LigninâCarbohydrate Complexes from Poplar Wood - ACS Publications

Chapter 21

Lignin—Carbohydrate Complexes from Poplar Wood Isolation and Enzymatic Degradation

Downloaded by UNIV OF PITTSBURGH on May 4, 2015 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch021

R. P. Overend and K. G. Johnson Division of Biological Sciences, National Research Council of Canada, Ottawa, OntarioK1AOR6, Canada

Four distinct lignin-carbohydrate complexes (LCCs) were isolated from a thermal hydrolysate of Populus deltoides wood by a preparative sequence involving extraction with ethyl acetate, ethyl alcohol, and exhaustive fractionation on a variety of gel filtration media. Purified LCCs presented peaks of superimposable carbohydrate content and ultraviolet absorption when subjected to gel filtration chromatography wherein each given species eluted within the included gel volume. While LCCs of apparent high molecular size were typified by high ultra-violet absorption, lower total carbohydrate, and critical micelle concentrations in the range of 20 to 35 micrograms dry weight, LCCs of apparent low molecular weight possessed less ultraviolet absorbance, significantly more carbohydrate, and critical micelle concentrations that were 10fold greater on average than the larger sized species. All LCC preparations were highly O-acetylated, and contained varying proportions of glucose, xylose, mannose, and uronic acids. Treatment of purified LCCs with a variety of enzymes including acetyl xylan esterase, xylanase, β-mannanase, and β-glucosidase independently and in combination, while not producing monomeric (carbohydrate) components, generated products of altered gel permeation and critical micelle concentration properties. Lignin-carbohydrate complexes (LCCs), covalent associations of lignin polymers with hemicellulose moieties, are typified by their extreme stability to physicochemical and possibly to biological agents. Since the first description of LCCs by Bjôrkman (i), many studies to characterize the linkages between carbohydrate and lignin components have been conducted. The task has been formidable in that the drastic conditions used in sufficiently degrading the cell wall network to facilitate LCC isolation can hydrolyze some lignin-carbohydrate linkages, while allowing other new linkage to be formed (2,3). Further complexity arises from the diversity 0097-6156/91/0460-0270$06.00/0 Published 1991 American Chemical Society

In Enzymes in Biomass Conversion; Leatham, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


21.

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Lignin-Carbohydrate ComplexesfromPoplar 271

of hemicellulose which may be composed of varying amounts of xylan, glucomannan, galactoglucomannan, and arabinan and which differs with the source of plant cell wall material (4,5). LCCs have been isolated from milled wood lignin and purified by alkaline treatment and/or differential extraction with organic solvents (4,6,7,8), and from rumen fluids (9,10,11). In these studies, even LCCs from a single source exhibited variable molecular weights, ligninxarbohydrate ratios, and hydrophobicity emphasizing that a major deterrent in L C C characterization arises from the difficulty in isolating homogeneous entities. In recent years, enzymic modification/removal of lignin from LCCs has received increased attention because of potential applications in the pulp and paper industry, waste treatments, fuel products, and animal feeds. While preliminary studies suggested that lignin peroxidases do not result in lignin removal (22), and likely result in lignin polymerization (73), use of hemicellulolytic enzymes has enhanced chemical pulp bleaching processes (14), with concomitant improvements in pulp beatability (25) and tear strength (22). However, chemical and enzymic treatments to remove the blockage by lignin of carbohydrate hydrolysis by rumen enzymes (16,17) have not been fully successful. Isolation, purification, and characterization of P. deltoïdes LCCs and the effect of hemicellulases upon these materials are reported below. Materials and Methods Preparation of LCCs. An aqueous 10% (w/v) suspension of P. deltoïdes wood (particle size < 0.5mm) was passed through a high shear valve and plug flow reactor combination for a total residence time of 2 minutes at 217°C. The reaction was quenched by flash discharge to atmospheric pressure, and the hemicellulose starting material was separated from the treated residue by centrifugation at 10,000 χ g for 15 minutes. Following concentration of the filtrate by evaporation under reduced pressure, the hemicellulose starting material was lyophilized. Gel Permeation Chromatography. Samples were filtered on columns of Bio-Gel P-6DG (Bio-Rad Laboratories), and columns of Sephadex G-10, G-25, and G-50 (Pharmacia Corp.) using deionized water as eluant. Gel filtration properties are expressed in terms of the distribution coefficient calculated from the relationship = (V - V J / (V - V ) where V the the volume at which a component elutes, V is the void volume, and V is the total volume of the system. Blue dextran 2,000 and xylose were used to determine V and V respectively. e

