconversion of lignocellulosic biomass into biofuels in future.

Plant cell wall is made up of the complex structures of cellulose, hemicellulose and lignin, and stores sustainable renewable energy captured through the process of photosynthesis. This lignocellulosic biomass can be converted into biofuels used as an alternative energy source to alleviate the growing energy demands worldwide, and decrease the impact of fossil fuel emissions on the atmosphere (Henry, 2010). Cellulose is the major structural component of plant cell wall. It is a fibrous, insoluble, and high molecular weight polysaccharide consisting of 2,000-20,000 glucose units linked by β-1,4 glycosidic bonds.

A number of fungi and bacteria express cellulase, enabling them to utilize cellulose as a carbon source. Because of high enzyme activity and production, the current extensively studied cellulases are from fungi species such as Aspergillus, Trichoderma, Penicillium, and Phanerochaete chrysosporium (Coral et al. 2002). Fungal cellulases are a consortium of three hydrolytic enzymes comprised of endo-(1,4)-β-D-glucanase (EC 3.2.1.4), exo-(1,4)-β-D-glucanase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21) (Goyal et al. 1991). They act together to hydrolyze cellulose into soluble sugars for cell metabolism. Cellulose can be converted to ethanol by simultaneous saccharification and fermentation (SSF) (Kádár et al. 2004). The critical problem with SSF is the difference between the optimal temperature of cellulase and microbial fermentation. Fermentation requires an operating temperature of 35ºC; however, the frequently used fungal cellulases have maximum hydrolysis efficiency at 50ºC (Kádár et al. 2004). A possible strategy to address this problem is to identify fungal cellulases capable of hydrolysis at lower temperatures.

A psychrotrophic organism has a capacity to survive at temperatures less than 7ºC and grow at temperatures around 20ºC. Several of these fungal species such as Cadophora malorum, Geomyces pannorum, Penicillium herbarum, and Thelebolus microspores, have been isolated from pristine soils of Antarctica (Kerry, 1990; Duncan et al. 2008). These strains may produce cellulase with high cellulytic activity at low and moderate temperatures. In this study, we screened ten fungal isolates from the soils of Antarctica for the presence of cellulase. Among them, the fungal strain with the highest cellulase activity, AnsX1, was identified as Verticillium sp. by morphological and molecular analyses. The biochemical characteristics of crude AnsX1 cellulase samples were determined by filter paper cellulase (FPase) assays. The effects of inoculum size, carbon and nitrogen sources, and the addition of elicitors on cellulase production were evaluated by measuring filter paper units (FPU) during fungal growth.

Antarctic soils were collected around the Great Wall Station at geographic location of 62º13'15" S, 58º57'45" W, during the 24^th Antarctic scientific expedition of China.

Ten grams of crude soil sample was extracted with 90 ml of autoclaved Mili-Q water at room temperature for 20 min. Five milliliters of soil extracts were transferred to a 250 ml flask with 45 ml enrichment culture (EC) medium containing 1% peptone, 0.5% yeast extract and 1% NaCl (pH 7.0), and cultured at 15ºC for 24 hrs on a rotary shaker (150 rpm). The gradient dilutions (10^-4, 10^-5, 10^-6) of the above enriched sample were smeared on the selection medium A containing 0.5% carboxylmethyl cellulose sodium salt (CMC-Na), 0.25% peptone, 0.05% yeast extract, 0.05% MgSO₄, 0.1% KH₂PO₄, 0.1% Na₂HPO₄ and 1.3% agar (pH 7.0), and cultured at 15ºC for 24 hrs. The separated single colonies were inoculated on selection medium B containing 2% CMC-Na, 0.25% peptone, 0.05% yeast extract, 0.15% KH₂PO₄, 0.25% Na₂HPO₄ and 1.3% agar (pH 7.0), and cultured at 15ºC for 24 hrs. The candidate strains were selected according to the Congo red staining assay.

