Streptomyces mobaraenesis transglutaminase
has been widely used in food processing. We here significantly improved
the catalytic properties of S2P-S23V-Y24N-S199A-K294L (TGm1), a highly
stabilized variant of the transglutaminase. First, a virtual proline
scan was performed based on folding free energy changes to obtain
TGm1 variants with enhanced thermostability. Second, the residues
within 15 Å of Cys64 in the enzyme–substrate complex of
TGm1 were subjected to virtual saturation mutagenesis to generate
the variants with reduced binding free energy and increased activity.
After combining the favorable mutations, we obtained the variant FRAPD-TGm1-E28T-A265P-A287P
(FRAPD-TGm2), exhibiting 66.9 min of half-life at 60 °C (t
1/2(60 °C)), 67.8 °C of melting temperature
(T
m), and 71.8 U/mg of specific activity,
which are 2-fold, 2.6 °C, and 43.8% higher than those of FRAPD-TGm1,
respectively. At last, to increase the surface negative net charge
of FRAPD-TGm2, we introduced the mutations N96E-S144E-N163D-R183E-R208E-K325E,
yielding FRAPD-TGm3. The latter’s t
1/2(60 °C), T
m, and specific activity
were 122.9 min, 68.6 °C, and 83.7 U/mg, which are 83.8%, 0.8
°C, and 16.6% higher than the former, respectively. FRAPD-TGm3
is thus a robust candidate for transglutaminase application.
The objective was to study the effects of microencapsulated organic acids (OA) and essential oils (EO) on growth performance, immune system, gut barrier function, nutrient digestion and absorption, and abundance of enterotoxigenic Escherichia coli F4 (ETEC F4) in the weaned piglets challenged with ETEC F4. Twenty-four ETEC F4 susceptible weaned piglets were randomly distributed to four treatments including (1) sham-challenged control (SSC; piglets fed a control diet and challenged with phosphate-buffered saline (PBS)); (2) challenged control (CC; piglets fed a control diet and challenged with ETEC F4); (3) antibiotic growth promoters (AGP; CC + 55 mg·kg-1 of Aureomycin); and (4) microencapsulated OA and EO [P(OA+EO); (CC + 2 g·kg-1 of microencapsulated OA and EO]. The ETEC F4 infection significantly induced diarrhea at 8, 28, 34, and 40 hours post-inoculation (hpi) (P < 0.05) in the CC piglets. At 28 days post-inoculation (dpi), piglets fed P(OA+EO) had a lower (P < 0.05) diarrhea score compared to those fed CC, but the P(OA+EO) piglets had a lower (P < 0.05) diarrhea score compared to those fed the AGP diets at 40 dpi. The ETEC F4 infection tended to increase in vivo gut permeability measured by the oral gavaging fluorescein isothiocyanate-dextran 70 kDa (FITC-D70) assay in the CC piglets compared to the SCC piglets (P = 0.09). The AGP piglets had higher FITC-D70 flux than P(OA+EO) piglets (P < 0.05). The ETEC F4 infection decreased mid-jejunal VH in the CC piglets compared to the SCC piglets (P < 0.05). The P(OA+EO) piglets had higher (P < 0.05) VH in the mid-jejunum than the CC piglets. The relative mRNA abundance of SGLT1 and B0AT1 was reduced (P < 0.05) by ETEC F4 inoculation when compared to the SCC piglets. The AGP piglets had a greater relative mRNA abundance of B0AT1 than the CC piglets (P < 0.05). The ETEC F4 inoculation increased the protein abundance of OCLN (P < 0.05), and the AGP piglets had the lowest relative protein abundance of OCLN among the challenged groups (P < 0.05). The supplementation of microencapsulated OA and EO enhanced intestinal morphology and showed anti-diarrhea effects at a one-time point in weaned piglets challenged with ETEC F4. Even if more future studies can be required for further validation, this study brings evidence that microencapsulated OA and EO combination can be useful within the tools to be implemented in strategies for alternatives to antibiotics in swine production.
Streptomyces transglutaminase
(TGase) is widely
used to improve food texture properties. In this study, random mutagenesis
and site-directed genetic modification were used to improve the production
of TGase in Streptomyces mobaraensis. First, S. mobaraensis DSM40587 (smWT)
was subjected to atmospheric and room-temperature plasma mutagenesis,
and then a mutant (smY2019) with a 5.5-fold increase in TGase yield
was screened from approximately 3000 × 25 (round) mutants. Compared
to smWT, smY2019 exhibits a 3.2-fold higher TGase mRNA level and two
site mutations within the −10 region of the TGase promoter.
The recombinant expression analysis in the TGase-deficient S. mobaraensis suggests that the mutated TGase promoter
is more robust than the wild-type one. Finally, we integrated two
additional TGase expression cassettes into the smY2019 genome, yielding
the recombinant strain smY2019-3C with a 103% increase in TGase production
compared to smY2019. The smY2019-3C strain with 40 U/mL of TGase yield
could be a suitable candidate for the industrial production of TGase.
In this study, the thermostability of an acid-resistant GH11 xylanase (xynA) from Aspergillus niger AG11 was enhanced through systematic modification of its four highly flexible regions (HFRs) predicted using MD simulations. Among them, HFR I (residues 92−100) and HFR II (residues 121−130) were modified by iterative saturation mutagenesis (ISM), yielding mutants G92F/G97S/G100K and T121V/A124P/I126V/T129L/A130N, respectively. For HFR III, the N-(residues 1−37) and Ctermini (residues 179−188) were, respectively, substituted with the corresponding sequences from thermophilic EvXyn11 TS and Nesterenkonia xinjiangensis xylanase. N-Glycosylation was introduced into HFR IV (residues 50−70) through site-directed mutation (A55N/D57S/S61N) and the recombinant expression in A. niger AG11. Combining these positive mutations from each HFR yielded the variant xynAm1 with 137.6-and 1.3-fold increases in half-life at 50 °C and specific activity compared to the wild-type xynA, respectively. With the highest thermostability at 80 and 90 °C in reports, xynAm1 could be a robust candidate for industrial applications in functional foods, feed products, and bioethanol production.
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