Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.

Production, purification and characterization of a double-tagged TEV protease.

Many recombinant proteins are products of great value in biomedical and industrial fields. The use of solubility and affinity tags are commonly used to increase yields and facilitate the purification process. However, it is of paramount importance in several applications to remove the fusion tag from the final product. In this regard, the Tobacco Etch Virus protease (TEV) is one of the most widely used for tag removal. The presence in the TEV of the same tag to be removed facilitates the separation of TEV and the tag from the cleaved recombinant protein in a single purification step. We generated a double-tagged (StrepTagII and HisTag) TEV variant with reported mutations that improve the activity, the expression yield in E.coli, and that decrease the auto-proteolysis. This TEV can be easily purified by two consecutive affinity chromatography steps with high yields and purity. The cleavage reaction can be done to almost completeness in as fast as 15 min at room temperature and the removal of the protease and tags is performed in a single purification step, independent of the previous presence of a StrepTagII or a HisTag on the target.

Exoproduction and Biochemical Characterization of a Novel Serine Protease from Ornithinibacillus caprae L9T with Hide-Dehairing Activity.

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9T) production from Ornithinibacillus caprae L9T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80°C) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70°C and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag+, Ca2+ and Sr2+, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9T protease in industrial applications, especially in leather processing.

Transcranial direct-current stimulation protects against cerebral ischemia-reperfusion injury through regulating Cezanne-dependent signaling.

Transcranial direct-current stimulation (tDCS) is proved safe and shows therapeutic effect in cerebral ischemic stroke in clinical trials. But the underlying molecular mechanisms remain unclear. Here we show that tDCS treatment reduces the infarct volume after rat cerebral ischemia-reperfusion (I/R) injury and results in functional improvement of stroke animals. At the cellular and molecular level, tDCS suppresses I/R-induced upregulation of Cezanne in the ischemic neurons. Cezanne inhibition confers neuroprotection after rat I/R and oxygen glucose deprivation (OGD) in the cortical neuronal cultures. Inhibiting Cezanne increases the level of SIRT6 that is downregulated in the ischemic neurons. Suppressing SIRT6 blocks Cezanne inhibition-induced neuroprotective effect and overexpressing SIRT6 attenuates OGD-induced neuronal death. We further show that downregulating Cezanne reduces DNA double-strand break (DSB) through upregulation of SIRT6 in OGD-insulted neurons. Together, this study suggests that Cezanne-dependent SIRT6-DNA DSB signaling pathway may mediate the neuroprotective effect of tDCS in ischemic neurons.

Genomic Analysis of a Novel Phage Infecting the Turkey Pathogen Escherichia coli APEC O78 and Its Endolysin Activity.

Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of maintaining a healthy digestive tract. However, some E. coli strains can cause serious zoonotic diseases, including diarrhea, pneumonia, urinary tract infections, and hemolytic uremic syndrome. In this study, we characterized a newly isolated Myoviridae phage, vB_EcoM_APEC. The phage vB_EcoM_APEC was able to infect E. coli APEC O78, which is the most common MDR E. coli serotype in turkeys. Additionally, the phage's host range included Klebsiella pneumoniae and other E. coli strains. The genome of phage vB_EcoM_APEC (GenBank accession number MT664721) was 35,832 bp in length, with 52 putative open reading frames (ORFs) and a GC content of 41.3%. The genome of vB_EcoM_APEC exhibited low similarity (79.1% identity and 4.0% coverage) to the genome of Acinetobacter phage vB_AbaM_IME284 (GenBank no. MH853787.1) according to the nucleotide Basic Local Alignment Search Tool (BLASTn). Phylogenetic analysis revealed that vB_EcoM_APEC was a novel phage, and its genome sequence showed low similarity to other available phage genomes. Gene annotation indicated that the protein encoded by orf11 was an endolysin designated as LysO78, which exhibited 64.7% identity (91.0% coverage) with the putative endolysin of Acinetobacter baumannii phage vB_AbaM_B9. The LysO78 protein belongs to glycoside hydrolase family 19, and was described as being a chitinase class I protein. LysO78 is a helical protein with 12 α-helices containing a large domain and a small domain in terms of the predicted three-dimensional structure. The results of site-directed mutagenesis indicated that LysO78 contained the catalytic residues E54 and E64. The purified endolysin exhibited broad-spectrum bacteriolytic activity against Gram-negative strains, including the genera Klebsiella, Salmonella, Shigella, Burkholderia, Yersinia, and Pseudomonas, as well as the species Chitinimonas arctica, E. coli, Ralstonia solanacearum, and A. baumannii. An enzymatic assay showed that LysO78 had highly lytic peptidoglycan hydrolases activity (64,620,000 units/mg) against E. coli APEC O78, and that LysO78 had lytic activity in the temperature range of 4-85 °C, with an optimal temperature of 28 °C and optimal pH of 8.0, and was active at pH 3.0-12.0. Overall, the results suggested that LysO78 might be a promising therapeutic agent for controlling MDR E. coli APEC O78 and nosocomial infections caused by multidrug-resistant bacteria.