The colonies selected by the Congo red staining assay were inoculated into 200 ml cellulase production (CP) medium containing 0.5% (NH₄)₂SO₄, 0.02% MgSO₄ and 2% wheat bran, and cultured at 15ºC for 24 hrs on a rotary shaker (150 rpm). Total cellulase activity was measured by FPase assay as described by the analytical procedure of the National Renewable Energy Laboratory (Adney and Baker, 1996). Briefly, crude cellulase was incubated with 50 mg Whatman No. 1 filter paper strip in 50 mM of citric acid/Na₂HPO₄ buffer for exactly 60 min at 50ºC. The glucose was released from filter paper and separated by centrifugation (4,000 rpm and 30 min). The supernatants were transferred to the new tubes. The glucose was then measured by DNS method (Miller, 1972). Briefly, the above tubes with 1.5 ml appropriate glucose dilutions and 3.0 ml dinitrosalicylic acid (DNS) reagent were boiled for 5.0 min and cooled down in the cold ice-water bath. The colour-developed reaction mixture was added with 2.5 ml Mili-Q water, mixed, and separated by brief centrifugation. The absorbance of the supernatant was measured at 540 nm. One FPU of enzyme activity is defined as the amount of enzyme required to release 1 μmol of glucose per ml under assay condition. The colony with highest enzyme activity was subcultured onto Potato Dextrose Agar (PDA) medium and cultured at 15ºC for 24 hrs to study its morphological characteristics. The changes in the edge and colour of the colony were observed for several days.

The above cultures were centrifuged at 4ºC with 18,000 g for 15 min to separate fungal strains from liquid medium. Genomic DNA was isolated from freeze-dried fungal strain pellets following the modified 2 x CTAB procedure (Doyle and Doyle, 1987). The universal primers were designed from the ribosomal DNA internal transcribed spacers (ITS). The PCR products were purified and sequenced. The sequencing results were aligned with known ITS homologues from the NCBI database to identify the taxonomy of fungal strain isolated from the soils.

AnsX1 was a cellulase-excreting fungal stain. To prepare crude AnsX1 cellulase, AnsX1 was inoculated into 200 ml CP medium and cultured at 10ºC for 4 days on a rotary shaker (150 rpm). The fungal cultures supernatant were harvested by centrifugation (4,000 rpm, 30 min and 4ºC). The supernatants were collected as crude cellulase samples for further studies.

In order to study the biochemical properties of AnsX1 cellulase, total cellulase activity of crude cellulase samples were analyzed under various conditions, such as different temperatures, pHs, metal ions, and ethanol concentration. To determine the optimal temperature of AnsX1 cellulase, crudecellulase samples and 50 mg Whatman No. 1 filter paper strip were incubated in 50 mM of citric acid/Na₂HPO₄ buffer with neutral pH (pH = 5.3) for 1 hr at 23, 33, 35, 38, 40, 43, 50, and 53ºC. To determine the optimal pH of AnsX1 cellulase, crudecellulase samples 50 mg Whatman No. 1 filter paper strip were incubated in 50 mM of citric acid/Na₂HPO₄ buffer with different pH values ranging from 3.0 to 9.0 at 38ºC for 1 hr. The effects of reaction temperature and pH value on AnsX1 cellulase stability were studied. Crude AnsX1 cellulase samples were incubated in 50 mM of citric acid/Na₂HPO₄ buffer (pH 5.3) without filter paper strip at different temperatures for 1 hr, and then measured by FPase assay under the standard conditions. The activity without treatment was taken as 100%, and the relative cellulase activity was measured to determine the stability of AnsX1 cellulase at different temperatures. To study effects of pH value on AnsX1 cellulase stability, crude AnsX1 cellulase samples were incubated in 50 mM of citric acid/Na₂HPO₄ buffer (pH 3.0-9.0) without filter paper strip at 38ºC for 60 min, and then measured by FPase assay under the standard conditions. The relative cellulase activity was measured to determine the stability of AnsX1 cellulase at different pH values. To study the effect of metal ions on the activity of AnsX1 cellulase, 10 mM of EDTA, Li⁺, K⁺, Na⁺, Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Mn²⁺ or Zn²⁺ was added into 50 mM of citric acid/ Na₂HPO₄ buffer with optimal pH. In addition, the effect of various concentrations of K⁺ or Ca²⁺ (1, 10 and 100 mM) was examined at optimal temperature and pH. To study the effect of ethanol on the activity of AnsX1 cellulase, enough ethanol was added to yield a final concentration of 5, 10, 15 or 20% in 50 mM of citric acid/ Na₂HPO₄ buffer. The reaction solution including crudecellulase samples 50 mg Whatman No. 1 filter paper strip was incubated at optimal temperature for 60 min. Total cellulase activity was measured by FPase assay.

