Cellulose conversion of corn pericarp without pretreatment

Cellulose conversion of corn pericarp without pretreatment

Accepted Manuscript Cellulose conversion of corn pericarp without pretreatment Daehwan Kim, David Orrego, Eduardo A. Ximenes, Michael R. Ladisch PII: ...

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Accepted Manuscript Cellulose conversion of corn pericarp without pretreatment Daehwan Kim, David Orrego, Eduardo A. Ximenes, Michael R. Ladisch PII: DOI: Reference:

S0960-8524(17)31470-0 http://dx.doi.org/10.1016/j.biortech.2017.08.156 BITE 18765

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

27 June 2017 23 August 2017 24 August 2017

Please cite this article as: Kim, D., Orrego, D., Ximenes, E.A., Ladisch, M.R., Cellulose conversion of corn pericarp without pretreatment, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.08.156

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Cellulose conversion of corn pericarp without pretreatment Daehwan Kima,b, David Orregoa,b, Eduardo A. Ximenesa,b, Michael R. Ladischa,b,c

a

Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette,

IN 47907-2032, United States b

Department of Agricultural and Biological Engineering, Purdue University, West

Lafayette, IN 47907-2032, United States c

Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN

47907-2032, United States



Corresponding author at: Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN 47907-2032, United States. Tel.: +1 765 494 7022; fax: +1 765 494 7023. E-mail address: [email protected] (M. Ladisch).

1

ABSTRACT

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We report enzyme hydrolysis of cellulose in unpretreated pericarp at a cellulase

3

loading of 0.25 FPU/g pericarp solids using a phenol tolerant Aspergillus niger

4

pectinase preparation. The overall protein added was 5 mg/g and gave 98% cellulose

5

conversion in 72 hours. However, for double the amount of enzyme from

6

Trichoderma reesei, which is significantly less tolerant of phenols, conversion was

7

only 16%. The key to achieving high conversion without pretreatment is combining

8

phenol inhibition-resistant enzymes (such as from A. niger) with unground pericarp

9

from which release of phenols is minimal. Size reduction of the pericarp, which is

10

typically carried out in a corn-to-ethanol process, where corn is first ground to a fine

11

powder, causes release of highly inhibitory phenols that interfere with cellulase

12

enzyme activity. This work demonstrates hydrolysis without pretreatment of large

13

particulate pericarp is a viable pathway for directly producing cellulose ethanol in

14

corn ethanol plants.

15 16 17 18 19

Keywords: Lignocellulosic biomass; Corn pericarp; Enzyme; Inhibition; Enzymatic

20

hydrolysis

21 22

23 24

1. Introduction Pericarp is derived from the outer covering of corn kernels, and is ordinarily

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ground together with the rest of the corn prior to cooking, hydrolysis, and

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fermentation in commercial corn-to-ethanol facilities. Current practice in some of

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these facilities involves recovering fermentation residuals after distillation, pretreating

28

them, and then carrying out cellulose hydrolysis and a second fermentation in order to

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produce cellulose ethanol. Distillers’ grains that include pericarp as well as other

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residual components has a composition of 12.6% cellulose, 14.9% xylan, 5.5%

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arabinan, 5.9% starch and 37% crude protein (Kim et al., 2008a) while pericarp itself

32

has a higher fraction of structural polysaccharides: 18% cellulose, 35% hemicellulose,

33

and 20% starch (in addition to oil, protein, and lignin) (Gáspár et al., 2007). Hence,

34

residual by-products from corn ethanol plants have significant potential for producing

35

cellulose ethanol.

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Pretreatment is usually required to reduce recalcitrance of cellulose to enzyme

37

hydrolysis. Pretreatment solubilizes hemicellulose and some lignin, disrupts cellulose

38

structure, and enhances accessibility of the cellulose to enzyme (Jönsson and Martin,

39

2016; Kim et al., 2015, 2013, 2011, 2009). Methods for corn fiber pretreatment

40

include alkali (Akin et al., 2008; Gáspár et al., 2007, 2005), heat extraction (Benkő et

41

al., 2007), and liquid hot water (Kim et al., 2008a, 2008b; Mosier et al., 2005).

