Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E.

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

Protein purification via consecutive histidine-polyphosphate interaction.

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.

Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

Nanoformulation of the Broad-Spectrum Hydrophobic Antiviral Vacuolar ATPase Inhibitor Diphyllin in Human Recombinant H-ferritin.

Background: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing. Methods: We produced human recombinant H- ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain. Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH. Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity.

Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

Self-Assembled Recombinant Elastin and Globular Protein Vesicles with Tunable Properties for Diverse Applications.

ConspectusVesicles are self-assembled structures comprised of a membrane-like exterior surrounding a hollow lumen with applications in drug delivery, artificial cells, and micro-bioreactors. Lipid or polymer vesicles are the most common and are made of lipids or polymers, respectively. They are highly useful structures for many applications but it can be challenging to decorate them with proteins or encapsulate proteins in them, owing to the use of organic solvent in their formation and the large size of proteins relative to lipid or polymer molecules. By utilization of recombinant fusion proteins to make vesicles, specific protein domains can be directly incorporated while also imparting tunability and stability. Protein vesicle assembly relies on the design and use of self-assembling amphiphilic proteins. A specific protein vesicle platform made in purely aqueous conditions of a globular, functional protein fused to a glutamate-rich leucine zipper (ZE) and a thermoresponsive elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) is discussed here. The hydrophobic conformational change of the ELP above its transition temperature drives assembly, and strong ZE/ZR binding enables incorporation of the desired functional protein. Mixing the soluble proteins on ice induces zipper binding, and then warming above the ELP transition temperature (Tt) triggers the transition to and growth of protein-rich coacervates and, finally, reorganization of proteins into vesicles. Vesicle size is tunable based on salt concentration, rate of heating, protein concentration, size of the globular protein, molar ratio of the proteins, and the ELP sequence. Increasing the salt concentration decreases vesicle size by decreasing the Tt, resulting in a shorter coacervation transition stage. Likewise, directly changing the heating rate also changes this time and increasing protein concentration increases coalescence. Increasing globular protein size decreases the size of the vesicle due to steric hindrance. By changing the ELP sequence, which consists of (VPGXG)n, through the guest residue (X) or number of repeats (n), Tt is changed, affecting size. Additionally, the chemical nature of X variation has endowed vesicles with stimuli responsiveness and stability at physiological conditions.Protein vesicles have been used for biocatalysis, biomacromolecular drug delivery, and vaccine applications. Photo-cross-linkable vesicles were used to deliver small molecule cargo to cancer cells in vitro and antigen to immune cells in vivo. pH-responsive vesicles effectively delivered functional protein cargo, including cytochrome C, to the cytosol of cancer cells in vitro, using hydrophobic ion pairing to improve cargo distribution in the vesicles and release. The globular protein used to make the vesicles can be varied to achieve different functions. For example, enzyme vesicles exhibit biocatalysis, and antigen vesicles induce antibody and cellular immune responses after vaccination in mice. Collectively, the development and engineering of the protein vesicle platform has employed amphiphilic self-assembly strategies and rational protein engineering to control physical, chemical, and biological properties for biotechnology and nanomedicine applications.

Self-Assembling Triple-Helix Recombinant Collagen Hydrogel Enriched with Tyrosine.

The self-assembly of collagen within the human body creates a complex 3D fibrous network, providing structural integrity and mechanical strength to connective tissues. Recombinant collagen plays a pivotal role in the realm of biomimetic natural collagen. However, almost all of the reported recombinant collagens lack the capability of self-assembly, severely hindering their application in tissue engineering and regenerative medicine. Herein, we have for the first time constructed a series of self-assembling tyrosine-rich triple helix recombinant collagens, mimicking the structure and functionality of natural collagen. The recombinant collagen consists of a central triple-helical domain characterized by the (Gly-Xaa-Yaa)n sequence, along with N-terminal and C-terminal domains featuring the GYY sequence. The introduction of GYY has a negligible impact on the stability of the triple-helical structure of recombinant collagen while simultaneously promoting its self-assembly into fibers. In the presence of [Ru(bpy)3]Cl2 and APS as catalysts, tyrosine residues in the recombinant collagen undergo covalent cross-linking, resulting in a hydrogel with exceptional mechanical properties. The recombinant collagen hydrogel exhibits outstanding biocompatibility and bioactivity, significantly enhancing the proliferation, adhesion, migration, and differentiation of HFF-1 cells. This innovative self-assembled triple-helix recombinant collagen demonstrates significant potential in the fields of tissue engineering and medical materials.

