Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Mixing of two or more solid polymers; mixing of solid...
Reexamination Certificate
2000-10-23
2002-08-13
Nutter, Nathan M. (Department: 1711)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Mixing of two or more solid polymers; mixing of solid...
C435S177000, C435S178000, C435S180000, C435S181000, C530S812000, C530S815000, C530S816000
Reexamination Certificate
active
06433078
ABSTRACT:
BACKGROUND OF THE INVENTION
Biocatalysts, i.e., free or conjugated enzymes, offer unique advantages over classical chemical methods for producing a wide variety of products. In particular, biocatalysts are highly selective, i.e., able to differentiate between similar molecules or fragments; mild, i.e., minimize side reactions; and are often more environmentally friendly than classical chemical catalysts. However, all biocatalysts on the market either cannot be reused or do not work efficiently in heterogeneous reaction systems. Consequently, biocatalysis, i.e., methods of organic chemistry employing biocatalysts, is often overlooked for large-scale industrial applications.
Biocatalysts are currently commercially available in three forms: free enzymes, immobilized enzymes, and cross-linked enzyme crystal (CLEC®; Altus, Cambridge, Mass.) biocatalysts. Free enzymes are soluble in aqueous reaction systems and act as any other homogeneous catalyst. Free enzymes work well in both homogeneous and heterogeneous reaction systems. However, problems associated with free enzymes include low stability under work-up conditions and significant surface activity of enzyme-containing solutions, especially at high concentrations. This makes the reuse of free enzymes practically impossible and complicates product purification for many large-scale industrial applications. Thus, most free enzymes are not cost-effective for large-scale industrial applications.
Immobilized enzymes are enzymes that are attached to a chemically inert solid carrier. Thus, immobilized enzymes are easier to separate from homogeneous reaction systems than free enzymes. However, most industrial processes involve heterogeneous reaction systems with insoluble starting materials or products or both. On the industrial scale, separating insoluble reaction products from insoluble immobilized enzymes is often the bottleneck in industrial processes and/or not cost-effective. Moreover, the activity of immobilized enzymes in heterogeneous reaction systems constitutes only a fraction of their activity in homogeneous reaction systems [Hayashi, T. & Ikada, Y.,
Biotechnol. Bioeng.
35:518 (1990)]. In addition, the active enzyme content in most commercially-available, covalently bound immobilized enzyme preparations is usually well below 1% by total weight (see Table 1).
TABLE 1
Active protein content of several common, commercially-available immobilized
enzyme preparations
Activity of
Enzyme
Activity of free
immobilized enzyme
content by
Enzyme
enzyme (U/g)
(U/g)
weight (%)
Source
Chirazyme L-2
>1,200,000
200
0.02
Roche, item #s
(Lipase from
3,000
0.25
1836021,
Candida
1663917,
antarctica
, type
1835807
B)
Chirazyme L-3
1,500,000
500
0.03
Roche, item #s
(Lipase from
1978659,
Candida rugosa
)
1965972
Chirazyme L-9
3,000,000
10
0.0003
Roche, item #s
(Lipase from
1831313,
Mucor miehei
)
1827308
b-Chymotrypsin
40,000-60,000
500-1,500
1.25-2.5
Sigma, item #s C
7762, C 5407
Penicillin
10,000
60-120
0.6-1.2
Sigma, item #s P
amidase
3319, P 3942
Penicillinase
1,500,000-
1,500-4,500
0.1-0.3
Sigma, item #s P
3,000,000
0389, P 8817
Protease from
500,000-
250-500
0.025-0.1
Sigma, item #s P
Staphylococcus
1,000,000
2922, P 6552
aureus
strain 8
Table 1 provides a comparison of the activities (U/g) of commercially-available free and immobilized enzyme preparations listed in their respective catalogs. The activities shown in Table 1 are corrected for protein content where that information is available in the same catalog for a given preparation. It was assumed that 100% of the protein in a given preparation is active enzyme. Comparisons between the activities of free and immobilized enzymes were made only where activity was measured using the same method. The Roche catalog in some cases gives lower limits of enzyme activity. Therefore, the calculated enzyme content of a particular immobilized enzyme preparation may represent a lower limit of its respective value. However, these data demonstrate that the actual amount of active enzyme in a particular immobilized enzyme preparation is quite low when compared to free enzyme preparations. As a result of the difficulty to reuse immobilized enzymes in heterogeneous reaction systems and the small percentages of active enzyme in commercially-available immobilized enzyme preparations, immobilized enzymes are less preferred than classical chemical techniques for many large-scale industrial applications.