42

This work shows enzymatic hydrolysis of cellulose in corn pericarp does not

43

require pretreatment. Comparison of three different enzyme preparations from

44

Trichoderma reesei and Aspergillus niger at high protein loadings (0.5 and 1.4 FPU

45

enzyme/g solids, which corresponds to 10 and 27 mg enzyme/g solids, respectively)

46

shows complete hydrolysis is possible for enzymes in an A. niger pectinase

47

preparation. The impact of phenolic inhibitors becomes pronounced at lower enzyme

48

levels of 1 - 5 mg protein/g solids, (equivalent to approximately 4.6 to 22 mg

49

protein/mg cellulose) particularly for enzymes from T. reesei, where cellulose

50

hydrolysis yields decrease. Grinding of pericarp to a smaller particle decreases

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conversion significantly, rather than resulting in an expected increase. This is

52

attributed to release of phenolic compounds that inhibited the enzymes. This paper

53

presents the role of phenolic inhibitors on cellulose from pericarp hydrolysis and

54

applies these findings to achieve cellulose enzymatic hydrolysis without pretreatment.

55

2. Materials and methods

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2.1. Materials

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Corn kernels were provided by Purdue Farms (Fall, 2013 harvest, 3x7 gallon

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buckets of Yellow #2 dent corn Dupont / Pioneer P1151AM1), dried at 45C for 24 h

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to a 9.42% moisture content. Cracked corn was acquired from Salamonie Mills

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(Warren, IN), and was referred to as “cracked pericarp derived intact,” and upon

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grinding “ground pericarp.” Whole corn from Purdue Farms was hand-cut into quarter

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and half sections in order to obtain defined particle sizes. Pericarp from these corn

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samples are referred to as quarter-size and half-size derived pericarp in this work.

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Spezyme CP (batch number: 3016295230, cellulase/hemicellulase from

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Trichoderma reesei) and Multifect Pectinase (batch number: A216235001, pectinase,

66

cellulase, and hemicellulase from Aspergillus niger) were donated by Genencor,

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Danisco Division (Palo Alto, CA). Promod 144GL (protease from papain) and Depol

68

692L (serial number: 11687615, cellulase/hemicellulase from T. reesei and A. niger)

69

were purchased from Biocatalysts (Wales, UK). Enzyme profiles and protein

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concentrations in these preparations are summarized in Table 1.

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Corn pericarp was collected after enzyme treatment of corn kernels, which

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removed the starch and gave pericarp as a residue. Other substrates utilized for

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enzyme activity measurement were Whatman filter paper No. 1 (Whatman

74

International Ltd, England, Cat. No. 1001125), carboxymethylcellulose (low viscosity,

75

Cat. No. C5678), oat spelt xylan (Cat. No. X0627), and para-nitrophenyl substrates

76

(para-nitropenyl β-D-glucopyranoside (p-NPG), Cat. No. N7006, and para-nitropenyl

77

β-D-xylopyranoside (p-NPX), Cat. No. N2123) from Sigma Aldrich (St. Louis, MO).

78

All other chemicals were from Fisher Scientific International, Inc. (Hampton, NH).

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All reagents were of analytical quality.

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2.2. Preparation of corn pericarp

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Cracked corn from Salamonie Mills yielded an average particle size of pericarp of

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5.1 mm, while kernels from Purdue farms that had been manually cut into halves or

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quarters, gave pericarp with a mean length of 13.7 mm and, 7.2 mm, respectively. 200

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g of the kernels (cracked and manually cut in halves or quarters) was mixed with 800

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mL citrate buffer (pH 5.0, (25% w/v), 0.2% (v/v) Spezyme CP (1.3 mg protein/g

86

solids corresponding to 0.84 FPU/g solids) and 0.15% (v/v) Promod 144GL (0.9 mg

87

protein/g solids), and incubated at 50C for 36 h at 100 rpm with an up-flow impeller

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with a 3 inch diameter. Fractionation was conducted in triplicate.

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The resulting slurry was filtered through a 35 mesh screen, which retained the

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pericarp fraction. This pericarp fraction was then washed with 500 mL distilled water

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and dried at room temperature overnight. The residual moisture of 6.6% was

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measured by a Halogen moisture analyzer (Mettler Toledo HB43). Ground pericarp

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was obtained from cracked pericarp ground to pass a 20-mesh screen using a hammer

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mill. The pericarp, thus isolated from other corn kernel components, was used to

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evaluate hydrolysis of cellulosic components in a corn kernel.