To optimize the fungal culture conditions, the effects of inoculum size, carbohydrate and nitrogen sources, and elicitors on cellulase production were studied by measuring total cellulase activity of crude AnsX1 samples from 2^nd day after inoculation at 1 day interval. To examine optimal inoculum size for cellulase production, seed germination (SG) medium was prepared as described below. Briefly, 200 g of wheat bran was extracted by boiling in 1 l Mili-Q H₂O for 30 min, and filtered through 0.5 mm filter. The total volume of wheat bran extract was brought to 1 l with Mili-Q H₂O. The single colony grown on the selection medium A plate was inoculated into 500 ml SG medium and pre-cultured for 4 days. The 2, 5 or 10% inoculum size of pre-cultured AnsX1 was inoculated into the CP medium and cultured at 15ºC for 7 days on a rotary shaker (150 rpm) for cellulase production. To examine the optimal carbohydrate source for cellulase production, carboxymethyl cellulose (CMC), microcrystalline cellulose (MCC), or 2% of starch, instead of wheat bran, was used as the carbohydrate resource in CP medium. To examine the optimal nitrogen source for cellulase production, either 0.5% of NH₄NO₃, urea or yeast nitrogen base (YNB) replaced (NH₄)₂SO₄and was used as the nitrogen resource in CP medium. To study the effect of elicitor on cellulase production, 0.1% of maltose, lactose, or sorbitol was add to the CP medium.

Triplicate samples were collected for each treatment. Data were subjected to analysis of variance (ANOVA). The significance of treatments was tested at the P < 0.05 level. Standard errors are provided in all figures as appropriate. All the statistical analyses were performed with the SPSS package (SPSS Inc., Chicago, IL, USA).

Morphological and molecular characteristics of fugal strains from Antarctic soils

Twenty putative fungal strains were isolated from the crude Antarctic soil extracts. Ten fungal strain colonies with detectable cellulase activity, AnsX1-10, were selected by using a rapid and sensitive Congo red staining assay system (Figure 1a). The strains were cultured in liquid CP medium and collected for FPase assay. Among them, AnsX1 showed the highest FPU and was selected for further analysis.

To study the morphological characteristics of AnsX1, the strains were cultured on the PDA flat plate by using the scratching method. Compared with other isolated fungal strains, AnsX1 had the thicker mycelial cushion. It developed a villiform colony with a whitish surface and yellowish back 2 weeks after inoculation (Figure 1b). The molecular characteristics of AnsX1 were determined by analyzing the 16S rDNA internal transcribed spacer (ITS) region sequences. Based on blast analysis, the ITS sequences of AnsX1 had 97% similarity to those of Verticillium sp. and thus was preliminarily identified as Verticillium sp.

The biochemical properties of cellulases depend on their origin. The temperature and pH of solution affect on the structure and activity of cellulase. The optimum temperature and pH for the activity of crude AnsX1 cellulase were examined.

FPase assays indicate that AnsX1 cellulase had the highest rate of enzyme activity (0.8 FPU/ml) at 38ºC which was 12ºC lower than that of current used commercial cellulase enzymes, and was stable at temperatures below 40ºC (Figure 2a and 2b). Compared with other fungal cellulases, AnsX1 cellulase showed comparable FPU activity to Trichoderma reesei Rut C-30 cellulase (Sun et al. 2008), and a lower activity than the commercially available cellulase produced from Aspergillus niger and Trichoderma reesei (Sigma). Neither the lower (23ºC) nor the higher temperature (53ºC) facilitated a high rate of cellulase activity (Figure 1a).

