Dataset Information

Comparative biochemical analysis of full-length and truncated BaqA α-amylases from Bacillus aquimaris MKSC 6.2.

ABSTRACT: BaqA is a raw starch degrading α-amylase produced by the marine bacterium Bacillus aquimaris MKSC 6.2, associated with soft corals. This α-amylase belongs to a new subfamily Glycoside Hydrolases (GH) 13_45 which has several unique characteristics, namely, a pair of tryptophan residues Trp201 and Trp202, a distinct LPDIx signature in the Conserved Sequence Region-V (CSR-V), and an elongated C-terminus containing five aromatic residues. The research aims to investigate physicochemical, kinetics, and biochemical properties of BaqA. In this study, the full-length enzyme (BaqA) and a truncated form of BaqA (designated as BaqAΔC), lacking the C-terminal 34 amino acids were constructed and expressed in Escherichia coli ArcticExpress (DE3). BaqA formed inclusion bodies, while BaqAΔC was produced as a soluble protein. Purified and refolded BaqA exhibited a catalytic efficiency (k _cat/K _m) of 53.1 ± 6.3 mL mg^-1 s^-1 at 40 °C and pH 7.5, whereas the purified BaqAΔC displayed k _cat/K _m of 11.4 ± 1.3 mL mg^-1 s^-1 under the optimum condition of 50 °C and pH 6.5. Moreover, BaqAΔC showed a slight reduction in the binding affinity towards sago granules. Interestingly, BaqAΔC displayed robust stability and halotolerant properties compared to BaqA. BaqAΔC maintained 50 % amylolytic activity for up to 6 h, whereas BaqA lost over 50 % of its activity within 90 min. Furthermore, BaqAΔC showed a remarkable increase in amylolytic activity upon the addition of NaCl, with an optimum concentration of 0.5 M. Even at a high salt concentration (1.5 M NaCl), BaqAΔC retained over 50 % of its residual activity. Taken together, its solubility, amylolytic activity, stability, ability to degrade raw starch, and moderate halotolerance make BaqAΔC a promising candidate for various starch processing industries.

SUBMITTER: Ulpiyana A

PROVIDER: S-EPMC11301243 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Comparative biochemical analysis of full-length and truncated BaqA α-amylases from <i>Bacillus aquimaris</i> MKSC 6.2.

Ulpiyana Ayra A Frima Fina Khaerunnisa FK Annisa Diandra Sekar DS Tan Josephine Claudia JC Puspasari Fernita F Aditama Reza R Ihsanawati Natalia Dessy D

Heliyon 20240705 14

BaqA is a raw starch degrading α-amylase produced by the marine bacterium <i>Bacillus aquimaris</i> MKSC 6.2, associated with soft corals. This α-amylase belongs to a new subfamily Glycoside Hydrolases (GH) 13_45 which has several unique characteristics, namely, a pair of tryptophan residues Trp201 and Trp202, a distinct LPDIx signature in the Conserved Sequence Region-V (CSR-V), and an elongated C-terminus containing five aromatic residues. The research aims to investigate physicochemical, kine ...[more]

PMID: 39108854

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Project description:BackgroundBt-cry1Ac gene has been reputedly effective against Helicoverpa armigera a notorious lepidopteran pest. Reports on the expression of full-length and truncated cry1Ac genes in plants for effective resistance against Helicoverpa sp. have been documented however, their performance is still ambiguous. Moreover, the question remains to be addressed that truncation of 3' end of the native gene was documented and suggested for active insecticidal toxin production while the most successful transgenic event(s) of commercialized-cotton are based on full-length of the cry gene. Therefore, we performed a comparative study on the efficacy of the two versions of cry1Ac genes (full-length: 3,510 bp and truncated: 1,845 bp) in T0 and T1 transgenic tomato plants and analyzed the extent of protection against H. armigera and also compared the results with our previous findings related to a successful transgenic tomato line Ab25E, expressing cry1Ab gene. The integration of cry1Ac gene(s) in T0 transgenic plants and its inheritance in T1 progeny was observed by PCR, RT-PCR and Southern blot hybridization analysis while, the toxin integrity, expression and toxicity was monitored by Western immunoassay, DAS-ELISA and insect bioassay respectively.ResultsAn average transformation frequency and Bt-Cry protein content of 16.93 ± 2.10 and 0.0020-0.0128% of total soluble protein (TSP) was obtained with pRD400 vector (Trcry1Ac) while, a much lower value of 9.30 ± 2.041 and 0.0001 - 0.0026% of TSP was observed with pNBRI-1 vector (Flcry1Ac), respectively. The promising Trcry1Ac T0 transgenic plants and their T1 progeny gave full protection from H. armigera. Although Flcry1Ac gene showed lower transformation frequency and lower expression, it showed higher toxicity to H. armigera when compared with truncated Trcry1Ac gene.ConclusionsThe full-length cry1Ac gene can be redesigned for higher expression and performance in dicots or a hybrid gene could be designed having a blend of strong receptor binding and stable expression characteristics for enhanced efficacy and toxicity to the susceptible insects.