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2.3. Particle size measurement

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The particle sizes used in this study ranged from 840 micron to 13.7 mm, and this

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size range exceeded the capabilities of the Malvern Zetasizer instrument. Hence, sieve

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analysis was carried out to determine the average particle size of pericarp. A sample

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of 50 g of fractionated pericarp at 6.6% moisture was sieved over a set of stainless

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steel screens ranging from 35 to 5/8 inches mesh, corresponding to opening sizes of

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0.5 mm to 16 mm, respectively (USA standard testing sieve, ASTM E11 specification,

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VWR, Philadelphia, PA). All tests were performed in triplicate in a sieve shaker

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(Ro-Tap Model E, Test Sieve Shaker, W.S. Tyler Mentor, OH) at room temperature for

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5 min. The samples were collected and weighed at 6.6% moisture to determine the

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size distribution. The pericarp was stored at -4C until further use.

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2.4. Analytical assays

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Cellulase activities in Spezyme CP (Spezyme), Depol 692L (Depol), Multifect

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Pectinase, and the carry-over liquid (filtered from fractionated corn kernels) were

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determined with Whatman number 1 filter paper strips, carboxymethylcellulose

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sodium salt (CMCase, endo-glucanase), oat spelt xylan (xylanase), and

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para-nitrophenyl substrates (β-glucosidase and β-xylosidase) as substrates (Dien et al.,

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2008). Specific enzyme activities were determined at 50C in 0.05 M sodium acetate

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buffer (pH 4.8) according to described protocols. One unit of enzyme activity refers to

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the amount of enzymes required to produce one micromole of substrate per min under

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the specified conditions.

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Amylase activity in each enzyme and for the carry-over liquid was measured

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according to Campos and Felix (1995). A starch solution (0.5% w/v) in 0.5 M sodium

119

acetate buffer (pH 6.0) was used as a substrate in order to test for this enzymatic

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activity at 50C. Released glucose was measured by HPLC. One unit of amylase was

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defined as the release of one micromole glucose per min from soluble starch. The

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protein concentration in each sample was measured using a Pierce BCA protein assay

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kit (Thermo Scientific, IL). The remaining starch content in the pericarp was

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determined using a starch assay kit (Megazyme, Wicklow, Ireland). Glucose formed

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from the remaining starch, as well as residual glucose in the carry-over liquid, was not

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counted in the total glucose concentration after the cellulose hydrolysis. Total phenols

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in the carry-over liquid were analyzed by Folin-Ciocalteu colorimetry assay at 765 nm.

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The goal here was to quantify the concentration of phenols in the different samples

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tested rather than individually identify them, and then correlate that to inhibitory

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effects. Our previous work (Kim et al., 2013, 2011; Ximenes et al., 2011, 2010) and

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that of others (Oliva-Taravilla et al., 2015; Tejirian and Xu, 2011) had identified

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individual phenols as a cause of major inhibitory effect on cellulases, hemicellulases

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and/or pectinases. Gallic acid was used as the standard in order to obtain a calibration

134

curve against which concentrations of phenols were determined based on Singleton et

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al (1999) and Waterhouse (2002). The control was the same buffer as used for

136

obtaining the standard curve except for that phenols were not added.

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2.5. Compositional and HPLC analyses

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Chemical composition of initial pericarp and extractive free solids were analyzed

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by NREL (National Renewable Energy Laboratory) using standard analytical

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protocols described by Sluiter et al. (2008, 2005a, 2005b). Sugars, acetate, and other

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solubles in hydrolyzed samples were determined by HPLC as described by Kim et al.

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(2013). Hydrolyzed samples were filtered through a 0.45 µm filter to remove solids.

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The HPLC system was equipped with a Bio-Rad Aminex HPX-87H ion exchange

144

column (300 mm x 7.8 mm, Bio-Rad Laboratories Inc., Hercules, CA) connected to

145

liquid chromatography system. 5 mM sulfuric acid diluted in distilled water was used

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as the mobile phase at 0.6 mL/min flow rate, and the column temperature was kept

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constant at 60C controlled by an Eppendorf TC-50 (Eppendorf, Wetbury, NY) (Kim

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et al., 2013, 2011).

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2.6. Enzyme hydrolysis of corn pericarp

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Pericarp was enzymatically hydrolyzed in the presence of liquid (pH 4.8) carried

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over from the pericarp preparation step. The carry-over liquid was clear with a light

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gray color. It contained residual enzyme activities, sugars, and inhibitors (phenols) at

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low concentrations, and was used to dilute the stock solutions of each enzyme to

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concentrations corresponding to protein loadings of 1-27 mg protein/g solids. This

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liquid simulates what we believe would represent conditions encountered in a

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dry-grind facility. In addition to cellobiose (0.1 g/L), glucose (2.5 g/L), acetate (1.1

157

g/L), and total phenols (0.3 mg/L), the carry over liquid also had low levels of

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residual enzyme activities of FPU (0.1), endoglucanase (6.2 U/mL), and xylanase (2.1

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OSX/mL), with a total protein concentration of 3.4 mg/mL. A total of 100 mL mixture

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(carry-over liquid combined with each enzyme preparation) and 15 g dry pericarp (15%

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w/v) were mixed into a 250 mL flask that was capped with a stopper. Enzyme

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activities corresponding to a loading of 10 mg/g solids are summarized in Table 1.

