Enzymatic Saccharification of Cassava Residues and Glucose

Nov 4, 2014 - Cassava residues are byproducts of the starch industry containing abundant cellulose for bioproduction of green fuel. ... The rate const...
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Enzymatic Saccharification of Cassava Residues and Glucose Inhibitory Kinetics on β‑Glucosidase from Hypocrea orientalis Xin-Qi Xu,† Xiao-Bing Wu,† Yi Cui,† Yi-Xiang Cai,† Rui-Wen Liu,† Min-Nan Long,‡ and Qing-Xi Chen*,† †

State Key Laboratory of Cellular Stress Biology and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems and ‡School of Energy Research, Xiamen University, Xiamen 361005, China ABSTRACT: Cassava residues are byproducts of the starch industry containing abundant cellulose for bioproduction of green fuel. To obtain maximum sugar yields from cassava residues, the optimal conditions for hydrolyzing the residues were determined using cellulase prepared from a novel Hypocrea orientalis strain. The optimal pH value and optimal temperature for the cellulase hydrolysis were 5.0 and 50 °C, respectively. The concentration of NaOH was determined to be 1% for pretreatment of cassava residues to gain enough soluble sugars suitably. The yield of released sugars was 10 mg/mL in the optimal conditions after 24 h of reaction, which was similar to that of bagasse and wheat grass. Inhibition kinetics of H. orientalis β-glucosidase (BG) by glucose was first studied using the progress-of-substrate-reaction method as described by Tsou (Tsou, C. L. Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381−436), and the microscopic inhibition rate constants of glucose were determined. The results showed that glucose could inhibit BG reversibly and competitively. The rate constants of forward (k+0) and reverse (k−0) reaction were measured to be 4.88 × 10−4 (mM·s)−1 and 2.7 × 10−4 s−1, respectively. Meanwhile, the inhibition was more significant than that of L-glucose, D-mannose, D-galactose, D-aminoglucose, acetyl-D-glucose, and D-fructose. This work reveals how to increase sugar yields and reduce product inhibition during enzymatic saccharification of cellulose. KEYWORDS: cassava residues, saccharification, β-glucosidase, glucose, inhibition kinetics



INTRODUCTION Biofuels, known as renewable and environmentally friendly, have attracted increasing attention because of the shortage problem of fossil fuel in modern society. Many resources of biomass, especially tonnes of agroindustrial wastes, have been exploited for green energy alternatives to fossil fuel.1 Cellulose is a major carbohydrate polymer in biomass and has been extensively used to produce glucose for bioethanol production via enzymatic saccharification, which requires mild conditions to avoid pollution.2 With application of biotechnology, cellulases were used for enzymatic hydrolysis of agroindustrial residues to achieve an adequate amount of glucose and, especially, realize the additional value of the wastes for easing environmental pressure.2 Cassava (Manihot esculenta Crantz) residues, containing abundant coarse fiber, are produced massively from starch factories every year in southern China and usually processed for methane fermentation, feeding animals, and preparation of edible food.3 Recently, cassava residues have become increasingly appreciated for bioproduction of ethanol to refrain from consumption of feedstuff to bioethanol.4 Effective enzymes from microorganisms have been exploited for the hydrolysis of cassava residues, including cellulase, xylase, and amylase. Researchers have already discovered many novel cellulosehydrolyzing microbes that produce efficient enzymes and have established efficient microbial or enzyme blends, such as Phanerochaete plus Trichoderma.5 Molecular directed mutation was another very effective approach to raise the activities of cellulytic enzymes and improve the glucose tolerance of active sites for increasing bioconversion of cellulose.4 On the other hand, pretreatment of residues was necessary for enzymatic hydrolysis of cellulosic materials according to previous studies.5 © 2014 American Chemical Society

