Highest Specific Activity Of Enzyme Research Articles

High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Identification and characterization of a novel high-activity amylosucrase from Salinispirillum sp. LH10-3-1.

In this study, a novel high-activity amylosucrase from Salinispirillum sp. LH10-3-1 (SaAS) was identified and characterized. The recombinant enzyme was determined as a monomer with a molecular mass of 75 kDa. SaAS protein exhibited the maximum total and polymerization activities at pH 9.0 and maximum hydrolysis activity at pH 8.0. The optimum temperature for total, polymerization, and hydrolysis activities were 40, 40, and 45 °C, respectively. Under the optimal pH and temperature, SaAS had a specific activity of 108.2 U/mg. SaAS also showed excellent salt tolerance and could retain 77.4% of its original total activity at 4.0 M NaCl. The addition of Mg2+, Ba2+, and Ca2+ enhanced the total activity of SaAS. When the conversion of 0.1 M and 1.0 M sucrose was catalyzed at pH 9.0 and 40 °C for 24 h, the ratios of hydrolysis, polymerization, and isomerization reactions were 11.9:77.4:10.7 and 15.3:53.5:31.2, respectively. The α-arbutin yield of 60.3% was achieved from 20 mM sucrose and 5 mM hydroquinone catalyzed by SaAS. KEY POINTS: • A novel amylosucrase from Salinispirillum sp. LH10-3-1 (SaAS) was characterized. • SaAS has the highest specific enzyme activity among all known amylosucrase. • SaAS has hydrolysis, polymerization, isomerization, and glucosyltransferase activities.

Treatment of lactose by fermentation: Production process on β-galactosidase using Kluyveromyces marxianus isolated from kefir grains

β-galactosidase is an enzyme that catalyzes the hydrolysis of lactose, a disaccharide, into glucose and galactose. β-galactosidase is a commercially essential enzyme for its catalytic properties and is widely used in various biotechnology processes. In this study, optimum production conditions were studied to produce the highest level of β-galactosidase using Kluyveromyces marxianus isolated from kefir grains, and changes in some fermentation metabolites during enzyme production were determined under optimum conditions. The pH, temperature, and incubation time were optimised to produce of β-galactosidase. The pH (4.0, 7.0, 8.0), temperature (25 - 37°C), and incubation time (0 - 60 h) were evaluated in the ranges. The highest specific enzyme activity was 47.31 U/mL at the end of 48 hour-incubation at 200 rpm, at pH 8.0, and 30°C with 2% (v/v) inoculation rate. A high-performance liquid chromatography system determined the changes in lactose, glucose, galactose, lactic acid, and ethanol concentrations at the optimum fermentation conditions by taking samples from liquid fermentation medium during enzyme production under these conditions. The amount of glucose and galactose formed by the lactose fermentation metabolism of K. marxianus decreased during the fermentation. The ethanol concentration reached its highest concentration (18.44 g/L) at the 36th hour of fermentation. When fermentation metabolites were examined, β-galactosidase and K. marxianus metabolism treatment indicated remarkable findings.

Amylolytic microorganisms from diverse tropical environments: Isolation, identification, and amylase production

AbstractThis study aimed to identify microbes producing amylases from diverse unexplored environments in Kenya. Screening based on microorganisms' ability to grow on minimal media (M9) supplemented with starch resulted in the selection of 70 amylolytic isolates. The isolates were then subjected to primary screening based on starch hydrolysis ratio (SHR) obtained by halo formation on starch agar plates after flooding plates with Lugol's iodine solution. SHR narrowed down the isolates' number to 17 (SHR > 1.5), which were used for subsequent experiments. The 17 isolates were grown on M9 starch broth for the production of amylases. Crude amylases and commercial enzyme (control) were assayed for amylase activity using the 3,5‐dinitrosalicylic acid method. Specific enzyme activities of crude enzymes ranged between 0.0079 and 0.0629 U g−1, which were lower than 1.7188 U g−1 of the control. Five enzymes with the highest specific enzyme activity were used for cassava hydrolysis. All crude enzymes exhibited higher specific enzyme activity (0.0707–0.1853 U g−1) than the control (0.0052 U g−1) in hydrolyzing cassava. Alkaline lakes had the highest number (4) of isolates whose enzymes had the highest specific enzyme activity, while isolates from potato processing plant waste, termite gut, and black soldier fly gut had a representative each. Isolates with enzymes having the highest specific enzyme activity were identified by DNA sequencing and belonged to Lysinibacillus sp. (four isolates), Alternaria sp. (one isolate), and Bacillus sp. (two isolates). The study demonstrated the presence of microorganisms in tropical environments with amylase activity that can be scaled and optimized for practical applications.