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Activities at other tested enzyme levels are proportionately higher or lower,

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depending on enzyme loadings.

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The enzymatic hydrolysis of pericarp was initially performed in flasks in a

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shaking incubator at 50C and 290 rpm, with an enzyme loading of 27 mg protein/g

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solids. The results showed 50 and 72% conversion at 12 and 24 h, respectively

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compared to 98% using a dual impeller mixing. Therefore, all the following

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enzymatic hydrolyses were carried out in triplicate at 50C and 290 rpm mixing with

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a dual impeller overhead mixer (IKA Eurostar power control-visc, Wilmington, NC)

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for 72 h in a water bath (VWR, Radnor, PA). The dual impeller had two elephant ear

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impeller blades (2 inch, 5.08 cm) with a 45 angle and a ¼ in shaft bushing

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(Cole-Parmer, Vernon Hills, IL). The appearance of pericarp after hydrolysis is

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illustrated in Supplementary data. Hydrolysis of pericarp in 0.05 M citrate buffer (pH

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4.8) and in carry-over liquid gave similar results indicating that differences in

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hydrolysis of the pericarp were due to the pericarp rather than other components that

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may have carried over from the corn kernel.

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2.7 Statistical Analysis

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Statistical analysis was performed with Minitab 16. The T-test was conducted,

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with 95% significant difference, for enzymatic hydrolysis of different particle sizes of

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pericarp.

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3. Results and discussion

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3.1. Compositional analysis of corn pericarp

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Chemical composition of initial pericarp and extractive free solids were analyzed

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by NREL (National Renewable Energy Laboratory) standard analytical protocols

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described by Sluiter et al. (2008, 2005a, 2005b). Corn pericarp, washed with distilled

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water after the fractionation step, was composed of glucan with 22.5% cellulose, and

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with 3.2% remaining starch (Table 2). After water and ethanol extractions, the

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pericarp was enriched in polysaccharide components with the composition of glucan,

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xylan, and arabinan increased by 60% (Table 2).

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Corn fiber includes 11-12% protein (free amino acids, globulins, and albumins),

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3-4% crude fat, 2% acetyl groups, and other components such as waxes, free fatty

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acids, sterols, sterol esters, sterol ferulates, tocopherols, and phyto sterols (Moreau et

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al., 1996; Saha et al., 1998; Watson and Ramstad, 1987; Wu and Norton, 2001). Of

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the total pericarp solids, water and ethanol steps extracted 14.1% and 25.6% of the

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dry weight, respectively. We used the initial (non-extractive free) corn pericarp for the

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study of conversion of glucan to sugars; extraction was only carried out to

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characterize compositional changes in the pericarp.

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3.2. Cellulose hydrolysis in corn pericarp with different enzyme preparations Hydrolysis of un-pretreated pericarp was initially carried out at high enzyme

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loadings (10 and 27 mg protein/g solids, respectively) (Fig. 1A and B) in order to

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facilitate comparison of different commercial enzyme preparations with activity

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profiles given in Table 1 A and B for pericarp compositions in Table 2. A loading of

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27 mg Multifect Pectinase protein/g solids (equivalent to 6.2 FPU/g cellulose)

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incubated with un-pretreated fiber gave 100% conversion compared to Spezyme CP

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(equivalent to 77.3 FPU/g cellulose, 120 mg protein/g cellulose) and Depol 692L (8.6

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FPU/g cellulose equivalent to 170 mg protein/g cellulose) where conversions were 22

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to 60% (Fig. 1B). The enzyme profile of Multifect Pectinase has been reported before

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(Dien et al., 2008), and it has accessory enzymes including α-arabinofuranosidase,

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α-galactosidase, feruloyl esterase, and p-coumaroyl esterease. These later two

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enzymes were not present in enzyme preparations derived from T. reesei. The known

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reduced sensitivity to inhibition of the enzymes derived from A. niger (Ximenes et al.,

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2011, 2010), together with the larger proportion of endoglucanase (EG) and

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β-glucosidase (β-G) relative to the other enzyme preparations coincide with the higher

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conversions.