Structural characteristics of alpha-fetoprotein, including N-glycosylation, metal ion and fatty acid binding sites.

Alpha-fetoprotein (AFP), a serum glycoprotein, is expressed during embryonic development and the pathogenesis of liver cancer. It serves as a clinical tumor marker, function as a carcinogen, immune suppressor, and transport vehicle; but the detailed AFP structural information has not yet been reported. In this study, we used single-particle cryo-electron microscopy(cryo-EM) to analyze the structure of the recombinant AFP obtained a 3.31 Å cryo-EM structure and built an atomic model of AFP. We observed and identified certain structural features of AFP, including N-glycosylation at Asn251, four natural fatty acids bound to distinct domains, and the coordination of metal ions by residues His22, His264, His268, and Asp280. Furthermore, we compared the structural similarities and differences between AFP and human serum albumin. The elucidation of AFP's structural characteristics not only contributes to a deeper understanding of its functional mechanisms, but also provides a structural basis for developing AFP-based drug vehicles.

Cloning and catalytic profile of Hyalomma dromedarii leucine aminopeptidase.

Ticks have harmful impacts on both human and animal health and cause considerable economic losses. Leucine aminopeptidase enzymes (LAP) play important roles during tick infestation to liberate vital amino acids necessary for growth. The aim of the current study is to identify, express and characterize the LAP from the hard tick Hyalomma dromedarii and elucidate its biochemical characteristics. We cloned an open reading frame of 1560 bp encoding a protein of 519 amino acids. The LAP full-length was expressed in Escherichia coli BL21 (DE3) and purified. The recombinant enzyme (H.d rLAP- 6×His) had a predicted molecular mass of approximately 55 kDa. Purification and the enzymatic characteristics of H.d rLAP- 6×His were studied. The purified enzyme showed maximum activity at 37 °C and pH 8.0-8.5 using Leu-p-nitroanilide as a substrate. The activity of H.d rLAP- 6×His was sensitive to ß-mercaptoethanol, dl-dithiothreitol, 1,10- phenanthroline, bestatin HCl, and EDTA and completely abolished by 0.05 % SDS. In parallel, the enzymatic activity was enhanced by Ni2+, Mn2+ and Mg2+, partially inhibited by Na+, Cu2+, Ca2+ and completely inhibited by Zn2+.

Enhancing the acid stability of the recombinant GH11 xylanase xynA through N-terminal substitution to facilitate its application in apple juice clarification.

The utilization of xylanase in juice clarification is contingent upon its stability within acidic environments. We generated a mutant xynA-1 by substituting the N-terminal segment of the recombinant xylanase xynA to investigate the correlation between the N-terminal region of xylanase and its acid stability. The enzymatic activity of xynA-1 was found to be superior under acidic conditions (pH 5.0). It exhibited enhanced acid stability, surpassing the residual enzyme activity values of xynA at pH 4.0 (53.07 %), pH 4.5 (69.8 %), and pH 5.0 (82.4 %), with values of 60.16 %, 77.74 %, and 87.3 %, respectively. Additionally, the catalytic efficiency of xynA was concurrently improved. Through molecular dynamics simulation, we observed that N-terminal shortening induced a reduction in motility across most regions of the protein structure while enhancing its stability, particularly Lys131-Phe146 and Leu176-Gly206. Furthermore, the application of treated xynA-1 in the process of apple juice clarification led to a significant increase in clarity within a short duration of 20 min at 35 °C while ensuring the quality of the apple juice. This study not only enhances the understanding of the N-terminal region of xylanase but also establishes a theoretical basis for augmenting xylanase resources employed in fruit juice clarification.

