Cloning and Expression of Levansucrase Gene of Bacillus velezensis BM-2 and Enzymatic Synthesis of Levan

Levan is a versatile and valuable fructose homopolymer, and a few bacterial strains have been found to produce levan. Although levan products have numerous specific functions, their application and promotion were limited by the production capacity and production cost. Bacillus velezensis BM-2 is a levan-synthesizing strain, but its levan production is too low to apply. In this study, the levansucrase gene of B. velezensis BM-2 was cloned to plasmid pET-32a-Acma-zz, and the recombinant plasmids were transferred to Escherichia coli BL21. A transformed clone was selected to express and secrete the fusion enzymes with an Acma-tag efficiently. The expressed products were further purified by a self-developed separating material called bacterial enhancer matrix (BEM) particles. The purification efficiency was 93.4%, with a specific activity of 16.589 U/mL protein. The enzymatic reaction results indicated that the optimal reaction temperature is 50 °C, the optimal pH of the acetate buffer is 5.6, and the buffer system greatly influenced the enzyme activity. The enzyme activity was enhanced to 130% in the presence of 5 mM Ca2+, K+, Zn2+, and Mn2+, whereas it was almost abolished in the case of Cu2+ and Fe3+. The values of Km, kcat, and kcat/Km were 17.41 mM, 376.83 s−1, and 21.64 mM−1s−1, respectively. The enzyme amount of 20 U/g sucrose was added to the system containing 400 g/L sucrose, and the levan products with a concentration of 120 g/L reached after an incubation of 18 h, which was 8 times that of the yield before optimization. The results of molecular docking analysis indicated that the Asp86 might act as a nucleophilic catalytic residue for sucrose, Arg246 and Asp247 act as transition state stabilizer of transfructosylation, and Glu340 and Arg306 were recognized as general acid donors. They formed the catalytic-groups triad. The unique properties and catalytic activity of the levansucrase suggest that it deserves further research and might have good industrial application prospects.

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Effects of music playing on biological molecules

MATEC Web of Conferences ◽

10.1051/matecconf/201821005006 ◽

2018 ◽

Vol 210 ◽

pp. 05006 ◽

Cited By ~ 1

Author(s):

Catia Algieri ◽

Claudio Guarnaccia ◽

Vincenzo Barone ◽

Maria Raffaella Gullo ◽

Laura Donato

Keyword(s):

Enzyme Activity ◽

Detrimental Effect ◽

Specific Activity ◽

Time Lag ◽

Enzymatic Reaction ◽

Biological Molecules ◽

National Council ◽

Musical Piece ◽

Tyrosinase Enzyme ◽

Positive Effect

In this work the effect of two different musical pieces (called “Reminiscenza” and “Pioggia”) on the tyrosinase enzyme activity was investigated. The l-DOPA production by the enzymatic reaction was measured during the continuous playing of these musical pieces. Experiments were performed in the laboratories of the Institute of Membrane Technology (ITM) of the Italian National Council for Research (CNR). The results showed that a positive effect was exercised by the “Reminiscenza” piece, which determined an increase of the specific activity about of 30 % with respect to the value measured in the absence of sound. On the contrary, “Pioggia” piece had a detrimental effect on the enzymatic process. In particular, a time lag on the l-DOPA production, during the first minutes of the reaction, was detected. After this period, an increase of the reaction velocity occurred even if the enzyme activity was lower than the value obtained in the absence of music. These results show that the peculiar characteristics of a musical piece can exercise a positive or negative action on biological elements and open the way to further studies in this area.

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A β-Mannanase from Bacillus subtilis B36: Purification, Properties, Sequencing, Gene Cloning and Expression in Escherichia coli

Zeitschrift für Naturforschung C ◽

10.1515/znc-2006-11-1212 ◽

2006 ◽

Vol 61 (11-12) ◽

pp. 840-846 ◽

Cited By ~ 17

Author(s):

Ya Nan Li ◽

Kun Meng ◽

Ya Ru Wang ◽

Bin Yao

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Bacillus Subtilis ◽

Specific Activity ◽

Ph Value ◽

High Specificity ◽

Apparent Molecular Weight ◽

Cloning And Expression ◽

Gene Cloning And Expression ◽

Reading Frame

Abstract MANB36, a secrete endo-β-1,4-D-mannanase produced by Bacillus subtilis B36, was purified to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 °C. The enzyme activity of MANB36 is remarkably thermostable at 60 °C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoetha- nol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted molecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.