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Since EG and β-G activities in Multifect Pectinase, Spezyme, and Depol were

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measured with respect to carboxymethylcellulose (CMC) and para-nitrophenyl-β-D-

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glucopyranoside (p-NPG) in buffer, respectively, the activities reported in Table 1

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were unaffected by inhibition. Although Spezyme showed higher filter paper activity

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in the absence of phenols (Table 1), its hydrolysis of un-pretreated pericarp, in the

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presence of phenols, was markedly lower. At 10 mg protein/g solids, yields after 72

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hours were distinctly lower for Spezyme (30% conversion) and Depol (18%

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conversion) compared to Multifect Pectinase (close to 100% conversion) (Fig. 1A).

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Corn fiber releases phenolics in the form of p-coumaric acid and ferulic acid

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(Akin et al., 2008; Carpita, 1996; Hartley and Ford, 1989). Akin et al. (2008) reported

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that corn fiber had ester-linked p-coumaric and ferulic acids (1.56 and 14.84 mg/g

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pericarp, respectively), with 80% of these phenolic compounds released after

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pretreatment with 4 M sodium hydroxide at 170C. Release of inhibitors was also

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noted for liquid hot water pretreated corn fiber i.e., pericarp. (Dien et al., 2008). Even

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without pretreatment, and using enzyme hydrolysis, phenols are still released.

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Inhibition is a key determinant of enzyme activity in the hydrolysis of pericarp.

232

3.3. Effect of cellulase loadings on hydrolysis in corn pericarp

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Considering previous results, Multifect Pectinase was selected for further

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experiments to test lower enzyme dosage (1 or 5 mg protein/g solids corresponding to

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0.24 FPU or 1.1 FPU/g cellulose) (Fig. 2). When this activity load, which is

236

equivalent to 5 mg protein/g solids (22 mg/g cellulose), was used at the same

237

experimental conditions 98% glucose conversion occurred in 72 h. The lowest

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enzyme dose at 1 mg protein/g solids (Fig. 2) gave 40% yield after 72 h, which was

239

higher than the results obtained with loadings of 10 mg protein/g solids for Spezyme

240

(16% conversion) and Depol (32% conversion) (Fig. 1A). There was no difference

241

between enzyme to buffer or in carry-over liquid both for protein loadings of 1 and 27

242

mg protein/g solids, respectively.

243

We also evaluated hydrolysis of the hemicellulosic fraction, however, xylan

244

hydrolysis was negligible despite the high amount of hemicellulase (mainly xylanase

245

and β-xylosidase) in Multifect Pectinase. The hemicellulase activity, without a

246

pretreatment step to increase enzyme accessibility to substrate, is insufficient to

247

effectively hydrolyze hemicellulose. Pericarp xylan has complex inter-linkages with

248

arabinan and ferulic acid and these may play a role in protecting the xylan backbone

249

from enzymatic hydrolysis (Dien et al., 2008).

250

3.4. Effect of particle size of pericarp on enzymatic hydrolysis

251

The difference in conversion between cracked, half, and quarter sized pericarp in

252

Fig. 3 was statistically insignificant, based on a T-test with 95% significant difference.

253

While a decrease in size from half (13.7 mm) to quarter (7.2 mm) to cracked (5.1 mm)

254

led to slightly lower rates of cellulose conversion at 27 mg Multifect Pectinase

255

protein/g solids loadings, cellulose hydrolysis was still 95% after 24 h for all three

256

cases (Fig. 3). In comparison, ground pericarp (0.84 mm) was 87% less, even when

257

the hydrolysis time was extended to 72 h. Deactivation due to shear has been noted in

258

the literature for amylase (van der Veen et al., 2004). However, when comparing

259

conversion of the pericarp with agitation by impeller to the conversion obtained in the

260

incubator shaker with agitation at 290 rpm, we observed significant improvement in

261

hydrolysis, indicating that loss in activity due to high stirring speed was not a major

262

factor. We observed an increased release of phenols with decreased pericarp particle

263

size contributed to the lower cellulose conversions. Ground pericarp released 22 times

264

more total phenols (0.44 mg/L) than pericarp half size (0.02 mg/L) and 1.7 than

265 266

cracked pericarp (Fig. 4). The inhibitory effect of phenols is not just a matter of their concentration but also

267

reflects chemical compositions as shown in our earlier work. Our previous work has

268

shown that strong enzyme inhibition occurred at low phenol concentration levels (for

269

instance, 80% loss of β-glucosidase activity with 1 mg tannins from wood/mg enzyme

270

protein, equivalent to 5 mg/mL in solution) (Ximenes et al., 2011; Kim et al., 2011;

271

2013). The inhibition effect became less pronounced as enzyme loadings increased.

