Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: Differential role of metal ions in stability and activity

Sinha, Rajeshwari; Khare, Sunil Kumar

doi:10.1016/j.biortech.2012.11.024

Cited by 71 publications

(41 citation statements)

References 19 publications

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“…Halophilic enzymes are thus uniquely adapted to function in low water/ non-aqueous media (Kumar and Khare, 2012). Significant solvent stability among halophilic enzymes has been reported by different groups (Shafiei et al, 2011; Li and Yu, 2012; Li et al, 2012; Sinha and Khare, 2013a). However, very little has been investigated about the structure of halophilic enzymes in organic solvents.…”

Section: Effect Of Denaturants On Halophilic Proteins: Protective Rolmentioning

confidence: 94%

“…Differential roles of divalent Ca 2+ and monovalent Na + in preventing unfolding and regulation of catalytic activity respectively was also reported recently for Bacillus sp. EMB9 protease (Sinha and Khare, 2013a). Salt and divalent metal ions were reported to independently stabilize and regulate catalysis and folding of RNase H1 from Hbt.…”

Section: Salt Is Essential For Maintaining Structure and Function Of mentioning

confidence: 99%

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Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

Sinha

Khare

2014

Front. Microbiol.

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Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants.

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Section: Effect Of Denaturants On Halophilic Proteins: Protective Rolmentioning

confidence: 94%

Section: Salt Is Essential For Maintaining Structure and Function Of mentioning

confidence: 99%

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

Sinha

Khare

2014

Front. Microbiol.

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“…For reactivation, the metal-free enzyme was incubated with divalent metal ions (Fe 2+ , Ba 2+ , Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ , Ca 2+ , Mg 2+ and Mn 2+ ) at a final concentration of 1 mM for 10 min, followed by the remaining activity determination. The procedure used to assess the metal ions was also used to determine the effects of chemical agents (Triton X-100, SDS, Tween20, Tween80 and urea) and organic reagents (methanol, ethanol, acetonitrile, isopropanol and DMSO) [22, 23]. The activity in the absence of any additives was used as the control (100%).…”

Section: Methodsmentioning

confidence: 99%

Heterologous expression of cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production

Jiang

Zhou

et al. 2018

BMC Biotechnol

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BackgroundCyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries.ResultsIn this study, the cgt gene encoding α-CGTase from Paenibacillus macerans was codon-optimized (opt-cgt) and cloned into pET-28a (+) for intracellular expression in E. coli BL21 (DE3). The Opt-CGT was purified by Ni2+-NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75 U·mg− 1. In addition, the enzyme was adopted to transform L-AA into 9.1 g/L of AA-2G. Finally, more economic substrates, including β-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively.ConclusionsN-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His6-tags on the C- and N-terminal, the recovery of purification by Ni2+-NTA resin is appreciably enhanced. α-cyclodextrin was the ideal glycosyl donor for AA-2G production. In addition, the selection of low cost glycosyl donors would make the process of AA-2G production more economically competitive.

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“…Understanding their biological roles; could allow proteases to be manipulated in many industries, in therapeutics and also academia [1]. Many studies have been carried out on the application of microbial proteases in detergents, foods, pharmaceuticals, leather dehairing, bio-waste disposal, silk degumming, and silver recovery [2,3,4,5,6,7,8]. There are several methods available to improve the yield and activity of proteases such as optimization in fermentation, molecular recombinant and site directed mutagenesis [9].…”

Section: Introductionmentioning

confidence: 99%

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2017

JESR

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Abstract:Protease is an enzyme that catalyses the hydrolysis of peptide bond in polypeptide chain and hold a wide range of applications in industry. The aim of this study is to clone and to express several genes encoding proteases from alkalitolerant bacteria Bacillus lehensis strain G1. A total of 13 genes encoding proteases have been selected using bioinformatics approach. These genes were then amplified using polymerase chain reaction (PCR) method. Subsequently, the PCR product was cloned into cloning vector pGEM®T easy and transformed into competent cell E. coli DH5α. The transformants were further verified by sequencing. The positive cloned were subcloned into the expression vector and were then expressed in Luria Bertani medium in the present of IPTG using E. coli BL21. The expressions of recombinant proteases were optimized for several hours at different temperatures, 16-37°C. Furthermore, structural prediction was performed using Modeller v9.18 for BleG1_1940. Each generated model was verified for overall completeness and bias, using PROCHECK, ERRAT, and Verify 3D. The overall quality of the model was relatively good with percentage of Ramachandran plot is 96.3%, PROCHECK is 86.2% and ERRAT score is 95%,.

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Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: Differential role of metal ions in stability and activity

Cited by 71 publications

References 19 publications

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

Heterologous expression of cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production

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