Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN′ and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.

Bacterial alkaline proteases: molecular approaches and industrial applications

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme ...

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Role of Biocatalysis in Sustainable Chemistry

The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.

Methods and compositions for cellular and metabolic engineering

Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment--high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60-->Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures--it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.

https://www.pnas.org/content/pnas/90/12/5618.full.pdf

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide

Enzyme activity is often dramatically reduced in polar organic solvents, even under conditions where the folded structures are stable. We have utilized random mutagenesis by polymerase chain reaction (PCR) techniques combined with screening for enhanced activity in the presence of dimethylformamide (DMF) to probe mechanisms by which improved enzymes for chemical synthesis in polar organic media might be obtained. Two amino acid substitutions which enhance subtilisin E activity in the presence of DMF, Q103R and D60N, were identified by screening on agar plates containing DMF and casein. The two substitutions are located near the substrate binding pocket or in the active site, and their effects on the catalytic efficiency kcat/KM for the hydrolysis of a peptide substrate are additive. The effects of D60N are apparent only in the presence of DMF, highlighting the importance of screening in the organic solvent. Protein engineering is an effective approach to enhancing enzyme activity in organic media: the triple mutant D60N + Q103R + N218S is 38 times more active than wild-type subtilisin E in 85% DMF. An evolutionary approach consisting of multiple steps of random mutagenesis and screening in continually higher concentrations of organic solvent should result in enzymes that are substantially more active in organic media.

Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN′, also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueoussolvent-compatible enzymes.

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media

In accordance with the present invention, there are provided novel, modified subtilisin enzyme(s) having improved catalytic activity and/or stability in organic media.

Subtilisin variants suitable for hydrolysis and synthesis in organic media

Subtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 --> Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface-charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 --> Asn + Asn218 --> Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.

Keqin Chen

Papers

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide

Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media.

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media

Subtilisin variants suitable for hydrolysis and synthesis in organic media

Stabilization of substilisin E in organic solvents by site-directed mutagenesis.