To maximize sugar yields, raw cellulosic wastes need pretreatment to strip lignin and hemicelluloses and decrease crystallinity of cellulose for enhancing accessibility of cellulases to substrates. Methods of pretreatment included stream explosion, high-heat− pressure, chop, acid plus alkaline treatment, and microbial fermentation.6 During hydrolysis, many factors influence the enzymatic saccharification of cellulosic residues, involving the pH value of the hydrolysate, temperature, dry matter ratio, liquid content (water, buffer, and ions), product accumulation, enzyme compositions, and pretreatments of raw materials,7,8 among which sugar product inhibition is one of the main factors impeding the enzymatic hydrolysis. Released sugars can competitively bind to enzymes depressing the activity of cellulases. Inhibition mechanisms of monosaccharides on hydrolysis yields at high concentrations have been investigated already,9,10 which showed that high concentrations monosaccharides, including xylose, mannose, galactose, and glucose, disturbed the liquid performance of hydrolysate and thus discounted the efficiency of the enzymes, but inhibitory kinetics of cellulase by sugar products had not been studied yet.10 Otherwise, hydrolysis of cellobiose and oligosaccharides by β-glucosidase (BG) in the progress of enzymatic hydrolysis of cellulose could turn down the inhibition of oligosaccharides on endoglucanase and cellobiohydrolase to work efficiently; removal of the inhibition of glucose on β-glucosidase could lead the yield of cellulose hydrolysis to rise.11,12 Various β-glucosidases from filament fungi had low KI values by glucose, which was an Received: Revised: Accepted: Published: 11512

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obstacle to improving the final amount of yields of cellulose hydrolysis.13 Besides, previous studies showed that addition of BG during enzymatic hydrolysis of cellulose could promote the sugar yield effectively and reduce inhibition of cellobiose on CBH or EG.13 Research also indicated that BG in hydrolysate having better glucose tolerance through directed mutation could also promote the yield of cellulose saccharification.14 A novel cellulytic strain, Hypocrea orientalis, has been isolated for enzymatic degradation of agricultural residues and production of bioethanol. Research found that the cellulase activity secreted by H. orientalis was obviously higher than that of H. jecorina, T. koningii, P. decumbens, and A. niger, and especially, H. orientalis secreted a relatively balanced cellulase system,15 which showed good application of H. orientalis enzymes in the bioethanol industry. In recent work, BG of H. orientalis has been purified and the inhibition mechanism of glucose on BG was investigated. This study provided the basis for directed mutation of BG to improve the glucose tolerance of H. orientalis BG.



The substrate reaction progress curve was analyzed to obtain the reaction rate constants as detailed below. The results showed that the inhibition was a reversible reaction with fractional residual enzyme activity. This catalytic reaction can be written as where E, S, I, and P

denote enzyme, substrate, inhibitor (glucose), and product, respectively. EI and ES are the respective compounds. k+0 and k−0 are the microscopic rate constants for the forward and reverse reactions of EI, respectively. As a rule, [S] ≫ [E0] and [I] ≫ [E0], and the product formation can be written as

