L-Malate

The enzyme fumarase catalyzes the stereospecific addition of water to fumarate to form L-malate. A standard solution of fumarase, with a concentration of 0.150 tM, gave a rate of reaction of 2.00 tM mim under conditions in which the concentration of the substrate was significantly greater than K. The rate of reaction for a sample, under identical conditions, was found to be 1.15 tM mimh What is the concentration of fumarase in the sample ... 

Fumarate is hydrated in a stereospecific reaction by fumarase to give L-malate (Figure 20.17). The reaction involves fraw5-addition of the elements of water across the double bond. Recall that aconitase carries out a similar reaction. 

CoA to form L-malate. The net effect is to conserve carbon units, using two acetyl-CoA molecules per cycle to generate oxaloacetate. Some of this is converted to PEP and then to glucose by pathways discussed in Chapter 23. 

The strongest known producer of j8-poly(L-malic acid) has been identified as Aureobasidium sp. providing 61 g of polymer from 1 liter of culture medium [5,6]. 8-Poly(L-malate, Ca -salt) of the culture broth was first separated from accompanying bulk pullulan by methanol precipitation. The water-redisolved precipitate was converted to the polymer acid by passage over Amberlite IR-120B (H -form). Thus, the best to-day producers of... 

The purification of j8-poly(L-malic acid) from A o-basiae has been reported involving methanol precipitation of the polymer in the form of the Ca salt [5]. This is possible because a high concentration of CaCOs is present in the growth medium. Unfortunately, the polymer acid is not soluble in aceton thus missing an additional purification step. In our hands, purification of jS-poly(L-malate) from several Aureobasidiae strains was unsatisfactory because of low yields and resisting impurities. 

A. Correlation of Growth and Synthesis of /3-Poly(L-Malate) in P. polycephalum... 

The production of the polymer depends on several factors such as the composition of the growth medium, the time of harvest, and the particular stage of the life-cycle of organism under consideration. Eor P. polycephalum only plasmodia are the producers of j8-poly(L-malate) neither amoebae nor spherules (specialized cell forms that can survive unfavorable environmental conditions)... 

Remove traces of ethanol and dissolve precipitate in a minimum (10 ml) of distilled water. Molecular sieving on a Sephadex G25 fine column (1300 ml, 0 5 cm). Assay fraction for /3-poly(L-malate) and chloride ions. Use only salt-free fractions. 

Obtain pure /3-poly(L-malate) potassium salt by lyophylization. Store in the freezer. 

The biochemistry of jS-poly(L-malate) synthesis has been investigated for P. polycephalum but is far from... 

Figure 1 )S-Poly(L-malate) released into the culture medium during plasmodial growth of P. polycephalum. (a) Effect of the nutrient (o-glucose) on the growth of plasmodia (— —), and the production of )S-poly(L-malate) (—O—). Growth conditions are otherwise as indicated in Table 1 footnote 2. (b) Content of )S-poly(L-malate) (— —) and l-malate (—O—) in the culture medium during growth of strain M3CVIII under conditions indicated in Table 1 footnote 2. Inoculation on day 0. Growth termination on day 4.

In the beginning of the investigation it was believed that enzymatic synthesis of j8-poly(L-malate) follows a route, which is similar to the synthesis of poly(j8-hydrox-ybutyrate) (for a review see [15]). However, enzymati-... 

Infrared spectra and the degree of specific rotation show typical features of the malic acid polyester (Table 3). Ultraviolet absorbance spectra of )3-poly(L-malate) from both P. polycephalum and Aureobasidium sp, A-91 are similar and are reminiscent of malate itself [4,5]. For a solution of 1.0 mg/ml polymer, absorbance increases from 0.40 units at 230 nm to 10 units at 190 nm wavelength. After saponification and pH-neutralization, the absorbance increased from 8.7 units at 230 nm to 100 units at 190 nm. 

Alkaline (and also acidic) ester hydrolysis of /3-poly(L-malate) is accompanied by side reactions leading to the formation of fumarate, maleate and/or racemiza-tion, especially at elevated temperatures. The above assays thus underestimate the polymer contents due to the formation of small amounts of 2-4% fumarate (unpublished results). This fraction of fumarate increases for the hydrolysis of more concentrated polymer solutions. 

Molecules of interest that contain free amino groups can be coupled in aqueous solution to /S-poIy(L-malate) as amides using carbodiimides such as the water-soluble l-ethyl-3(3-dimethyIaminopropyl)carbodiimide hydrochloride (EDC) [2,12,20,21]. By this method, the molecules are attached randomly. A selective amide bond formation at the carboxylate terminus can be achieved... 

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