simple equation for enzymatic hydrolysis of cellulose

1. Introduction

Bioethanol is currently employed as a renewable energy to replace gasoline and addresses the issue of fossil fuel depletion. The main feedstocks used to pro-duce bioethanol are sugar and starchy materials, with corn, sugarcane, and cassava typically being employed in the USA, Brazil, and Indonesia, respectively, as these three materials contain sugar or starch compounds that can be fermented to produce ethanol after hydrolysis when necessary. However, the use of such sugary and starchy materials competes with the demand for food supply, resulting in increased prices and the threatening of food security.

In this context, lignocellulosic materials from agri-cultural and forest residues, such as rice straw, sawdust, and palm oil empty fruit bunch, have attracted growing attention as alternative feedstocks for bioethanol pro-duction, as they are both abundant and unutilized1). In the case of lignocellulosic materials, the cellulose present in these materials can be hydrolyzed to give glucose, which can then be employed as a substrate for ethanol fermentation. However, due to the presence of lignin and hemicellulose in the lignocellulosic structure, this potential feedstock must go through an appropriate pre-treatment method prior to hydrolysis2). For example,

methods such as ammonia explosion, dilute acid treat-ment, lime pretreatment, and hydrothermal pretreatment are among a number of techniques reported to date3).

Following such pretreatment, hydrolysis must then be carried out to convert cellulose to glucose, and so this stage is particularly important when considering the production costs of bioethanol2). For example, hydrolysis by enzymes has been identified as a “green” and environmentally friendly process4) in which the cel-lulose polymer is degraded by cellulase to give the monomer glucose, which can in turn be naturally fer-mented by the yeast Saccharomyces cerevisiae to yield ethanol5). As such, a number of synergistic studies have been conducted to decrease the enzyme costs for the commercial production of ethanol6). One potential strategy is low-cost enzyme production and subsequent recovery and reuse of the enzyme. For example, Nojiri et al.7) reported that alkaline-treated woody bio-mass can be used as a low-cost material for an enzyme production medium. In addition, Kobayashi et al.8) employed the functional lignin-based material lignocre-sol, which was synthesized from hinoki wood meal, to produce immobilized cellulase.

An alternative strategy involves the optimization of an enzyme cocktail for saccharification. Currently, several commercial cellulases have been identified to produce cellobiose and glucose as its main products. The composition of these cellulases can be divided into three categories of enzyme, namely endo-1,4-β - g l u c a n a s e s , c e l l o b i o h y d r o l a s e s , a n d

322 Journal of the Japan Petroleum Institute, 60, (6), 322-328 (2017)

J. Jpn. Petrol. Inst., Vol. 60, No. 6, 2017

[Regular Paper]

Simple Equation for Enzymatic Hydrolysis of Cellulose Using Cellulase Complex and β-Glucosidase Mixture

Novi SYAFTIKA and Yukihiko MATSUMURA＊

Dept. of Mechanical Science and Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8527, JAPAN

(Received June 12, 2017)

We herein report the first study into the use of an enzyme mixture of cellulase and β-glucosidase for cellulose hydrolysis, with the overall aim of expressing the effect of β-glucosidase addition using a simple equation. To achieve this goal, different amounts of commercial cellulase and β-glucosidase were added to a cellulose slurry, and hydrolysis experiments were carried out at 40 ℃ and 50 ℃ over 0-48 h in a shaker incubator using a 0.1 mol/dm3 sodium acetate solution to maintain a solution pH of 5. The glucose yield got higher for the larger amount of cellulase and larger amount of β-glucosidase. The yield of glucose recovered at 50 ℃ was lower than that recovered at 40 ℃ . Finally, an equation to estimate glucose production using such a cellulase and β-glucosidase mixture was then developed for the first time, and this equation correlated well with the experimental data. The obtained equation expressed the hydrolysis using another bottle of cellulase, too.

