Improving the Thermostability of Serine Protease PB92 from Bacillus alcalophilus via Site-Directed Mutagenesis Based on Semi-Rational Design.

Proteases have been widely employed in many industrial processes. In this work, we aimed to improve the thermostability of the serine protease PB92 from Bacillus alcalophilus to meet the high-temperature requirements of biotechnological treatments. Eight mutation sites (N18, S97-S101, E110, and R143) were identified, and 21 mutants were constructed from B-factor comparison and multiple sequence alignment and expressed via Bacillus subtilis. Among them, fifteen mutants exhibited increased half-life (t1/2) values at 65 °C (1.13-31.61 times greater than that of the wild type). Based on the composite score of enzyme activity and thermostability, six complex mutants were implemented. The t1/2 values of these six complex mutants were 2.12-10.05 times greater than that of the wild type at 65 °C. In addition, structural analysis revealed that the increased thermal stability of complex mutants may be related to the formation of additional hydrophobic interactions due to increased hydrophobicity and the decreased flexibility of the structure. In brief, the thermal stability of the complex mutants N18L/R143L/S97A, N18L/R143L/S99L, and N18L/R143L/G100A was increased 4-fold, which reveals application potential in industry.

Enhanced Extracellular Expression of a Ca2+- and Mg2+-Dependent Hyperthermostable Protease EA1 in Bacillus subtilis via Systematic Screening of Optimal Signal Peptides.

Proteases have been widely applied in various industries, including tanning, silk, feed, medicine, food, and environmental protection. Herein, the protease EA1 (GenBank accession no. U25630.1) was successfully expressed in Bacillus subtilis and demonstrated to function as a Ca2+- and Mg2+-dependent hyperthermostable neutral protease. At 80 °C, its half-life (t1/2) in the presence of 10 mM Mg2+ and Ca2+ was 50.4-fold longer than that in their absence (7.4 min), which can be explained by structural analysis. Compared with the currently available commercial proteases, protease EA1 has obvious advantages in heat resistance. The largest peptide library was used to enhance the extracellular expression of protease EA1 via constructing and screening 244 signal peptides (SPs). Eleven SPs with high yields of protease EA1 were identified from 5000 clones using a high-throughput assay. Specifically, the enzyme activity of protease produced by the strain (217.6 U/mL) containing the SP XynD was 5.2-fold higher than that of the strain with the initial SP. In brief, the protease is a potential candidate for future use in the high-temperature industry.

Discovery of the Key Mutation Site Influencing the Thermostability of Thermomyces lanuginosus Lipase by Rosetta Design Programs.

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications.

Effect of dietary Bacillus coagulans on the performance and intestinal microbiota of weaned piglets.

The performance of weaned piglets suffers from severe limitations resulting from diarrhoea. Therefore, this trial was performed to investigate the effects of Bacillus coagulans as an alternative to antibiotics on piglet growth performance and intestinal health. Ninety (initial BW = 7.70 ± 0.17 kg, weaning age of 26 days) healthy weaned piglets with similar BWs were selected and randomised into three treatment groups. Pigs in the negative control (NC) group were fed a basal diet, pigs in the positive control (PC) group were fed the basal diet plus antibiotics, and pigs in the test group (BC) were fed the basal diet plus Bacillus coagulans at 600 g/t; the trial lasted for 28 days. The results showed that the ratios of feed to gain (F:G) of both the BC and PC groups from 1 to 21 days were significantly lower (P < 0.05), and the average daily weight gain (ADG) of the BC group from 22 to 28 days was significantly higher (P < 0.05) than that of the NC group in terms of growth performance. The diarrhoea index was lowest in the PC group, followed by the BC group, and highest in the NC group. The BC group had a lower diarrhoea index at the later stage. We performed 16S rRNA sequencing to measure the intestinal bacteria and found that the BC group had a higher intestinal bacteria diversity than the NC and PC groups (P < 0.05). From days 1 to 21, the main differential species were Ruminococcaceae_UCG-014 and Faecalibacterium (P < 0.05); from days 22 to 28, the main differential species were Prevotella_9, unclassified_f__Lachnospiraceae, Anaerovibrio, and Ruminococcaceae_UCG-002 (P < 0.05). The correlation analysis between growth performance and species revealed specific gut microorganisms responsible for variation in F:G, ADG, and diarrhoea index, such as Prevotellaceae, Clostridium_sensu_stricto_1, Ruminococcaceae, Phascolarctobacterium, and Anaerovibrio. In conclusion, Bacillus coagulans changed the microbial composition in the faeces of weaned piglets, which had positive effects on growth performance and the diarrhoea index. Therefore, our study provided new insight into the future application of Bacillus coagulans as an additive.

Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2.

Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure's stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.

Transcriptomic Analysis of Pichia pastoris (Komagataella phaffii) GS115 During Heterologous Protein Production Using a High-Cell-Density Fed-Batch Cultivation Strategy.

Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast that is widely used in industry as a host system for heterologous protein expression. Heterologous gene expression is typically facilitated by strongly inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters. However, protein production is usually accomplished by a fed-batch induction process, which is known to negatively affect cell physiology, resulting in limited protein yields and quality. To assess how yields of exogenous proteins can be increased and to further understand the physiological response of P. pastoris to the carbon conversion of glycerol and methanol, as well as the continuous induction of methanol, we analyzed recombinant protein production in a 10,000-L fed-batch culture. Furthermore, we investigated gene expression during the yeast cell culture phase, glycerol feed phase, glycerol-methanol mixture feed (GM) phase, and at different time points following methanol induction using RNA-Seq. We report that the addition of the GM phase may help to alleviate the adverse effects of methanol addition (alone) on P. pastoris cells. Secondly, enhanced upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was observed in P. pastoris following methanol induction. The MAPK signaling pathway may be related to P. pastoris cell growth and may regulate the alcohol oxidase1 (AOX1) promoter via regulatory factors activated by methanol-mediated stimulation. Thirdly, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways were not significantly upregulated during the methanol induction period. These results imply that the presence of unfolded or misfolded phytase protein did not represent a serious problem in our study. Finally, the upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes but not to the production of phytase. This work describes the metabolic characteristics of P. pastoris during heterologous protein production under high-cell-density fed-batch cultivation. We believe that the results of this study will aid further in-depth studies of P. pastoris heterologous protein expression, regulation, and secretory mechanisms.

Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison and computational simulation.

BACKGROUND: Phytase supplied in feeds for monogastric animals is important for improving nutrient uptake and reducing phosphorous pollution. High-thermostability phytases are particularly desirable due to their ability to withstand transient high temperatures during feed pelleting procedures. A comparison of crystal structures of the widely used industrial Aspergillus niger PhyA phytase (AnP) with its close homolog, the thermostable Aspergillus fumigatus phytase (AfP), suggests 18 residues in three segments associated with thermostability. In this work, we aim to improve the thermostability of AnP through site-directed mutagenesis. We identified favorable mutations based on structural comparison of homologous phytases and molecular dynamics simulations. RESULTS: A recombinant phytase (AnP-M1) was created by substituting 18 residues in AnP with their AfP analogs. AnP-M1 exhibited greater thermostability than AnP at 70 °C. Molecular dynamics simulations suggested newly formed hydrogen bonding interactions with nine substituted residues give rise to the improved themostability. Thus, another recombinant phytase (AnP-M2) with just these nine point substitutions was created. AnP-M2 demonstrated superior thermostability among all AnPs at ≥70 °C: AnP-M2 maintained 56% of the maximal activity after incubation at 80 °C for 1 h; AnP-M2 retained 30-percentage points greater residual activity than that of AnP and AnP-M1 after 1 h incubation at 90 °C. CONCLUSIONS: The resulting AnP-M2 is an attractive candidate in industrial applications, and the nine substitutions in AnP-M2 are advantageous for phytase thermostability. This work demonstrates that a strategy combining structural comparison of homologous enzymes and computational simulation to focus on important interactions is an effective method for obtaining a thermostable enzyme.

Improving the thermostability of a fungal GH11 xylanase via site-directed mutagenesis guided by sequence and structural analysis.

