Evaluate the potential use of TonB-dependent receptor protein as a subunit vaccine against Aeromonas veronii infection in Nile tilapia (Oreochromis niloticus).

Aeromonas veronii is an emerging bacterial pathogen that causes serious systemic infections in cultured Nile tilapia (Oreochromis niloticus), leading to massive deaths. Therefore, there is an urgent need to identify effective vaccine candidates to control the spread of this emerging disease. TonB-dependent receptor (Tdr) of A. veronii, which plays a role in the virulence factor of the organism, could be useful in terms of protective antigens for vaccine development. This study aims to evaluate the potential use of Tdr protein as a novel subunit vaccine against A. veronii infection in Nile tilapia. The Tdr gene from A. veronii was cloned into the pET28b expression vector, and the recombinant protein was subsequently produced in Escherichia coli strain BL21 (DE3). Tdr was expressed as an insoluble protein and purified by affinity chromatography. Antigenicity test indicated that this protein was recognized by serum from A. veronii infected fish. When Nile tilapia were immunized with the Tdr protein, specific antibody levels increased significantly (p-value <0.05) at 7 days post-immunization (dpi), and peaked at 21 dpi compared to antibody levels at 0 dpi. Furthermore, bacterial agglutination activity was observed in the fish serum immunized with the Tdr protein, indicating that specific antibodies in the serum can detect Tdr on the bacterial cell surface. These results suggest that Tdr protein has potential as a vaccine candidate. However, challenging tests with A.veronii in Nile tilapia needs to be investigated to thoroughly evaluate its protective efficacy for future applications.

Are phenolic compounds produced during the enzymatic production of prebiotic xylooligosaccharides (XOS) beneficial: a review.

Phenolics produced during xylooligosaccharide production might inhibit xylanases and enhance the antioxidant and antimicrobial activities of XOS. The effects of phenolic compounds on xylanases may depend on the type and concentration of the compound, the plant biomass used, and the enzyme used. Understanding the effects of phenolic compounds on xylanases and their impact on XOS is critical for developing viable bioconversion of lignocellulosic biomass to XOS. Understanding the complex relationship between phenolic compounds and xylanases can lead to the development of strategies that improve the efficiency and cost-effectiveness of XOS manufacturing processes and optimise enzyme performance.

A multi-epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia.

Edwardsiella ictaluri infects several fish species and protection of the all the susceptible fish hosts from the pathogen using a monovalent vaccine is impossible because the species is composed of host-based genotypes that are genetic, serological and antigenic heterogenous. Here, immunoinformatic approach was employed to design a cross-immunogenic chimeric EiCh protein containing multi-epitopes. The chimeric EiCh protein is composed of 11 B-cell epitopes and 7 major histocompatibility complex class II epitopes identified from E. ictaluri immunogenic proteins previously reported. The 49.32 kDa recombinant EiCh protein was expressed in vitro in Escherichia coli BL-21 (DE3) after which inclusion bodies were successfully solubilized and refolded. Ab initio protein modelling revealed secondary and tertiary structures. Secondary structure was confirmed by circular dichroism spectroscopy. Antigenicity of the chimeric EiCh protein was exhibited by strong reactivity with serum from striped catfish and Nile tilapia experimentally infected with E. ictaluri. Furthermore, immunogenicity of the chimeric EiCh protein was investigated in vivo in Nile tilapia juveniles and it was found that the protein could strongly induce production of specific antibodies conferring agglutination activity and partially protected Nile tilapia juveniles with a relative survival percentage (RPS) of 42%. This study explored immunoinformatics as reverse vaccinology approach in vaccine design for aquaculture to manage E. ictaluri infections.

Comparative genomics of Edwardsiellaictaluri revealed four distinct host-specific genotypes and thirteen potential vaccine candidates.