KeywordsBioethanol, Cellulose, Hydrolysis, Enzyme, Cellulase, β-Glucosidase

DOI: doi.org/10.1627/jpi.60.322 ＊ To whom correspondence should be addressed. ＊ E-mail: [email protected]

β-glucosidases. The general mechanism of cellulase action firstly involves random hydrolysis of the internal 1,4-β-linkages in the amorphous regions of cellulose by endoglucanases, which rapidly decreases the degree of polymerization. Cellobiohydrolases then hydrolyze the cellulose polymer at the free ends to release cellobi-ose as the main product, and finally, β-glucosidases hy-drolyze the produced cellobiose to yield glucose9). However, it should be noted that cellobiose is not a fer-mentable sugar, and as such, its presence strongly inhibits cellulase10). It has been considered that β-glucosidase is the key enzyme component present in cellulase, as it completes the final step in cellulose hy-drolysis by convert ing cel lobiose to glucose4). However, although the majority of commercial cellu-lases contain the three enzyme components outlined above, they tend to lack the optimum amount of β - g l u c o s i d a s e11). A s s u c h , t h e a d d i t i o n o f β-glucosidase as an accessory enzyme can enhance cel-lulase hydrolysis12). Indeed, Shimokawa et al.13) employed a mixture of cellulase from Trichoderma reesei and β-glucosidase from Aspergillus tubingensis to ob-tain a high activity of 5 FPU (filter paper unit)/mL, in which the enzyme cocktail contained 10 wt% A. tubin-gensis, and the cocktail effect was also demonstrated in the simultaneous saccharification and fermentation (SSF) process. Although it is difficult to determine all enzymes that contribute to the saccharification yield, the supplementation of β-glucosidase certainly in-creased the yield13).

Few studies have been published to date regarding enzyme optimization for cellulose hydrolysis, and a cel-lulase formulation containing additional β-glucosidase has yet to be developed. We expect that elucidation of the effect of β-glucosidase addition to conventional cel-lulase and the expression of this effect using a simple equation will provide an excellent guideline for design-ing an efficient hydrolysis reactor. We therefore aim to determine the effect of β-glucosidase addition on cel-lulose hydrolysis and to derive a simple equation to ex-press this effectiveness in this study.

2. Materials and Methods

2. 1. General ExperimentalThe hydrolysis reaction was conducted using crystal-

line cellulose (1 g) in an aqueous sodium acetate buffer solution (60 mL), as Matsumura et al. reported that real biomass samples (e.g., eucalyptus and palm oil empty fruit bunch) exhibited different glucose production characteristics1). As such, a fundamental study em-ploying pure cellulose is carried out herein.

Subsequently, cellulase and β-glucosidase were added to the cellulose slurry in the desired quantities (see Table 1), and the resulting mixture was shaken in a low temperature incubator at 120 rpm (Model IN604,

Yamato) to promote glucose production. This experi-ment was conducted at both 40 ℃ and 50 ℃, as enzy-matic hydrolysis is usually performed under mild con-ditions (i.e., pH 4.5-5 and temperatures of 40-50 ℃)7). Specific details for each run can be found in Table 1. The hydrolysis reaction was stopped after 48 h, and samples were taken at 0, 24, and 48 h.2. 2. Analytical Method

Following hydrolysis, the glucose content of each sample was determined using high-performance liquid chromatography (HPLC, Shimadzu Prominence HPLC system, Shimadzu), where the system was equipped with a Shodex SUGAR series KS-802 GPC (gel perme-ation chromatography) column (Shodex). To obtain a transparent solution, the liquid effluent was filtered prior to analysis using a syringe filter (RC-membrane 15, pore size＝0.20 μm). Finally, the glucose yield was calculated according to Eq. (1):

glucose yield( ) -[ ]

�� = gluose amount after hydrolysis( ) mol[ ]theoretical glucose amount in the feedstock( ) mol[ ]

(1)

2. 3. Reagents and MaterialsAll chemicals used in this study were of high purity

and were used without further treatment or purification. Sigmacell crystalline cellulose type 20 was employed as the substrate. Cellulase from Trichoderma reesei ATCC 26921 (C2730 Sigma) and β-glucosidase BGH-201 were purchased from Sigma-Aldrich (US) and Toyobo Co., Ltd. (Japan), respectively. Cellulase was a mixture of several enzymes, and thus is expressed as “cellulase complex A” below. β-Glucosidase was obtained as a light yellow amorphous powder from sweet almond, and was in its lyophilized form with an enzyme activity of 16.0 units/mg. Here, 1 unit of β-glucosidase is the amount of enzyme that can hydro-lyze 1 μmol of p-nitrophenyl-β-D-glucopyranoside in 1 min under pH 5.0. To prepare the buffer solution, ana-lytical grade sodium acetate was used. Cellulase com-plex of different bottle was also used. The activity of this cellulase complex was lower than cellulose com-plex A maybe due to storage under not completely de-sirable condition. This cellulase complex is expressed as “cellulase complex B” below. The cellulase employed was in the form of an aqueous solution, and according to the information provided by the manufac-turer, it had an enzyme activity of ≥ 700 units/g. Here 1 unit of cellulase is the amount of enzyme that is mea-sure relative to a Novozyme enzyme standard. Using this value, the relation between the enzyme amount and its effectiveness could be determined as 62 μL of the enzyme complex had the activity of 60 units. The amount of cellulase complex is expressed by nominal units calculated using this relationship in the following sections.

