Short-Fiber Formation During Cellulose Degradation by Trichoderma

Chapter 12

Downloaded by UNIV MASSACHUSETTS AMHERST on October 7, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch012

Short-Fiber Formation During Cellulose Degradation by Trichoderma pseudokoningii S38 Jie Liu and Peiji Gao State Key Laboratory of Microbial Technology, Institute of Microbiology, Shandong University, Jinan 250100, China

A low-molecular weight component, which could cause depolymerization of native cellulose to form insoluble short fibres without production of soluble reducing sugar, was separated from the cellfree culture of cellulolytic fungal strain Trichoderma pseudokoningii S38. It is termed Short Fibre Generating Factor (SFGF) according to its function. Its essential features, as well as the process of short fibre formation, which is the intermediate state of cellulose degradation, were studied.

Much attention has been given to study the mechanisms of biological degradation of cellulose. Due to the structural complexity of native cellulose materials, the degradation of cellulose is a quite complicated process. Insoluble crystalline microfibrils are highly resistant to enzymatic hydrolysis. Cellulase systems contain a multiplicity of enzyme components showing a marked synergism against crystalline cellulose (7,2). But the degrading efficiency of native cellulose is still quite low, which partly lies in the uncertainty of degradation mechanism. The appearance of short, insoluble fibres during the cellulose degradation was regarded as the initial stage or the first step, which was not accompanied by the formation of soluble reducing sugar, but which made the substrate more accessible to endoglucanase attack. Evidence (3,4) suggested that besides the hydrolytic mode, oxidative degradation is also a part of the degradation process. Although the former mode has the main effect on cellulose degradation, the latter helps to accelerate the process and make use of cellulosic materials. Comparatively speaking, hydrolytic degradation is better understood than oxidative degradation. The present paper shows the isolation of a kind of non-enzymatic component in filmentous fungi and its functions during cellulose degradation. It is termed short fibre generating factor (SFGF).

0097-6156/96/0655-0166$15.00/0 © 1996 American Chemical Society

In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

12. LIU & GAO

167 Short-Fiber Formation During Cellulose Degradation

Isolation ofSFGF


Media and Culture Conditions. The cellulolytic fungal strain Trichoderma pseudokoningii S38 (5) was maintained at 30 °C for 6 days in a solid-layer fermentation system, consisting of (W:W%) corn stalk powder 80, wheat bran 20, and water 250 supplemented with Mandels' mineral salts (6). Assay of Cellulase Activity. Cellulase activity (filter paper activity, FPA) was measured by incubating enzyme solution with 50 mg filter paper (Whatman No.l) in a 1.5 ml acetate buffer (100 m M , pH 4.8) system at 50 °C for one hour. The activity was expressed as equivalent of reducing sugar produced, which was assayed by DNS (3,5-dinitrosalicylic acid) reagent (7). And one unit (U) was defined as the amount ( μ mol) of glucose produced by enzymes per minute under given condition. Assay of short fibre formation. 50 mg filter paper taken as substrate and the sample solution supplemented with phosphate buffer (20 m M , pH 6.0) upto 10 ml, were incubated in a 50 ml flask at 45 C , 30 rpm for one day. The turbidity of the suspension was determined using an integrating sphere attachment (Spectrophoto meter UV-VIS-240, Shimadzu). The increase ratio of turbidity was defined as following: e

T% = (Ts-Tc)/Tc Ts,Tc represented the turbidity of sample and control respectively. Separation of SFGF from Crude Enzyme. The crude cell-free culture filtrate (crude enzyme) was collected by centrifugation from acetate buffer extracts (50 mM, pH 4.8) of solid-layer culture. The crude enzyme had a relatively high ability to degrade cellulose, including both reducing-sugar production (1.2 U cellulase activity per milliliter) and short-fibre formation. The components with molecular weights above 10,000 Da, such as cellobiohydrolase (CBH), endo-glucanase (EG) and β -glucosidase ( β G), were removed from the crude enzyme broth by ultra filtration (Amicon, PM-10 with cut-off of 10,000 Da, Pharmarcia). The permeate from ultrafiltration had little cellulase activity (CBH, E G , β G) that could be neglected, but it could decompose native cellulose to produce short fibres, which were still insoluble. After concentrated by ultrafiltration (Amicon, YM-1 with cut-off of 1,000 Da), the filtrate was applied in turn to columns of Sephadex LH-20, DEAE-Sephadex A-25, Bio-gel P-4, and HPLC (Supelcosil, LC-18-DB), eluted with appropriate buffers indicated respectively in Section A , B , C and D of Figure 1. In each purification step, the component having the ability of generating short fibre (shown in Figure 1) was collected and prepared for the next step. In the step of HPLC, a single peak was obtained.

In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


168

ENZYMES FOR PULP AND PAPER PROCESSING

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Short-Fiber Formation During Cellulose Degradation by Trichoderma

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