Edwardsiella ictaluri has been considered an important threat for catfish aquaculture industry for more than 4 decades and an emerging pathogen of farmed tilapia but only 9 sequenced genomes were publicly available. We hereby report two new complete genomes of E. ictaluri originated from diseased hybrid red tilapia (Oreochromis sp.) and striped catfish (Pangasianodon hypophthalmus) in Southeast Asia. E. ictaluri species has an open pan-genome consisting of 2615 core genes and 5592 pan genes. Phylogenetic analysis using core genome MLST (cgMLST) and ANI values consistently placed E. ictaluri isolates into 4 host-specific genotypes. Presence of unique genes and absence of certain genes from each genotype provided potential biomarkers for further development of genotyping scheme. Vaccine candidates with high antigenic, solubility and secretion probabilities were identified in silico from the core genes. Microevolution within the species is brought about by bacteriophages and insertion elements and possibly drive host adaptation.

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding.

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded ß-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.

Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviously, detecting reagents such as a specific antibody is essential for the development of immune-based detection methods for C. jejuni or C. coli. In this study, in silico techniques were used to design a chimeric recombinant antigen, named multiepitope antigen (MEA), for the production of specific polyclonal antibody. To design MEA polypeptide based on C. jejuni fibronectin-binding protein or CadF, four conserved and unique antigenic peptides were identified and fused together directly. The C. jejuni CadF-based MEA polypeptide fused with two single six-histidine tags at both C- and N-terminal ends was expressed under Escherichia coli expression system. The recombinant MEA was successfully produced and purified by Ni-NTA resin with a high satisfactory yield. Indirect ELISA results showed that anti-MEA polyclonal antibody derived from rabbit serum had a titer of 16,000, indicating high antigenicity of MEA polypeptide. Dot blot results also confirmed that the produced anti-MEA antibody could specifically recognize both C. jejuni and C. coli whole cells as expected while there was no cross-reactivity to non-Campylobacter spp. tested in this study.

Refolded recombinant major capsid protein (MCP) from Infectious Spleen and Kidney Necrosis Virus (ISKNV) effectively stimulates serum specific antibody and immune related genes response in Nile tilapia (Oreochromis niloticus).

Infectious spleen and kidney necrosis virus (ISKNV) is a causative agent of high mortality in fish resulting in significant economic loss to the fish industry in many countries. The major capsid protein (MCP) (ORF006) is an important structural component that mediates virus entry into the host cell, therefore it is a good candidate antigen of ISKNV for subunit vaccine development. In this study, MCP of ISKNV was successfully produced in Escherichia coli strain Ril and was purified as the soluble form by refolding recombinant MCP using urea in combination with dialysis process. The refolded recombinant MCP protein had ability of oligomerization to become trimer like native MCP protein. Fish immunized with refolded recombinant MCP showed significantly higher serum antibody titer than fish immunized with insoluble form of the protein (p < 0.05) at 21, 28- and 35-day post-immunization (dpi). Analysis of immune-related genes response in spleen and kidney of fish immunized with refolded recombinant MCP suggested that MHC-I, MHC-II, IL-1ß and IL-4 genes were also significantly expressed relative to the group immunized with insoluble protein (p < 0.05) at 14, 21, 28- and 35-day post immunization. The highest serum antibody and immune related genes response were found at 28 day post immunization. Therefore, refolded recombinant MCP should be better than previously reported insoluble form as the candidate subunit vaccine to prevent infection of Nile tilapia from ISKNV.

Antigenicity of hypothetical protein HP33 of Vibrio harveyi Y6 causing scale drop and muscle necrosis disease in Asian sea bass.