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3. Results and Discussion

3. 1. Effect of Temperature on HdrolysisFigures 1 and 2 show the yields of glucose obtained

at 40 ℃ and 50 ℃ using cellulase complex A, respec-tively. As shown, the overall glucose yield at 40 ℃ was higher than that at 50 ℃ for the same incubation time. This result is comparable with a previous study by Tengborg et al., where the optimal conditions for glucose hydrolysis were 38 ℃, pH 4.9, and a 144 h res-idence time using the Celluclast 2 L enzyme prepara-tion14). These results imply that each enzyme has a specific optimum temperature in addition to specific properties. Moreover, as discussed by Taherzadeh et al., the optimum temperature and pH are functions of the raw material, the enzyme source, and the hydrolysis duration15).

Although the standard temperature for enzymatic hydrolysis is 50 ℃14), this study shows that 40 ℃ is suffi-cient to obtain high glucose yields. Such a low tem-

perature requirement may be caused by the specific properties of β-glucosidases, as indicated by Farinas et al., who examined the activities of endoglucanases and β-glucosidases from A. niger16). They found that all cellulase enzymes exhibited high stability at 37 ℃, whereas the activities of β-glucosidases decreased by approximately 28 % after 48 h incubation at 50 ℃. In the same study, 40 % and 60 % losses in activity after 24 h and 96 h incubation, respectively, at 50 ℃ were also reported for endoglucanases, suggesting that tem-perature is a key variable. Furthermore, Calderon et al. examined the precipitate obtained from centrifuga-tion of the cellulase samples prior to protein quantifica-tion, and found that high temperatures promoted protein aggregation, causing the concentration of the enzyme protein in the liquid phase to decrease17). From our perspective, a lower temperature is more desirable when considering the lower energy required for the heating process.

We also observed that upon the use of cellulase

324


Table 1 Enzyme Amount

Run numberCellulase

complex typea)Temperature

[℃]

Enzyme amount [unit]

cellulase complex β-glucosidase

1 A 40 0 0 2 A 40 60 0 3 A 40 60 60 4 A 40 60 180 5 A 40 60 300 6 A 40 120 60 7 A 40 180 60 8 A 40 300 60 9 A 40 300 30010 A 50 0 011 A 50 60 012 A 50 60 6013 A 50 60 18014 A 50 60 30015 A 50 120 6016 A 50 180 6017 A 50 300 6018 A 50 300 30019 B 40 60 6020 B 40 120 021 B 40 180 022 B 40 300 023 B 40 120 18024 B 40 180 18025 B 40 300 18026 B 40 120 30027 B 40 60 028 B 40 60 6029 B 40 120 6030 B 40 180 6031 B 40 300 6032 B 40 60 18033 B 40 60 30034 B 40 300 300

a) Cellulase complex A: Cellulase from Trichoderma reesei ATCC 26921. Cellulase complex B: Same as A but from different bottle.

alone, glucose was recovered from the reaction. This result implies that cellulase complex contains enzyme that catalyzes the hydrolysis of cellobiose. In contrast, when only β-glucosidase was employed, no glucose was produced.3. 2. Effect of Enzyme Amount on Hydrolysis

One of the main aims of this study was to derive an equation for glucose production using a mixture of cel-lulase and β-glucosidase. As shown in Figs. 1 and 2, glucose yield increases with time. However, the in-crease from 24 to 48 h is much less than that from 0 to 24 h, and this fact indicates that an incubation time of 48 h was sufficient to obtain a suitable final yield. Thus, we attempted to derive a simple equation to esti-mate the glucose yield after 48 h based on the quantities of cellulase and β-glucosidase employed during hydro-lysis. The yield of glucose obtained after 48 h using the various amounts of cellulose complex A is shown in Figs. 3 and 4 for 40 ℃ and 50 ℃ using cellulase com-plex A, respectively. In addition, Fig. 5 shows the glucose yield for the cellulase complex B for 40 ℃.

