Hemicellulose hydrolysis enzymes

In direct microbial conversion of lignocellulosic biomass into ethanol that could simplify the ethanol production process from these materials and reduce ethanol production costs, Clostridium thermocellum, a thermoanaerobe was used for enzyme production, hydrolysis and glucose fermentation (755). Cofermentation with C thermosaccharolyticum simultaneously converted the hemicellulosic sugars to ethanol. However, the formations of by-products such as acetic acid and low ethanol tolerance are some drawbacks of the process. Neurospora crassa produces extracellular cellulase and xylanase and has the ability to ferment cellulose to ethanol 139). 

Platform compounds are further converted to products by microbial fermentation processes. Saccharification and fermentation can be accomplished in a sequential process by separate hydrolysis and fermentation (SHF), or in a consolidated one-pot process known as simultaneous saccharification and fermentation (SSF) of single sugars or simultaneous saccharification and co-fermentation (SSCF) of all monosaccharides. Future developments might even combine the production of saccharolytic enzymes, the hydrolysis of cellulose, and hemicellulose to monomeric sugars and the fermentation of hexose and pentose sugars in a single process, the so-called consolidated biomass processing (CBP) (Menon and Rao 2012). 

To break up cellulose/hemicellulose, it is treated physically (milling), with heat, and hydrolyzed (sulfuric acid + enzymes). Also in this case, improved (bio)cata-lytic hydrolysis processes for cellulose/hemicellulose are needed. The sugar can then serve as feedstock for standard fermentation plants. 

Attempts to remove hemicellulose for production of dissolving pulps with very low hemicellulose contents have shown that complete enzymatic hydrolysis of hemicellulose within the pulp is difficult to achieve. The xylan content in delignified mechanical aspen pulp was reduced from approximately 20 to 10%, whereas in bleached hardwood sulphite pulp the xylan content was decreased from 4 to only 3.5% even at very high enzyme dosages (50). The complete removal of residual hemicellulose seems thus unattainable, apparently due to modification of the substrate or to structural barriers. 

Hemicellulose is so vulnerable to acid attack that it does not constitute an accessibility retarding agent for acid hydrolysis. However, it appears that hemicellulose does protect cellulose from enzyme attacks by a shielding action. 

The foregoing observations confirm the conclusions derived from former experiments with beechwood holocellulose (10) (1) A partial degradation of the hemicelluloses is imperative before the cellulose fibrils can be attacked. (2) The hemicelluloses seem to be deposited between the cellulose fibrils or even to be encrusting them. (3) The enzymatic hydrolysis of the cellulose is governed by the porosity of the tissue (enzyme diffusion), the impediment of the hemicelluloses, and the properties of the cellulose (e.g., crystallinity). 

The current observations confirm previous studies on beechwood and sprucewood holocellulose (7,10,19). The attack of the hemicellulose proceeds from the primary wall/Si as well as from the tertiary wall into S2 the pit chambers constitute preferred paths of enzyme diffusion into the walls. Also, substances of the middle lamella, especially in the cell corners, are removed by the xylanase and the mannanase treatments. Parallel to the removal of hemicelluloses, the fibrillar structure of the cellulose and its lamellar arrangement in transections of cell walls became obvious. In samples treated with cellulases, the cellulose fibrils were often completely hydrolyzed in the Si layer, occasionally accompanied by complete dissolution of cell-wall portions. This is also in conformity with the previous conclusion that the cellulases hydrolyze highly ordered zones of cellulose and remove hemicelluloses by hydrolysis or by detachment. 

In contrast to cellulose, which is crystalline, strong, and resistant to hydrolysis, hemicellulose has a random, amorphous structure with little strength. It is easily hydrolyzed by dilute acid or base, but nature provides an arsenal of hemicellulase enzymes for its hydrolysis. Hemicellulases are commercially important because they open the structure of wood for easier bleaching and thus support the introduction of ECF or TCF methods. Many different pentoses are usually present in hemicellulose. Xylose, however, is always the predominating sugar. The pentoses are also present in rings (not shown) that can be five- or six-membered. 

The production of fuel ethanol from renewable lignocellulosic material ("bioethanol") has the potential to reduce world dependence on petroleum and to decrease net emissions of carbon dioxide. The lignin-hemicellulose network of biomass retards cellulose biodegradationby cellulolytic enzymes. To remove the protecting shield of lignin-hemicellulose and make the cellulose more readily available for enzymatic hydrolysis, biomass must be pretreated (1). 

A major problem in the commercialization of this potential is the inherent resistance of lignocellulosic materials toward conversion to fermentable sugars (4). To improve the efficiency of enzymatic hydrolysis, a pretreatment step is necessary to make the cellulose fraction accessible to cellulase enzymes. Delignification, removal of hemicellulose, and decreasing the crystallinity of cellulose produce more accessible surface area for cellulase enzymes to react with cellulose (5). 

The complete degradation of hemicellulose becomes more complex than that of cellulase, since substituent-hydrolyzing activities are also necessary. With heteroxylans, apart from endo-l,4- 3-xylanase, which catalyzes the hydrolysis of internal 3-l,4-xylan links and P-xylosidase, which catalyzes the hydrolysis of xylooligossacharides, mainly xylobiose into xylose, other enzymes must act to accomplish complete hydrolysis, such as acetyl xylan esterase, a-glucuronidase, and a-L-arabinofuranosidase (1). 

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