[Ρ]t =

MATERIALS AND METHODS

vB A[I] A[I] ×t+ − (A[I ] + B) (A[I] + B)2 (A[I] + B)2 × e−(A[I] + B)t

Cassava Residues, Enzymes, and Reagents. Cassava residues were generated in a local factory following starch production, and residual starch was stripped for the next process. The analysis of cassava residues was carried out according to the protocols of NREL.16 H. orientalis strain was cultured in modified MS medium (1% cassava residues as carbon source).7 Crude enzyme was prepared from the culture with centrifugation at 10000 rpm for 15 min at 4 °C. Homogeneous BG was isolated from the fermentation liquid sequentially through ammonium sulfate precipitation, DEAE chromatography, and Sephacryl S200 filtration. p-Nitrophenyl-β-D-glucopyranoside (pNPG) was from Sigma (St. Louis, MO, USA). Cellulase DF was purchased from Lizhudongfeng Biotechnology Co., Shanghai, China, and Cellulase R-10 was a product of Yakult in Japan. All other chemicals were local products of analytical grade. Enzymatic Activity and Hydrolysis of Cassava Residues. Cassava residues were pretreated with NaOH for 30 min at 121 °C in an autoclave. After cooling, the residues was filtered and washed to neutral pH. Then the cassava residues were dried in an oven and chopped for hydrolysis. The hydrolysis reactions were run in 100 mL of 50 mM sodium acetate buffer (pH 5.0) containing 1.5% (w/v) residues and 0.1 mg/mL enzymes with incubation in a shaking water bath (160 rpm) for up to 24 h at the given temperature. Samples of hydrolysate were taken periodically and centrifuged (5000g, 5 min) for quantification of reducing sugars by DNS assay.17 An Aminex HPX-87H column was used to determine the composition and quantity of the products with 5 mM H2SO4 as flow phase and pure glucose, cellobiose, and xylose as standards. Activities of CMCase, CBH, xylase and amylase were determined according to the method given in ref 15. One unit (U) of enzyme activity was defined as the amount of enzyme liberating 1 μmol of glucose, xylose, p-nitrophenol, or maltose from the appropriate substrates per minute per milliliter of crude filtrate under assay conditions. Glucose Inhibition Assays. β-Glucosidase activity was measured by adding 40 μL of enzyme (0.03 μg) into 960 μL of substrate solution containing 0.16 mM pNPG and 50 mM NaAc buffer at pH 5.0. The mixture was incubated in a 60 °C water bath for 15 min, and then 2 mL of 0.5 M NaOH was added to stop the reaction; the adsorption at 405 nm of the reaction solution was measured by using a Beckman DU800 to determine the amount of liberated p-nitrophenolate using a standard curve. To determine the effect of D-glucose on BG, activity was measured in substrate solution containing different concentrations of glucose. Determination of Microscopic Inhibition Rate Constants. The progress-of-substrate-reaction method described by Tsou17 was used to study the inhibitory kinetics of BG by glucose. In this method, 40 μL of enzyme was added to 1.0 mL of assay system 0.16 mM pNPG in the presence of different concentrations of glucose.

A=

k+0 × K m K m+ [S]

(1)

(2)

B = k −0

(3)

where [P]t is the concentration of the product formed at time t, which is the reaction time; A and B are the apparent rate constants of inhibition; [S] and [I] are the concentrations of the substrate and inhibitor, respectively; v is the initial rate of reaction in the absence of the inhibitor, where v = (Vm × [S])/(Km + [S]). When t is sufficiently large, the curves become straight lines and the product concentration is written as [P]calcd: [P]calcd =

Bv A[I] ×t+ A[I] + B (A[I] + B)2

(4)

Combining eqs 1 and 4 yields

[P]calcd − [P]t =

A[I] × e−(A[I] + B)t (A[I] + B)2

ln([P]calcd − [P]t ) = constant − (A[I] + B) × t

(5) (6)

where [P]calcd is the product concentration to be expected from the straight-line portions of the curves as calculated from eq 4 and [P]t is the product concentration actually observed at time t. Plots of ln([P]calcd − [P]t) versus t give a series of straight lines at different concentrations of inhibitor ([I]) with slopes of −(A[I] + B). A secondary plot of the slopes versus [I] gives a straight line. The apparent forward and reverse rate constants, A and B, can be obtained from the slope and intercept of this straight line. The value of B directly gives the reverse rate constant k−0. From eq 2 and the Michaelis−Menten equation we can get

K × k+0 A 1 = m × v Vm [S]

(7)

A plot of A/v versus 1/[S] gives a straight line with (k+0 × Km/Vm) as the slope of the straight line; as Km and Vm are obtained from measurement of the substrate reaction in the absence of glucose at different substrate concentrations, the rate constant k+0 can be easily determined from the slope of the straight line.