272

This is consistent with our current work where enzyme loadings of 1, 5, and 10 mg

273

Multifect Pectinase protein/g ground solids gave 33%, 57%, and 74% conversion

274

yields, respectively (Fig. 5A, B, and C).

275

These data are opposite of the observations of Kim et al. (2015) for hydrolysis of

276

liquid hot water pretreated mixed hardwood, where conversion increased by 50%

277

when the average particle size of substrate decreased by 33% from 3 mm to 2 mm.

278

Differences in the biomass material help to explain this. Lignin in pericarp is less than

279

5% (Table 2), while hardwood after pretreatment is more than 30% (Kim et al., 2015;

280

Ko et al., 2015a). Lignin adsorbs cellulases and decreases conversion (Kim et al.,

281

2015; Ko et al., 2015a, 2015b). Also, in these referred studies with hardwood the

282

pretreated materials were exhaustively washed to remove inhibitors, such as phenols,

283

and a lignin blocking agent such as bovine serum albumin, BSA, was used to

284

minimize enzyme adsorption on lignin. This effectively minimized enzyme adsorption

285

on lignin exposed by pretreatment and directed the cellulolytic enzymes to the

286

cellulose substrate whose exposure increased with decreased particle size (Kim et al.,

287 288

2015; Ko et al., 2015a). In the case of pericarp, lignin content is only 5%, so its effect is minimal.

289

However, grinding to a smaller particle size released more soluble inhibitors (mainly

290

phenols), which were not washed away from the pericarp after it was ground. The

291

increase in phenol concentration in the liquid and decreased conversions indicate an

292

inhibitory effect by the released phenols. Kim et al. (2013, 2011) showed hydrolysis

293

of lignin-free cellulose (Solka Floc) in buffer gave 70% conversion at 1 mg protein/g

294

glucan enzyme loading, while Solka Floc at the same conditions in liquid containing

295

soluble phenol inhibitors gave a conversion of less than 40%. Hence as lignin content

296

increases, cellulase activity decreases due to adsorption on lignin.

297

4. Conclusions

298

Aspergillus niger cellulolytic enzymes in a pectinase preparation effectively

299

hydrolyzed pericarp, with a 5 mg protein/g pericarp resulting in 98% yield in 72 h, but

300

only if the pericarp size was at 5.1 mm. When pericarp was ground to a smaller size

301

of 0.84 mm, conversion decreased. This was found to coincide with the release of

302

phenols by the ground material. Confirming previous reports, phenols were identified

303

as the major potential enzyme inhibitors, whose effect could be minimized by using

304

enzymes from A. niger and a larger particle size of pericarp where phenol release was

305

minimal.

306

Acknowledgements

307

This research was supported by Indiana Corn Marketing Council grant 209346,

308

Hatch fund 199225 and 0205217, the Department of Agricultural and Biological

309

Engineering, and Purdue University Agricultural Research Programs. We thank

310

Genencor for providing Spezyme and Multifect Pectinase enzymes. We also thank

311

Ximing Zhang, Iman Beheshti, and Raymond RedCorn for internal review, and Carla

312

Carie for excellent assistance in preparing this manuscript.

313 314

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inhibitory by-products and strategies for minimizing their effects. Bioresour.

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10. Kim, Y., Kreke, T., Ko, J.K., Ladisch, M.R., 2015. Hydrolysis-determining

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substrate characteristics in liquid hot water pretreated hardwood. Biotechnol.

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11. Kim, Y., Kreke, T., Hendrickson, R., Parenti, J., Ladisch, M.R., 2013.

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Fractionation of cellulase and fermentation inhibitors from steam pretreated mixed

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hardwood. Bioresour. Technol. 135, 30-38.

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inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.

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13. Kim, Y., Hendrickson, R., Mosier, N.S., Ladisch M.R., 2009. Liquid hot water pretreatment of cellulosic biomass. Methods Mol. Biol. 581, 93-102.

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14. Kim, Y., Mosier, N., Ladisch, M.R., 2008a. Process simulation of modified dry

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grind ethanol plant with recycle of pretreated and enzymatically hydrolyzed

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distillers’ grains. Bioresour. Technol. 99, 5177-5192.

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AFEX pretreated distillers’ grains at high-solids loadings. Bioresour. Technol. 99,

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pretreatment severity on properties of hardwood lignin and enzymatic hydrolysis

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of cellulose. Biotechnol. Bioeng. 122, 252-262.