As is pointed above, when no cellulase complex was added, no glucose was obtained. With increase in the cellulase complex amount, the glucose yield increased, but it got saturated. When the addition of cellulase complex is more than 150 units, effect of increasing cellulase complex amount is negligible for all cases. It

is to be noted that the glucose yields for 50 ℃ is lower than those for 40 ℃.3. 3. Simple Equation for the Effect of Enzyme

ContentTo derive this simple equation, the mechanism of cel-

lulose hydrolysis by cellulase C2730 should first be considered. Indeed, the general mechanism of cellu-lose hydrolysis can be explained as the synergic action

325


Fig. 1 Glucose Yield at 40 ℃ (cellulase complex A)

Fig. 2 Glucose Yield at 50 ℃ (cellulase complex A)

Fig. 3● Glucose Yield (40 ℃, cellulase complex A, β-glucosidase, circle: 0 unit, triangle: 60 unit, square: 180 unit, diamond: 300 unit)

Fig. 4● Glucose Yield (50 ℃, cellulase complex A, β-glucosidase, circle: 0 unit, triangle: 60 unit, square: 180 unit, diamond: 300 unit)

Fig. 5● Glucose Yield (40 ℃, cellulase complex B, β-glucosidase, circle: 0 unit, triangle: 60 unit, square: 180 unit, diamond: 300 unit)

of the cellulase enzyme complex, which typically con-tains endo-1,4-β-glucanases, cellobiohydrolases, and β-glucosidases. During the hydrolysis process, endo-1,4-β-glucanases and cellobiohydrolases attach to the cellulose chain and hydrolyze the bonds to produce cel-lobiose. In addition to these two enzymes, all cellulo-lytic organisms also produce free/membrane-bound β-glucosidase, which acts on cellobiose and cellodex-trins to produce glucose18). We herein employed com-mercial β-glucosidases (BGH-201) for addition to the cellulase complex (cellulase C2730), and the overall mechanism can be understood as shown in Fig. 6.

Based on Fig. 6, the following equations were there-fore derived. Firstly, we expressed the glucose yield obtained using the cellulase complex alone, ηgc, as a function of cellulase quantity:

hgc = f xc( ) (2)

As the cellobiose yield ηbc obtained from cellulose is assumed to be proportional to ηgc because it is also pro-duced by the same cellulase complex, the relationship between ηbc and the cellulase quantity can be expressed as:

hbc = k f xc( ) (3)

where k is a constant. This cellobiose is further hydrolyzed by commercial β-glucosidase, and so the yield of glucose obtained by commercial β-glucosidase is proportional to ηbc and the function of β-glucosidase quantity, xb:

hgb = k f xc( )g xb( ) (4)

Therefore, the total glucose yield ηg can be expressed as:

hg = f xc( ) + k f xc( )g xb( ) (5)

For a constant β-glucosidase amount, the curves

shown in Figs. 3-5 exhibit the form of exponential curves passing through the origin. Taking this to be true, then the function f(xc) can be expressed as:

f xc( ) = b 1 - exp -axc( )[ ] (6)

Similarly, the function g(xb) can be expressed as:

g xb( ) = d 1 - exp -cxb( )[ ] (7)

and the total glucose yield can be expressed as:

hg = b 1 - exp -axc( )[ ] 1 + kd 1 - exp -cxb( )[ ]{ } (8)

It is a good assumption that when an infinite amount of the cellulase complex and β-glucosidase are added, the glucose yield reaches unity, and so:

1 = b 1 + kd( ) (9)

or

k = 1 - b( ) bd (10)

As such, Eq. (8) can be simplified as:

hg = b 1 - exp -axc( )[ ] b + 1 - b( ) 1 - exp -cxb( )[ ]{ } (11)

Based on Eq. (11), the parameters that must be deter-mined are a, b, and c, and so these parameters were de-termined by fitting the calculated value to experimental data. Least square error method was employed for this purpose. The resulting values of a, b, and c are shown in Table 2. The curves for Eq. (11) obtained using these determined parameters are shown in Figs. 3-5, where it is clear that the exponential curve correlates well with the experimental data, including the results obtained using cellulase complex B.