RESULTS AND DISCUSSION Enzymatic Hydrolysis of Cassava Residues. The composition of cassava residue enzymes secreted by H. orientalis was previously determined to ensure what enzyme could be used for the hydrolysis (Tables 1 and 2). The optimum conditions for 11513

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Table 1. Main Components of Cassava Residuesa

a

NaOH for pretreatment

cellulose

hemicellulose

lignin

starch

ash

reducing sugars

0% (nontreated) 1% 4%

25.73 63.08 68.08

17.02 11.22 10.95

16.0 9.12 9.03

31.6 b 

8.21 3.08 3.70

0.21  

Grams per 100 g dry weight matter. Each value is average of three independent experiments. bHard to detect.

Table 2. Enzymatic Activity in H. orientalis Fermentation Liquida activity (IU/mL)

a

enzyme

CMCase

CBH

BG

Xylnase

α-amylase

protein

H. orientalis cellulase DF cellulase R-10

20.42 37.5 38.15

7.375 1.4 1.55

21.75 3.15 21.8

8530.55 4028.82 9253.15

2.17 ndb nd

0.5 mg/mL

The values are average of the results of four independent tests. bNot detected.

saccharification yields of cassava residues were studied with 0.1 mg/mL enzymes and 1.5% matter. The results in Figure 1A show that, at pH 5.0, highest sugar yield (9.7 mg/mL) of enzymatic saccharification was obtained for 24 h of reaction. The hydrolysis yields at different temperatures are presented in Figure 1B. As seen from the reaction curves, the highest yield of reducing sugar was obtained 10 mg/mL for 24 h of hydrolysis at 50 °C. Reaction kinetic study (data not shown) found that although enzymatic hydrolysis at 55 and 60 °C released sugars more quickly and more sugars were obtained at the initial stage, much less sugars were produced finally than at other temperatures due to thermo-inactivation of enzymes, which was in accordance with a previous study.7 Effects of different concentrations of NaOH on sugar yields were determined. The results in Figure 1C indicate that alkali pretreatment can effectively and obviously improve yields of cellulase hydrolysis of cassava residues. Enzymatic hydrolysis of cassava residues pretreated with 0.5% (w/v) NaOH yielded 3-fold the amount of sugar of 0% NaOH pretreated residues (curve 3, from 2.5 to 7.5 mg/mL). However, there is a less significant increase in the amount of released sugar even alkaline up to 4%, showing the performances of NaOH above 0.5% were similar on removal of lignin of cassava residues. Hydrolysis products were then separated and identified through Aminex HPX-87H (Figure 1D) chromatography. Major glucose (8.74 min) was found far more than xylose (9.36 min) and cellobiose (7.20 min), and the concentrations of the three sugars were 7.6, 0.75, and 0.34 mg/mL, respectively. Meanwhile, in a later phase of hydrolysis, a small amount of cellobiose emerged due to inhibition on β-glucosidase by accumulated glucose, with the yield of saccharification of cassava residues maintained but not increased. Hydrolysis of Cassava Residues by Different Enzymes. Two resources of cellulases were used to hydrolyze cassava residues as compared with H. orientalis cellulase. Enzyme compositions of these three cellulases are presented in Table 2. It can be easily seen from the data that cellulase R-10 occupied the highest activity of each enzyme activity and that the BG activity of cellulase DF was lower much than those of the other two enzymes. The CMCase activity of H. orientalis was relatively small. When hydrolyzing cassava residues, CMCases of enzymes were all adjusted to 15 U/mL in the hydrolysis systems (pH 5.0, 50 °C). The yields of 24 h of hydrolysis catalyzed by H. orientalis enzyme and cellulase R-10 (Figure 2, curves 1 and 2) were close (9 mg/mL) and greater than that of cellulase DF (curve 5), which may be due to relatively small