Identification and recombinant expression of a cutinase from Papiliotrema laurentii that hydrolyzes natural and synthetic polyesters.

Given the multitude of extracellular enzymes at their disposal, many of which are designed to degrade nature's polymers (lignin, cutin, cellulose, etc.), fungi are adept at targeting synthetic polyesters with similar chemical composition. Microbial-influenced deterioration of xenobiotic polymeric surfaces is an area of interest for material scientists as these are important for the conservation of the underlying structural materials. Here, we describe the isolation and characterization of the Papiliotrema laurentii 5307AH (P. laurentii) cutinase, Plcut1. P. laurentii is basidiomycete yeast with the ability to disperse Impranil-DLN (Impranil), a colloidal polyester polyurethane, in agar plates. To test whether the fungal factor involved in this clearing was a secreted enzyme, we screened the ability of P. laurentii culture supernatants to disperse Impranil. Using size exclusion chromatography (SEC), we isolated fractions that contained Impranil-clearing activity. These fractions harbored a single ~22 kD band, which was excised and subjected to peptide sequencing. Homology searches using the peptide sequences identified, revealed that the protein Papla1 543643 (Plcut1) displays similarities to serine esterase and cutinase family of proteins. Biochemical assays using recombinant Plcut1 confirmed that this enzyme has the capability to hydrolyze Impranil, soluble esterase substrates, and apple cutin. Finally, we confirmed the presence of the Plcut1 in culture supernatants using a custom antibody that specifically recognizes this protein. The work shown here supports a major role for the Plcut1 in the fungal degradation of natural polyesters and xenobiotic polymer surfaces.IMPORTANCEFungi play a vital role in the execution of a broad range of biological processes that drive ecosystem function through production of a diverse arsenal of enzymes. However, the universal reactivity of these enzymes is a current problem for the built environment and the undesired degradation of polymeric materials in protective coatings. Here, we report the identification and characterization of a hydrolase from Papiliotrema laurentii 5307AH, an aircraft-derived fungal isolate found colonizing a biodeteriorated polymer-coated surface. We show that P. laurentii secretes a cutinase capable of hydrolyzing soluble esters as well as ester-based compounds forming solid surface coatings. These findings indicate that this fungus plays a significant role in biodeterioration through the production of a cutinase adept at degrading ester-based polymers, some of which form the backbone of protective surface coatings. The work shown here provides insights into the mechanisms employed by fungi to degrade xenobiotic polymers.

Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import.

The bacterial Efe system functions as an importer of free Fe2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

Expression and purification of fluorinated proteins from mammalian suspension culture.

The site-specific encoding of noncanonical amino acids allows for the introduction of rationalized chemistry into a target protein. Of the methods that enable this technology, evolved tRNA and synthetase pairs offer the potential for expanded protein production and purification. Such an approach combines the versatility of solid-phase peptide synthesis with the scalable features of recombinant protein production. We describe the large scale production and purification of eukaryotic proteins bearing fluorinated phenylalanine in mammalian suspension cell preparations. Downstream applications of this approach include scalable recombinant protein preparation for ligand binding assays with small molecules and ligands, protein structure determination, and protein stability assays.

Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence.

Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.

Production of native recombinant proteins using a novel split intein affinity technology.

Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a CC-intein tag is engineered into a protein of interest for binding to a NC-intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the NC-intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the CC-intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems.

AviTrap: A novel solution to achieve complete biotinylation.

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.

A highly bioactive THPC-crosslinked recombinant collagen hydrogel implant for aging skin rejuvenation.

