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Enhanced catalytic activity of Bacillus aryabhattai P1 protease by modulation with nanoactivator.

ABSTRACT: In the developing area of modern nanobiotechnology, the research is being focused on enhancement of catalytic performance in terms of efficiency and stability of enzymes to fulfill the industrial demand. In the context of this interdisciplinary era, we isolated and identified alkaline protease producer Bacillus aryabhattai P1 by polyphasic approach and then followed one variable at a time approach to optimize protease production from P1. The modified components of fermentation medium (g/L) were wheat bran 10, soybean flour 10, yeast extract 5, NaCl 10, KH2PO4 1, K2HPO4 1 and MgSO4·7H2O 0.2 (pH 9). The optimum alkaline protease production from P1 was recorded 75 ± 3 U/mg at 35 °C and pH 9 after 96 h of fermentation period. Molecular weight of partially purified P1 alkaline protease was 26 KDa as revealed by SDS-PAGE. Calcium based nanoceramic material was prepared by wet chemical precipitation method and doped in native P1 protease for catalytic activity enhancement. Catalytic activity of modified P1 protease was attained by nanoactivator mediated modulation was more by 5.58 fold at pH 10 and 30 °C temperature. The nanoceramic material named as nanoactivator, with grain size of 40-60 nm was suitable to redesign the active site of P1 protease. Such types of modified proteases can be used in different nanobiotechnological applications.

SUBMITTER: Pathak AP

PROVIDER: S-EPMC7276444 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Enhanced catalytic activity of <i>Bacillus aryabhattai</i> P1 protease by modulation with nanoactivator.

Pathak Anupama P AP Rathod Mukundraj G MG Mahabole Megha P MP Khairnar Rajendra S RS

Heliyon 20200604 6

In the developing area of modern nanobiotechnology, the research is being focused on enhancement of catalytic performance in terms of efficiency and stability of enzymes to fulfill the industrial demand. In the context of this interdisciplinary era, we isolated and identified alkaline protease producer <i>Bacillus aryabhattai</i> P1 by polyphasic approach and then followed one variable at a time approach to optimize protease production from P1. The modified components of fermentation medium (g/L ...[more]

PMID: 32529068

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Project description:Biosurfactants are eco-friendly surface-active molecules recommended for enhanced oil recovery techniques. In the present study, a potential lipopeptide (biosurfactant) encoding the iturin A gene was synthesized from Bacillus aryabhattai. To improvise the yield of the lipopeptide for specific applications, current research tends toward engineering and expressing recombinant peptides. An iturin A gene sequence was codon-optimized, amplified with gene-specific primers, and ligated into the pET-32A expression vector to achieve high-level protein expression. The plasmid construct was transformed into an E. coli BL21 DE3 host to evaluate the expression. The highly expressed recombinant iturin A lipopeptide was purified on a nickel nitrilotriacetic acid (Ni-NTA) agarose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the purity and molecular mass of iturin A was 41 kDa. The yield of recombinant iturin A was found to be 60 g/L with a 6.7-fold increase in comparison with our previously published study on the wild strain. The approach of cloning a functional fragment of partial iturin A resulted in the increased production of the lipopeptide. When motor oil was used, recombinant protein iturin A revealed a biosurfactant property with a 74 ± 1.9% emulsification index (E24). Purified recombinant protein iturin A was characterized by mass spectrometry. MALDI-TOF spectra of trypsin digestion (protein/trypsin of 50:1 and 25:1) showed desired digested mass peaks for the protein, further confirming the identity of iturin A. The iturin A structure was elucidated based on distinctive spectral bands in Raman spectra, which revealed the presence of a peptide backbone and lipid. Recombinant iturin A was employed for enhanced oil recovery through a sand-packed column that yielded 61.18 ± 0.85% additional oil. Hence, the novel approach of the high-level expression of iturin A (lipopeptide) as a promising biosurfactant employed for oil recovery from Bacillus aryabhattai is not much reported. Thus, recombinant iturin A demonstrated its promising ability for efficient oil recovery, finding specific applications in petroleum industries.

Project description:BackgroundHumic substances (HS) form the largest proportion among all the constituents of soil organic matter and are a key component of the terrestrial ecosystem. HS plays a multifunctional role in the environment by controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants. The rate of formation of HS in soils determines its productivity and carbon sequestration capacity. Enhancement of HS synthesis in the soil through the microbial route not only increases CO2 sequestration but also mitigates the greenhouse gas emissions in the environment.ResultIn this study, we attempted to understand the mechanism of formation and enhancement of HS from coir pith wastes using the tyrosinase produced by Bacillus aryabhattai TFG5. The bacterium TFG5 isolated from the termite garden produced the tyrosinase (1.34 U mL-1) and laccase (2.1 U mL-1) at 48 h and 60 h of fermentation, respectively. The extracellular tyrosinase from B. aryabhattai TFG5 was designated as TyrB. Homology modeling of TyrB revealed a structure with a predicted molecular mass of 35.23 kDa and two copper ions in the active center with its conserved residues required for the tyrosinase activity. TyrB efficiently transformed and polymerized standard phenols, such as p-cresol, p-hydroxyl benzoic acid, Levo DOPA, and 2,6 DMP, besides transforming free phenols in coir pith wash water (CWW). Additionally, UV-Vis and FT-IR spectra of the degradation products of the coir pith treated with TyrB revealed the formation of HS within 3 days of incubation. Furthermore, the E472/664 ratio of the degradation products revealed a higher degree of condensation of the aromatic carbons and the presence of more aliphatic structures in the HS.ConclusionThe results confirmed the influence of TyrB for the effective synthesis of HS from coir pith wastes. The results of the present study also confirm the recently accepted theory of humification proposed by the International Humic Substances Society.

Dataset Information

Enhanced catalytic activity of Bacillus aryabhattai P1 protease by modulation with nanoactivator.

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Enhanced catalytic activity of <i>Bacillus aryabhattai</i> P1 protease by modulation with nanoactivator.

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