Application of Outtrup

• Decision Date: 01 April 1976
• Docket Number: Patent Appeal No. 76-507.
• Citation: 531 F.2d 1055
• Parties: Application of Helle OUTTRUP et al.
• Court: U.S. Court of Customs and Patent Appeals (CCPA)

531 F.2d 1055

Application of Helle OUTTRUP et al.

Patent Appeal No. 76-507.

United States Court of Customs and Patent Appeals.

April 1, 1976.

Joseph F. Nakamura, Washington, D.C., for the Commissioner of Patents; Jack E. Armore, Washington, D.C., of counsel.

Morris Fidelman, Fidelman, Wolffe & Leitner, Washington, D.C., atty. of record, for appellants.

Before MARKEY, Chief Judge, and RICH, BALDWIN, LANE and MILLER, Associate Judges.

RICH, Judge.

This appeal is from the decision of the Patent and Trademark Office (PTO) Board of Appeals affirming the final rejection under 35 U.S.C. § 103 of claims 1-3, 11, and 13, the remaining claims in appellants' application serial No. 40,725, filed May 26, 1970, entitled "Preparation of Microbial Enzymes." We affirm.

The Invention

Appellants claim to have discovered that certain strains of the bacterium Bacillus licheniformis (BL), when suitably cultured, produce an alpha-amylase¹ which has excellent heat stability, is suitable for use in cleaning agents at high pH values, e. g., 9, and is stable in the presence of high concentrations of Ca‡-sequestering agents. Appellants assert that their alpha-amylase is unexpectedly superior to the prior art alpha-amylase produced by Bacillus subtilis. The claims on appeal read:

1. A process for the production of an enzyme product containing an alpha-amylase having a good activity and stability at high pH values, at low concentrations of Ca‡ and at high temperature, which comprises cultivating Bacillus licheniformis in a suitable nutrient medium for a period in excess of about 1 day until a high level of alpha-amylase activity has been induced in the fermentation broth, whereafter a high alpha-amylase content enzyme product is isolated from the fermentation broth.

2. A process as claimed in claim 1, wherein a strain of Bacillus licheniformis NCIB 8061 is used.

3. A process as claimed in claim 1, wherein a strain of Bacillus licheniformis selected from the group consisting of NCIB 8059, ATCC 6634, ATCC 6598, ATCC 11945, ATCC 8480 and ATCC 9945a is used.

11. The alpha-amylase obtained by cultivating a culture of Bacillus licheniformis for in excess of about one day and thereafter isolated from the fermentation broth.

13. A process for hydrolyzing starch which comprises treating starch in solution with the alpha-amylase obtained by cultivating a culture of Bacillus lichenformis sic for in excess of about one day and thereafter isolated from the fermentation broth.

                The Prior Art
                Keay et al. (Keay)          3,592,737      July 13, 1971
                                       (application filed Aug. 14, 1968)
                Roald et al. (Roald)        3,451,935      June 24, 1969
                Bernlohr, Postlogarithmic Phase Metabolism of Sporulating
                          Microorganisms, 239 J.Biol.Chem. 538
                          (1964)
                

Keay discloses a method for separating amylase and protease and recovering each fraction from an enzyme solution produced by the culturing, preferably, of a mutated strain of B. subtilis. Keay employs organic solvents to precipitate the amylase present, stating:

After precipitating proteinaceous impurities using the soluble calcium salt, which is employed at a moderately high level, any amylase present may be precipitated either after removing the precipitate or without prior removal of the precipitate, as desired, by addition of a sufficient amount of an organic solvent which is water-miscible and in which the amylase is itself not soluble.

Roald is essentially cumulative to Keay and need not be separately discussed.