A unique strain of Vibrio harveyi is the causative agent of scale drop and muscle necrosis disease (SDMND) in Asian sea bass (Lates calcarifer). This study investigated the protein profiles of SDMND-causing Vibrio harveyi isolates compared to the reference V. harveyi ATCC 14126 strain. A distinct protein band of 33 kDa, namely HP33, found from only V. harveyi SDMND was subjected to analysis by LC-MS/MS and the identified peptide sequences matched to an unknown hypothetical protein. Detection of HP33 coding sequence was investigated at both genomic and transcriptional levels and the results consistently supported the protein analysis. Recombinant HP33 protein was then produced using Escherichia coli system. The rHP33 protein did not cause mortality or visible clinical signs to Asian sea bass. However, the rHP33 protein was able to stimulate antibody response in Asian sea bass as evidenced by Western blotting and agglutination tests. Here, we proposed that rHP33 might be a good protein target for development of subunit vaccine and/or immunostimulant to protect Asian sea bass from SDMND.

Synergistic effects between the additions of a disulphide bridge and an N-terminal hydrophobic sidechain on the binding pocket tilting and enhanced Xyn11A activity.

Synergistic effect of distal site-directed mutations and molecular mechanisms on the enhanced thermostability of GH11 xylanase from B. firmus Strain K-1 (xyn11A) was investigated through enzyme activity assays and atomistic molecular dynamics (MD) simulation. From the experiment, single N-terminal leucine substitution at K40L caused a significant drop in enzymatic activity. However, the addition of a disulphide bond at S100C/N147C, along with the K40L mutation enhanced the enzymatic activity at room temperature. Molecular mechanisms on the improvement of enzymatic activity were addressed through atomistic molecular dynamics (MD) simulations of enzyme-substrate complexes. Conformational analysis of the right-hand-shaped GH11 protein structures showed that K40L mutation 'tilted' the Palm region away from the Pinky finger at N-terminus and S100C/N147C tilted the Palm region towards the Pinky finger at N-terminus, which destabilized the binding complexes. The extended hydrophobic cluster formed within the K40L/S100C/N147C mutant stabilized the loops associated with the N-terminus and the Thumb region, which facilitated substrate binding and corresponded to the enhanced activity. This proposed mechanism could serve as a scheme for protein engineering to enhance enzymatic activity of GH11 enzymes at low temperatures.

Blood and liver biopsy for the non-destructive screening of tilapia lake virus.

Detection of tilapia lake virus (TiLV) in tilapines is mainly from visceral organs of killed fish. However, lethal sampling might not be viable to broodstock and economically important ornamental cichlids. To contribute towards screening of the virus in asymptomatic infected fish, a subclinically infected population of Nile tilapia adults obtained from a local farm was preliminarily tested to compare different non-lethal sampling methods, for example liver biopsy, gill biopsy, fin clip, mucus, faeces and blood for detection of TiLV. Only liver and blood samples gave positive results by PCR. Since blood sampling is relatively simpler, it was further used for five naturally co-cultured juvenile fish species from above-mentioned farm including 40 red tilapia broodstock and 20 Nile tilapia adults from two other different farms. The results showed that from the tested fish, 4 of 5 Nile tilapia, 2 of 5 hybrid red tilapia and 3 of 5 giant gourami blood samples tested positive, while 38 of 40 blood samples of red tilapia tested positive for TiLV in second-step PCR. Sequencing representative PCR amplicons of positive samples confirmed sequence identity to TiLV. In conclusion, both blood and liver biopsy are practical non-destructive sampling platforms for TiLV screening in cichlids with blood being more convenient, especially for tilapia broodstock.

C-terminal domain of WSSV VP37 is responsible for shrimp haemocytes binding which can be inhibited by sulfated galactan.