Skin aging, a complex physiological progression marked by collagen degradation, poses substantial challenges in dermatology. Recombinant collagen emerges as a potential option for skin revitalization, yet its application is constrained by difficulties in forming hydrogels. We have for the first time developed a highly bioactive Tetrakis(hydroxymethyl) phosphonium chloride (THPC)-crosslinked recombinant collagen hydrogel implant for aging skin rejuvenation. THPC demonstrated superior crosslinking efficiency compared to traditional agents such as EDC/NHS and BDDE, achieving complete recombinant collagen crosslinking at minimal concentrations and effectively inducing hydrogel formation. THPC's four reactive hydroxymethyl groups facilitate robust crosslinking with triple helical recombinant collagen, producing hydrogels with enhanced mechanical strength, excellent injectability, increased stability, and greater durability. Moreover, the hydrogel exhibited remarkable biocompatibility and bioactivity, significantly promoting the proliferation, adhesion, and migration of human foreskin fibroblast-1. In photoaged mice skin models, the THPC-crosslinked collagen hydrogel implant notably improved dermal density, skin elasticity, and reduced transepidermal water loss, creating a conducive environment for fibroblast activity and healthy collagen regeneration. Additionally, it elevated superoxide dismutase (SOD) activity and displayed substantial anti-calcification properties. The THPC-crosslinked recombinant collagen hydrogel implant presents an innovative methodology in combating skin aging, offering significant promise in dermatology and tissue engineering.

Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

Characterization of a highly thermostable recombinant xylanase from Anoxybacillus ayderensis.

Xylanases are the main enzymes to hydrolyze xylan, the major hemicellulose found in lignocellulose. Xylanases also have a wide range of industrial applications. Therefore, the discovery of new xylanases has the potential to enhance efficiency and sustainability in many industries. Here, we report a xylanase with thermophilic character and superior biochemical properties for industrial use. The new xylanase is discovered in Anoxybacillus ayderensis as an intracellular xylanase (AAyXYN329) and recombinantly produced. While AAyXYN329 shows significant activity over a wide pH and temperature range, optimum activity conditions were determined as pH 6.5 and 65 °C. The half-life of the enzyme was calculated as 72 h at 65 °C. The enzyme did not lose activity between pH 6.0-9.0 at +4 °C for 75 days. Km, kcat and kcat/Km values of AAyXYN329 were calculated as 4.09824 ± 0.2245 µg/µL, 96.75 1/sec, and 23.61/L/g.s -1, respectively. In conclusion, the xylanase of A. ayderensis has an excellent potential to be utilized in many industrial processes.

Enzymatic characterization and thermostability improvement of an acidophilic endoxylanase PphXyn11 from Paenibacillus physcomitrellae XB.

GH11 enzyme is known to be specific and efficient for the hydrolysis of xylan. It has been isolated from many microorganisms, and its enzymatic characteristics and thermostability vary between species. In this study, a GH11 enzyme PphXyn11 from a novel xylan-degrading strain of Paenibacillus physcomitrellae XB was characterized, and five mutants were constructed to try to improve the enzyme's thermostability. The results showed that PphXyn11 was an acidophilic endo-ß-1,4-xylanase with the optimal reaction pH of 3.0-4.0, and it could deconstruct different kinds of xylan substrates efficiently, such as beechwood xylan, wheat arabinoxylan and xylo-oligosaccharides, to produce xylobiose and xylotriose as the main products at the optimal reaction temperature of 40 °C. Improvement of the thermal stability of PphXyn11 using site-directed mutagenesis revealed that three mutants, W33C/N47C, S127C/N174C and S49E, designed by adding the disulfide bonds at the N-terminal, C-terminal and increasing the charged residues on the surface of PphXyn11 respectively, could increase the enzymatic activity and thermal stablility significantly and make the optimal reaction temperature reach 50 °C. Molecular dynamics simulations as well as computed the numbers of salt bridges and hydrogen bonds indicated that the protein structures of these three mutants were more stable than the wild type, which provided theoretical support for their improved thermal stability. Certainly, further research is necessary to improve the enzymatic characteristics of PphXyn11 to achieve the bioconversion of hemicellulosic biomass on an applicable scale.