To further extend the practical application of a thermostable and acidic resistance β-mannanase (ManAK) in animal feed additives, an effective strategy that combined directed evolution and metabolic engineering was developed. Four positive mutants (P191M, P194E, S199G and S268Q) with enhanced specific activity (25.5%-60.9%) were obtained. The S199G mutant exhibited 56.7% enhancement of specific activity at 37°C and good thermostability, and this was selected for high-level expression in P. pastoris X33. A multi-functional and scarless genetic manipulation system was proposed and functionally verified (gene deletion, substitution/insertion and point mutation). This was then subjected to Rox1p (an oxygen related transcription regulator) deletion and Vitreoscilla haemoglobin (VHb) co-expression for high enzyme productivity in P. pastoris X33VIIManAK S199G . An excellent strain, named X33VIIManAK S199G Δrox1:: VHb, was achieved by combining these two factors, and then the maximum enzymatic activity was further increased to 3753 U ml -1 , which was nearly twice as much as the maximum production of ManAK in P. pastoris. This work provides a systematic and effective method to improve the enzymatic yield of β-mannanase, promotes the application of ManAK in feed additives, and also demonstrated that a scarless genetic manipulation tool is useful in P. pastoris.
In order to improve the catalytic efficiency of a thermostable and acidophilic β-mannanase (ManAK; derived from marine Aspergillus kawachii IFO 4308), three mutants were designed by amino acid sequence consensus analysis with a second β-mannanase (ManCbs), which also belongs to the glycoside hydrolase family 5 (GH5) and has excellent catalytic efficiency. Three mutants were constructed and their biochemical characteristics were measured after heterologous expression in Pichia pastoris. The results revealed that the kcat/Km values of the three recombinant mannanases ManAKC292V, ManAKL293V, and ManAKL294H were enhanced by 303.0, 280.4, and 210.1%, respectively. Furthermore, ManAKL293V showed greater thermostability than ManAK, retaining 36.5% of the initial enzyme activity after incubation at 80°C for 5min. This study therefore provides a rational design strategy based on consensus sequence analysis to develop industrially valuable β-mannanase for future applications in marine aquafeed.
Aims The aim was to isolate a neotype bifidobacteria strain and evaluate its in vitro probiotic potential. Methods and Results Bifidobacterium pseudolongum YY‐26 (CGMCC 24310) was isolated from faeces of mice treated with low‐molecular‐weight hydrolyzed guar gum (GMPS) and identified based on 16S rRNA sequence and genome sequence. Whole‐genome sequencing obtained using PacBio's single‐molecular and Illumina's paired‐end sequencing technology. A genome of 2.1 Mb in length, with 1877 predicted protein‐coding sequences was obtained. Carbohydrate‐Activity enZyme analysis revealed that YY‐26 encodes 66 enzymes related to carbohydrate metabolism. Whole genome sequence analysis revealed the typical probiotic characteristics of YY‐26, including safety in genetic level and ability to produce beneficial metabolites and extracellular polysaccharides. Ability of extensive carbon source utilization and short‐chain fatty acid production was observed with single YY‐26 cultivation. Considerable acetic acids and lactic acids were determined in GMPS utilization. YY‐26 showed tolerance to simulated gastrointestinal tract and displayed appreciable antioxidant activity of free radical scavenging. Conclusions B. pseudolongum YY‐26 was identified with numerous probiotic‐associated genes and its probiotic characteristics were verified in vitro. Significance and Impact of Study This study supplemented with limited publicly information regarding the genomes of B. pseudolongum strains and revealed the probiotic potential of YY‐26.
Antimicrobial peptides (AMPs) serve as alternative candidates for antibiotics and have attracted the attention of a wide range of industries for various purposes, including the prevention and treatment of piglet diarrhea in the swine industry. Escherichia coli, Salmonella, and Clostridium perfringens are the most common pathogens causing piglet diarrhea. In this study, the antimicrobial peptide gloverin2 (BMGlv2), derived from Bombyx mandarina, was explored to determine the efficient prevention effect on bacterial piglet diarrhea. BMGlv2 was heterologously expressed in Trichoderma reesei Tu6, and its antimicrobial properties against the three bacteria were characterized. The results showed that the minimum inhibitory concentrations of the peptide against E. coli ATCC 25922, S. derby ATCC 13076, and C. perfringens CVCC 2032 were 43.75, 43.75, and 21.86 μg/mL, respectively. The antimicrobial activity of BMGlv2 was not severely affected by high temperature, salt ions, and digestive enzymes. It had low hemolytic activity against rabbit red blood cells, indicating its safety for use as a feed additive. Furthermore, the measurements of the leakage of bacterial cell contents and scanning electron microscopy of C. perfringens CVCC 2032 indicated that BMGlv2 exerted antimicrobial activity by destroying the cell membrane. Overall, this study showed the heterologous expression of the antimicrobial peptide BMGlv2 in T. reesei and verified its antimicrobial properties against three common pathogenic bacteria associated with piglet diarrhea, which can provide a reference for the applications of AMPs as an alternative product in industrial agriculture.
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