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Isolation of Xylanase Producing Strains, Optimization of Fermentation Conditions and Research on Enzymatic Properties

Journal of Biology and Life Science ◽

10.5296/jbls.v12i2.18483 ◽

2021 ◽

Vol 12 (2) ◽

pp. 1

Author(s):

Ji Huilong ◽

Gao Xin ◽

WU Wenxuan ◽

Ma Zhuang ◽

Qing Qing

Keyword(s):

Enzyme Activity ◽

Specific Activity ◽

Ph Value ◽

Fermentation Broth ◽

Sodium Dodecyl ◽

Alkali Resistance ◽

Initial Ph ◽

Acid And Alkali Resistance ◽

Optimal Reaction Temperature ◽

Optimization Of Fermentation

In this study, we successfully isolated a strain of Aspergillus oryzae TR08, which produced xylanase secreted to the outside of the cell productively. The enzyme activity and specific activity in the fermentation broth of this strain reached peak values of 451 IU/mL and 1963 IU/mg after 156 h of fermentation. A single factor experiment was designed, and it was found that the strain was adjusted to the initial pH of the fermentation broth to 7.5 in a shaker at 180 rpm and 32 °C. After 156 h of fermentation, the enzyme activity reached a maximum of 1264 IU/mL. The optimal reaction temperature and pH value of the xylanase were 55 °C and 7.5, respectively, and it had excellent acid and alkali resistance and a wide pH activity range. The xylanase was increased the catalytic activity by 15% in 0.25 mM Fe3+, and the biological activity of the enzyme was not affected in the sodium dodecyl sulfate environment.

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PRODUKSI DAN PENENTUAN KONDISI OPTIMUM ENZIM XILANASE Bacillus amyloliquefaciens FUKUMOTO PADA SUBSTRAT XILAN JERAMI

Jurnal Riset Kimia ◽

10.25077/jrk.v2i1.115 ◽

2015 ◽

Vol 2 (1) ◽

pp. 74

Author(s):

Widiyanti Sekatresna ◽

Abdi Dharma ◽

Periadnadi

Keyword(s):

Enzyme Activity ◽

Rice Straw ◽

Incubation Time ◽

Substrate Concentration ◽

Bacillus Amyloliquefaciens ◽

Specific Activity ◽

Enzymatic Reaction ◽

Optimal Conditions ◽

Enzyme Specific Activity

ABSTRACT The production and determination of optimal condition of xylanase produced by Bacillus amyloliquefaciens on rice straw xylan were investigated in this study. The parameters to be observed were optimal conditions of pH, temperature, substrate concentration and incubation time. Xilanase activity was determined by measuring the amount of reducing sugar formed in the enzymatic reaction based on Somogyi Nelson method. Optimal conditions needed for the production of xylanase were at pH 7, temperature 27°C and six days of incubation time. While optimal conditions of xylanase action were reached at pH 8.2, temperature 45°C, substrate concentration 3.5%(w/w) and 15 minutes of incubation time with enzyme activity and enzyme specific activity of 1.285 U/mL and 0.738 U/mg respectively. As a comparison, xylanase was also produced on pure xylan (birchwood), enzyme activity and enzyme specific activity obtained were 2.701 U/mL and 1.658 U/mg respectively. Cellulase content in enzyme produced on rice straw xilan showed the enzyme activity of 0.094 U/mL. Keywords : xylanase, Bacillus amyloliquefaciens, rice straw xilan

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Optimization of Cultural Conditions for Maximum Production of Fibrinolytic Enzymes from the Local Marine Bacterial Isolates and Evaluation of their Wound Healing and Clot Dissolving Properties

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i28a31528 ◽

2021 ◽

pp. 246-255

Author(s):

K. Gowthami ◽

R. Jaya Madhuri

Keyword(s):

Wound Healing ◽

Enzyme Activity ◽

Incubation Time ◽

Specific Activity ◽

Blood Clot ◽

Inoculum Size ◽

Fibrinolytic Enzyme ◽

Physical Parameters ◽

Bacterial Strains ◽

Fibrinolytic Enzymes

Fibrinolytic enzymes find necessary applications to treat and prevent cardiovascular diseases. In this study, optimal conditions for enhancing the production of fibrinolytic enzyme from local marine bacterial strains were evaluated. The present study also focuses on screening of wound healing efficacy of the isolated fibrinolytic enzymes.Various physical parameters such as temperature, pH, incubation time and medium components viz. inoculum size, substrate (nitrogen and carbon) concentrations were optimized. A cultivation medium was designed using optimized conditions for mass production of fibrinolytic enzyme and specific activity of enzyme was analyzed. The maximum enzyme production was observed at 37 °C temperature, 8.0 pH,substrate concentration with 3 ml inoculum size and 32 h. of incubation time. Among the different carbon sources tested, Mannitol showed maximum enzyme activity i.e 538 U/ml. yeast extract was found to be the best nitrogen source with an enzyme activity of 498 U/ml. The best substrate for the production fibrinolytic enzyme was found to be kernelwith high activity of 1056U/ml. The crude enzyme displayed potent activity and digested blood clot completely in in vitro condition and exhibited potent activity on wound healing property in macrophages.

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Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽

10.3390/catal8080325 ◽

2018 ◽

Vol 8 (8) ◽

pp. 325 ◽

Cited By ~ 1

Author(s):

He Chen ◽

Jie Huang ◽

Binyun Cao ◽

Li Chen ◽

Na Song ◽

...