Oncogenesis, Microenvironment Modulation and Clinical Potentiality of FAP in Glioblastoma: Lessons Learned from Other Solid Tumors.

Currently, glioblastoma (GBM) is the most common malignant tumor of the central nervous system in adults. Fibroblast activation protein (FAP) is a member of the dipeptidyl peptidase family, which has catalytic activity and is engaged in protein recruitment and scaffolds. Recent studies have found that FAP expression in different types of cells within the GBM microenvironment is typically upregulated compared with that in lower grade glioma and is most pronounced in the mesenchymal subtype of GBM. As a marker of cancer-associated fibroblasts (CAFs) with tumorigenic activity, FAP has been proven to promote tumor growth and invasion via hydrolysis of molecules such as brevican in the extracellular matrix and targeting of downstream pathways and substrates, such as fibroblast growth factor 21 (FGF21). In addition, based on its ability to suppress antitumor immunity in GBM and induce temozolomide resistance, FAP may be a potential target for immunotherapy and reversing temozolomide resistance; however, current studies on therapies targeting FAP are still limited. In this review, we summarized recent progress in FAP expression profiling and the understanding of the biological function of FAP in GBM and raised the possibility of FAP as an imaging biomarker and therapeutic target.

TGF-ß1-activated cancer-associated fibroblasts promote breast cancer invasion, metastasis and epithelial-mesenchymal transition by autophagy or overexpression of FAP-α.

Cancer-associated fibroblasts (CAFs) play an important role in the initiation, metastasis, and invasion of breast cancer. However, whether autophagy acts as a tumor promotion mechanism by inducing epithelial-mesenchymal transition (EMT) is still controversial and remains undefined at the mechanistic levels. In this study, we investigated whether autophagy or FAP-α is required for the invasion, pulmonary metastasis and EMT of breast cancer cells and underlying mechanism. We employed an in vitro model of NIH3T3 fibroblasts treated with H2O2 and confirmed that TGF-ß1 could convert fibroblasts into CAFs through autophagy under oxidative stress in the tumor microenvironment. Modulation of autophagy by rapamycin, 3-methyladenine or ATG-5 knockdown regulated the expression of CAFs markers, suggesting a role of autophagy in the tumor promotion mechanism of TGF-ß1-induced CAFs activation. Furthermore, we established an indirect co-culture model and a mixed xenograft as a corresponding in vivo model. We demonstrated that TGF-ß1-activated CAFs promote tumor invasion, pulmonary metastasis and EMT, which act through autophagy and overexpression of FAP-α in both models, while autophagy inhibitor 3-methyladenine blocked these effects induced by TGF-ß1-activated CAFs. Moreover, the co-localization of LC3ß and EMT marker vimentin in mixed xenograft also revealed that TGF-ß1-activated CAFs promote tumor growth, pulmonary metastasis, and EMT program partly through autophagy. In addition, knockdown of FAP-α resulted in reversed EMT and abolished tumor invasion and pulmonary metastasis induced by TGF-ß1-activated CAFs. Taken together, we conclude that both autophagy and FAP-α are required for breast cancer cell invasion and metastasis. Targeting autophagy or FAP-α rather than both can serve as a potential approach to improve the prognosis for human breast cancer.

A Novel Thermostable and Alkaline Protease Produced from Bacillus stearothermophilus Isolated from Olive Oil Mill Sols Suitable to Industrial Biotechnology.

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 108 CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca2+/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca2+ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.