Q

t

Q

e

t

G

t

Enzyme Treatment of LCCs. Purified Bacillus circulans xylanase (specific activity, 110 units/mg protein), purified Coriolus versicolor 0-mannanase (specific activity 179 units/mg protein), purified Thermoascus aurianticum 0-glucosidase (specific activity, 150 units/mg protein), and purified Schizophyllum commune acetyl xylan esterase (AXE; specific activity, 180 units/mg protein) were used. Unit activity for xylanase and 0-mannanase was defined as the amount of enzyme catalyzing the release of 1 μπιοί reducing power per minute at 50°C and pH 6.0 using oat spelts


272

ENZYMES IN BIOMASS CONVERSION


xylan (Sigma) and konjacu root glucomannan substrates, respectively. Unit activity for acetyl xylan esterase and 0-glucosidase was defined as the amount of enzyme catalyzing the release of 1 μπιοί acetic acid or /7-nitrophenol per minute at 50°C and pH 6.0 using larch wood acetyl xylan and /?-nitrophenyl-0glucopyranoside (Sigma) substrates, respectively. Purified LCCs (10 mg dry weight) were incubated at 50°C for 3 h with 50 units of each enzyme alone and in specified combinations in a total volume of 1.0 mL. Reaction mixtures contained a final concentration of 20 mM potassium phosphate buffer, pH 6.0. Enzyme digests were then fractionated on columns of Sephadex G-50 or Sephadex G-10 as described above. Analytical Techniques. Total carbohydrate and reducing groups were estimated by the phenol-sulfuric acid technique (18) and the Somogyi modification (19) of the Nelson technique, respectively. Neutral sugars were quantitated following trifluoroacetic acid hydrolysis (20), by hplc on either a Bio-Rad HPX-8P or three HPX-8H column(s) in series using water or 10 mM sulfuric acid, respectively, as eluants. Uronic acids were estimated with the m-hydroxydiphenyl reagent (22). Acetyl groups were quantitated by hplc after alkaline hydrolysis (22). Lignin was determined with acetyl bromide reagent (23) using ethylene glycol soluble lignin from P. deltoïdes as standard. Critical micelle concentration (cmc) was determined using the interaction of pinocyanol chloride (Sigma) with increasing concentrations of LCCs as previously described (24). The concentration at which rapid change in absorption occurs at 547 and 598 nm was defined as the critical micelle concentration. Elemental analyses were performed on a Perkin-Elmer 240C Elemental Analyzer. Spectroscopy. Fourier transform infrared (FTIR) analysis was performed as follows: 5 to 7 mg of sample was mixed with KBr in a grinder and the sample having approximately 1 to 2% (w/v) of L C C was mounted in the diffuse reflectance accessory (DRIFT) of a Nicolet DB-5 FTIR spectrometer. Four hundred scans were accumulated at a resolution of 4 cm" . Absorptions for C 0 and H 0 were subtracted and baseline correction applied to eliminate the ramping effect of the DRIFT technique. C-Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker CXP-180 spectrometer operating at a resonance frequency of 45.267 MHz. Spinning speed was 3.5 kHz. CPMAS measurements were run at an ambient temperature (297° K) with a 1 H decoupling field of 60 kHz. Typically 3,600 scans (sweep width setting of 20 kHz) were taken with a delay time of 2 seconds between acquisition cycles. Contact time was 2 ms. Free induction decays were 512 words, which were zero filled to 4 k prior to Fourier transformation. Electron spin resonance (ESR) spectra were recorded and measured with a Varian E-12 spectrometer, a Systron-Donner frequency counter, and a Bruker D35M gaussmeter. For the range 100-300 K, temperature control inside the rectangular resonant cavity was achieved with a stream of pre-cooled N and a proportional controller. 1