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lignins of liquid hot water pretreated hardwoods. Biotechnol. Bioeng. 112,

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2008. National Renewable Energy Laboratory, Golden, Colorado.

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403 404

Figure captions

405

Fig. 1. Enzymatic hydrolysis of un-pretreated pericarp (15 g dry solids/100 mL) in the

406

presence of three different enzymes at (A) 10 and (B) 27 mg enzyme protein/g solids.

407

Experimental conditions: Spezyme CP, Depol 692L, and Multifect Pectinase were

408

incubated with pericarp slurry in carry-over liquid in an agitated vessel submerged in

409

a water bath to maintain temperature at 50°C, pH 4.8, for 72 h. Agitation provided by

410

a dual head impeller at 290 rpm.

411

Fig. 2. Enzymatic hydrolysis of un-pretreated pericarp (15 g dry solids/ 100 mL) with

412

or without carry-over liquid at different enzyme loadings (equivalent to 1, 5, 10, and

413

27 mg Multifect Pectinase protein/g total pericarp solids). Hydrolysis tests were

414

carried out at 50°C, pH 4.8, for 72 h with a dual head impeller; mixing at 290 rpm.

415

Fig. 3. Comparison of cellulose conversion of different sizes of un-pretreated pericarp

416

(15 g dry solids/100 mL) for enzyme hydrolysis. Enzymatic hydrolysis was performed

417

in the presence of carry-over liquid combined with Multifect Pectinase (27 mg

418

protein/g solids) at 50°C, pH 4.8, for 72 h with a dual impeller head mixing of 290

419

rpm. Control: no enzyme treatment with cracked pericarp. Half particle size pericarp:

420

13.7 mm; Quarter particle size pericarp: 7.2 mm; Cracked particle size pericarp: 5.1

421

mm; Ground particle size pericarp: 0.84 mm.

422

Fig. 4. Total phenols released as a function of particle size of corn pericarp (ground,

423

cracked, quarter, and half) after hydrolysis. Enzymatic hydrolysis (15 g dry solids/100

424

mL) was performed in the presence of carry-over liquid combined with Multifect

425

Pectinase (27 mg enzyme protein/g solids) at 50°C, pH 4.8, for 72 h with a dual head

426

impeller mixing of 290 rpm. Half particle size pericarp: 13.7 mm; Quarter particle

427

size pericarp: 7.2 mm; Cracked particle size pericarp: 5.1 mm; Ground particle size

428

pericarp: 0.84 mm.

429

Fig. 5. Enzymatic hydrolysis of cracked pericarp and ground pericarp (15 g dry

430

solids/100 mL) with different Multifect Pectinase loadings at (A) 1, (B) 5, and (C) 10

431

mg protein/g total solids. All runs were performed at 50°C, pH 4.8, for 72 h with a

432

dual impeller head mixing of 290 rpm. Cracked particle size pericarp: 5.1 mm;

433

Ground particle size pericarp: 0.84 mm.

434

435

Fig. 1. A. 10 mg protein/g solids

100 80 60 40

436 437 438 439 440 441 442 443 444 445 446 447 448

Cellulose conversion to glucose (%)

20 0

24

100

48

72

B. 27 mg protein/g solids

80 60

Fig. 2.

40 20 0 24

48 Hydrolysis time (h)

Spezyme CP

Depol 692L

72

Multifect Pectinase

27 mg protein

27 mg protein in buffer 5 mg protein

80

30

10 mg protein

60 20

1 mg protein in buffer 1 mg protein

40

10

20 0

0 0

24

48

72

Hydrolysis time (h)

449 450 451 452 453 454 455 456 457 458 459 460 461 462

27 mg protein in carry-over liquid

27 mg protein in buffer

10 mg protein in carry-over liquid

5 mg protein in carry-over liquid

1 mg protein in carry-over liquid

1 mg protein in buffer

Glucose concentration (g/L)

Cellulose conversion to glucose (%)

40

100

Fig. 3. 40

5.1 mm

100 80

30

0.84 mm 7.2 mm 13.7 mm

60

20

40 10 20 Control (no enzyme) 0

0 0

Control 464 465 466 467

12

24

13.7 mm

36 Time (h) 7.2 mm

48

5.1 mm

60

0.84 mm

72

Glucose concentration (g/L)

Cellulose conversion to glucose (%)

463

468

Fig. 4.