An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a cell-surface expressed type II glycoprotein that has a unique proteolytic activity. FAP has active soluble forms that retain the extracellular portion but lack the transmembrane domain and cytoplasmic tail. FAP expression is normally very low in adult tissue but is highly expressed by activated fibroblasts in sites of tissue remodelling. Thus, FAP is a potential biomarker and pharmacological target in liver fibrosis, atherosclerosis, cardiac fibrosis, arthritis and cancer. Understanding the biological significance of FAP by investigating protein structure, interactions and activities requires reliable methods for the production and purification of abundant pure and stable protein. We describe an improved production and purification protocol for His6-tagged recombinant soluble human FAP. A modified baculovirus expression construct was generated using the pFastBac1 vector and the gp67 secretion signal to produce abundant active soluble recombinant human FAP (residues 27-760) in insect cells. The FAP purification protocol employed ammonium sulphate precipitation, ion exchange chromatography, immobilised metal affinity chromatography and ultrafiltration. High purity was achieved, as judged by gel electrophoresis and specific activity. The purified 82 kDa FAP protein was specifically inhibited by a FAP selective inhibitor, ARI-3099, and was inhibited by zinc with an IC50 of 25 µM. Our approach could be adopted for producing the soluble portions of other type II transmembrane glycoproteins to study their structure and function.

The Increased Accumulation of Staphylococcus aureus Virulence Factors Is Maximized in a purR Mutant by the Increased Production of SarA and Decreased Production of Extracellular Proteases.

Mutation of purR was previously shown to enhance the virulence of Staphylococcus aureus in a murine sepsis model, and this cannot be fully explained by increased expression of genes within the purine biosynthesis pathway. Rather, the increased production of specific S. aureus virulence factors, including alpha toxin and the fibronectin-binding proteins, was shown to play an important role. Mutation of purR was also shown previously to result in increased abundance of SarA. Here, we demonstrate by transposon sequencing that mutation of purR in the USA300 strain LAC increases fitness in a biofilm while mutation of sarA has the opposite effect. Therefore, we assessed the impact of sarA on reported purR-associated phenotypes by characterizing isogenic purR, sarA, and sarA/purR mutants. The results confirmed that mutation of purR results in increased abundance of alpha toxin, protein A, the fibronectin-binding proteins, and SarA, decreased production of extracellular proteases, an increased capacity to form a biofilm, and increased virulence in an osteomyelitis model. Mutation of sarA had the opposite effects on all of these phenotypes and, other than bacterial burdens in the bone, all of the phenotypes of sarA/purR mutants were comparable to those of sarA mutants. Limiting the production of extracellular proteases reversed all of the phenotypes of sarA mutants and most of those of sarA/purR mutants. We conclude that a critical component defining the virulence of a purR mutant is the enhanced production of SarA, which limits protease production to an extent that promotes the accumulation of critical S. aureus virulence factors.

A dynamic soft senor modeling method based on MW-ELWPLS in marine alkaline protease fermentation process.

The vital state variables in marine alkaline protease (MP) fermentation are difficult to measure in real-time online, hardly is the optimal control either. In this article, a dynamic soft sensor modeling method which combined just-in-time learning (JITL) technique and ensemble learning is proposed. First, the local weighted partial least squares algorithm (LWPLS) with JITL strategy is used as the basic modeling method. For further improving the prediction accuracy, the moving window (MW) is used to divide sub-dataset. Then the MW-LWPLS sub-model is built by selecting the diverse sub-datasets according to the cumulative similarity. Finally, stacking ensemble-learning method is utilized to fuse each MW-LWPLS sub-models. The proposed method is applied to predict the vital state variables in the MP fermentation process. The experiments and simulations results show that the prediction accuracy is better compared to other methods.

Ubiquitin-specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1-dependent pathway.

Ubiquitin-specific protease 19 (USP19) belongs to USP family and is involved in promoting skeletal muscle atrophy. Although USP19 is expressed in the heart, the role of USP19 in the heart disease remains unknown. The present study provides in vivo and in vitro data to reveal the role of USP19 in preventing pathological cardiac hypertrophy. We generated USP19-knockout mice and isolated neonatal rat cardiomyocytes (NRCMs) that overexpressed or were deficient in USP19 to investigate the effect of USP19 on transverse aortic constriction (TAC) or phenylephrine (PE)-mediated cardiac hypertrophy. Echocardiography, pathological and molecular analysis were used to determine the extent of cardiac hypertrophy, fibrosis, dysfunction and inflammation. USP19 expression was markedly increased in rodent hypertrophic heart or cardiomyocytes underwent TAC or PE culturing, the increase was mediated by the reduction of Seven In Absentia Homolog-2. The extent of TAC-induced cardiac hypertrophy, fibrosis, dysfunction and inflammation in USP19-knockout mice was exacerbated. Consistently, gain-of-function and loss-of-function approaches that involved USP19 in cardiomyocytes suggested that the down-regulation of USP19 promoted the hypertrophic phenotype, while the up-regulation of USP19 improved the worsened phenotype. Mechanistically, the USP19-elicited cardiac hypertrophy improvement was attributed to the abrogation of the transforming growth factor beta-activated kinase 1 (TAK1)-p38/JNK1/2 transduction. Furthermore, the inhibition of TAK1 abolished the aggravated hypertrophy induced by the loss of USP19. In conclusion, the present study revealed that USP19 and the downstream of TAK1-p38/JNK1/2 signalling pathway might be a potential target to attenuate pathological cardiac hypertrophy.