Biological indicators affected by land use change, soil resource availability and seasonality in dry tropics

Abstract Land use change (LUC) in tropics, explicitly from forest to conventional agriculture, is negatively affecting soil health and productivity. However, effect of such LUC on soil biological properties are poorly known in dry tropical environment. This study aimed to determine the impact of such LUC and climate seasonality on soil biological properties (microbial and enzyme activities), and to also explore the role of soil resources (C, N and P) in driving soil biological properties during this environmental change. Soil biological indicators of topsoil (0–15 cm) were measured on a seasonal basis in the natural forest, fallow and agricultural land. Soil microbial biomass C (Cmic), CO2 efflux (Cefflux) and hydrolytic enzyme activities (acid phosphatase, alkaline phosphatase, β-glucosidase, dehydrogenase, fluorescein diacetate) were generally higher in the forest followed agriculture and fallow. The lower level of these biological variables in agriculture soils were possibly due to lower total soil organic carbon (SOC), labile SOC (particulate OC; POC), total N and P, and cropping disturbance (i.e., fertilization) induced higher inorganic N and P. Whereas, lower biological activities in fallow soils were mainly attributed lower plant activity (i.e. litter production and root activity). In contrast, oxidative enzyme (particularly peroxidase) activities were higher in agriculture followed by the forest and fallow, which might be attributed to higher soil oxygenation from tillage in agriculture and higher persistent SOC (non-particulate OC; NPOC). Seasonal variation in soil biological properties was identical among land uses, though its extent was greater in the forest than fallow and agriculture, indicating LUC can alter the degree of seasonality in biological properties. In addition, the higher specific enzyme activities (i.e., enzyme activities per unit of Cmic) and microbial metabolic quotient (qCO2) in the fallow followed by agriculture and forest, indicating the higher degree of stress on soil microbes after the deforestation than cropping. Whereas, the higher specific enzyme activities and qCO2 in agriculture possibly attributed to higher microbial nutrients demand and lower SOC accumulation. Collectively, our results reveal the significant effects of deforestation and agriculture on soil biological activities and improve our understanding of the potential mechanism driving these effects.

A novel fungal beta-propeller phytase from nematophagous Arthrobotrys oligospora: characterization and potential application in phosphorus and mineral release for feed processing

Phytases are widely utilized in feed industry to increase the utilization of phosphorus, minerals, and amino acids for improvement of animal and human nutrition. At present, all known β-propeller phytases (BPP) have been generated from bacteria, particularly Bacillus. In this work we report for the first time a new fungal-derived BPP phytase. We identified a phytase highly differentially expressed only in the parasitic stage of a nematophagous fungus, Arhtrobotrys oliogospora, during the development of the 3D traps. We found that this phytase was homologous to the known bacterial BPP phytase, thus we referred the new phytase to Aophytase. The heterologous expression of codon-optimized Aophytase gene in Pichia pastoris was successfully investigated to yield recombinant Aophytase (r-Aophytase) with high specific enzyme activity of 74.71 U/mg, much higher than those of recombinant BPP phytases derived bacteria. The kinetic parameters of the r-Aophytase, the optimum pH and temperature, as well as the effects of surfactant, EDTA and different ions on its enzyme activity were further investigated. The potential utilization of r-Aophytase in feed processing was finally explored. We found that the optimal pH value was about 7.5, and the optimal temperature was 50 °C.; r-Aophytase significantly increased the release of inorganic phosphorus from soybean meal, and improved the release of soluble minerals from the durum wheat flour and finger millet flour. The findings indicate its potential utilization in the feed processing to ameliorate nutritional value of cereals and animal feed in the future.