Figure 1. Enzymatic hydrolysis yield of pretreated cassava residues at different pH values (A) and temperatures (B) for 24 h of incubation at 50 °C with 0.1 mg/mL cellulase and 1.5% residues. (C) Enzymatic hydrolysis of cassava residues pretreated by different doses of NaOH. The hydrolysis times for curves 1−4 were 0.5, 2, 9, and 12 h, respectively. The values were means of duplicated experiments. (D) HPLC spectra of hydrolysate at three different hydrolysis times, 0.5 (dashed line), 9, and 12 h, respectively. The products were released from hydrolysis of 4% NaOH treated residues. 11514

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Figure 2. Enzymatic hydrolysis of cassava residues by different enzymes (50 °C, pH 5.0). The hydrolyzed residues were pretreated by 4% NaOH. Curves: 1, cellulase R-10 at 15 U/mL CMCase; 2, H. orientalis enzymes at 15 U/mL CMCase; 3, cellulase R-10 at 15 U/mL CMCase; 4, 2 U/mL CMCase H. orientalis enzymes with 7.5 U/mL cellulase R-10; 5, cellulase DF at 15 U/mL CMCase; 6, H. orientalis enzymes at 2 U/mL CMCase. The values are the average of three replicates.

Figure 3. Enzymatic hydrolysis yields of pretreated wastes for 24 h of incubation at 50 °C and pH 5.0 with 0.1 mg/mL cellulase and 1.5% dry matter.

On the basis of the results above, with pretreatment, each gram of nonstarch cassava residues hydrolyzed by 0.1 mg/mL cellulase at 50 °C, pH 5.0, for 24 h can release the highest amount of 660 mg of reducing sugars, equal to that of bagasse and wheat grass. H. orientalis secreted enzymes were deficient to degrade peanut hulls and stems of swainsona salsula efficiently. The yield was similar to that of cellulase R-10. Glucose Gave Reversible and Competitive Inhibition on BG. The relationship between the activity of H. orientalis BG and the concentrations of glucose is shown in Figure 4A.

activities of BG and xylase. Meanwhile, hydrolysis by H. orientalis enzyme reached the maximum yield faster than the other two enzymes, which could effectively and remarkably reduce the production costs. To determine whether BG promotes the enzymatic hydrolysis, different enzyme blends were made to hydrolyze the residues. H. orientalis enzymes at 2 U/mL CMCase was respectively mixed with cellulase DF and cellulase R-10, both at 7.5 U/mL CMCase (total 9.5 U/mL CMCase with similar activity of xylase). Hydrolyses by the enzyme blends were run in the same conditions as above, respectively. The yields are shown in Figure 2 (curves 3 and 4) compared with respective enzymes. Results presented in the figure suggested that the sugar yields of the blends containing cellulase R-10 decreased significantly more than that of cellulase R-10 hydrolysis due to lower EG activity than that of cellulase R-10, whereas another enzyme blend containing H. orientalis enzymes and cellulase DF yielded a similar amount of sugars as that of cellulase DF, even though CMCase was relatively smaller. This result was in accordance with previous studies, which pointed out that adding BG could promote the yield of cellulose hydrolysis.18,19 Hydrolysis of Various Residues by H. orientalis Enzymes. Various resources of agricultural lignocelluloses producing sugars catalytically with H. orientalis cellulase (0.1 mg/mL enzymes loaded) were also studied. These cellulosic substrates were prepared from wheat grass, sugar cane bagasse, waste papers, peanut hulls, and stem dregs of swainsona salsula taubert (Sphaerophysa salsula, Chinese traditional medicine), respectively, which were all treated with 4% NaOH. Stems of swainsona salsula taubert were processed for extraction of flavonoids before this work. Hydrolysis yields of these materials are shown in Figure 3. The results showed that, without pretreatment, saccharification of cassava residues can produce more sugars than the other cellulose materials due to residual starch. With 4% NaOH pretreatment, sugar cane bagasse, wheat grass, and cassava residues produced similar amounts of sugars far greater than that of waste papers, peanut hulls, and stems of swainsona salsula taubert. Due to condensed lignin cortex, peanut hulls and stems of swainsona salsula taubert cannot release significant amounts of sugars by H. orientalis cellulase hydrolysis, whether 4% NaOH pretreated or not, and other effective treatment methods and enzymes were required for that saccharification.