Marine microbial alkaline protease: An efficient and essential tool for various industrial applications.

With the modern world focusing on environmental friendly products, more and more chemical processes are being replaced by enzymatic methods. Alkaline proteases (APases) place more than 50% of the total world enzyme production. Marine microorganisms are capable of producing an extensive spectrum of APases which have important ecological roles and promising industrial applications. Marine microbial APases can meet the required market demand for various industrial processes due to their strong specificity, mild reaction conditions, environmental friendliness and easy inactivation or control in comparison with chemical catalysts. In this review, a bird's-eye view on recent research works in the field of APase production from marine microorganisms as well as their potential industrial applications. The effect of various physical and chemical parameters on marine microbial APase is discussed. Isolation, purification, optimum pH and temperature of marine microbial APases are also reported. We anticipate that this review will provide an outline of potential industrial application of marine microbial APases and open new avenues to help the academicians, researchers and industrialists.

Efficient matrix-assisted refolding of the recombinant anti-staphylococcal truncated endolysin LysKCA and its structural and enzymatic description.

The recombinant truncated endolysin LysK consisting of two catalytic domains, N-terminal CHAP and amidase-2 (LysKCA) was overexpressed in E. coli in the form of inclusion bodies (IBs). These IBs were dissolved in 6 M solution of urea followed by the refolding process. The refolding efficacy of the dilution and matrix-assisted renaturation method on SP Sepharose was compared at different purification stages of LysKCA. Solubilizate of IBs, DEAE Sepharose flowthrough, and SP Sepharose elution fractions were examined. The presence of negatively charged nucleic acids (NA) in the solution has shown a decrease in the recombinant LysKCA refolding yield (less than 11.5 ± 1.3% for both renaturation methods) due to their non-specific interaction with the positively charged endolysin. The renaturation efficiency of the enzyme purified from NA (SP elution fraction) was about 29.5 ± 6.7% and 28.2 ± 3.75% for dilution and matrix-assisted methods respectively. The later approach allows conducting one-step LysKCA refolding, purification and collection, and also noticeably cuts time and material expenses. The analysis of CD spectroscopy data of LysKCA, renatured on the resin matrix, revealed alpha helices and beta strands content similar to that of the modeled 3D structure. The theoretical 3D model with two predicted domains (CHAP and amidase-2) agrees well with the differential scanning calorimetry (DSC) results of the renatured LysKCA showing two well-resolved peaks corresponding to the two calorimetrically-revealed domains with the midpoint transition temperature (Tm) of 40.1 and 65.3°Ð¡. The enzyme so obtained exhibited in vitro anti-staphylococcal activity with 2.3 ± 0.45 × 103 U/mg and retained it for at least one year.

High cell-density fermentation, expression and purification of bacteriophage lysin TSPphg, a thermostable antimicrobial protein from extremophilic Thermus bacteriophage TSP4.

Recently, high cell-density (HCD) cultivation has become an important tool for production of many microbial products. However, to the best of our knowledge, no study regarding HCD fermentation, overproduction and purification of thermostable bacteriophage lysin has been reported. Here, by employing a glucose-limited fed-batch strategy, we performed high density fermentation of the host Escherichia coli BL21(DE3) cells, compared the efficiency of high pressure homogenization, ultrasonication and thermolysis in bacterial cell disruption after HCD cultivation, and purified TSPphg, a thermostable lysin derived from extremophilic bacteriophage TSP4. On the 20-L scale, the overproduction level of TSPphg was up to 67.8 ± 0.7%. In total, we obtained a broth titer of 3322.8 ± 26 mg/L TSPphg with a purity of 95.5 ± 0.7% from a bacterial cell mass of 86.3 ± 4.9 g/L after 26 h of fermentation. The overall productivity of TSPphg was 127.8 ± 1 mg/L/h. Additionally, the antimicrobial activity of purified TSPphg against both Gram-negative (Escherichia coli O157) and Gram-positive (Staphylococcus aureus) pathogenic bacteria was further confirmed by scanning electron microscope analysis. Summarily, for the first time, we have established a relatively stable and efficient HCD cultivation and purification process for recovery of thermostable lysins from extremophilic Thermus bacteriophages. Our results provide insights into the strategies for time-saving and cost-effective production of antimicrobial proteins to replace or supplement antibiotics in the current age of mounting antibiotic resistance.

