Crystalline cellulase and method for producing same

#The present invention is related to the crystallization of cellulase enzymes. More particularly, the present invention relates to selective crystallization using salt of cellulase enzymes in an aqueous solution produced by, for example, the fermentation of microbial organisms such as filamentous fungi, yeast or bacteria.

#Intensive research efforts have been directed to the precipitation and crystallization of enzymes as a means of purification and preparation of enzyme products. For example, in U.S. Pat. No. 4,659,667, a process is disclosed for the recovery of an enzyme from solution by concentrating to supersaturation the enzyme-containing solution at pH near the isoelectric point of the enzyme, inducing crystallization and recovering the crystallized final product. Inducing crystallization is achieved by allowing the enzymes to spontaneously crystallize upon concentration or by seeding, sound, stirring or scratching the inner surface of the container. Crystallization of alpha-amylase is exemplified.

#In PCT Publication No. WO 89/08703, a process is described for the crystallization of subtilisin by adding a halide salt, such as sodium chloride or calcium chloride, to a concentrated subtilisin solution of at least about 40 grams per liter at temperatures less than 10° C.

#FIG. 1 illustrates the proposed tertiary structure of an exocellobiohydrolase enzyme derived from Trichoderma longibrachiatum (CBHI) showing the catalytic domain which is connected via the linker to the cellulose binding domain.

#“Cellulase,” “cellulolytic enzymes” or “cellulase enzymes” means bacterial, plant or fungal exoglucanases or exocellobiohydrolases, endoglucanases, and β-glucosidases. These three different types of cellulase enzymes act synergistically to convert cellulose and its derivatives to glucose. A cellulase composition produced by a naturally occurring source and which comprises one or more cellobiohydrolase-type and endoglucanase-type components wherein each of these components is found at the ratio produced by the source is sometimes referred to as a “complete cellulase system” or a “complete cellulase composition” to distinguish it from the classifications and components of cellulase isolated therefrom, from incomplete cellulase compositions produced by bacteria and some fungi, or from a cellulase composition obtained from a microorganism genetically modified so as to overproduce, underproduce, or not produce one or more of the cellobiohydrolase-type and/or endoglucanase-type components of cellulase. The different components, i.e., the various endoglucanases and exocellobiohydrolases in a whole cellulase or incomplete multi-component cellulase, generally have different properties, such as isoelectric point, molecular weight, degree of glycosylation, substrate specificity and enzymatic action patterns.

#The term “cellulose binding domain” refers herein to a peptide or group of related peptides responsible in large part for the cellulose binding activity of a cellulase or derivative thereof. Cellulose binding domains generally function by non-covalently binding the cellulase to cellulose, a cellulose derivative or other polysaccharide equivalent of cellulose. Cellulose binding domains as defined herein are believed to attach the enzyme to cellulose in a manner which permits or facilitates hydrolysis of cellulose fibers by the structurally distinct catalytic core region, but nonetheless functioning in an independent manner from the catalytic core. Thus, a cellulose binding domain will not possess the significant hydrolytic activity attributable to a catalytic core. Moreover, the cellulose binding domain as defined herein should be distinguished from a cellulose binding region which is integral to an enzymatic subunit which possesses catalytic activity. While such a cellulose binding region can, in fact, provide some cellulose binding activity, such a feature of a cellulase would not be equivalent to a cellulose binding domain, which domain is a distinct structural feature of the enzyme. In other words, a cellulose binding domain is a structural element of the cellulase enzyme protein tertiary structure which is distinct from the structural element which possesses catalytic activity. For illustrative purposes, an example of a cellulase incorporating a cellulose binding domain as defined herein is provided in FIG. 1. Analysis of the genes coding for CBHI, CBHII, EGI, EGII and EGV in Trichoderma longibrachiatum shows a domain structure comprising a catalytic core region or domain (CCD) and a hinge or linker region (used interchangeably herein) which connects the catalytic core region to a cellulose binding domain (CBD).

#While not wishing to be bound by theory, it is believed that the cellulose binding domain interferes with the crystallization of cellulases in part due to its tertiary structure, which is believed to protrude from the main body of the enzyme, and because of the flexibility of the binding domain as conferred by the linker region. These characteristics can result in significantly unfavorable crystallization kinetics, and thus prevent the crystallization of many cellulases. “Linker or hinge region” means a short peptide region that links together structurally distinct catalytic core and cellulose binding domains of a cellulase. These domains in T. longibrachiatum cellulases, for example, are linked by a peptide rich in Ser, Thr and Pro.