Figure 4. Inhibition of BG by glucose. Methods were described in the text. (A) Relative activity of BG at different concentrations of glucose. (B) Effects of BG concentrations on activity for the hydrolysis of pNPG with different concentrations of glucose. The concentrations of glucose for curves 0−4 were 0, 0.1, 0.2, 0.4, and 0.8 mM, respectively. (C) Lineweaver−Burk plots for the hydrolysis of pNPG catalyzed by BG in the presence of different concentrations of glucose. The glucose concentrations for lines 0−4 were 0, 0.1, 0.2, 0.4, and 0.8 mM, respectively. (Inset) Plot of Km versus glucose concentrations to determine the inhibition constant.

The effect of glucose on BG was dose-dependent inhibition, and the activity of BG was sharply reduced to 20% following the increase of glucose to 5 mM (0.9 mg/mL); the IC50 value of glucose was estimated to be 0.87 ± 0.1 mM in the presence of 0.16 mM pNPG. 11515

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Plots of BG activity versus enzyme concentrations under various doses of glucose gave a family of straight lines all passing through the origin (Figure 4B), indicating that D-glucose was a reversible inhibitor of H. orientalis BG. Meanwhile, Lineweaver− Burk plots of 1/v versus 1/[S] under different concentrations of glucose gave a family of lines on a common intercept with slopes ascending along with the increase of glucose (Figure 4C), showing glucose can competitively inhibit BG activity in the progress of cellulose hydrolysis. The results indicated that adding enough enzymes or substrates can effectively reduce depression by glucose products during cellulose saccharification. From curve 0, values of Km and Vm for pNPG hydrolysis in the absence of inhibitor were determined as 0.28 mM and 14.92 μM/min, respectively. The inhibition constant (KI) of glucose was calculated to be 0.56 mM from the plot of Km versus concentrations of glucose as presented in the inset of Figure 4C, which was higher than BG from P. purpurogenum, T. harzianum C-4, Daldinia eschscholzii but lower than another six Aspergillus species.20−25

Figure 6. Courses of substrate reaction of BG in the presence of 0.1 mM glucose with different concentrations of pNPG. (A) Substrate reaction course. The concentrations of pNPG for curves 1−5 were 0.16, 0.24, 0.32, 0.48, and 0.64 mM, respectively. (B) Semilogrithmic plots of ln ([P]calcd − [P]t) against time. Data were taken from curves 1−5 in panel a.

Figure 7. Determination of microscopic inhibition rate constants of BG by glucose. (A) Secondary plots of the slopes versus glucose concentrations for a series of fixed substrate concentrations. The concentrations pNPG for lines 1−5 were 0.16, 0.24, 0.32, 0.48, and 0.64 mM, respectively. (B) Plot of A/v versus 1/[S]. The A value was calculated from the slopes of the straight lines in panel A.

Figure 5. Courses of substrate reaction of BG in the presence of different concentrations of glucose. The substrate concentration was 0.16 mM in 0.1 M NaAc buffer (pH 5.0). (a) Substrate reaction course. The concentrations of glucose for curves 0−4 were 0, 0.1, 0.2, 0.4, and 0.8 mM, respectively. (b) Semilogrithmic plots of ln ([P]calcd − [P]t) against time. Data were taken from curves 0−4 in panel a.