2

2

13

2


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Results LCC Purification. The sequence for LCC purification and component yield appears in Figure 1. The lyophilized hemicellulose starting material (40 g as an aqueous 10% w/v suspension) underwent two cycles of extraction with equal volumes of ethyl acetate. Resultant aqueous (i.e., ethyl acetate insoluble) material was sequentially subjected to two cycles of precipitation at 4°C with 40 volumes of 95% ethanol, evaporation under reduced pressure to remove ethanol, and lyophilization. Water-resuspended precipitate (6 g) was fractionated according to the scheme illustrated in Figure 1. Fractionation strategies involved successive rechromatography of materials on permeation gels of greater exclusion limits until components having superimposable and carbohydrate content eluted in the included gel volume. For this and all subsequent filtration steps, pooled fractions were concentrated by lyophilization, and dried samples were dissolved with water at concentrations of not less than 25 mg/mL dry weight. Isolation of components having superimposable A ^ and carbohydrate content was dependent upon maintenance of high sample concentrations and avoidance of ionic environments. After initial fractionation of higher and lower molecular weight fractions "A" and "Β" using Bio-Gel P-6 D G (Fig 2a), fractions were pooled as indicated. Further representative fractionations of low molecular weight LCC material into the key components B l , B2, and B3, as well as the penultimate fractionation of ARB1 on Sephadex G-50 generating LCC 1, and the penultimate fractionation generating LCCs 2 and 3 are illustrated in Figures 2b, 2c, and 2d respectively. For LCC 4 (Figure 1), alternate filtration on a gel of lower exclusion limit was necessary to remove non-carbohydrate associated A ^ material. Characterization of LCCs. Properties of the four LCCs appear in Table I. Table I. Properties of Populus deltoïdes Lignin-carbohydrate Complexes Lignin-carbohydrate Complex 3 1 2

Property b

cmc ^g/mL)

4 0.039 260-320

0.100 20-30

0.067 25-35

0.685 250-350

13.40

17.7

1.00

1.60

204

188

748

398

925°

893

223

383

^280

(units/mg) Carbohydrate (Mg/mg) Lignin (Mg/mg)

c

a

All values were obtained using Sephadex G-50 gel filtration media with the exception of LCC 4 where Sephadex G-10 was employed. cmc; critical micelle concentration. Values for lignin content were beyond the range of the assay. b


274


Thermal Hydrolysate of

Popv/us deltoid**

Wood (40

g)

I 2 cycles extraction with Ethyl acetate

Ethyl acetate phase (discard)

Aqueous phase


2 cycles extraction with 96% Ethanol Ethsnol Soluble (discard)

Ethsnol Insoluble

Filtration on Bio-Gel P-6 I Ά Fraction' I Filtration on Sephadex Q-25

(6g)

DQ

I *B Fraction*

I

Reftitration on Bio-Gel P-6 DQ r B1

AR ARB1

I B3

B2

Filtration on Sephadex

Q-25

rzj L

r Filtration on Sephadex

Q-60

B2A

B2B :

B3A î B3B

L. LCC / (142 mg)

Re-filtration on Sephadex' Q-26 —

Filtration on Sephadex G-50

I LCC C I A

•

Re-filtration on Bio-Gel P-6

1

:

LCC C1B

Re-filtration on Sephadex

LCC 2 (33.4 mg)

1 C2

C1

C2A

G-50

LCC 3 (372 mg)

DQ

:

C2B (disoard)

2 cycles filtrstion on Sephadex Q-10

LCC 4 (671 mg)

Figure 1. Scheme used for the purification of Populus deltoïdes LCCs.


:

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Lignin-Carbohydrate Complexes from Poplar 275


21.

Figure 2. Representative fractionations employed in the purification of Populus deltoïdes LCCs. a, initial fractionation of high ("A") and low ("B") molecular weight components through Bio-Gel P-6; b, re-fractionation of "B" through Bio-Gel P-6; c, penultimate fractionation of ARB1 on Sephadex G50; d, penultimate fractionation of CI components on Sephadex G-50. A ^ , (O—O); carbohydrate, ( · — · ) .


276


The least abundant moieties, LCCs 1 and 2, were relatively high molecular weight species having lower cmc values than the more abundant, smaller LCCs 3 and 4, which exhibited about 10-fold higher cmc values. Elemental analyses of the thermal hydrolysate, ethanol insoluble material, and purified LCCs appear in Table II. The ash content of starting material was highly conserved in the ethanol insoluble fraction, and particularly in LCCs 1 and 2. After adjusting for ash content, contents of C, H , and Ο were comparable in the four LCC preparations. 3

Table II.