The Bernlohr article is primarily concerned with the production of protease by culturing the A-5 strain of BL. The portion of the article particularly relied upon below states:

Routinely, culture supernatant solutions containing at least 50 units per ml of enzyme were concentrated about 100-fold by lyophilization before preparative work was performed. Attempts to purify the protease activity by conventional methods yielded poor results, apparently because most of the extracellular protein had similar physical characteristics, even after dialysis at 0°. Several other enzyme activities, including alpha anylase, were recovered in the same fraction as the protease after ammonium sulfate or cold ethanol fractionation.1

The protease was purified several fold, however, simply by dialyzing the concentrated enzyme solution against distilled water at 37°. Thirty milliliters of solution were dialyzed against 1 liter of water, and the water was changed each hour. The protein content and the activity of the enzyme were monitored, and it was found that after 7 hours, the protease had hydrolyzed the other proteins and then had begun autolysis. At this point, the enzyme solution was lyophilized and stored at -20°. Fig. 2 diagrams the time course of this dialysis procedure. The total activity of the protease remains fairly constant until the 6th hour of dialysis and then decreases. During this time, the protein concentration drops precipitously, resulting in an increase in the specific activity of the protease. At the 7th hour of dialysis, the enzyme is freeze-dried and stored. Emphasis ours.

Footnote 1 reads: "Unpublished experiments." Bernlohr's Fig. 1 is a graph showing the increase in protease activity in the supernatant solution over the 24-hour culturing period he employs. The graph shows that protease activity increased approximately 55 units/ml of supernatant solution between hour 11 and hour 12, but only about 40 units/ml over the 12-hour period up to hour 24, or about 3½ units/hr.

The Rejection

The examiner's final rejection states:

Claims 1 to 3 and 11 to 13 are rejected under 35 USC 103 as unpatentable over Bernlohr (1964) taken with Keay et al and Roald et al, all of record. Bernlohr teaches that Bacillus licheniformis (the same strain employed in applicant's sic Example 6) produces alpha-amylase as well as protease which were recovered in the same fraction. (Page 539). Keay et al and Roald et al each teach that amylases (carbohydrases and diastase) are often produced in addition to proteases when the latter is produced by culturing microorganisms including Bacilli to produce enzymes. Keay et al further teaches the separation of amylase from protease when it is the desired objective. Furthermore it is not apparent that the claims require the separation of protease from alpha amylase. Applicants' examples reveal the presence of substantial proteolytic activity with amylase-containing culture fluid. Applicants' arguments have been carefully considered. However they are not pursuasive sic that the claimed subject matter is unobvious over the prior art. Figure 1 of the 1964 Bernlohr reference indicates an increase in enzyme (protease) activity as culture time increases. There is no suggestion in this reference that enzyme activity after 24 hours will decrease. As a matter of fact it would be obvious from Figure 1 that enzyme activity would continue to increase after 24 hours cultivation. The fact that Bernlohr had difficulty separating the enzymes would not disuade sic one skilled in the art from isolationg sic alpha amylase from the fermentation broth, particularly in view of Keay et al (subsequent to Bernlohr) who recognize the problem and difficulties related to the fractionation of amylase and protease and provide a solution to this problem.

The board adopted this reasoning, commenting that appellants appeared to have admitted in their specification that the method of claim 13 was old in the art. The board also held that two declarations by appellant Aunstrup, which the examiner entered after the final rejection, did not overcome the rejection. These declarations will be discussed in detail infra.

OPINION

Appellants attack the rejection on the ground that Bernlohr does not disclose the production of alpha-amylase by culturing BL in a manner sufficient to put alpha-amylase from this source "in the hands of the public." For that reason, argue appellants, persons of ordinary skill in the art would not have been motivated by the Bernlohr disclosure to use BL as a source of alpha-amylase that may be fractionated by the Keay process.² Appellants refer to both the Bernlohr disclosure itself and to the Aunstrup declarations to support their position.

Appellants' legal theory is based on In re Coker, 463 F.2d 1344, 59 CCPA 1185 (1972); In re Sheppard, 339 F.2d 238, 52 CCPA 859 (1964); and In re Brown, 329 F.2d 1006, 51 CCPA 1254 (1964). Coker and Sheppard stand for the principle that a reference does not "describe" an invention...