the ordinate intercept, equal to the microscopic rate constant k−0. Then, the plot of A/v versus 1/[S] was obtained, a straight line that passed through the origin (Figure 7B) with the slope of (Kmk+0/Vm). The microscopic forward rate constant k+0 was obtained according to eq 7. The results are summarized in Table 3. Relative BG Activities with Different Monosaccharides. Effects of some monosaccharides (derivatives or isomers of glucose) on BG activity were studied as compared with glucose. Residual activities of BG at different concentrations of monosaccharides were assayed as described previously. It can be seen from the results (Table 4) that, within low concentrations of monosaccharides, BG was far more sensitive to D-glucose than to the other sugars. Meanwhile, the inhibitory effect of L-glucose, a chiral isomer of D-glucose, declined evidently with IC50 to >5 mM. Moreover, mannose showed lower IC50 than galactose, perhaps because the 2′-OH in the sugar molecule was more accessible to BG active residues than 3′-OH to block the catalytic site. Additionally, a 20% loss of BG activity caused by aminoglucose was detected at 200 mM, and no significant loss of BG activity was found in the presence of N-acetylglucose or fructose at a range below 200 mM. Some of these sugars are components of many agricultural and food wastes,12 which can be hydrolyzed catalytically

Determination of Microscopic Rate Constants of Glucose Inhibition on BG. Reaction kinetics hydrolysis of pNPG by BG in the presence of different concentrations of D-glucose was measured as shown in Figure 5a. The results indicated that the reaction rate at each concentration of glucose decreased with increasing time until a straight line (identical with dash line) was approached. Plots of ln([P]calcd − [P]t) versus t at different concentrations of glucose are shown in Figure 5b with slopes of −(A[I] + B). Time courses of the reaction at different concentrations of pNPG are shown in Figure 6a in the presence of 0.1 mM glucose. Plots of ln([P]calcd − [P]t) versus t in Figure 6b gave a family of straight lines with slopes of −(A[I] + B). The kinetic curves of substrate reaction at other concentrations of glucose can also be obtained with the same method. With the presence of various substrate concentrations, plots of the values of (A[I] + B) versus concentrations of glucose presented a series of straight lines with slopes of A, respectively, and had a common intercept on the ordinate (Figure 7A). The value of the apparent reverse rate constant B was obtained from

Table 3. Inhibition Equilibrium Constants and Microscopic Rate Constants for BG by D-Glucose IC50

KI

k+0

k−0

0.87 ± 0.1 mM

0.56 ± 0.02 mM

4.88 × 10−4 (mM· s)−1

2.7 × 10−4 s−1

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Table 4. Relative Activities of BG in the Presence of Different Concentrations of Monosaccharide percentage of β-glucosidase activity 1 mM D-glucose L-glucose D-mannose D-galactose D-aminoglucose