Viral envelope proteins play an important role in facilitating the attachment of viruses to the surface of host cells. Here, we investigated the binding of White Spot Syndrome Virus (WSSV) VP37 to haemocytes of whiteleg shrimp, Litopenaeus vannamei. Three versions of recombinant VP37 proteins, including full length VP37 (VP37(1-281)), C-terminal domain VP37 (VP37(111-281)) and C-terminal domain disrupted VP37 (VP37(1-250)) were individually expressed and tested for their haemocytes binding ability. Through an ELISA-based binding assay, we found that VP37(111-281) bound to shrimp haemocytes in a similar way to VP37(1-281), while VP37(1-250) exhibited a significantly weaker binding. This suggests that the C-terminal domain of VP37 is required for the binding of VP37 to shrimp haemocytes. Furthermore, we found that the binding of VP37 to shrimp haemocytes was impaired by pre-incubation of VP37 with sulfated galactan (SG), a sulfated polysaccharide derived from red seaweed (Gracilaria fisheri). Previously, it has been shown that a type of sulfated polysaccharide, heparin, is also present in L. vannamei. To investigate the role of heparin as a receptor for VP37, the binding of VP37 to porcine heparin, whose structure is similar to that found in L.vannamei, was investigated in a Surface Plasmon Resonance (SPR) system. The results showed that VP37 bound strongly to heparin with binding affinity (KD) of 1.0 µM and the binding was significantly blocked by SG. These findings have lead us to propose that the attachment of WSSV might be mediated by the interaction between VP37 and a heparin-like molecule presented on the shrimp cells.

The expression and purification of WSSV134 from white spot syndrome virus and its inhibitory effect on caspase activity from Penaeus monodon.

WSSV134 or VP36A protein of white spot syndrome virus was previously reported to be able to reduce apoptosis in Sf-9 cells transfected with caspase of Penaeus monodon (PmCasp). The protein was therefore believed to have a role in supporting the survival of WSSV inside the host cells during infection. However, the anti-apoptosis activity of WSSV134 involved in the inhibition of PmCasp is still unclear. In this study, we produced a recombinant WSSV134 (rWSSV134) and tested for its ability to inhibit PmCasp in vitro. The results from a caspase inhibition assay revealed that rWSSV134 could inhibit PmCasp in a dose-dependent manner. Since WSSV134 was predicted to contain three potential caspase binding sites, corresponding to the D54, D104 and D259, we then employed site-directed mutagenesis to investigate the involvement of these sites in PmCasp inhibition. D54A and D259A mutants could still inhibit PmCasp while D104A mutant lacks this activity. Our results confirmed that the WSSV134 is an inhibitor for PmCasp and that residue D104 is important for PmCasp inhibition.

The interaction between PmHtrA2 and PmIAP and its effect on the activity of Pm caspase.

Apoptosis is an essential mechanism in multicellular organisms which results in the induction of cell death. Important apoptotic proteins, including high temperature requirement A2 (PmHtrA2; also known as serine protease), inhibitor of apoptosis protein (PmIAP) and Pm caspase, have been previously identified in black tiger shrimp, Penaeus monodon. However, the relevance among these proteins in apoptosis regulation has not been established yet in shrimp. Here, we showed that PmHtrA2 was able to interact with PmIAP and the binding of the two proteins was mediated by the BIR2 domain of PmIAP. In addition, the BIR2 of PmIAP was shown to be able to inhibit Pm caspase activity. The inhibitory effect of the BIR2 domain on Pm caspase was impaired under the presence of the IBM peptide of PmHtrA2, implying a role for PmHtrA2 in apoptosis activation. Our combined results suggested that P. monodon possesses a conserved mechanism by which the caspase-3 activity is modulated by HtrA2 and IAP, as previously seen in insects and mammals.

Allosteric Signal within the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein Mediated by a Class 3 Monoclonal Antibody Revealed through Molecular Dynamics Simulations and Protein Residue Networks.

This study investigated the allosteric action within the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein caused by class 3 monoclonal antibody (mAb) binding. As the emergence of SARS-CoV-2 variants has raised concerns about the effectiveness of treatments by antibodies, targeting the highly conserved class 3 epitopes has become an alternative strategy of antibody design. Simulations of explicitly solvated RBD of the BA.2.75 omicron subvariants were carried out both in the presence and in the absence of bebtelovimab, as a model example of class 3 monoclonal antibodies against the RBD of the SARS-CoV-2 spike protein. The comparative analysis showed that bebtelovimab's binding on two α helices at the epitope region disrupted the nearby interaction network, which triggered a denser interaction network formation on the opposite side of the receptor-binding motif (RBM) region and resulted in a "close" conformation that could prevent the ACE2 binding. A better understanding of this allosteric action could lead to the development of alternative mAbs for further variants of concern. In terms of computational techniques, the communicability matrix could serve as a tool to visualize the effects of allostery, as the pairs of amino acids or secondary structures with high communicability could pinpoint the possible sites to transfer the allosteric signal. Additionally, the communicability gain/loss matrix could help elucidate the consequences of allosteric actions, which could be employed along with other allostery quantification techniques in some previous studies.

