10 research outputs found

    Property Improvement of α-Amylase from Bacillus stearothermophilus by Deletion of Amino Acid Residues Arginine 179 and Glycine 180

    Get PDF
    Proizveden je mutant AmySΔR179-G180 delecijom arginina (Arg179) i glicina (Gly180) u α-amilazi iz bakterije Bacillus stearothermophilus (AmyS) pomoću ciljane mutageneze, a radi poboljšanja njezinih svojstava. Amilaze AmyS i AmySΔR179-G180 eksprimirane su u bakteriji Bacillus subtilis i pročišćene taloženjem pomoću amonijeva sulfata, te su im opisana i uspoređena svojstva. Delecijom aminokiselina Arg179 i Gly180 poboljšala se termostabilnost α-amilaze AmySΔR179-G180, a vrijeme poluraspada pri 100 °C bitno se produljilo s 24 na 33 min. Osim toga, ova je amilaza otpornija na djelovanje kiselina i treba manje kalcija za održavanje aktivnosti. Sekrecijska svojstva rekombinantnog soja ispitana su šaržnom fermentacijom u fermentoru od 7,5 L, te je dobivena velika aktivnost α-amilaze. Postignuta je najveća aktivnost od 3300 U/mL i produktivnost od 45,8 U/(mL•h).To improve the properties of α-amylase from Bacillus stearothermophilus (AmyS), a deletion mutant AmyS∆R179-G180 was constructed by deleting arginine (Arg179) and glycine (Gly180) using site-directed mutagenesis. AmyS and AmyS∆R179-G180 were expressed in Bacillus subtilis and purified by ammonium sulfate precipitation, after which the enzymatic properties were characterized and compared. By deleting amino acids Arg179 and Gly180, the thermostability of α-amylase AmyS∆R179-G180 was enhanced and the half-life at 100 °C significantly increased from 24 to 33 min. In addition, AmyS∆R179-G180 exhibited greater acid resistance and lower calcium requirements to maintain α-amylase activity. The secretory capacity of the recombinant strain was evaluated by fed-batch fermentation in a 7.5-litre fermentor in which high α-amylase activity was obtained. The highest activity reached 3300 U/mL with a high productivity of 45.8 U/(mL•h)

    Assessment of Spoilage Microbiota of Rainbow Trout (Oncorhynchus mykiss) during Storage by 16S rDNA Sequencing

    No full text
    Due to the high contents of protein and fat in rainbow trout, it is highly susceptible to spoilage, which limits the storage and transportation processes. Exploring the spoilage microbial community during rainbow trout storage is essential to develop an effective preservation method. Here, the changes in the total bacterial colony and total volatile base nitrogen (TVB-N) during the storage of rainbow trout were investigated. Storage at 0 °C can effectively slow down the spoilage process with bacterial counts and TVB-N contents decreased from 8.7 log CFU/g and 18.7 mg/100 g obtained at 4 °C to 5.6 log CFU/g and 14.5 mg/100 g, respectively. 16S rDNA high-throughput sequencing results showed that the diversity of microbial genera decreased during storage. Acinetobacter, Pseudomonas, and Shewanells gradually became the dominant spoilage genera with contents of 59.9%, 18.6%, and 1.7%, respectively, in the late stage of storage. The spoilage abilities of bacteria belonging to the Pseudomonas and Shewanells genera were analyzed. Shewanella sp. S5-52 showed the highest level of TVB-N content (100.6 mg/100 g) in sterile fish juice, indicating that it had a strong spoilage ability. This study confirmed the dominant spoilage bacterial genera and evaluated the spoilage abilities of isolated strains during the storage of rainbow trout, which laid the foundation for further investigation of the spoilage mechanism of rainbow trout and other aquatic products

    A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker

    No full text
    Abstract Background Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d -psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis . Results In this study, the d -alanine racemase-encoding gene dal was deleted from the chromosome of B. subtilis 1A751 using Cre/ lox system to generate the food-grade host. Subsequently, the plasmid-coded selection marker dal was complemented in the food-grade host, and RDPE was thus successfully expressed in dal deletion strain without addition of d -alanine. The selection appeared highly stringent, and the plasmid was stably maintained during culturing. The highest RDPE activity in medium reached 46\ua0U/ml at 72\ua0h which was comparable to RDPE production in kanamycin-based system. Finally, the capacity of the food-grade B. subtilis 1A751D2R was evaluated in a 7.5\ua0l fermentor with a fed-batch fermentation. Conclusion The alanine racemase-encoding gene can be used as a selection marker, and the food-grade expression system was suitable for heterologous proteins production in B. subtilis

    Evolution of E. coli Phytase for Increased Thermostability Guided by Rational Parameters

    No full text

    Electrophoretic Mobility of carp erythrocyte on Microfluidic Chip

    No full text
    The electrophoretic mobility of three groups of common carp erythrocyte on polymethal methacrylate chip has been studied by singe-cell imaging. The values of cell electrophoretic mobility were 1.138 × 10-4, 0.1279 × 10-4, -0.8520 × 10-4 cm2V -1s-1 respectively. It is demonstrated that the variation of the cell migration velocity is originated from the change of water quality and Lambda-cyhalothrin. The cell electrophoretic mobility being used as a parameter for cell classification have also been discussed

    Electrophoretic Mobility of Carp Erythrocyte on Microfluidic Chip

    No full text
    The electrophoretic mobility of three groups of common carp erythrocyte on polymethal methacrylate chip has been studied by singe-cell imaging. The values of cell electrophoretic mobility were 1.138 × 10-4, 0.1279 × 10-4, -0.8520 × 10-4 cm2V -1s-1 respectively. It is demonstrated that the variation of the cell migration velocity is originated from the change of water quality and Lambda-cyhalothrin. The cell electrophoretic mobility being used as a parameter for cell classification have also been discussed

    Comparative functional analysis of Jembrana disease virus Tat protein on lentivirus long terminal repeat promoters: evidence for flexibility at its N-terminus

    No full text
    Abstract Background Jembrana disease virus (JDV) encodes a potent regulatory protein Tat that strongly stimulates viral expression by transactivating the long terminal repeat (LTR) promoter. JDV Tat (jTat) promotes the transcription from its own LTR as well as non-cognate LTRs, by recruiting host transcription factors and facilitating transcriptional elongation. Here, we compared the sequence requirements of jTat for transactivation of JDV, bovine immunodeficiency virus (BIV) and human immunodeficiency virus (HIV) LTRs. Results In this study, we identified the minimal protein sequence for LTR activation using jTat truncation mutants. We found that jTat N-terminal residues were indispensable for transactivating the HIV LTR. In contrast, transactivation of BIV and JDV LTRs depended largely on an arginine-rich motif and some flanking residues. Competitive inhibition assay and knockdown analysis showed that P-TEFb was required for jTat-mediated LTR transactivation, and a mammalian two-hybrid assay revealed the robust interaction of jTat with cyclin T1. In addition, HIV LTR transactivation was largely affected by fusion protein at the jTat N-terminus despite the fact that the cyclin T1-binding affinity was not altered. Furthermore, the jTat N-terminal sequence enabled HIV Tat to transactivate BIV and JDV LTRs, suggesting the flexibility at the jTat N-terminus. Conclusion This study showed the distinct sequence requirements of jTat for HIV, BIV and JDV LTR activation. Residues responsible for interaction with cyclin T1 and transactivation response element are the key determinants for transactivation of its cognate LTR. N-terminal residues in jTat may compensate for transactivation of the HIV LTR, based on the flexibility.</p
    corecore