Highlights in Biocatalysis – Historical Landmarks and Current Trends

Biocatalysis has ancient roots, yet it is developing into a key tool for synthesis in a wide range of applications. Important events in the history of enzyme technology from the 19^th century onwards are highlighted. Considering the most relevant progress steps, the production of penicillanic acid and the impact of genetic engineering are traced in more detail. Applied biocatalysis has been defined as the application of a biocatalyst to achieve a desired conversion selectively, under controlled, mild conditions in a bioreactor. Biocatalysts are currently used to produce a wide range of products in the fields of food manufacture (such as bread, cheese, beer), fine chemicals (e.g., amino acids, vitamins), and pharmaceuticals (e.g., derivatives of antibiotics). They not only provide access to innovative products and processes, but also meet criteria of sustainability. In organic synthesis, recombinant technologies and biocatalysts have greatly widened the scope of application. Examples of current applications and processes are given. Recent developments and trends are presented as a survey, covering new methods for accessing biodiversity with new enzymes, directed evolution for improving enzymes, designed cells, and integrated downstream processing.     

High hydrostatic pressure and the dissection of fertilization responses : I. The relationship between cortical granule exocytosis and proton efflux during fertilization of the sea urchin egg

High hydrostatic pressure applied between sperm attachment and the onset of cortical granule exocytosis will inhibit this exocytotic event in sea urchin eggs. Such pressure-treated zygotes, nevertheless, are activated and capable of development. Thus, this technique can be used as a tool to study the relationship between cortical granule breakdown and other fertilization-related responses. We have studied whether the exocytosis of cortical granules is necessary for proton efflux (acid release) to occur. Our results indicate that although Ca²⁺ is released while the eggs are under pressure (a prerequisite for the following events to take place), cortical granule exocytosis and acid release are pressure-sensitive and completely inhibited at pressures above 400 atm (6000 psi) and 275 atm (4000 psi), respectively. However, upon decompression, acid release is initiated which amounts to 65–70% of that seen in the unpressurized controls, suggesting that the efflux mechanism does not require cortical granule exocytosis and must result from some modification of the original plasma membrane of the egg. The remaining 30–35% of the acid release is related to cortical granule exocytosis, since it can be obtained upon induction of the cortical granule fusion 30 min later under atmospheric pressure. The initiation of acid release after decompression indicates that the efflux mechanism is not transiently turned on at fertilization, but undergoing long-term modification; the recovery of the ability to induce cortical granule fusion after fertilization under pressure suggests a refilling of cytoplasmic Ca²⁺ stores within this time course.     

Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties.

Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall.     

Glycolytic activity of rat aorta after exercise

Activities of glycolytic enzymes in the aorta were investigated in female Wistar rats. There were two groups of rats; one served as the control (sedentary rats), while the other group was forced to run on a treadmill for 10 weeks. In the control animals, the activities of hexokinase, phosphofructokinase and aldolase were relatively lower than those of the other glycolytic enzymes (phosphoglucose isomerase, lactate dehydrogenase and pyruvate kinase). After exercise, the activity of phosphofructokinase increased by 15%, whereas the other enzymatic activities were much the same as in the controls. Within the limits of the experiments, the increased percentage of phosphofructokinase was statistically significant (p less than 0.05). Since phosphofructokinase is a putative rate limiting enzyme, this enzymatic activation may indicate that glycolytic activity in the rat aorta is enhanced during and after running exercise.     

Protein kinase activity of FSV (Fujinami sarcoma virus) P130gag-fps shows a strict specificity for tyrosine residues

A number of oncogenic viruses encode transforming proteins with protein kinase activities apparently specific for tyrosine residues. Recent evidence has raised questions as to the substrate specificity of these kinases in general and the physiological relevance of tyrosine phosphorylation in particular. The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) is strongly phosphorylated at 2 tyrosine residues in FSV-transformed cells of which 1 (Tyr-1073) is also the major site of P130gag-fps intermolecular autophosphorylation in vitro. We have investigated the specificity of the protein kinase activity intrinsic to FSV P130gag-fps by using site-directed mutagenesis to change the codon for Tyr-1073 to those for the other commonly phosphorylated hydroxyamino acids, serine and threonine. This approach has some advantages over the use of synthetic peptides to define protein kinase recognition sites in that the protein containing the altered target site can be expressed in intact cells. In addition it allows higher order as well as primary structure of the enzyme recognition site to be considered. Neither serine nor threonine were phosphorylated when substituted for tyrosine at position 1073 of P130gag-fps indicating a stringent specificity for tyrosine as a substrate of the P130gag-fps protein kinase autophosphorylating activity. Consistent with the suggestion that tyrosine phosphorylation is of functional significance we find that these and other FSV Tyr-1073 mutants have depressed enzymatic and oncogenic capacities.