Project description:Accumulation of amyloid-β peptides is a dominant feature in the pathogenesis of Alzheimer's disease; however, it is not clear how individual amyloid-β species accumulate and affect other neuropathological and clinical features in the disease. Thus, we compared the accumulation of N-terminally truncated amyloid-β and full-length amyloid-β, depending on disease stage as well as brain area, and determined how these amyloid-β species respectively correlate with clinicopathological features of Alzheimer's disease. To this end, the amounts of amyloid-β species and other proteins related to amyloid-β metabolism or Alzheimer's disease were quantified by enzyme-linked immunosorbent assays (ELISA) or theoretically calculated in 12 brain regions, including neocortical, limbic and subcortical areas from Alzheimer's disease cases (n = 19), neurologically normal elderly without amyloid-β accumulation (normal ageing, n = 13), and neurologically normal elderly with cortical amyloid-β accumulation (pathological ageing, n = 15). We observed that N-terminally truncated amyloid-β42 and full-length amyloid-β42 accumulations distributed differently across disease stages and brain areas, while N-terminally truncated amyloid-β40 and full-length amyloid-β40 accumulation showed an almost identical distribution pattern. Cortical N-terminally truncated amyloid-β42 accumulation was increased in Alzheimer's disease compared to pathological ageing, whereas cortical full-length amyloid-β42 accumulation was comparable between Alzheimer's disease and pathological ageing. Moreover, N-terminally truncated amyloid-β42 were more likely to accumulate more in specific brain areas, especially some limbic areas, while full-length amyloid-β42 tended to accumulate more in several neocortical areas, including frontal cortices. Immunoprecipitation followed by mass spectrometry analysis showed that several N-terminally truncated amyloid-β42 species, represented by pyroglutamylated amyloid-β11-42, were enriched in these areas, consistent with ELISA results. N-terminally truncated amyloid-β42 accumulation showed significant regional association with BACE1 and neprilysin, but not PSD95 that regionally associated with full-length amyloid-β42 accumulation. Interestingly, accumulations of tau and to a greater extent apolipoprotein E (apoE, encoded by APOE) were more strongly correlated with N-terminally truncated amyloid-β42 accumulation than those of other amyloid-β species across brain areas and disease stages. Consistently, immunohistochemical staining and in vitro binding assays showed that apoE co-localized and bound more strongly with pyroglutamylated amyloid-β11-x fibrils than full-length amyloid-β fibrils. Retrospective review of clinical records showed that accumulation of N-terminally truncated amyloid-β42 in cortical areas was associated with disease onset, duration and cognitive scores. Collectively, N-terminally truncated amyloid-β42 species have spatiotemporal accumulation patterns distinct from full-length amyloid-β42, likely due to different mechanisms governing their accumulations in the brain. These truncated amyloid-β species could play critical roles in the disease by linking other clinicopathological features of Alzheimer's disease.

Dataset Information

Comparative biochemical analysis of full-length and truncated BaqA α-amylases from Bacillus aquimaris MKSC 6.2.

Publications

Comparative biochemical analysis of full-length and truncated BaqA α-amylases from <i>Bacillus aquimaris</i> MKSC 6.2.

Similar Datasets

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