N-acetyl-D-glucose D-fructose

a

48.0 97.6 93.6 100.1 99.0 96.4 97.1

± ± ± ± ± ± ±

0.9 2.8 3.1 3.4 1.6 2.8 5.1

5 mM 16.2 86.1 87.1 96.8 98.8 95.7 99.0

± ± ± ± ± ± ±

0.7 3.5 2.0 3.0 5.6 2.3 2.4

50 mM a

nd 33.6 48.8 71.5 88.4 95.0 97.9

± ± ± ± ± ±

1.3 3.0 1.7 1.3 6.3 6.7

200 mM nd 8.5 18.6 42.2 81.4 95.9 94.3

± ± ± ± ± ±

4.8 1.2 11.0 3.5 4.0 5.5

nd, not determined. endoglucanase in urea solution. J. Agric. Food Chem. 2011, 59, 10971− 10975. (8) Kovacs, K.; Macrelli, S.; Szakacs, G.; Zacchi, G. Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house. Biotechnol. Biofuels 2009, 2, 14−24. (9) Murphy, L.; Bohlin, C.; Baumann, M. J.; Søren, N. O.; Trine, H. S.; Lars, A.; Kim, B.; Peter, W. Product inhibition of five Hypocrea jecorina cellulases. Enzyme Microb. Technol. 2013, 52, 163−169. (10) Hsieh, C. C.; Cannella, D.; Jørgensen, H.; Felby, C.; Thygesen, L. G. Cellulase inhibition by high concentrations of monosaccharides. J. Agric. Food Chem. 2014, 62, 3800−3805. (11) Ma, S. J.; Leng, B.; Xu, X. Q.; Zhu, X. Z.; Shi, Y.; Tao, Y. M.; Chen, S. X.; Long, M. N.; Chen, Q. X. Purification and characterization of β-1,4-glucosidase from Aspergillus glaucus. Afr. J. Biotechnol. 2011, 10 (84), 19607−19614. (12) Tao, Y. M.; Zhu, X. Z.; Huang, J. H.; Ma, S. J.; Wu, X. B.; Long, M. N.; Chen, Q. X. Purification and properties of endoglucanase from a sugar cane bagasse hydrolyzing strain, Aspergillus glaucus XC9. J. Agric. Food Chem. 2010, 58, 6126−6130. (13) Ma, L.; Zhang, J.; Zou, G.; Wang, C. S.; Zhou, Z. H. Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a β-glucosidase gene from Penicillium decumbens. Enzyme Microb. Technol. 2011, 366−371. (14) Liu, J. J.; Zhang, X. C.; Fang, Z. M.; Fang, W.; Peng, H.; Xiao, Y. Z. The 184th residue of β-glucosidase Bgl1B plays an important role in glucose tolerance. J. Biosci. Bioeng. 2011, 112 (5), 447−450. (15) Long, C. N. Study on Cellulose Degradation Enzymes and Gene Expression of Hypocrea orientalis EU7-22. Ph.D. dissertation, Xiamen University, 2013. (16) NREL. Chemical Analysis and Testing Laboratory Analytical Procedures, 1998. (17) Miller, G. L. Use of the dinitrosalicylic acid reagent for the determination of reducing sugars. Anal. Chem. 1959, 31, 426−428. (18) Tsou, C. L. Kinetics of substrate reaction during irreversible modification of enzyme activity. Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381−436. (19) Maeda, R. N.; Serpa, V. I.; Rocha, V. A. L.; Mesquita, R. A. A.; Santa Anna, L. M. M.; de Castro, A. M.; Driemeier, C. E.; Pereira, N., Jr.; Polikarpov, I. Enzymatic hydrolysis of pretreated sugar cane bagasse using Penicillium f uniculosum and Trichoderma harzianum cellulases. Process Biochem. 2011, 1196−1201. (20) Paripok, P.; Kazuo, S.; Khanok, R. Improvement of lignocellulosic biomass in planta: a review of feedstocks, biomass recalcitrance, and strategic manipulation of ideal plants designed for ethanol production and processability. Biomass Bioenrg. 2013, 58, 390−405. (21) Jeya, M.; Joo, A. R.; Lee, K. M.; Manish, K. T.; Lee, K. M.; Kim, S. H.; Lee, J. K. Characterization of β-glucosidase from a strain of Penicillium purogenum KJS506. Appl. Microbiol. Biotechnol. 2010, 86, 1473−1484. (22) Decker, C. H.; Visser, J.; Schreier, P. β-Glucosidases from five black Aspergillus species: study of their physico-chemical and biocatalytic properties. J. Agric. Food Chem. 2000, 48 (10), 4929−4936.

accompanied with cellulosic materials for production of biofuels or other chemicals. This work provides theoretical support for that application. Furthermore, comparisons of effects on BG by these saccharides proved that the combination between the active site and chemicals required strict structural specificity, which guided molecular modification to improve sugar tolerance of cellulase.14



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*(Q.-X.C.) Phone/fax: +86-592-2185487. E-mail: chenqx@ xmu.edu.cn. Funding

The present research was financially supported by the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant J1310027/J0106) and a grant of the National Key Basic Research Program (NKBRP) (No. 2010CB732201). Notes

The authors declare no competing financial interest.



ABBREVIATIONS USED BG, β-glucosidase; CBH, cellobiohydrolase; DNS, 3,5-dinitrosalicylic; NaAc, sodium acetate; pNPG, p-nitrophenyl-β-Dglucopyranoside; Km, Michaelis−Menten constant



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