The identification and expression of the full-length HtrA2 gene from Penaeus monodon (black tiger shrimp).

HtrA2 is an apoptosis-activating protein to enhance the apoptotic process by preventing the formation of the IAP-caspase complex, thus freeing caspase to trigger the apoptosis pathway. Here, we presented the full-length sequence of HtrA2 from the black tiger shrimp (PmHtrA2). The full-length PmHtrA2 transcript was 1403 bp with a 1338 bp open reading frame encoding 445 amino acids and contains 5 conserved domains, namely, a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a serine protease domain, and a PDZ domain normally found in HtrA2 proteins of other organisms. The mature form of PmHtrA2 was cloned into the pET28b(+) and pET15bThio vectors, and the expression of the protein was compared in Escherichia coli BL21 DE3 and BL21 RIL (CodonPlus) strains. Greater quantities of stable and soluble PmHtrA2 were expressed as a thioredoxin fusion protein in E. coli BL21 RIL (CodonPlus) cells with the recombinant pET15bThio-PmHtrA2 vector. To investigate the expression of PmHtrA2 in shrimp, the crude proteins from several shrimp tissues were imaged by Western blot using the polyclonal antibody specific to the recombinant PmHtrA2. The expression of the 47-kDa immature PmHtrA2 protein could be detected in shrimp lysates from the gills and the muscles. This study is the first to report the full-length PmHtrA2 gene, which is functional in black tiger shrimp and will lead to more focused studies on the function of PmHtrA2 in apoptosis regulation during immune responses to viral infection in shrimp.

A computational study on the molecular mechanisms of panduratin A as a potential inhibitor on SARS-CoV-2 protein targets.

Panduratin A from Boesebergia rotunda was recently reported as a potent anti-SARS-CoV-2 compound. However, the molecular mechanisms underlying the inhibition by Panduratin A and its target remained unclear. Molecular docking calculations were performed between panduratin A and five important proteins, i.e., main protease (Mpro), papain-like protease (PLpro), receptor binding domain (RBD) of spike proteins, RNA-dependent-RNA-polymerase (RdRp), and 2'-O-methyltransferase (MTase). The estimated binding free energy and the interaction networks extracted from the best docking mode for each complex suggested that MTase was the most probable target for panduratin A inhibition. To further validate the ability of panduratin A to inhibit MTase, molecular dynamics (MD) simulations and binding free energy calculations were performed for panduratin A-MTase complex, in comparison with another MTase complex with sinefungin as a positive control. Chemical features of panduratin A and sinefungin were compared for their contribution in MTase binding. It was found that both molecules could bind to the S-Adenosyl methionine (SAM) binding pocket and prevent the SAM entrance co-substrate, which could eventually halt the function of MTase. Despite a slightly weaker binding free energy, the equilibrated positional binding of panduratin A was found at a closer distance to the active sites. Therefore, this study proposed MTase as a possible target of panduratin A, along with the mechanisms of inhibition, prompting another future in vitro study as a verification.

Comparative Genomic Analysis of Virulent Vibrio (Listonella) anguillarum Serotypes Revealed Genetic Diversity and Genomic Signatures in the O-Antigen Biosynthesis Gene Cluster.