The effect of pH on the cellulase enzyme activity was studied by measuring the FPU activity at 38ºC. AnsX1 cellulase had the highest enzymatic activity at pH 5-6 and lowest at pH > 8 or < 3 (Figure 2c). The predicted optimum pH was 5.3. AnsX1 cellulase had comparable stability at pH 5-6 and was able to retain more than 80% of maximum activity even after 1 hr treatment at 38ºC (Figure 2d). A cellulase previously isolated from an alkali-tolerant Verticillium strainshowed the high cellulase activities at neutral and alkaline pH values (Solovyeva et al. 1999). Compared with the abovecellulase, AnsX1 cellulase had highest enzymatic activity at low temperature (38ºC) in an acidic environment (pH 5.3). It supports our hypothesis that the environmental factors had an important impact on the biochemical properties of cellulase, and the cellulase with capability of hydrolysis at lower temperatures can be identified from the extreme environment.

Metal ions have an effect on the activity and adsorption of cellulase on cellulose. To study the impacts of metal ions on AnsX1 cellulase activity, we first measured cellulase activity in the presence of EDTA at 38ºC and pH 5.3. Compared with the control, AnsX1 cellulase activity was reduced to 30% in the 10 mM concentration of EDTA (Figure 3a). This suggests that the presence of trace metal ions in solution was required to maintain AnsX1 cellulase activity (Ferchak and Pye, 1983). Furthermore, we assessed AnsX1 cellulase activity in the presence of various cations. The ions were independently added as their chlorides. The final concentration of each ion was 10 mM. AnsX1 cellulase activity assays demonstrate a significant inhibitory effect in the presence of Li⁺, Fe³⁺ and Zn²⁺, respectively (Figure 3a). In contrast, a 27% increase in AnsX1 cellulase activity was achieved in the presence of K⁺ or Ca²⁺ (Figure 3a). No effects were observed in the presence of Na⁺, Co²⁺, Cu²⁺, Fe²⁺, Mg²⁺ and Mn²⁺. These data imply that AnsX1 cellulase might use some metal ions as cofactors for activation or stabilization during the interaction with cellulose.

Both K⁺ and Ca²⁺ addition resulted in a 27% increase in AnsX1 cellulase activity and were selected for further studies. Compared with the control, the highest cellulase activity was obtained with 10 mM K⁺. The presence of 100 mM Ca²⁺ resulted in a severe inhibition of cellulase activity (Figure 3b). Similar AnsX1 cellulase activity was observed in the presence of various concentrations of K⁺ (Ferchak and Pye, 1983). However, AnsX1 cellulase had a tolerance to higher concentrations of K⁺ (Figure 3b).

Cellulases are inhibited by glucose which is the end-product of cellulose hydrolysis. To overcome the feedback inhibitory effects, simultaneous saccharification and fermentation (SSF) has been developed to fulfill the concurrent conversion of sugar into ethanol. Although ethanol has less inhibition on cellulase than cellobiose and glucose, it can denature protein structure and has a negative influence on cellulase activity. In this work, AnsX1 cellulase demonstrated 54-63% enzymatic activity at ethanol concentrations of 5-10% (Figure 4). In contrast, the cellulase prepared from a Trichoderma reesi retained 36% of its original activity at an ethanol concentration of 6.0% (Wu and Lee, 1997). Similarly, about 50% of P. decumbens cellulase activity was reduced at an ethanol level of 7% (Chen and Jin, 2006). Our data suggest that AnsX1 cellulase was able to tolerate high ethanol concentration and had potential to use for SSF.

We studied the effect of inoculum size on AnsX1 cellulase activity. The highest cellulase activity was reached on the 4^th day after inoculation with 5% inoculum (Figure 5). The increased inoculum size reduced the time required to attain the maximum cellulase activity. For example, the maximum activity was obtained on the 6^th day after inoculation with 2% inoculum while it was reached on the 3^rd day with 10% inoculum (Figure 5). The larger inoculum size, however, dramatically decreased the production of AnsX1 cellulase due to a diffusion barrier in the liquid medium. This suggests that there is an inoculum threshold for AnsX1 and it has critical effects on cellulase production (Domingues et al. 2000). Thus, we selected a 5% inoculum for further studies.

