scholarly journals a PRODUCTION OF PROTEASE FROM BACILLUS SUBTILIS UNDER SSF AND EFFECT OF ORGANIC SOLVENTS ON LYOPHILIZED PROTEASE PREPARATIONS

2020 ◽  
pp. 1-17
Author(s):  
SELMIHAN SAHIN ◽  
YASEMİN DEMIR ◽  
ISMAIL OZMEN
Keyword(s):  
Bacillus Subtilis ◽  
Organic Solvent ◽  
Organic Solvents ◽  
Specific Activity ◽  
Moisture Level ◽  
Protease Production ◽  
Kinetic Properties ◽  
Buffer Solution ◽  
Fermentation Time ◽  
Increased Activity

Objective: In the present work, protease was produced from Bacillus subtilis under solid-state fermentation (SSF). The effect of lyophilization with different additives on the activity of protease in an organic solvent and kineteci properties was investigated. Methods: Production conditions of protease (fermentation time, moisture level, initial pH, temperature) were optimized. After production, it was partially purified and then, lyophilized with different additives from an aqueous buffer solution containing 98% (w/w) of different additives (pumice, KCl, without additive) for 72 h after freezing in liquid nitrogen. After that, the effect of organic solvents (2.5% and 5% of DCM, ethanol, hexane, toluene) on these lyophilized protease preparations was determined and their kinetic properties were determined. Results: Optimum protease production was obtained with 40% of moisture level, at pH 7.5, 37 °C after 24 h fermentation. It was partially purified by using ammonium sulphate precipitation (20-80%) with 5.8-fold and specific activity of 38 U/mg and then dialysed with 6.4-fold and a specific activity of 35 U/mg. Co-lyophilization of protease with pumice and KCl was increased activity of an enzyme in aqueous organic solvents when compared lyophilized protease without additive. Used solvents, except DCM, were increased activity of lyophilized protease with pumice/KCl. It was found that the lyophilization with pumice and KCl resulted in an increasing in the catalytic efficiency, while it was decreased in Km and Vmax values. Conclusion: The obtained findings demonstrated that protease from B. subtilis can effectively be produced under SSF by using wheat bran and used in industrial applications because of showing improved activity in an organic solvent by co-lyophilization with pumice/KCl.

scholarly journals Characterization of XynC from Bacillus subtilis subsp. subtilis Strain 168 and Analysis of Its Role in Depolymerization of Glucuronoxylan

2006 ◽  
Vol 188 (24) ◽  
pp. 8617-8626 ◽  
Author(s):  
Franz J. St. John ◽  
John D. Rice ◽  
James F. Preston
Keyword(s):  
Bacillus Subtilis ◽  
Specific Activity ◽  
Glycosyl Hydrolase ◽  
Hydrolysis Product ◽  
Genetic System ◽  
Product Formation ◽  
Subtilis Strain ◽  
Kinetic Properties ◽  
Product Analysis ◽  
Flight Mass Spectrometry

ABSTRACT Secretion of xylanase activities by Bacillus subtilis 168 supports the development of this well-defined genetic system for conversion of methylglucuronoxylan (MeGAXn [where n represents the number of xylose residues]) in the hemicellulose component of lignocellulosics to biobased products. In addition to the characterized glycosyl hydrolase family 11 (GH 11) endoxylanase designated XynA, B. subtilis 168 secretes a second endoxylanase as the translated product of the ynfF gene. This sequence shows remarkable homology to the GH 5 endoxylanase secreted by strains of Erwinia chrysanthemi. To determine its properties and potential role in the depolymerization of MeGAXn, the ynfF gene was cloned and overexpressed to provide an endoxylanase, designated XynC, which was characterized with respect to substrate preference, kinetic properties, and product formation. With different sources of MeGAXn as the substrate, the specific activity increased with increasing methylglucuronosyl substitutions on the β-1,4-xylan chain. With MeGAXn from sweetgum as a preferred substrate, XynC exhibited a V max of 59.9 units/mg XynC, a Km of 1.63 mg MeGAXn/ml, and a k cat of 2,635/minute at pH 6.0 and 37°C. Matrix-assisted laser desorption ionization—time of flight mass spectrometry and 1H nuclear magnetic resonance data revealed that each hydrolysis product has a single glucuronosyl substitution penultimate to the reducing terminal xylose. This detailed analysis of XynC from B. subtilis 168 defines the unique depolymerization process catalyzed by the GH 5 endoxylanases. Based upon product analysis, B. subtilis 168 secretes both XynA and XynC. Expression of xynA was subject to MeGAXn induction; xynC expression was constitutive with growth on different substrates. Translation and secretion of both GH 11 and GH 5 endoxylanases by the fully sequenced and genetically malleable B. subtilis 168 recommends this bacterium for the introduction of genes required for the complete utilization of products of the enzyme-catalyzed depolymerization of MeGAXn. B. subtilis may serve as a model platform for development of gram-positive biocatalysts for conversion of lignocellulosic materials to renewable fuels and chemicals.

