Variant EGIII-like cellulase compositions

Details Variant EGIII-like cellulase compositions Variant EGIII-like cellulase compositions

1998-12-18

2001-07-31

Gupta, Yogendra N. (Department: 1751)

Cleaning compositions for solid surfaces, auxiliary compositions

Cleaning compositions or processes of preparing

Enzyme component of specific activity or source

C510S320000, C510S321000, C510S530000, C008S181000, C008S401000, C008S137000, C008S116100, C162S095000, C435S209000

Reexamination Certificate

active

06268328

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to novel mutant cellulase compositions which have improved stability. More specifically, the present invention relates to a family of mutant cellulase enzymes from fungi and bacteria which are related in sequence to EGIII produced by
Trichoderma reesei
, but which have certain mutations which provide resistance to, for example, temperature stress.
2. State of the Art
Cellulases are enzymes which are capable of hydrolysis of the &bgr;-D-glucosidic linkages in celluloses. Cellulolytic enzymes have been traditionally divided into three major classes: endoglucanases, exoglucanases or cellobiohydrolases and &bgr;-glucosidases (Knowles, J. et al., (1987),
TIBTECH
5, 255-261); and are known to be produced by a large number of bacteria, yeasts and fungi.
Primary among the applications that have been developed for the use of cellulolytic enzymes are those involving degrading (wood)cellulose pulp into sugars for (bio)ethanol production, textile treatments like ‘stone washing’ and ‘biopolishing’, and in detergent compositions. Thus, cellulases are known to be useful in the treatment of mechanical pulp (see e.g., PCT Publication No. WO 92/16687). Additionally, cellulases are known to be useful as a feed additive (see e.g., PCT Publication No. WO 91/04673) and in grain wet milling.
Of primary importance, however, cellulases are used in the treatment of textiles, i.e., in detergent compositions for assisting in the removal of dirt or grayish cast (see e.g., Great Britain Application Nos. 2,075,028, 2,095,275 and 2,094,826 which illustrate improved cleaning performance when detergents incorporate cellulase) or in the treatment of textiles prior to sale to improve the feel and appearance of the textile. Thus, Great Britain Application No. 1,358,599 illustrates the use of cellulase in detergents to reduce the harshness of cotton containing fabrics and cellulases are used in the treatment of textiles to recondition used fabrics by making their colors more vibrant (see e.g., The Shizuoka Prefectural Hammamatsu Textile Industrial Research Institute Report, Vol. 24, pp. 54-61 (1986)). For example, repeated washing of cotton containing fabrics results in a grayish cast to the fabric which is believed to be due to disrupted and disordered fibrils, sometimes called “pills”, caused by mechanical action. This greyish cast is particularly noticeable on colored fabrics. As a consequence, the ability of cellulase to remove the disordered top layer of the fiber and thus improve the overall appearance of the fabric has been of value.
Thus, cellulases have been shown to be effective in many industrial processes. Accordingly, there has been a trend in the field to search for specific cellulase compositions or components which have particularly effective performance profiles with respect to one or more specific applications. In this light, cellulases produced (expressed) in fungi and bacteria have been subject of attention. For example, cellulase produced by certain fungi such as Trichoderma spp. (especially
Trichoderma longibrachiatum
) have been given much attention because a complete cellulase system capable of degrading crystalline forms of cellulose is readily produced in large quantities via fermentation procedures. This specific cellulase complex has been extensively analyzed to determine the nature of its specific components and the ability of those components to perform in industrial processes. For example, Wood et al., “Methods in Enzymology”, 160, 25, pages 234 et seq. (1988), disclose that complete fungal cellulase systems comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (EC 3.2.1.91) (“CBH”), endoglucanases (EC 3.2.1.4) (“EG”), and &bgr;-glucosidases (EC 3.2.1.21) (“BG”). The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. U.S. Pat. No. 5,475,101 (Ward et al.) discloses the purification and molecular cloning of one particularly useful enzyme called EGIII which is derived from