Cellulose is hydrolyzed by cellulase and offers the carbon source for fungi cell metabolism in the form of glucose. Cellulase biosynthesis can be induced in the presence of cellulose or cellobiose, but repressed by glucose (Xiao et al. 2004). Thus, the type of carbon sources in the medium influence cell metabolism, cellulase biosynthesis, and play a vital role in the growth of fungi. CMC, MCC, 2% starch, and wheat bran as carbon sources influenced AnsX1 cellulase production. Among the various carbon sources, the highest cellulase activity and shortest time required for maximum activity were achieved by using wheat bran as the carbon source (Figure 6a). In contrast, less than 30% cellulase activity was obtained by using either CMC or MCC as the carbon source.

CMC and MCC were the best carbon sources for A. niger cellulase production while sucrose was the best for Trichoderma sp. (Gautam et al. 2011). The cellulase of P. chrysogenum PCL501 was stimulated by cellulose and sawdust in the absence of glucose (Nwodo-Chinedu et al. 2007). In this work, wheat bran was the best carbon source for the production of AnsX1 cellulase (Figure 6a). Starch, MCC and CMC used as the sole carbon source had potential catabolite repression effects on fungi cellulase (Berry and Paterson, 1990). Wheat bran is known as a complex carbon source including cellulose and hemicellulose. Hemicellulose can be depolymerized by most microorganisms because it has greater solubility than cellulose. The simple sugars derived from hemicellulose can furnish the AnsX1 metabolism but less repression on its cellulase express. Given the low cost of wheat bran together, wheat bran is an attractive carbon source for the production of AnsX1 cellulase.

Nitrogen is another limiting factor that influences the production of AnsX1 cellulase. The effect of various nitrogen sources on AnsX1 cellulase activity was examined. The AnsX1 strain had access to organic (YNB and urea) or inorganic ((NH₄)₂SO₄ and NH₄NO₃) forms of nitrogen (Figure 6b). In the presence of the various nitrogen sources, the cellulase activity of AnsX1 increased in the following order: urea (minimum) < NH₄NO₃ < (NH₄)₂SO₄ = YNB (maximum). Furthermore, the time required to reach the maximum cellulase activity in the medium with YNB and (NH₄)₂SO₄ was less than that with urea and NH₄NO₃ (Figure 6b). Compared with YNB, (NH₄)₂SO₄ had a low cost and was selected as an effective nitrogen source for further studies.

Small soluble compounds such as cellobiose, sophorose, and lactose can induce cellulase genes expression in fungi (Llmén et al. 1997). The effect of various elicitors on AnsX1 cellulase activity was studied.

A significant increase of cellulase activity was also obtained by adding maltose or lactose in the medium. However, the time required to reach the maximum enzymatic activity was delayed in the presence of maltose or lactose (Figure 7). The above results suggest that poor carbon sources (lactose and maltose) could provoke the production of fungi cellulase. The addition of sorbitol resulted in a 200% increase in cellulase activity compared with the control (Figure 7). As reported previously, sorbitol has been recognized as a carbon source which neither promotes nor inhibits cellulase expression (Llmén et al. 1997). However, our results clearly showed that sorbitol had impacts on AnsX1 cellulase biosynthesis. It is possible that unknown molecular mechanisms may regulate AnsX1 cellulase genes expression. It is also possible that sorbitol plays a role in AnsX1 cell metabolism. The above alternatives are supported by the fact that the most carbon sources promoting cellulase expression are also used for growth by the fungi.

In summary, the fungal strain with highest cellulase activity (AnsX1) was isolated from Antarctic soils and identified by morphological and molecular analyses. The biochemical properties of crude cellulase demonstrate that AnsX1 cellulase was a cold-adapted enzyme in an acidic environment with metal ions and had a comparable tolerance to ethanol. Furthermore, the effects of inoculum size, carbohydrate and nitrogen sources, and elicitors on AnsX1 cellulase activity were studied to establish the optimum culture parameters for cellulase production. The optimal culture conditions for AnsX1 cellulase production were 5% inoculum, 2.0% wheat bran as carbon source, 0.5% (NH₄)₂SO₄as nitrogen source, and 0.1% sorbitol added in the medium. Together with the development of genetic manipulation of cellulase structure, our present work will enable the development of an economic and efficient cold-adapted cellulase system for the bioconversion of lignocellulosic biomass into biofuels.

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