Improvement of enzyme activity, stability and selectivity via immobilization techniques

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

https://science.sciencemag.org/content/sci/253/5022/872.full.pdf

Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein

Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.

Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic

True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.

A serine protease triad forms the catalytic centre of a triacylglycerol lipase.

Molecular and cellular biology of cholinesterases

ARhizomucor miehie cDNA library constructed inEscherichia coli was screened with synthetic oligonucleotides designed from knowledge of a partial amino acid sequence of the secreted triglyceride lipase (triacyl-glycerol acylhydrolase EC 3.1.1.3) from this fungus. Lipase-specific recombinants were isolated and their insert sequenced. Unlike characterized bacterial and mammalian triglyceride lipases, the fungal enzyme is synthesized as a precursor, including a 70 amino acid residue propeptide between the 24 amino acid residues of the signal peptide and the 269 residues of the mature enzyme. The precursor processing mechanism, which involves cleavage between a methionine and a serine residue, is unknown. By sequence comparison with other lipases, a serine residue involved in substrate binding was identified in the fungal lipase. The sequence around this residue is well-conserved among characterized lipases. Conservation of an intron in an isolated cDNA recombinant and immunoprecipitation of in vitro synthesizedR. miehei translation products indicates that the expression of the lipase gene might involve inefficient mRNA splicing.

Rhizomucor miehei triglyceride lipase is synthesized as a precursor.

An extracellular lipase, a glycoprotein, produced by fermentation with a selected strain ofMucor miehei has been partially purified in two forms, A and B. The two forms have a high degree of antigenic identity and have similar pH-activity profiles with tributyroylglycerol as a substrate with optima at pH 7. They differ as follows: A, in contrast to B, requires activation at alkaline pH before analysis; A binds with concanavalin-A more completely than B; the net charges are slightly different at pH 8; and the isoelectric points are different. Our results indicate that the B lipase is formed by partial deglycosylation of the A lipase and that this influences the activity toward emulsions.In addition, the two enzymes have been immobilized by adsorption. These preparations and the soluble forms were highly specific for primary esters of triacylglycerols (TG); they usually hydrolyzed TG of 12∶0, 14∶0, 16∶0, and 18∶1 more rapidly than those of 4∶0, 6∶0, and 8∶0 and 10∶0 in mixtures of monoacid TG (4∶0 to 18∶1); and they were not stereospecific for TG. Immobilization altered the specificity of the preparations somewhat, in that slightly more 14∶0 and 16∶0 were released.

/pdf/partial-purification-and-characterization-of-free-and-59wzuqcc7w.pdf

Partial purification and characterization of free and immobilized lipases fromMucor miehei

The cDNA encoding the precursor of theRhizomucor miehei triglyceride lipase was inserted in anAspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of theAspergillus oryzae α-amylase gene promoter and theAspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced intoAspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted fromA. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the nativeRhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted fromAspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

Rhizomucor miehei triglyceride lipase is processed and secreted from transformedAspergillus oryzae

An extracellular lipase produced by fermentation with a selected strain ofMucor miehei has been purified partially in two forms: A and B. The forms have a high degree of antigenic identity and have similar pH-activity profiles with tributyroylglycerol as the substrate with optima at pH 7. The differences are A, in contrast to B, requires activation at alkaline pH before analysis; A binds with concanavalin A more completely than B, the net charges are slightly different at pH 8; the isoelectric points are different. Our results indicate that the B lipase is formed by partial deglycosylation of the A lipase and that this influences the activity toward emulsions. The two enzymes have been immobilized by adsorption. These preparations and the soluble forms were highly specific for primary ester of triacylglycerols (TG), usually hydrolyzed TG of 12:0, 14:0, 16:0 and 18:1 more rapidly than those of 4:0, 6:0, 8:0 and 10:0 in mixtures of monoacid TG (4:0 to 18:1), and were not stereospecific for TG. Immobilization altered the specificity of the preparations somewhat in that slightly more 14:0 and 16:0 were released.

Birgitte Huge-Jensen

Papers

A serine protease triad forms the catalytic centre of a triacylglycerol lipase.

Rhizomucor miehei triglyceride lipase is synthesized as a precursor.

Partial purification and characterization of free and immobilized lipases fromMucor miehei

Rhizomucor miehei triglyceride lipase is processed and secreted from transformedAspergillus oryzae

Studies on Free and Immobilized Lipases from Mucor miehei