Rational Design of the Soluble Variant of l-Pipecolic Acid Hydroxylase using the α-Helix Rule and the Hydropathy Contradiction Rule.

The production of recombinant proteins in Escherichia coli is an important application of biotechnology. 2-Oxoglutarate-dependent l-pipecolic acid hydroxylase derived from Xenorhabdus doucetiae (XdPH) is an excellent biocatalyst that catalyzes the hydroxylation of l-pipecolic acid to produce cis-5-hydroxy-l-pipecolic acid. However, the enzyme tends to form aggregates in the E. coli expression system. Our group established two rules, namely, the "α-helix rule" and the "hydropathy contradiction rule," to select residues to be altered for improving the heterologous recombinant production of proteins, by analyzing their primary structure. We rationally designed XdPH variants that are expressed in highly soluble and active forms in the E. coli expression system using these hotspot prediction methods, and the L142R variant showed a remarkably high soluble expression level compared to the wild-type XdPH. Further mutations were introduced into the L142R gene by site-directed mutagenesis. Moreover, the I28P/L142R and C76Y/L142R double variants displayed improved soluble expression levels compared to the single variants. These variants were also more thermostable than the wild-type XdPH. To analyze the effect of the alteration on one of the hotspots, L142 was replaced with various hydrophilic and positively charged residues. The remarkable increase in soluble protein expression caused by the alterations suggests that the decrease in the hydrophobicity of the protein surface and the enhancement of the interaction between nearby residues are important factors determining the solubility of the protein. Overall, this study demonstrated the effectiveness of our protocol in identifying aggregation hotspots for recombinant protein production and in basic biochemical research.

Effect of Escherichia coli on phospholipid monolayers: surface tensiometry and Brewster angle microscopy measurements.

The effect of Escherichia coli (E. coli) cells on two phospholipids [dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC)] monolayers at the surface of a 1.5 wt% NaCl salt solution has been investigated using surface tension measurement and Brewster angle microscopy. The results showed that a DPPC monolayer that has an elastic structure was changed in morphology by interaction with E. coli cells, whereas a DMPC monolayer that has an expandable structure did not change in morphology. In particular, the morphology changed significantly around the liquid-expanded (LE)-liquid-condensed (LC) phase transition point for the DPPC monolayer. It was found that the LE-LC phase transition range in a DPPC monolayer was sensitive to influence from the outside of the monolayer such as the action of E. coli cells. Such a monolayer has the potential for application as a membrane sensor for detecting a small amount of bacteria in a short time.

In vitro assembly and cellulolytic activity of a ß-glucosidase-integrated cellulosome complex.

The cellulosome is a supramolecular multi-enzyme complex formed by protein interactions between the cohesin modules of scaffoldin proteins and the dockerin module of various polysaccharide-degrading enzymes. In general, the cellulosome exhibits no detectable ß-glucosidase activity to catalyze the conversion of cellobiose to glucose. Because ß-glucosidase prevents product inhibition of cellobiohydrolase by cellobiose, addition of ß-glucosidase to the cellulosome greatly enhances the saccharification of crystalline cellulose and plant biomass. Here, we report the in vitro assembly and cellulolytic activity of a ß-glucosidase-coupled cellulosome complex comprising the three major cellulosomal cellulases and full-length scaffoldin protein of Clostridium (Ruminiclostridium) thermocellum, and Thermoanaerobacter brockii ß-glucosidase fused to the type-I dockerin module of C. thermocellum. We show that the cellulosome complex composed of nearly equal numbers of cellulase and ß-glucosidase molecules exhibits maximum activity toward crystalline cellulose, and saccharification activity decreases as the enzymatic ratio of ß-glucosidase increases. Moreover, ß-glucosidase-coupled and ß-glucosidase-supplemented cellulosome complexes similarly exhibit maximum activity toward crystalline cellulose (i.e. 1.7-fold higher than that of the ß-glucosidase-free cellulosome complex). These results suggest that the enzymatic ratio of cellulase and ß-glucosidase in the assembled complex is crucial for the efficient saccharification of crystalline cellulose by the ß-glucosidase-integrated cellulosome complex.

Comparative Biochemical Analysis of Cellulosomes Isolated from Clostridium clariflavum DSM 19732 and Clostridium thermocellum ATCC 27405 Grown on Plant Biomass.

The cellulosome is a supramolecular multienzyme complex formed via species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Here, we report a comparative analysis of cellulosomes prepared from the thermophilic anaerobic bacteria Clostridium (Ruminiclostridium) clariflavum DSM 19732 and Clostridium (Ruminiclostridium) thermocellum ATCC 27405 grown on delignified rice straw. The results indicate that the isolated C. clariflavum cellulosome exhibits lower activity for insoluble cellulosic substrates and higher activity for hemicellulosic substrates, especially for xylan, compared to the isolated C. thermocellum cellulosome. The C. clariflavum cellulosome was separated into large and small complexes by size exclusion chromatography, and the high xylanase activity of the intact complex is mainly attributed to the small complex. Furthermore, both C. clariflavum and C. thermocellum cellulosomes efficiently converted delignified rice straw into soluble sugars with different compositions, whereas a mixture of these cellulosomes exhibited essentially no synergy for the saccharification of delignified rice straw. This is the first study to report that isolated C. clariflavum cellulosomes exhibit greater xylanase activity than isolated C. thermocellum cellulosomes. We also report the effect of a combination of intact cellulosome complexes isolated from different species on the saccharification of plant biomass.