Keyword(s):

Enzyme Activity ◽

Lactobacillus Plantarum ◽

Industrial Development ◽

Extraction Efficiency ◽

Specific Activity ◽

Cell Envelope ◽

Serine Proteinase ◽

Kinetic Studies ◽

Predicted Values ◽

Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

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Enzymatic condensation of dopamine and acetaldehyde: a salsolinol synthase from rat brain

Biologia ◽

10.2478/s11756-011-0134-y ◽

2011 ◽

Vol 66 (6) ◽

Cited By ~ 12

Author(s):

Xuechai Chen ◽

Abida Arshad ◽

Hong Qing ◽

Rui Wang ◽

Jianqing Lu ◽

...

Keyword(s):

Enzyme Activity ◽

Substantia Nigra ◽

Human Subjects ◽

Enzymatic Reaction ◽

Brain Regions ◽

Activity Detection ◽

Reaction Products ◽

Sprague Dawley ◽

Sprague Dawley Rats ◽

Salsolinol Synthase

AbstractSalsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; Sal) is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is supposed to have a role in the development of Parkinson-like syndrome in both human and non-human subjects. In the human brain, the amount of (R)-enantiomer of Sal is much higher than (S)-enantiomer, suggesting that a putative enzyme may participate in the synthesis of (R)-salsolinol, called (R)-salsolinol synthase. In this study, the (R)-salsolinol synthase activity in the condensation of dopamine and acetaldehyde was investigated in the crude extracts from the brains of Sprague Dawley rats. Identification of the enzymatic reaction products and enzyme activity detection were achieved by HPLC-electrochemical detection. The discovery of this enzyme activity in rat’s brain indicates the natural existence of (R)-salsolinol synthase in the brains of humans and rats, and it is distributed in most brain regions of rat with higher activity in soluble proteins extracted from striatum and substantia nigra.

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Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

Functional Plant Biology ◽

10.1071/fp05055 ◽

2005 ◽

Vol 32 (9) ◽

pp. 839

Author(s):

Rui Zhou ◽

Lailiang Cheng

Keyword(s):

Heat Treatment ◽

Thermal Stability ◽

Enzyme Activity ◽

Inorganic Phosphate ◽

Thermal Inactivation ◽

Specific Activity ◽

Apple Leaves ◽

Apparent Homogeneity ◽

Adp Glucose Pyrophosphorylase ◽

High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

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5α-Reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue

Acta Endocrinologica ◽

10.1530/acta.0.1030273 ◽

1983 ◽

Vol 103 (2) ◽

pp. 273-281 ◽

Cited By ~ 8

Author(s):

Ole Djøseland ◽

Nicholas Bruchovsky ◽

Paul S. Rennie ◽

Navdeep Otal ◽

Sian Høglo

Keyword(s):

Enzyme Activity ◽

Specific Activity ◽

Reductase Activity ◽

Major Change ◽

Growth Stimulation ◽

Total Activity ◽

Prostatic Tissue ◽

Ventral Prostate ◽

Total Enzyme Activity ◽

Abnormal Growth

Abstract. The 5α-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37°C for 30 min in the presence of 50 nm [1,2-3H]testosterone and a NADPH-generating system started with 5 × 10−4 m NADP. The yield of 5α-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5α-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5α-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5α-reductase underwent a major change in specific activity, the latter peaking after 8–12 days of treatment. Furthermore, while the total activity of 5α-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

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SELECTED PROTEOLYTIC ACTIVITY BY EXTRACTS OF DERMESTID LARVAE

Southern Brazilian Journal of Chemistry - Southern Brazilian Journal of Chemistry, Volume 28, No. 28, 2020 ◽

10.48141/sbjchem.v19.n19.2011.82_2011.pdf ◽

2011 ◽

Vol 19 (19) ◽

pp. 79-83

Author(s):

Lavinel G. IONESCU

Keyword(s):

Enzyme Activity ◽

Ammonium Sulfate ◽

Proteolytic Activity ◽

Specific Activity ◽

Proteolytic Enzymes ◽

High Specific Activity ◽

Water Extract ◽

Dermestes Maculatus ◽

Crude Preparation

The larvae of the Beetle Dermestes maculatus De Geer can subsist on a diet consisting largely of protein. Studies have been undertaken to investigate the nature of proteolytic enzymes. A water extract of the larvae yielded a crude preparation that hydrolyzes gelatin, bide powder, hemoglobin substrate, benzoyl-DL-arginine p-nitroamilide, and glutaryl-L-phenylalanine p-nitroanilide. Enzyme activity was found in a non-dialyzable, heat- and acid0labile portion of the extract yielded two fractions with high specific activity towards gelatin. These are precipitated between 40% to 60% saturation of ammonium sulfate and 60% to 80% saturation. The higher specific activity was observed in the 40%-60% fraction. These results suggest that the larvae of these dermestids contain proteolytic enzymes with actions similar to mammalian trypsin and chymotrypsin. The results also suggest that other proteolytic enzymes may be present as well.

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