Effects of forest conversion on carbon-degrading enzyme activities in subtropical China

The mineralization of soil organic carbon (SOC) is primarily mediated by carbon (C) degrading enzyme. In the current study, we determined how the activities of four soil C-degrading enzymes, the hydrolases β-glucosidase (BG) and cellobiohydrolase (CBH) and the oxidases polyphenol oxidase (PPO) and peroxidase (POD), responded to forest conversion of natural broadleaf forests (BF) to secondary forests (SF) and plantation forests (PF) in subtropical China. We also quantified SOC, dissolved organic C (DOC), permanganate oxidase organic C (PXC), recalcitrant C (RC), microbial biomass C (MBC), mineral-associated C (MOC), soil particle-sizes distribution, pH, and moisture content, and C: nitrogen (N) ratio. Results showed that, the activities of all four C-degrading enzymes (BG, CBH, PPO and POD) decreased by 23.1, 9.5, 6.9 and 1.8%, respectively by forest conversion of BF to SF and 30.5, 15.3, 28.1 and 27.8%, respectively by conversion of BF to PF and were higher in the topsoil than in the subsoil. Relative to SF and PF, BF had higher hydrolase activities, which were related to its higher concentrations of MBC, DOC, and PXC, and to its lower C:N ratio. The BF also had higher oxidase activities, which were related to its higher concentrations of MBC, RC, and MOC, and to its lower C:N ratio. PF had higher specific enzyme activities (i.e., enzyme activities per unit of SOC) than BF and SF, indicating faster C turnover rates in PF. In addition to being affected by the concentrations of SOC and SOC components, forest conversion-induced changes in soil enzyme activities were affected by clay content and soil moisture content. These results revealed the different underlying mechanisms between soil hydrolases and oxidases in their responses to forest conversion.

Production of “biobetter” variants of glucarpidase with enhanced enzyme activity

Glucarpidase, also known as carboxypeptidase G2, is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels “biobetter” variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. Km, Vmax and Kcat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase.Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase.

Thermostability and Specific-Activity Enhancement of an Arginine Deiminase from Enterococcus faecalis SK23.001 via Semirational Design for l-Citrulline Production.

l-Citrulline is a nonessential amino acid with a variety of physiological functions and can be enzymatically produced by arginine deiminase (ADI, EC 3.5.3.6). The enzymatic-production approach is of immense interest because of its mild conditions, high yield, low cost, and environmental benignity. However, the major hindrances of l-citrulline industrialization are the poor thermostability and enzyme activity of ADI. Hence, in this work, directed evolution and site-directed mutagenesis aided with in silico screening, including the use of b-factor values and HoTMuSiC, were applied to a previously identified ADI from Enterococcus faecalis SK23.001 ( EfADI), and a triple-site variant R15K-F269Y-G292P was obtained. The triple-site variant displays a 2.5-fold higher specific enzyme activity (333 U mg-1), a lower Km value of 6.4 mM, and a 6.1-fold longer half-life ( t1/2,45°C = 86.7 min) than wild-type EfADI. This work provides a protein-engineering strategy to improve enzyme activity and thermostability, which might be transferrable to other ADIs and enzymes.

Optimizing of protease purification from bacillus cereus td5b by ammonium sulfate precipitation

The aim of this research was to optimize the protease purification from Bacillus cereus TD5B by ammonium sulfate precipitation. The material used was protease enzyme from Bacillus cereus TD5B. It was produced and purified in a liquid medium using ammonium sulfate precipitation. The optimization was performed using various levels (50, 60, 70 and 80 %) of ammonium sulfate as precipitation agent of crude microbial enzymes. Separation process of precipitate and supernatant was conducted by 3,500 rpm centrifugation for 30 min at 4°C. The specific activity of protease enzyme, and protein content was observed on precipitate and supernatant of each levels treatment of the ammonium sulfate. Based on specific enzyme activity and protein content, the measurement showed that 70 % ammonium sulfate level treatment has the highest specific enzyme activity (78.296 U/mg) and highest protein content (2.689 mg/mL). This specific enzyme activity was three times higher compared to specific enzyme activity before purification treatment (24.12 U/mg). In conclusion, the purification of crude Bacillus cereus TD5B enzyme by ammonium sulfate purification can increase the specific enzyme activity.