Benchmark Analysis of Native and Artificial NAD+-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data.

The expansion of protein sequence databases has enabled us to design artificial proteins by sequence-based design methods, such as full-consensus design (FCD) and ancestral-sequence reconstruction (ASR). Artificial proteins with enhanced activity levels compared with native ones can potentially be generated by such methods, but successful design is rare because preparing a sequence library by curating the database and selecting a method is difficult. Utilizing a curated library prepared by reducing conservation energies, we successfully designed two artificial l-threonine 3-dehydrogenases (SDR-TDH) with higher activity levels than native SDR-TDH, FcTDH-N1, and AncTDH, using FCD and ASR, respectively. The artificial SDR-TDHs had excellent thermal stability and NAD+ recognition compared to native SDR-TDH from Cupriavidus necator (CnTDH); the melting temperatures of FcTDH-N1 and AncTDH were about 10 and 5 °C higher than that of CnTDH, respectively, and the dissociation constants toward NAD+ of FcTDH-N1 and AncTDH were 2- and 7-fold lower than that of CnTDH, respectively. Enzymatic efficiency of the artificial SDR-TDHs were comparable to that of CnTDH. Crystal structures of FcTDH-N1 and AncTDH were determined at 2.8 and 2.1 Å resolution, respectively. Structural and MD simulation analysis of the SDR-TDHs indicated that only the flexibility at specific regions was changed, suggesting that multiple mutations introduced in the artificial SDR-TDHs altered their flexibility and thereby affected their enzymatic properties. Benchmark analysis of the SDR-TDHs indicated that both FCD and ASR can generate highly functional proteins if a curated library is prepared appropriately.

Enzymatic diversity of the Clostridium thermocellum cellulosome is crucial for the degradation of crystalline cellulose and plant biomass.

The cellulosome is a supramolecular multienzyme complex comprised of a wide variety of polysaccharide-degrading enzymes and scaffold proteins. The cellulosomal enzymes that bind to the scaffold proteins synergistically degrade crystalline cellulose. Here, we report in vitro reconstitution of the Clostridium thermocellum cellulosome from 40 cellulosomal components and the full-length scaffoldin protein that binds to nine enzyme molecules. These components were each synthesized using a wheat germ cell-free protein synthesis system and purified. Cellulosome complexes were reconstituted from 3, 12, 30, and 40 components based on their contents in the native cellulosome. The activity of the enzyme-saturated complex indicated that greater enzymatic variety generated more synergy for the degradation of crystalline cellulose and delignified rice straw. Surprisingly, a less complete enzyme complex displaying fewer than nine enzyme molecules was more efficient for the degradation of delignified rice straw than the enzyme-saturated complex, despite the fact that the enzyme-saturated complex exhibited maximum synergy for the degradation of crystalline cellulose. These results suggest that greater enzymatic diversity of the cellulosome is crucial for the degradation of crystalline cellulose and plant biomass, and that efficient degradation of different substrates by the cellulosome requires not only a different enzymatic composition, but also different cellulosome structures.

Improved reaction pattern of an endoglycanase from Paenibacillus cookii for chitosan oligosaccharide production.

The reaction pattern of an endoglycanase from Paenibacillus cookii SS-24 (Pgl8A) was improved to facilitate chitosan oligosaccharide production. Based on the sequence alignment with chitosanase of a known structure, we performed site-directed mutagenesis of possible substrate-binding residues in Pgl8A. The mutants were expressed in Escherichia coli cells, and their cellulase and chitosanase activities were then characterised. Our results indicated that three amino acid residues (W139, W208 and E285) were important for the substrate specificity of Pgl8A. D156 and Y390 were also essential for the cellulase and chitosanase activities of Pgl8A. The products of chitosan degradation by W139A, W208A and E285Q mutants were different from those by the wild type. A chitosan pentamer accumulated following chitosan degradation by W139A, W208A and E285Q mutants. Thus, the mutants obtained in this study are potentially useful for the production of biofunctional chitosan oligosaccharides.

Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme.

An endoglycanase gene of Paenibacillus cookii SS-24 was cloned and sequenced. This Pgl8A gene had an open reading frame of 1,230 bp that encoded a putative signal sequence (31 amino acids) and mature enzyme (378 amino acids: 41,835 Da). The enzyme was most homologous to a ß-1,3-1,4-glucanase of Bacillus circulans WL-12 with 84% identity. The recombinant enzyme hydrolyzed carboxymethyl cellulose, swollen celluloses, chitosan and lichenan but not Avicel, chitin powder or xylan. With chitosan as the substrate, the optimum temperature and hydrolysis products of the recombinant enzyme varied at pH 4.0 and 8.0. This is the first report that characterizes chitosanase activity under different pH conditions.