Properties of the reversible nonoxidative vanillate / 4-hydroxybenzoate decarboxylase from Bacillus subtilis

2008 ◽  
Vol 54 (1) ◽  
pp. 75-81 ◽  
Author(s):  
Boguslaw Lupa ◽  
Delina Lyon ◽  
Lindsey N. Shaw ◽  
Magdalena Sieprawska-Lupa ◽  
Juergen Wiegel
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gene Sequence ◽  
Specific Activity ◽  
Kinetic Properties ◽  
Carboxylase Activity ◽  
Native Form ◽  
Streptomyces Sp ◽  
Sequence Identification ◽  
Bacillus Subtilis Atcc

Bacillus subtilis (ATCC 6051) reversibly decarboxylates vanillate and 4-hydroxybenzoate under both aerobic and anoxic conditions. Thus, we have identified on the basis of gene sequence homology with Sedimentibacter hydroxybenzoicus and Streptomyces sp. strain D7, a putative B. subtilis hydroxybenzoate decarboxylase. The native form of this enzyme is encoded by 3 genes yclBCD (GI Sequence Identification Nos.: 2632649, 2632650, 2632651) that we have renamed during this research as bsdBCD to align with existing nomenclature. The bsdD gene is reported in the database to be 690 bp; however, our sequence analysis revealed that the size of this gene is in fact 228 bp, an observation that results in a shortening of YclD (i.e., BsdD) from 229 to 75 aa. The corresponding bsdBCD genes were cloned into Escherichia coli , and the heterologously expressed enzyme was assayed for activity. The decarboxylase exhibited a narrow substrate range, with only 2 of the tested substrates, vanillate (Kmapp = 4 mmol·L–1) and 4-hydroxybenzoate (Kmapp = ~1 mmol·L–1), being decarboxylated. The recombinant enzyme had properties similar to that of the native enzyme in respect to specific activity, kinetic properties, bidirectional decarboxylase–carboxylase activity, oxygen insensitivity, and substrate specificity.

scholarly journals Glycerol Carbonate: A Novel Biosolvent with Strong Ionizing and Dissociating Powers

2012 ◽  
Vol 2012 ◽  
pp. 1-6
Author(s):  
Guangnan Ou ◽  
Biyan He ◽  
Xuejing Li ◽  
Jianhui Lei
Keyword(s):  
Bacillus Subtilis ◽  
Organic Solvent ◽  
Organic Solvents ◽  
Protein Denaturation ◽  
Nonaqueous Solvents ◽  
Ionization Constants ◽  
Glycerol Carbonate ◽  
Protonation State ◽  
Liquid Media ◽  
Ionizable Groups

The activity of biocatalysts in nonaqueous solvents is related to the interaction of organic solvents with cells or enzymes. The behavior of proteins is strongly dependent on the protonation state of their ionizable groups, which ionization constants are greatly affected by the solvent. Due to the weak ionizing and dissociating powers of common organic solvents, the charge of the protein will change significantly when the protein is transferred from water to common organic solvents, resulting in protein denaturation. In this work, glycerol carbonate (GC) was synthesized, which ionizing and dissociating abilities were very close to those of water. Transesterification activities ofCandida antarcticalipase B (CALB) in GC were comparable to those in water and remained constant during 4-week storage.Bacillus subtilisandSaccharomyecs cerevisiaewere cultured in liquid media containing GC with test tubes. In the medium containing low GC concentration,Bacillus subtilisandSaccharomyecs cerevisiaegrew well as in a medium containing no organic solvent, but, in the medium containing high GC concentration, the growth ofBacillus subtilisandSaccharomyecs cerevisiaewas suppressed. The results suggested that GC is a potential biosolvent, which has great significance to biocatalysis in nonaqueous solvents.

scholarly journals Laccase and Its Mutant Displayed on the Bacillus subtilis Spore Coat for Oxidation of Phenolic Compounds in Organic Solvents

Catalysts ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 606
Author(s):  
Silu Sheng ◽  
Edgardo T. Farinas
Keyword(s):  
Bacillus Subtilis ◽  
Organic Solvents ◽  
Environmental Changes ◽  
Sinapic Acid ◽  
Product Yield ◽  
Spore Coat ◽  
Subtilis Spore ◽  
Bacillus Subtilis Spore ◽  
Cota Laccase ◽  
Inert Surface

