Evolution of Protein Molecules

This review describes a multidimensional treatment of molecular recognition phenomena involving aromatic rings in chemical and biological systems. It summarizes new results reported since the appearance of an earlier review in 2003 in host-guest chemistry, biological affinity assays and biostructural analysis, data base mining in the Cambridge Structural Database (CSD) and the Protein Data Bank (PDB), and advanced computational studies. Topics addressed are arene-arene, perfluoroarene-arene, S⋅⋅⋅aromatic, cation-π, and anion-π interactions, as well as hydrogen bonding to π systems. The generated knowledge benefits, in particular, structure-based hit-to-lead development and lead optimization both in the pharmaceutical and in the crop protection industry. It equally facilitates the development of new advanced materials and supramolecular systems, and should inspire further utilization of interactions with aromatic rings to control the stereochemical outcome of synthetic transformations.

Aromatic rings in chemical and biological recognition: energetics and structures.

Cation-π interaction: its role and relevance in chemistry, biology, and material science.

Nothing is solid about proteins. Governing rules and established laws are constantly broken. As an example, the last decade and a half have witnessed the fall of one of the major paradigms in structural biology. Contrarily to the more than a century-old belief that the unique function of a protein is determined by its unique structure, which, in its turn, is defined by the unique amino acid sequence, many biologically active proteins lack stable tertiary and/or secondary structure either entirely or at their significant parts. Such intrinsically disordered proteins (IDPs) and hybrid proteins containing ordered domains and functional IDP regions (IDPRs) are highly abundant in nature, and many of them are associated with various human diseases. Such disordered proteins and regions are very different from ordered and well-structured proteins and domains at a variety of levels and possess well-recognizable biases in their amino acid compositions and amino acid sequences. A characteristic feature of these proteins is their exceptional structural heterogeneity, where different parts of a given polypeptide chain can be ordered (or disordered) to different degrees. As a result, a typical IDP/IDPR contains a multitude of potentially foldable, partially foldable, differently foldable or not foldable structural segments. This distribution of conformers is constantly changing in time, where a given segment of a protein molecule has different structures at different time points. The distribution is also constantly changing in response to changes in the environment. This mosaic structural organization is crucial for their functions and many IDPs are engaged in biological functions that rely on high conformational flexibility and that are not accessible to proteins with unique and fixed structures. As a result, the functional repertoire of IDPs complements that of ordered proteins, with IDPs/IDPRs being often involved in regulation, signaling and control. This Sprenger Briefs volume is dedicated to IDPs and IDPRs and an attempt is made to compress a massive amount of knowledge and into a digest that aims to be of use to those wishing a fast entry into this promising field of structural biology.

Intrinsically Disordered Proteins

This review paper provides an overview regarding the main aspects of microbial lipases production. The most important microbial lipase-producing strains for submerged and solid-state fermentations are reviewed as well as the main substrates, including the use of agroindustrial residues. Current process techniques (batch, repeated-batch, fed-batch, and continuous mode) are discussed and the main bioreactors configurations are also presented. Furthermore, the present review paper shows a general overview about the development of mathematical models applied to lipase production. Finally, some future perspectives on lipase production are discussed with special emphasis on lipase engineering and the use of mathematical models as a useful tool for process improvement and control.

A Review on Microbial Lipases Production

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70°C and pH 9, respectively. It was stable up to 65°C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na+, Ca2+, Mn2+, K+ and Mg2+ , but inhibited by Cu2+, Fe3+ and Zn2+. Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10–C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T m for T1 lipase was around 72.2°C, as revealed by denatured protein analysis of CD spectra.

/pdf/a-thermoalkaliphilic-lipase-of-geobacillus-sp-t1-4l17sadqha.pdf

A thermoalkaliphilic lipase of Geobacillus sp. T1.

Background
Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78°C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5–99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.

/pdf/geobacillus-zalihae-sp-nov-a-thermophilic-lipolytic-28uc6qvvox.pdf

Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia

Novel cation-p interaction revealed by crystal structure of thermoalkalophilic lipase Hiroyoshi Matsumura,* Takahiko Yamamoto, Thean Chor Leow, Tadashi Mori, Abu Bakar Salleh, Mahiran Basri, Tsuyoshi Inoue, Yasushi Kai, and Raja Noor Zaliha Raja Abd Rahman* 1Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan 2CREST (Sosho Project), JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan 3 Enzyme and Microbial Technology Research, University Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

https://onlinelibrary.wiley.com/doi/pdf/10.1002/prot.21799

Novel cation-pi interaction revealed by crystal structure of thermoalkalophilic lipase

A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg−1 which corresponds to 2927.15 Ug−1 of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65°C and pH 9, respectively. It was stable up to 65°C at pH 7 and active over a wide pH range (pH 6–11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.

/pdf/high-level-expression-of-thermostable-lipase-from-2pa37izoi3.pdf

High level expression of thermostable lipase from Geobacillus sp. strain T1.

Psychrophilic microorganisms are cold-adapted with distinct properties from other thermal classes thriving in cold conditions in large areas of the earth's cold environment. Maintenance of functional membranes, evolving cold-adapted enzymes and synthesizing a range of structural features are basic adaptive strategies of psychrophiles. Among the cold-evolved enzymes are the cold-active lipases, a group of microbial lipases with inherent stability-activity-flexibility property that have engaged the interest of researchers over the years. Current knowledge regarding these cold-evolved enzymes in psychrophilic bacteria proves a display of high catalytic efficiency with low thermal stability, which is a differentiating feature with that of their mesophilic and thermophilic counterparts. Improvement strategies of their adaptive structural features have significantly benefited the enzyme industry. Based on their homogeneity and purity, molecular characterizations of these enzymes have been successful and their properties make them unique biocatalysts for various industrial and biotechnological applications. Although, strong association of lipopolysaccharides from Antarctic microorganisms with lipid hydrolases pose a challenge in their purification, heterologous expression of the cold-adapted lipases with affinity tags simplifies purification with higher yield. The review discusses these cold-evolved lipases from bacteria and their peculiar properties, in addition to their potential biotechnological and industrial applications.

Thean Chor Leow

Papers

A thermoalkaliphilic lipase of Geobacillus sp. T1.

Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia

Novel cation-pi interaction revealed by crystal structure of thermoalkalophilic lipase

High level expression of thermostable lipase from Geobacillus sp. strain T1.

Adaptational properties and applications of cold-active lipases from psychrophilic bacteria.