Figure 7 displays the parity plot comparing the experimental data with the calculated values obtained using the developed model. The average error of the calculation was only 7.7 %, and the developed model describing the production of glucose via cellulose hydrolysis agreed well with the experimental data, which indicates the effectiveness of Eq. (11).

As observed in Fig. 3, the conditions that produced the highest glucose yield from cellulose was a 300 : 300 amount of cellulose : β-glucosidase incubated at 40 ℃ for 48 h. Although an increase in the quantity of enzyme added resulted in higher yields, the values obtained did not reach 100 % of the theoretical value. However, the value predicted using the equation shown

326


Fig. 6 Scheme of Cellulose Hydrolysis

Table 2 Determined Parameters for the Simple Equation

ParametersCellulase complex Aa) Cellulase complex Ba)

40 ℃ 50 ℃ 40 ℃

a [1/unit-(cellulose complex)] 0.0268 0.0318 0.0228b [-] 0.5008 0.4564 0.1740c [1/unit-(β-glucosidase)] 0.0023 0.0012 0.0013

a) Cellulase complex A: Cellulase from Trichoderma reesei ATCC 26921. Cellulase complex B: Same as A but from different bottle.

in Fig. 3 indicates that a mixture containing 60 units of cellulase is sufficient for mixing with β-glucosidase. Furthermore, the use of β-glucosidase quantities ex-ceeding 60 units did not significantly increase the yield. It is therefore clear that the simple equation proposed herein is useful for determining the optimum enzyme quantities required and the corresponding glucose yields obtained following hydrolysis.

For other variety of the cellulase complex and β-glucosidase, the parameters in Eq. (11) must be re-determined. However, it should be noted that to deter-mine these parameters theoretically, only 3 experimental runs are required, where the quantities of enzymes employed each time should be varied. As such, com-pared to conventional approaches using Michaelis-Menten or Langmuir models where 2 or 3 parameters are required for each enzyme, the advantages of our proposed equation are clear.

4. Conclusion

We reported the successful enzymatic hydrolysis of cellulose using a mixture of a cellulase complex and β-glucosidase, and the various glucose yields obtained using different enzymatic ratios were determined. To express the obtained glucose yields, a simple equation was derived, which contained only 3 parameters that required experimental determination. Upon fitting the experimental result with the determined parameters, a

good agreement with the experimental data was ob-tained. These results suggest that the simple equation developed herein is useful for determining the optimum amounts of enzyme addition required for cellulose hydrolysis, and is advantageous over conventional approaches in that only a few experimental parameters are required.

AcknowledgmentNS would like to express gratitude to Ministry of

Education, Culture, Sports, Science and Technology Japan for the financial support during her study. This work was supported by JSPS KAKENHI Grant Number JP 25249142.

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Fig. 7● Parity Plot between Experimental Data and Calculated Value of Glucose Yield

328


要　　　旨

セルラーゼ複合体と β-グルコシダーゼ混合物を用いたセルロースの酵素加水分解のための簡易式

Novi SYAFTIKA，松村　幸彦

広島大学大学院工学研究院エネルギー・環境部門，739-8527 広島県東広島市鏡山1-4-1

簡易な式を用いて β-グルコシダーゼの追加効果を表すことを目的として，セルラーゼと β-グルコシダーゼの混合物を用いてセルロースを加水分解した初の試みを報告する。この目的のために，市販のセルラーゼと β-グルコシダーゼを量を変えてセルローススラリーに添加し，加水分解実験を40 ℃および50 ℃で，0.1 mol/dm3の酢酸ナトリウムで pHを5に保った水溶液中で，0～48 h，震とう培養器を用いて行った。セルラーゼ

量と β-グルコシダーゼ量が多いほどグルコース収率は高かった。また，50 ℃のグルコース収率は40 ℃のものよりも低かった。さらに，このセルラーゼと β-グルコシダーゼ混合物を用いた場合のグルコース収率の簡易推算式を初めて導出し，この式が実験データをよく表すことを示した。得られた式は，別のボトルのセルラーゼを用いた場合にもよく成立した。

simple equation for enzymatic hydrolysis of cellulose

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