Elemental Analysis of Populus deltoïdes L C C Components


Sample Thermal hydrolysate Ethanol precipitate LCC 1 LCC 2 LCC 3 LCC 4 a

5

C

H

N

Ô

Âsh

44.89

5.82

2.79

41.94

4.56

34.90 29.47 29.43 40.95 33.47

4.21 3.31 3.63 5.21 5.21

0.47 1.12 1.67 0.19 0.37

37.87 66.10 39.00 53.65 60.56

22.55 31.50 26.27 1.91 11.65

Data are expressed as percentages of dry weight. Oxygen was calculated by difference.

b

Carbohydrate/0-acetyl content of P. deltoïdes LCCs appears in Table III. All purified LCCs were O-acetylated moieties containing varying amounts of glucose, mannose, xylose, and uronic acids. Relative to xylose, the highest levels of uronic acid and Ô-acetyl content were encountered in LCCs 1 and 2. LCC 1 was the sole moiety to contain galactose. Xylose was the most abundant sugar found in both LCCs 3 and 4, both of which also contained nearly equimolar amounts of glucose and mannose. FTIR spectra of various P. deltoïdes preparations in the frequency range of 800 to 1,900 cm" appear in Figure 3. Spectra were examined to 4,000 cm" , but no essential differences were encountered with the exception of LCC 3 which presented a prominent C-H band at approximately 2,900 cm" . Spectra for LCCs 1 and 3 were the most distinct. LCC 1 displayed a prominent diffuse band in the 1,500 - 1,700 cm" region possibly associated with either aromatic ring stretches or conjugated keto-carbonyl groups. LCC 3 possessed a prominent peak at 1,736 cm" apparently associated with xylan carbonyl stretching and a 1,047 cm' band ascribed to the C-O stretch in hemicelluloses. The 987 cm" hemicellulose stretch was more clearly evident in LCC 3 spectra. All four preparations exhibited absorption increases in the 800-850 cm" regions ascribed to aromatic band absorption. A small peak at 875 cm" present in LCCs 1 and 3 was assigned to lignin. 1

1

1

1

1

1

1

1

1


21.

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1 3

CP-MAS C N M R spectra of LCCs 3 and 4 appear in Figure 4a and 4b respectively. LCC 3 displayed a prominent resonance at approximately 174 ppm which was assigned to the acetyl group in acetyl xylan and which confirmed the assignment of the 1,736 cm' FTIR absorption peak. LCC 4 presented a broad resonance in this region with major peaks at 183 and 176 ppm, and shoulders at 171 and 169 ppm. Both spectra presented peaks associated with the C - l of sugars centered on 103-104 ppm, and peaks associated with the secondary hydroxyl groups on carbons centered on 75 ppm and primary hydroxyl groups at about 65 ppm. LCC 3 spectra clearly contained much more carbon with primary hydroxyl groups and had much more methyl-CH signal at 22 ppm than did LCC 4 spectra. 1


3

Table III. Chemical Composition of Populus deltoïdes Lignin-carbohydrate Complexes μιηοΐ/mg

Molar Ratio

Glucose Mannose Xylose Galactose Uronic acids O-acetyl Glucose Mannose Xylose Uronic acids O-acetyl

0.050 0.027 0.133 0.039 0.233 0.320 0.061 0.022 0.153 0.144 0.133

0.38 0.20 1.00 0.29 2.40 1.75 0.46 0.17 1.00 0.94 0.87

3

Glucose Mannose Xylose Uronic acids O-acetyl

0.206 0.222 2.300 0.480 1.580

0.09 0.10 1.00 0.21 0.69

4

Glucose Mannose Xylose Uronic acids O-acetyl

0.211 0.211 0.993 0.356 0.400

0.21 0.21 1.00 0.36 0.40

LCC

1

2

Component



1900

- 1

)

Figure 3. FTIR spectra of Populus deltoïdes LCCs 1,3, and 4, and the ethanolinsoluble fraction used in their preparation.

WAVENUMBERS ( c m

1800 1700 1600 1500 1400 1300 1200 1100 1000 900


800

Ώ

GO

Ο Ο

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21.


280


ESR spectra of LCCs 3 and 4 appear in Figure 5. A strong free radical component at g = 2.0045 observed in the spectrum of LCC 3 (Figure 5a) was evident but diminished in the spectrum of LCC 4 (Figure 5b). Relative spins (Mn ) of the thermal hydrolysate, LCC 3 and LCC 4 were 16,

LigninâCarbohydrate Complexes from Poplar Wood - ACS Publications

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LigninâCarbohydrate Complexes from Poplar Wood - ACS Publications