Functional characterization of the endolysins derived from mycobacteriophage PDRPxv.

Bacteriophage-derived endolysin enzymes play a critical role in disintegration of the host bacterial cell wall and hence have gained considerable attention as possible therapeutics for the treatment of drug-resistant infections. Endolysins can target both dividing and non-dividing cells and given the vital role peptidoglycan plays in bacterial survival, bacteria are less likely to modify it even if continuously exposed to lysins. Hence, probability of bacteria developing resistance to lysins appear bleak. Endolysins from mycobacteriophages offer great potential as alternative therapeutics for the drug-resistant TB. However, considering that a large number of mycobacteriophages have been discovered so far, the information on endolysins come from only a few mycobacteriophages. In this study, we report the structural and functional characterization of endolysins (LysinA and LysinB) encoded by mycobacteriophage PDRPxv which belongs to B1 sub cluster. On in silico analysis, we found LysinA to be a modular protein having peptidase domain at the N-terminal (104 aa), a central amidase domain (174 aa) and the peptidoglycan binding domain (62 aa) at the C-terminal. Additionally, 'H-X-H', which is a conserved motif and characteristic of peptidase domains, and the conserved residues His-His-Asp, which are characteristic of amidase domain were also observed. In LysinB enzyme, a single α/ß hydrolase domain having a catalytic triad (Ser-Asp-His) and G-X-S-X-G motif, which are characteristic of the serine esterase enzymes were predicted to be present. Both the enzymes were purified as recombinant proteins and their antimycobacterial activity against M. smegmatis was demonstrated through turbidimetric experiments and biochemical assay. Interesting observation in this study is the secretory nature of LysinA evident by its periplasmic expression in E.coli, which might explain the ability of PDRPxv to lyse the bacterial host in the absence of transmembrane Holin protein.

A simplified method for producing laboratory grade recombinant TEV protease from E. coli.

The tobacco etch virus (TEV) protease has become a popular choice for cleaving fusion proteins because of its high stringency in sequence recognition. Procedures for isolating recombinant protein from the cytoplasm of E. coli require rupturing of the cell wall via enzymatic treatment combined with sonication or French press. Here we present an expedited method for producing laboratory-grade TEV protease in E. coli using a freeze-thaw method, followed by purification with immobilized metal affinity chromatography. Protease is obtained by expression from the pDZ2087 plasmid in BL21 (DE3) cells. Proteolysis resulting from this product, cleaves a maltose-binding protein fusion to completion at a fusion-to-protease molar ratio of 50:1.

Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709.

BACKGROUND: Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. RESULT: Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. CONCLUSION: We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.

Optimization of alkaline protease production by rational deletion of sporulation related genes in Bacillus licheniformis.

BACKGROUND: Our laboratory has constructed a Bacillus licheniformis strain that secretes alkaline protease (AprE) with excellent enzymatic properties. B. licheniformis is generally regarded as safe and has a high industrial exoenzyme secretion capacity, but the host retains some undomesticated characteristic that increase its competitiveness and survival, such as spore-formation, which increases the requirements and difficulties in industrial operations (e.g. sterilization and enzyme activity control). Furthermore, the influence of sporulation on alkaline protease production in B. licheniformis has not been elucidated in detail. RESULT: A series of asporogenic variants of the parent strain were constructed by individually knocking out the master regulator genes (spo0A, sigF and sigE) involved in sporulation. Most of the variants formed abortively disporic cells characterized by asymmetric septa at the poles and unable to survive incubation at 75 °C for 10 min. Two of them (ΔsigF and ΔsigE) exhibited superior characteristics in protease production, especially improving the expression of the aprE gene. Under the currently used fermentation conditions, the vegetative production phase of ΔsigF can be prolonged to 72 h, and the highest protease production of ΔsigF reached 29,494 ± 1053 U/mL, which was about 19.7% higher than that of the wild-type strain. CONCLUSION: We first constructed three key sporulation-deficient strain to investigate the effect of sporulation on alkaline protease synthesis. The sigF mutant retained important industrial properties such as facilitating the sterilization process, a prolonged stable phase of enzyme production and slower decreasing trend, which will be superior in energy conservation, simpler operations and target product controlling effect. In summary, the work provides a useful industrial host with preferable characteristics and a novel strategy to enhance the production of protease.

Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges.

Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.