BACKGROUND: Xylanases have been widely employed in many industrial processes, and thermophilic xylanases are in great demand for meeting the high-temperature requirements of biotechnological treatments. In this work, we aim to improve the thermostability of XynCDBFV, a glycoside hydrolase (GH) family 11 xylanase from the ruminal fungus Neocallimastix patriciarum, by site-directed mutagenesis. We report favorable mutations at the C-terminus from B-factor comparison and multiple sequence alignment. RESULTS: C-terminal residues 207-NGGA-210 in XynCDBFV were discovered to exhibit pronounced flexibility based on comparison of normalized B-factors. Multiple sequence alignment revealed that beneficial residues 207-SSGS-210 are highly conserved in GH11 xylanases. Thus, a recombinant xylanase, Xyn-MUT, was constructed by substituting three residues (N207S, G208S, A210S) at the C-terminus of XynCDBFV. Xyn-MUT exhibited higher thermostability than XynCDBFV at ≥70 °C. Xyn-MUT showed promising improvement in residual activity with a thermal retention of 14% compared to that of XynCDBFV after 1 h incubation at 80 °C; Xyn-MUT maintained around 50% of the maximal activity after incubation at 95 °C for 1 h. Kinetic measurements showed that the recombinant Xyn-MUT had greater kinetic efficiency than XynCDBFV (Km, 0.22 and 0.59 µM, respectively). Catalytic efficiency values (kcat/Km) of Xyn-MUT also increased (1.64-fold) compared to that of XynCDBFV. Molecular dynamics simulations were performed to explore the improved catalytic efficiency and thermostability: (1) the substrate-binding cleft of Xyn-MUT prefers to open to a larger extent to allow substrate access to the active site residues, and (2) hydrogen bond pairs S208-N205 and S210-A55 in Xyn-MUT contribute significantly to the improved thermostability. In addition, three xylanases with single point mutations were tested, and temperature assays verified that the substituted residues S208 and S210 give rise to the improved thermostability. CONCLUSIONS: This is the first report for GH11 recombinant with improved thermostability based on C-terminus replacement. The resulting Xyn-MUT will be an attractive candidate for industrial applications.

The 340-cavity in neuraminidase provides new opportunities for influenza drug development: A molecular dynamics simulation study.

Influenza neuraminidase (NA) is a pivotal target for viral infection control. However, the accumulating of mutations compromise the efficacy of NA inhibitors. Thus, it is critical to design new drugs targeted to different motifs of NA. Recently, a new motif called 340-cavity was discovered in NA subtypes close to the calcium binding site. The presence of calcium is known to influence NA activity and thermostability. Therefore, the 340-cavity is a putative ligand-binding site for affecting the normal function of NA. In this study, we performed molecular dynamics simulations of different NA subtypes to explore the mechanism of 340-loop formation. Ligand-binding site prediction and fragment library screening were also carried out to provide evidence for the 340-cavity as a druggable pocket. We found that residues G342 and P/R344 in the 340-loop determine the size of the 340-cavity, and the calcium ion plays an important role in maintaining the conformation of the 340-loop through contacts with G345 and Q347. In addition, the 340-cavity is predicted to be a ligand-binding site by metaPocket, and a sequence analysis method is proposed to predict the existence of the 340-cavity. Our study shows that the 340-cavity is not an occasional or atypical domain in NA subtypes, and it has potential to function as a new hotspot for influenza drug binding.

Molecular and Biochemical Characterization of a Novel Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti.

Xylanases sourced from different bacteria have significantly different enzymatic properties. Therefore, studying xylanases from different bacteria is important to their applications in different fields. A potential xylanase degradation gene in Massilia was recently discovered through genomic sequencing. However, its xylanase activity remains unexplored. This paper is the first to report a xylanase (XynRBM26) belonging to the glycosyl hydrolase family (GH10) from the genus Massilia. The gene encodes a 383-residue polypeptide (XynRBM26) with the highest identity of 62% with the endoxylanase from uncultured bacterium BLR13. The XynRBM26 expressed in Escherichia coli BL21 is a monomer with a molecular mass of 45.0 kDa. According to enzymatic characteristic analysis, pH 5.5 is the most appropriate for XynRBM26, which could maintain more than 90% activity between pH 5.0 and 8.0. Moreover, XynRBM26 is stable at 37°C and could maintain at least 96% activity after being placed at 37°C for 1 h. This paper is the first to report that GH10 xylanase in an animal gastrointestinal tract (GIT) has salt tolerance, which could maintain 86% activity in 5 M NaCl. Under the optimum conditions, Km, Vmax, and kcat of XynRBM26 to beechwood xylan are 9.49 mg/ml, 65.79 µmol/min/mg, and 47.34 /sec, respectively. Considering that XynRBM26 comes from an animal GIT, this xylanase has potential application in feedstuff. Moreover, XynRBM26 is applicable to high-salt food and seafood processing, as well as other high-salt environmental biotechnological fields, because of its high catalytic activity in high-concentration NaCl.