Vibrio anguillarum is the most frequent pathogen affecting fish worldwide. The only known virulent strains of V. anguillarum are serotypes O1, O2, and O3. Genetic differences between the serotypes that could shed insight on the evolution and serotype differences of this marine pathogen are unknown. Here, we fully sequenced and characterized a strain of V. anguillarum O1 (J382) isolated from winter steelhead trout (Oncorhynchus mykiss irideus) in British Columbia, Canada. Koch's postulates using the O1 strain were replicated in naïve lumpfish (Cyclopterus lumpus) and compared to O2. Phenotypic and genotypic comparisons were conducted for serotypes O1, O2, and O3, using biochemical tests and bioinformatic tools, respectively. The genome of V. anguillarum O1 (J382) contains two chromosomes (3.13 Mb and 1.03 Mb) and two typical pJM1-like plasmids (65,573 and 76,959 bp). Furthermore, V. anguillarum O1 (J382) displayed resistance to colistin sulphate, which differs from serotype O2 and could be attributed to the presence of the ugd gene. Comparative genomic analysis, among the serotypes, showed that intra-species evolution is driven by insertion sequences, bacteriophages, and a different repertoire of putative ncRNAs. Genetic heterogeneity in the O-antigen biosynthesis gene cluster is characterized by the absence or the presence of unique genes, which could result in differences in the immune evasion mechanisms employed by the respective serotypes. This study contributes to understanding the genetic differences among V. anguillarum serovars and their evolution.

Chondroitin sulfate A-adhering Plasmodium falciparum-infected erythrocytes express functionally important antibody epitopes shared by multiple variants.

Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes to chondroitin sulfate A in the intervillous space. Although interclonal variation of the var2csa gene is lower than that among var genes in general, VAR2CSA-specific Abs appear to target mainly polymorphic epitopes. This has raised doubts about the feasibility of VAR2CSA-based vaccines. We used eight human monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas peripheral isolates from children were essentially nonreactive. The mAb reactivity of peripheral isolates from pregnant women indicated that some were placental, whereas others had alternative sequestration foci. Most of the mAbs were comparable in their reactivity with bound infected erythrocytes (IEs) and recombinant VAR2CSA and interfered with IE and/or VAR2CSA binding to chondroitin sulfate A. Pair-wise mAb combinations were more inhibitory than single mAbs, and all of the mAbs together was the most efficient combination. Each mAb could opsonize IEs for phagocytosis, and a combination of the eight mAbs caused phagocytosis similar to that of plasma IgG-opsonized IEs. We conclude that functionally important Ab epitopes are shared by the majority of polymorphic VAR2CSA variants, which supports the feasibility of VAR2CSA-based vaccines against placental malaria.

Investigation into the effects of CbXyn10C and Xyn11A on xylooligosaccharide profiles produced from sugarcane bagasse and rice straw and their impact on probiotic growth.

This comparative study investigated the effects of CbXyn10C and Xyn11A on xylooligosaccharide profiles produced from sugarcane bagasse (SCB) and rice straw (RS) and their impact on probiotic growth. Generally, CbXyn10C produced more xylose and a higher total phenolic content than Xyn11A. Interestingly, XOS obtained from SCB with CbXyn10C contained significantly more gallic acid than that produced by Xn11A. All selected probiotics thrived in RS-derived XOS, regardless of the enzyme used. However, probiotics grew differently on SCB-derived XOS depending on the enzyme used. All probiotics thrived in Xyn11A-derived XOS from SCB. Only Lactobacillus plantarum thrived on CbXyn10C-derived XOS, while the other two were inhibited. Gallic acid in CbXyn10C-derived XOS from SCB has been linked to probiotic retardation, and gallic acid-enriched broth has been found to inhibit Bifidobacterium longum and Bacillus subtilis, but not L. plantarum. Consequently, the selection of enzymes and plant biomass is crucial for XOS properties and prebiotic effects.

Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde dehydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.).

Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD(+). Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 Å resolution after being cooled in a stream of N(2) immediately prior to X-ray diffraction experiments. The crystals belonged to space group C222(1), with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 Å. An initial molecular-replacement solution has been obtained and refinement is in progress.