CLEC® biocatalysts are fine enzyme crystals that are cross-linked and, therefore, insoluble in aqueous media. Because CLEC® biocatalysts are insoluble in aqueous media, CLEC® biocatalysts can be more easily separated from soluble starting materials and products than free enzymes. Further, because of their fine structure, CLEC® biocatalyst activity is close to the activity of free enzymes. However, CLEC® biocatalysts are expensive and possess most of the shortcomings of immobilized enzymes. Thus, a need exists for low-cost, reusable industrial enzyme biocatalysts that are able to work in heterogeneous reaction systems with the same activity as free enzymes.
Too fill this void, a few attempts have been made to design reversibly soluble enzyme-biocatalysts. These biocatalysts are reversibly soluble dependent upon minor changes in the reaction environment, such as temperature, salt concentration, pH, etc. Thus, when in the soluble state, reversibly soluble enzyme biocatalysts are able to function effectively in heterogeneous reaction systems. Further, the reversibly soluble nature of these biocatalysts permits precipitation recovery and reuse. Thus, reversibly soluble enzyme biocatalysts overcome the disadvantages of free enzymes, immobilized enzymes and CLEC® biocatalysts with respect to reuse and the ability to function in heterogeneous reaction systems. In all cases, reversibly soluble enzyme biocatalysts are conjugates of an enzyme with a reversibly soluble polymer i.e., reversibly soluble enzyme-polymer conjugates.
Reversibly soluble enzyme-polymer conjugates have been made with the following enzymes: chymotrypsin [Chen, J.-P. & Hsu, M.-S.,
J. Molec. Catalysis B: Enzymatic
2:233 (1997)], trypsin [Shiroya, T., Yasui, M., Fujimoto, K. & Kawaguchi, H.,
Colloid Surfaces B: Biointerfaces,
4:275 (1995)], &bgr;-D-glucosidase [Chen, G. & Hoffman, A. S.,
J. Biomater. Sci. Polym. Edn.,
5:371 (1994)], lactate dehydrogenase [Galaev, I. Yu. & Mattiasson, B,
Biotechnol. Bioeng.,
41:1101 (1993)], thermnolysin [Liu, F., Tao, G. & Zhuo, R.,
Polymer J.,
25:561 (1993)] and lipase [Takeuchi, S., Omodaka, I., Hasegawa, K., Maeda, Y. & Kitano, Y.,
Makromol. Chem.,
194:1991 (1993)]. However, current methods for producing reversibly soluble enzyme-polymer conjugates produce biocatalysts with enzyme activities, on a weight basis, that are usually significantly lower than those of free enzymes.
Significant loss of the enzyme during binding is one of the main shortcomings of current methods for producing enzyme-polymer conjugates. For example, the binding efficiency of a current method for conjugating chymotrypsin to a copolymer of N-iso-propylacrylamide (NIPAAM) and N-acrylosuccinimide is only 30-40% [Chen, J.-P. & Hsu, M.-S.,
J. Molec. Catalysis B: Enzymatic,
2: 233 (1997)]. When conjugating thermolysin to the same copolymer, the binding efficiency is about twice as low as with chymotrypsin [Liu, F., Tao, G. & Zhuo, R.,
Polymer J.,
25: 561 (1993)]. Only 43% of enzyme binds with the polymer in a reaction between thermolysin and a NIPAAM-based copolymer containing oxirane groups [Vorlop, K.-D., Steinke, K., Wullbrandt, D., Schlingmann, M., U.S. Pat. No. 5,310,786 (1994)]. Binding of chymotrypsin to a polymerized liposome gives much better results, i.e., between 36% and 89% of the enzyme was coupled [Suh, Y., Jin, X,-H., Dong, X.-Y., Yu, K. & Zhou, X. Z.,
Appl. Biochem. Biotechnol.,
56: 331 (1996)], but the procedure is quite cumbersome. When enzyme-polymer conjugates were prepared
Gololobov Mikhail Y.
Ilyashenko Victor M.
McAndrews Held & Malloy Ltd.
Polium Technologies, Inc.
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