Trichoderma longibrachiatum.
PCT Publication No. WO 94/14953 discloses endoglucanases which are encoded by a nucleic acid which comprises any one of a series of DNA sequences, each having 20 nucleotides.
Ooi et al.,
Curr. Genet
., Vol. 18, pp. 217-222 (1990) disclose the cDNA sequence coding for endoglucanase F1-CMC produced by
Aspergillus aculeatus
which contains the amino acid strings NNLWG, ELMIW and GTEPFT. Sakamoto et al.,
Curr. Genet
., Vol. 27, pp. 435-439 (1995) discloses the cCNA sequence encoding the endoglucanase CMCase-1 From
Aspergillus kawachii
IFO 4308 which contains the amino acid strings ELMIW and GTEPFT. Ward et al., discloses the sequence of EGIII having the amino acid strings NNLWG, ELMIW and GTEPFT. Additionally, two cellulase sequences, one from
Erwinia carotovara
and
Rhodothermus marinus
are disdosed in Saarilahti et al.,
Gene
, Vol.90, pp.9-14 (1990) and Hreggvidsson et al.,
Appl. Environ. Microb
., Vol. 62, No. 8, pp.3047-3049 (1996) which contain the amino acid string ELMIW.
Despite knowledge in the art related to many cellulase compositions having applications in some or all of the above areas, there is a continued need for new cellulase compositions which have improved stability under conditions present in applications for which cellulases are useful, i.e., household detergents, stonewashing compositions or laundry detergents.
SUMMARY OF THE INVENTION
It is an object of the invention to provide for novel variant EGIII or EGIII-like cellulase compositions which have improved stability.
It is a further object of the invention to provide for novel variant EGIII or EGIII-like cellulase compositions which have improved performance under conditions of thermal stress.
It is a further object of the invention to provide for novel variant EGIII or EGIII-like cellulase containing compositions which will provide excellent performance in detergent applications, including laundry detergents.
It is a further object of the invention to provide for novel variant EGIII or EGIII-like cellulase containing compositions which have improved performance attributes for use in the textiles treatment field.
It is a further object of the invention to provide for novel variant EGIII or EGIII-like cellulase composition which have improved characteristics for the reduction of biomass, as an additive in animal feed, in starch processing and in baking applications.
According to the present invention, a variant EGIII or EGIII-like cellulase is provided wherein one or more amino acids are modified or deleted to confer improved performance, including stability in the presence of thermal and/or surfactant mediated stress. Preferably, the amino acids to be modified correspond in position to residues T2, S3, A8, F10, S18, A24, S25, F30, G31, V36, L38, A42, A46, D47, Q49, Q61, Q64, I65, A66, Q69, A83, S86, S90, V109, T110, Y111, K123, D126, S133, Q134, G135, V139, T145, Q162, N164, T166, Y168, N174, R180, K183, N186, A188, G189, V192, L193, S205, G206, N209, A211, T214 and/or I217 in EGIII from
Trichoderma reesei
. In another preferred embodiment, the amino acids to be modified correspond in position to residues T2S, S3(L/F), A8(S/D/G), F10(Y/E/A/W), S18(N/Y/L), A24(R/K/Q), S25((N/T), F30(N/E/S/W), G31Q, V36(Y/E/G), L38((S/N), A42((V/I), A46((V/T), D47(N/E/T/A), Q49(N/S/E), Q61(P/A), Q64(G/V/A), I65(R/V/Y/K), A66(Q/E), Q69(T/E/R), A83(V/W), S86(N/T/Q), S90(N/T), V109(P/E/A), T110(N/S/G), Y111(S/G/W), K123(R/A), D126(N/G), S133(Q/D/T/F), Q134(V/G/H), G135(A/S), V139(I/L), T145(N/K/S/D), Q162(P/E/S), N164(Q/D/T), T166(N/E/R), Y168F/W, N174D, R180(Q/V/A/E), K183(R/H/Q), N186(P/S), A188(D/R), G189(S/E), V192L, L193(I/Q/T), S205(N/D/P), G206A, N209T, A211(R/S/N), T214(S/H/R) and/or 1217(Q/V/L) in EGIII. Most preferably, the modified amino acids correspond to A24(K/Q/R), G31Q, Q64(G/V/A), V139L, Y168F, N174D, V192L, G206A and/or N209T.
In another embodiment, the present invention provides for a varia

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