Total phenols at 765 nm (mg/L)

0.5 0.84 mm

0.4

5.1 mm

0.3

7.2 mm 0.2

0.1 y = -0.0318x + 0.4529 R² = 0.9846

13.7 mm

3

15

0 0 469 470

6 9 12 Corn pericarp particle size (mm)

18

471

Fig. 5. A. 1 mg protein/g solids

B. 5 mg protein/g solids

26

Cellulose conversion to glucose (%)

40

100 80

30

Ground cracked

60 20

Cracked 40

10

Ground cracked

20 0

0 0

472

12

24

36

48

60

72 40

100 80

30

60 20 40 10

20 0 473

0 0

Cracked

12

24

36

48

60

72

27

40

100 80

30

60

20 40 10

20 0 474 475 476

0 0

12

24

36

48

60

72 Hydrolysis time (h)

477 478 479 480

28

481

Table 1. (A) Commercial enzyme profiles of Spezyme CP, Depol 692L, and Multifect

482

Pectinase, and (B) normalized enzyme activities (unit/g pericarp solid) in Spezyme CP,

483

Depol 692, and Multifect Pectinase at 10 mg protein/g solids enzyme loadings.

484

(A) Amy (U/ml)

FPU

EG (U/ml)

β-G (CBU/ml)

Xyl (OSX/ml)

β-X (U/ml)

Protein (mg/ml)

Origin

5.4

52.8

163

104.5

2622

7.3

82

T. reesei

1

Spezy me

485 486 487 488 489 490 491 492

493 494 495 496 497

2

Depol

26.1

5.9

44

11

1510

18.2

116

T. reesei +A. niger

3

MP

1.5

4.2

577

176

947

35

82

A. niger

1

Spezyme: Spezyme CP. Depol: Depol 692L. 3 MP: Multifect Pectinase. Amy: amylase; EG: endo-glucanase activity with respect to CMC; β-G: β-glucosidase activity with respect to p-NPG; Xyl: xylanase activity with respect to oat spelt xylan; β-X: β-xylosidase activity with respect to p-NPX. 2

(B) Amy

FPU

EG

β-G

Xyl

β-X

Origin

Spezyme CP

0.04

0.43

1.33

0.85

21.32

0.06

T. reesei

Depol 692L

0.15

0.03

0.25

0.06

8.69

0.1

T. reesei +A. niger

Multifect Pectinase

0.01

0.03

4.69

1.43

7.7

0.28

A. niger

Amy: amylase; EG: endo-glucanase; β-G: β-glucosidase; Xyl: xylanase; β-X: β-xylosidase. Normalized enzyme activity is calculated by the enzyme unit (unit/mg protein) times the enzyme loading concentration dividing by the total pericarp solids. enzyme activity mg protein

ormalized enzyme activity (unit g solid 29

enzyme dose mg protein g solid

498 499 500 501

Table 2. Composition (%) of initial corn pericarp and extractive free of initial pericarp.

502

Corn pericarp was collected after enzymatic fractionation of corn kernels at solid

503

concentration of 25% (w/v) in the presence of 0.2% (v/v) Spezyme CP and 0.15%

504

(v/v) Promod 144GL (protease). Compositional analysis was done in triplicate. Composition (% dry weight) Component Glucan

1

Initial

Extractive free

25.7 ± 0.61

42.6 ± 0.72

Cellulose (22.5 ± 0.11) Starch (3.2 ± 0.12) Xylan/galactan

505 506 507 508 509 510

15.5 ± 0.04

25.8 ± 0.61

Arabinan

8.2 ± 0.3

13.7 ± 0.30

Acetyl

1.8 ± 0.03

3.0 ± 0.02

Acid Insoluble Lignin

4.5 ± 0.15

7.1 ± 0.65

Acid Soluble Lignin

0.2 ± 0.01

0.3 ± 0.00

Ash

0.1 ± 0.02

0.1 ± 0.8

Extractives (water + ethanol)

39.7 ± 0.12

-

Mass Closure

95.7 ± 0.16

92.6 ± 0.44

1

20.0% (dry basis) corn pericarp was recovered after enzymatic fractionation of corn kernels.

30

511 512

HIGHLIGHTS:

513



Cellulose hydrolysis of corn pericarp is feasible without pretreatment.

514



A. niger cellulases in pectinase enzyme fraction tolerate phenol inhibitors.

515



Ground pericarp (0.84 mm) releases more phenols than cracked pericarp (5.1 mm).

516



Phenols from ground pericarp strongly inhibit enzymatic hydrolysis of pericarp.

517

31