Оптимізація синтезу та розробка схеми очищення D-лактат : цитохром с оксидоредуктази рекомбінантного штаму дріжджів Ogataea (Hansenula) polymorpha “tr6”

The optimization of conditions for synthesis of D-lactate : cytochrome c oxidoreduc­tase (EC 1.1.2.4; DLDH) by recombinant yeast strain Ogataea ( Hansenula ) polymorpha “ tr6” ( gcr1 catX Δcyb2/DLD1 ) was performed. The strain is characterized by impairement of glucose catabolic repression, deletion of the CYB2 gene, responsible for the synthesis of L-lactate: cytochrome c oxidoreductase, and six-fold overproduction of DLDH. Moreover, an isolation and purification of the target enzyme using ion exchange chromatography was proposed. The optimization of the cultural medium composition and conditions for the yeast cells’ cultivation to obtain the biomass with the highest specific activity of DLDH have been performed. Biomass of the cells O. polymorpha “tr6” is the highest in a medium with 1 % glycerol and 0.5 % racemic lactate, while the best specific activity of DLDH and the lowest non-specific ferricyanide reductase activity are observed in a medium with 1 % ethanol and 0.5% racemic lactate. The influence of some ions of divalent metals on the activity of DLDH in permeabilized cells and induction of the enzyme’ synthesis in the cells of O. polymorpha “tr6” were investigated. It has been shown that the addition of Zn 2+ ions to the cultural medium significantly increases the activity of DLDH in the cell-free extracts of the yeast cells. The isolation of the membrane-bound DLDH was carried out using methods of physical destruction of the cells. DLDH purification was performed on the DEAE-Toyopearl ion-exchange cellulose 650 M. The yield of the enzyme after chromatographic purification was about 50 %. The highest specific enzyme activity in purified fractions with a 7-fold purification degree was reached up to 1.1 U·mg -1 . Some of the physicochemical properties of the purified preparation of DLDH of recombinant O. polymorpha yeast strain “tr6” have been investigated.

Purification and characterization of alginate lyase from locally isolated marinePseudomonas stutzeriMSEA04

An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).

Regulation of phosphatidylinositol 4-kinase activity in rat pancreatic acini.

Previous data showed that carbachol (CCh) elicits a concentration-dependent increase in phosphatidylinositol (PtdIns) 4-kinase activity in homogenates derived from agonist-stimulated pancreatic acini. In the present study CCh elicited a time-dependent increase in PtdIns 4-kinase activity, which was blocked by n-methylscopolamine and mimicked by muscarine. A membrane-associated PtdIns 4-kinase activity was identified that displayed maximal activity in the pH range of 5.5-8.5. The zymogen granule fraction possessed relatively high specific enzyme activity, which was sensitive to CCh stimulation. The enzyme had apparent Km values for PtdIns and ATP of 4 and 60 microM, respectively. CCh caused no discernible change in the Km for either PtdIns or ATP but increased Vmax. Dioctanoylglycerol and oleoyl acetylglycerol augmented PtdIns 4-kinase activity, which was blocked by staurosporine, thus suggesting a possible role for protein kinase C in the regulation of PtdIns 4-kinase. These collective findings demonstrate a muscarinic receptor-mediated regulation of PtdIns 4-kinase activity in exocrine pancreas, involving a protein kinase C-dependent process.