Enzymes displayed on the Bacillus subtilis spore coat have several features that are useful for biocatalysis. The enzyme is preimmobilized on an inert surface of the spore coat, which is due to the natural sporulation process. As a result, protein stability can be increased, and they are resistant to environmental changes. Next, they would not lyse under extreme conditions, such as in organic solvents. Furthermore, they can be easily removed from the reaction solution and reused. The laboratory evolved CotA laccase variant T480A-CotA was used to oxidize the following phenolic substrates: (+)-catechin, (−)-epicatechin, and sinapic acid. The kinetic parameters were determined and T480A-CotA had a greater Vmax/Km than wt-CotA for all substrates. The Vmax/Km for T480A-CotA was 4.1, 5.6, and 1.4-fold greater than wt-CotA for (+)-catechin, (−)-epicatechin, and sinapic acid, respectively. The activity of wt-CotA and T480A-CotA was measured at different concentrations from 0–70% in organic solvents (dimethyl sulfoxide, ethanol, methanol, and acetonitrile). The Vmax for T480A-CotA was observed to be greater than the wt-CotA in all organic solvents. Finally, the T480A-CotA was recycled 7 times over a 23-h period and up to 60% activity for (+)-catechin remained. The product yield was up to 3.1-fold greater than the wild-type.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

scholarly journals Effects of Organic Solvent on Curing Behavior and Storage Stability for Waterborne Paint Containing Catalyst Encapsulated in Micelles

Coatings ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 722
Author(s):  
Shuji Yomo
Keyword(s):  
Organic Solvent ◽  
Organic Solvents ◽  
Storage Stability ◽  
Dibutyltin Dilaurate ◽  
Film Properties ◽  
Surfactant Micelles ◽  
Catalytic Function ◽  
Hydrophilic Lipophilic Balance ◽  
Before And After ◽  
And Storage

In this study, a 2-pack isocyanate curing waterborne paint (without organic solvents) encapsulating dibutyltin dilaurate (hereinafter, DBTL) in nonionic surfactant micelles with an hydrophilic–lipophilic balance of 13–14 in advance releases DBTL when the micelles are collapsed at 80 °C or higher, whereby the curing progresses rapidly. On the other hand, the viscosity levels of the paint before and after being left at 40 °C for 1 h are almost the same. Organic solvents are mandatory for waterborne paints to provide paint and film properties, but they might collapse the micelles when they are formulated in the paint. In this study, we investigate whether the abovementioned paint containing organic solvents can develop switching functionality in terms of maintaining the storage stability at 40 °C and expressing a catalytic function at 80 °C to progress the curing. As a result, we find that if the solubility of the organic solvent in water at 20 °C is at least 10 g/100 mL and the boiling point is ≤200 °C, both curing and storage stability can be achieved.

Protease production by Bacillus subtilis in oxygen-controlled, glucose fed-batch fermentations

1988 ◽  
Vol 28 (4-5) ◽  
pp. 404-408 ◽  
Author(s):  
Manoj M. Kole ◽  
Indira Draper ◽  
Donald F. Gerson
Keyword(s):  
Bacillus Subtilis ◽  
Protease Production ◽  
Fed Batch

Purification to homogeneity and characterization of nonproteolyzed potato (Solanum tuberosum) tuber hexokinase 1

Botany ◽  
2011 ◽  
Vol 89 (5) ◽  
pp. 289-299 ◽  
Author(s):  
Marie-Claude Moisan ◽  
Jean Rivoal
Keyword(s):  
Solanum Tuberosum ◽  
Specific Activity ◽  
Solanum Tuberosum L ◽  
Extraction Procedure ◽  
Hydrophobic Interaction Chromatography ◽  
Solanum Chacoense ◽  
Kinetic Properties ◽  
Chromatographic Separations ◽  
The Stability ◽  
Fast Flow

We have developed an extraction procedure that improves the stability of potato ( Solanum tuberosum L.) tuber hexokinase (HK) after extraction. Using this protocol, we showed that at least four HK isoforms are present in this tissue, and they can be separated by hydrophobic-interaction chromatography on a butyl-Sepharose™ 4 Fast Flow column. One of the main HK isoforms was purified to homogeneity using further chromatographic separations on red dye, DEAE Fractogel, hydroxyapatite, cibacron blue, and MonoQ matrices. HK-specific activity of this fraction (10.2 U·mg protein–1) corresponds to an enrichment of more than 5500-fold, with a yield of 0.9%. This is the highest reported HK-specific activity from a plant source. The purified enzyme consisted of a monomer with a subunit apparent Mr of 51 kDa when analyzed by SDS–PAGE. This polypeptide was recognized by affinity-purified anti- Solanum chacoense Bitt. recombinant HK IgGs. The protein was digested with trypsin and its digestion products were subjected to MS – MS sequencing after HPLC separation. The sequences of these tryptic peptides matched the predicted coding sequence of the S. tuberosum HK1 gene with a coverage of 57%. Examination of the kinetic properties of the purified protein HK1 indicates that it may be regulated by the internal O2 concentration of the tuber because of its sensitivity to acidic pHs and inhibition by ADP.

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