Malic acid, a petroleum-derived C4-dicarboxylic acid that is used in the food and beverage industries, is also produced by a number of microorganisms that follow a variety of metabolic routes. Several members of the genus Aspergillus utilize a two-step cytosolic pathway from pyruvate to malate known as the reductive tricarboxylic acid (rTCA) pathway. This simple and efficient pathway has a maximum theoretical yield of 2 mol malate/mol glucose when the starting pyruvate originates from glycolysis. Production of malic acid by Aspergillus oryzae NRRL 3488 was first improved by overexpression of a native C4-dicarboxylate transporter, leading to a greater than twofold increase in the rate of malate production. Overexpression of the native cytosolic alleles of pyruvate carboxylase and malate dehydrogenase, comprising the rTCA pathway, in conjunction with the transporter resulted in an additional 27 % increase in malate production rate. A strain overexpressing all three genes achieved a malate titer of 154 g/L in 164 h, corresponding to a production rate of 0.94 g/L/h, with an associated yield on glucose of 1.38 mol/mol (69 % of the theoretical maximum). This rate of malate production is the highest reported for any microbial system.
The physiological role of the fibrinolytic system in maintaining the fluidity of the blood is now generally accepted [2]. The persistence of intravascular clots may be associated in part with some deficiency of the normal fibrinolytic mechanism. I n view of the frequency of thrombotic episodes the need for an effective, rational fibrinolytic therapy is very great, particularly in dangerous situations where clots form or lodge in the pulmonary, cerebral or coronary vascular systems. There are three fundamental approaches t o thrombolysis. The first is based on the induction of a fibrinolytic state b y adding amounts of fibrinolysin more than sufficient t o overcome the antifibrinolysin. The second approach is based on the ability of a n "activator" t o induce a fibrinolytic state. The third approach is to use a combination of a n activator and the fibrinolysin. A number of materials are being studied as activators to induce a fibrinolytic state: such as, streptokinase [7], urokinase [14], pyrogens [8], and chemicals [27].Our report deals with a study involving the preparation for clinical evaluation of human fibrinolysin with and without the activator, urokinase. The source of our profibrinolysin was the Fraction I11 paste usually discarded in the preparation of gamma globulin from outdated blood [S, 131. The source of the activator, urokinase, was stable a t neutral pH a t 25" C. These solutions are readily prepared in sterile condition for clinical use. All protein components have been subjected to a heat treatment period of 10 hours a t 60" C. during their purification. MaterialsBlood protein source. Human plasma Fraction I11 paste was kindly provided by the Blood Program of the American National Red Cross. This fraction was prepared b y Method 6 of Cohn, et al. [5] and Method 9 of Oncley, et al. [13] by E. R. Squibb & Sons, New Brunswick, New Jersey. Glycerol. Shell Chemical Corporation, 99.5 % U.S.P. synthetic glycerol. Phosphate-saline bufler. p H 7.4, 0.1 M sodium phosphate -0.9% NaCl buffer.Streptokinase. Lederle Varidase(R) was dissolved in phosphatesaline buffer a t 3,000 units per milliliter and stored a t 0-2" C.Casein solution. Devitaminized and high nitrogen caseins were kindly supplied by the Sheffield Company+. Stock solutions of 6% (w/v) casein were prepared by dissolving the material in 0.1 M phosphate-saline buffer and adjusting the p H to 7.4 with 1 N NaOH. The solution was heated in a boiling water bath for 15-30 minutes t o destroy any proteolytic activity present. The final solution was filtered, adjusted to p H 7.40 and stored in 50 ml. volumes in the frozen state (-30" C.).
A procedure is described for the separation and purification of 7-globulin from human placental extracts and postpartum blood. Frozen placentas together with the postpartum blood are thawed, chopped and extracted with isotonic saline. The initial separation of a fraction containing 7-globulin is accomplished by the addition of ethanol in the cold. A large part of the -globulins are separated from the 7-globulin fraction by the precipitation of the former at pH 4.8, and ethanol 8%. This is followed by the precipitation of a 7-globulin-rich (70%) fraction at pH 7.0-7.2 and ethanol 25%. A fraction rich (91%) in a-and /3-globulins is separated at pH 5.1 and ethanol 17%, leaving a supernatant fluid from which the 7-globulin is again precipitated with a final purity of 96%.
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