COMPARISON OF DIFFERENT CELL DISRUPTION METHODS AND CELL EXTRACTANT BUFFERS FOR RECOMBINANT BROMELAIN EXPRESSED IN E.COLI BL21-A1

The often-encountered problem such as protein degradation has driven various methods of cell lysis in obtaining recombinant protein post fermentation. In this paper, we compare methods such as homogenization, sonication, sonication with lysozyme and chemical lysis using B-PER reagent with lysozyme to extract the recombinant bromelain from E. coli BL21-AI. The sonication process is found to be the most effective in releasing recombinant bromelain without any pre-treatment. To obtain the high quality of protein from sonication method, the influence of different extractant buffer was investigated including phosphate buffer saline (PBS), PBS containing cysteine and EDTA (PBS-CE), and sodium phosphate buffer containing cysteine and EDTA (EB-CE). The highest specific enzyme activity was obtained when it was extracted with EB-CE buffer. Under sodium dodecyl sulfate polyacrylamide gel electrophoresis, the recombinant bromelain showed protein band at 55kDa. In conclusion, the sonication method with extractant buffer containing 100mM phosphate buffer pH7.0 with 15 mM cysteine and 2 mM EDTA (EB-CE) was shown to give high specific activity of recombinant bromelain.

Purification and characterisation of the extracellular cholesterol oxidase enzyme from Enterococcus hirae.

BackgroundRecently many efforts are being carried out to reduce cholesterol in foods. Out of the 50 selected isolates that were tested using the agar well diffusion method to assess their ability to decompose cholesterol, 24 bacterial isolates were screened based on their cholesterol-decomposition ability in liquid media.ResultsThe bacterial isolate that displayed the highest cholesterol oxidase activity was identified as Enterococcus hirae. The maximal growth and cholesterol decomposition were achieved with a 1-day incubation under static conditions at 37 °C in cholesterol basal medium adjusted to pH 7 supplemented with 1 g/l cholesterol as the substrate, no additional carbon or nitrogen sources and 0.5 % CaSO4. The cholesterol oxidase enzyme (ChoX) produced by E. hirae was extracted at an (NH4)2SO4 saturation level of 80 % and purified with 79 % yield, resulting in 2.3-fold purification. The molecular weight of (ChoX) was 60 kDa. The optimal conditions required for the maximal activity of the purified COD enzyme produced by E. hirae were 30 min, 40 °C, pH 7.8, substrate concentration of 1 g/l and 200 ppm of MgCl2. The enzyme maintained approximately 36 % and 58.5 % of its activity after 18 days of storage at 4–8 °C. Also, the enzyme loss its activity by gradual thermal treatment, but it maintained 58.5 % of its activity at 95 °C for 2 hr.ConclusionsE. hirae Mil-31 isolated from milk had a great capacity to decompose cholesterol in basal medium supplemented with cholesterol under its optimal growth conditions. Decomposition process of cholesterol by this strain results from its production of cholesterol oxidase enzyme (ChoX). The highest specific enzyme activity and highest purification fold of purified enzyme were achieved after using Sephadex G-100.

Surfactant-activated magnetic cross-linked enzyme aggregates (magnetic CLEAs) of Thermomyces lanuginosus lipase for biodiesel production

Novel surfactant-activated magnetic cross-linked enzyme aggregates of Thermomyces lanuginosus lipase (TLL-magnetic-CLEAs) were developed and provided an efficient approach to improve the activity and stability of lipase for biodiesel production. In the methanolysis of Jatropha oil for biodiesel synthesis, the maximum yield in isopropyl ether was 88% after 48h at 40°C, representing 3.5-fold and 2.5-fold higher activity than that exhibited by free TLL and TLL CLEAs, respectively. Moreover, Tween 80-activated TLL-magnetic-CLEAs retained their activity during storage at 4°C for 11 weeks and 10 cycles of repeated 48h biodiesel reactions at 40°C (over 30 days). Additionally, the surface morphology, particle size and loading of lipase aggregates were confirmed by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The combination of interfacial activation, high specific enzyme activity, improved stability and easy recovery of magnetic CLEAs presents an attractive process for lipase immobilization and provides a promising catalyst for biodiesel production.

