Molecular Dynamics (MD) simulations have been used to understand how protein structure, dynamics, and flexibility are affected by adaptation to high temperature for several years. We report here the results of the high temperature MD simulations of Bacillus stearothermophilus L1 (L1 lipase). We found that the N-terminal moiety of the enzyme showed a high flexibility and dynamics during high temperature simulations which preceded and followed by clear structural changes in two specific regions; the small domain and the main catalytic domain or core domain of the enzyme. These two domains interact with each other through a Zn(2+)-binding coordination with Asp-61 and Asp-238 from the core domain and His-81 and His-87 from the small domain. Interestingly, the His-81 and His-87 were among the highly fluctuated and mobile residues at high temperatures. The results appear to suggest that tight interactions of Zn(2+)-binding coordination with specified residues became weak at high temperature which suggests the contribution of this region to the thermostability of the enzyme.
A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.
An all-atom level MD simulation in explicit solvent at high temperature is a powerful technique to increase our knowledge about the structurally important regions modulating thermal stability in thermenzymes. In this respect, two large-sized thermoalkalophilic enzymes from Bacillus stearothermophilus L1 (L1 lipase) and Geobacillus zalihae strain T1 (T1 lipase) are well-established representatives. In this paper, comparative results from temperature-induced MD simulations of both model systems at 300 K, 400 K and 500 K are presented and discussed with respect to identification of highly flexible regions critical to thermostability. From our MD simulation results, specific regions along the L1 lipase and T1 lipase polypeptide chain including the small domain and the main catalytic domain or core domain of both enzymes show a marked increase in fluctuations and dynamics followed by clear structural changes. Overall, the N-terminal moiety of both enzymes and their small domains exhibit hyper-sensitivity to thermal stress. The results appear to propose that these regions are critical in determining of the overall thermal stability of both organisms.
Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL(-1) at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg(-1). The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0-8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t₁/₂) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification.
The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme's capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.
Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).
A novel α-amylase was isolated successfully from Glaciozyma antarctica PI12 using DNA walking and reverse transcription-polymerase chain reaction (RT-PCR) methods. The structure of this psychrophilic α-amylase (AmyPI12) from G. antarctica PI12 has yet to be studied in detail. A 3D model of AmyPI12 was built using a homology modelling approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9.9. Analysis of the AmyPI12 model revealed the presence of binding sites for a conserved calcium ion (CaI), non-conserved calcium ions (CaII and CaIII) and a sodium ion (Na). Compared with its template-the thermostable α-amylase from Bacillus stearothermophilus (BSTA)-the binding of CaII, CaIII and Na ions in AmyPI12 was observed to be looser, which suggests that the low stability of AmyPI12 allows the protein to work at different temperature scales. The AmyPI12 amino acid sequence and model were compared with thermophilic α-amylases from Bacillus species that provided the highest structural similarities with AmyPI12. These comparative studies will enable identification of possible determinants of cold adaptation.