Activity and Spatial Distribution of Candida antarctica Lipase B Immobilized on Macroporous Organic Polymeric Adsorbents

A systematic study of the influence of carrier particle size (500-850 μm) and enzyme load (26,200-66,100 lipase activity units (LU)/g dry carrier) on the content and activity of Candida antarctica lipase B (CALB) immobilized by adsorption onto macroporous poly(methyl methacrylate) (PMM) and polystyrene (PS) carriers was conducted. Furthermore, localization of CALB on the carrier was investigated by light and fluorescence microscopy of freeze microtome sliced catalyst particles. Fluorescence microscopy showed localization of enzyme in an outer rim of 50-85 and 10-20 μm thickness for the PMM and PS catalysts, respectively, whereas no rim was observed in the absence of enzyme. Statistical analyses showed that carrier type was the major effect in determining the activities of the catalysts, with enzyme load being the second most significant effect and particle size also exerting a significant, yet smaller, effect. The PMM catalysts showed higher activities compared to PS catalysts, possibly indicating that the microenvironment interactions of CALB with the PMM are more favorable than with the PS carrier, resulting in a higher specific enzyme activity. Furthermore, smaller particles and higher enzyme load had a positive influence on the activities within the investigated ranges, and the carrier type and enzyme load interaction was statistically significant (p < 0.001).

Conformation and Activity of Sol-Gels Encapsulated Cross-Linked Enzyme Aggregates of Lipase from &lt;i&gt;Burkholderia cepacia&lt;/i&gt;

Cross-linked enzyme aggregates (CLEAs) of lipase from Burkholderia cepacia (BCL) were explored using different precipitants and different amount of glutaraldehyde as cross-linkers. The ‘fresh’ and ‘mature’ CLEAs-BCL was successfully encapsulated with sol-gel support. The latter showed the highest specific enzyme activity, which was 1.7 and 13.2-fold over CLEAs-BCL before encapsulation with sol-gel and free BCL, respectively. It performed high transesterification activity with the biodiesel yield of 88.5%. The ‘mature’ CLEAs-BCL presented high stability in the ranges of temperature (30-70 ºС) and pH (pH = 5-10). Scanning electron microscopy (SEM) and circular dichroism (CD) studies showed that the morph secondary structure of ‘fresh’ and ‘mature’ CLEAs-BCL was variant to some extent, which may be responsible for the catalytic activity variance.

Vascular smooth muscle cells: a major source of the semicarbazide-sensitive amine oxidase of the rat aorta.

Several methods have been used to study the distribution of the semicarbazide-sensitive amine oxidase (SSAO) within the wall of the rat aorta. After separation of the smooth muscle-containing layers of the tunica media from the connective tissue of the tunica adventitia, much higher specific enzyme activity (measured with 1 microM benzylamine) was found in homogenates of the media than of adventitia. Similar results were obtained for MAO-A (with 1 mM 5-HT as substrate). SSAO activity was also considerably higher in homogenates of cells (predominantly smooth muscle) isolated from medial tissue by enzymatic dissociation with collagenase and elastase compared with homogenates of cells (mostly of connective tissue origin) from the adventitia. Histochemical staining resulting from SSAO activity (with benzylamine as substrate) occurred predominantly and intensely over the tunica media in rat aortic sections, although some occasional staining of adventitial sites was also observed. Staining was prevented by the SSAO inhibitors hydroxylamine (1 microM) and semicarbazide (1 mM), but not by the MAO inhibitor, clorgyline (1 mM). These results indicate that SSAO is associated predominantly, although not exclusively, with the smooth muscle cells in the rat aorta. Our findings that beta-aminopropionitrile (BAPN) is a reversible, competitive inhibitor (Ki around 2 X 10(-4)M) of SSAO, in contrast to the irreversible inhibition of the connective tissue lysyl oxidase by BAPN reported by others, provides further evidence that these enzymes are not identical.

Purification and characterization of a Na +/K + dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k cat/ K m value for alginate (heterotype) was 1.7 × 10 6 s −1 M −1. The enzyme activity was completely lost by dialysis and restored by addition of Na + or K +. The optimal activity exhibited in 0.1 M of Na + or K +. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).