ABSTRACT
INTRODUCTION: Radiographers must be aware of the needs and expectations of women to be able to involve them in the decision making on their own care pathway. The purpose of the study is to describe experiences of women with breast cancer in their individual care pathway. METHODS: Data was collected with qualitative open-ended online questionnaires via national breast cancer patient organizations in four countries. The subjects were women who had completed their breast cancer treatments at a maximum of six months before responding. Data was analysed using thematic analysis. RESULTS: Women responding to the survey questionnaire (N = 14) reported 11 main meaningful events in their care pathways. According to respondents, being well informed about the treatment process, a smooth flow of the care process, being treated individually and having a properly organized follow-up were the most important aspects for the optimal breast cancer care pathway. CONCLUSIONS: The subjects perceived their breast cancer care pathways somewhat differently from the way the pathways are usually described from the health care organizations' viewpoint. In different stages of their individual breast cancer care pathway, positive and less positive experiences of women were somewhat similar, yet contrasting. IMPLICATIONS FOR PRACTICE: In addition to general forms of support, targeted interventions should be planned to improve the quality of breast cancer care specific to different stages of the treatment process. The findings can be used to promote education for radiographers and other cancer care staff, as well as to develop patient-centred breast cancer care.
Subject(s)
Breast Neoplasms , Breast Neoplasms/therapy , Critical Pathways , Female , Humans , Male , Patient-Centered Care , Qualitative Research , Surveys and QuestionnairesABSTRACT
INTRODUCTION: With the introduction of digital radiography, the feedback between image quality and over-exposure has been partly lost which in some cases has led to a steady increase in dose. Over the years the introduction of exposure index (EI) has been used to resolve this phenomenon referred to as 'dose creep'. Even though EI is often vendor specific it is always a related of the radiation exposure to the detector. Due to the nature of this relationship EI can also be used as a patient dose indicator, however this is not widely investigated in literature. METHODS: A total of 420 dose-area-product (DAP) and EI measurements were taken whilst varying kVp, mAs and body habitus on two different anthropomorphic phantoms (pelvis and chest). Using linear regression, the correlation between EI and DAP were examined. Additionally, two separate region of interest (ROI) placements/per phantom where examined in order to research any effect on EI. RESULTS: When dividing the data into subsets, a strong correlation between EI and DAP was shown with all R-squared values > 0.987. Comparison between the ROI placements showed a significant difference between EIs for both placements. CONCLUSION: This research shows a clear relationship between EI and radiation dose which is dependent on a wide variety of factors such as ROI placement, body habitus. In addition, pathology and manufacturer specific EI's are likely to be of influence as well. IMPLICATIONS FOR PRACTICE: The combination of DAP and EI might be used as a patient dose indicator. However, the influencing factors as mentioned in the conclusion should be considered and examined before implementation.
Subject(s)
Radiation Dosage , Radiation Exposure , Radiographic Image Enhancement , Humans , Phantoms, Imaging , Reproducibility of ResultsABSTRACT
OBJECTIVES: To identify the roles of health care staff in interprofessional work related to breast cancer detection and diagnosis. KEY FINDINGS: A comprehensive search was performed using PICO to support inclusion and exclusion criteria. A shortened version of the STROBE checklist ensured evaluation of the studies. 21 included studies resulted in three main categories describing the role of health care professionals; (1) Communicating breast cancer awareness; (2) The Professional's tasks; (3) Efficacy of Interprofessional Teamwork relative to the profession and the individuals. CONCLUSIONS: Health care professionals' roles in the breast cancer diagnostic process were described mostly from each professional's viewpoint. Support from leadership and management is needed in order to promote interprofessional work, which will benefit health care professions, professionals, and the patient.
Subject(s)
Breast Neoplasms/diagnosis , Early Detection of Cancer/methods , Patient Care Team , Female , Health Promotion , Humans , Interdisciplinary Communication , Mammography , Physician's RoleABSTRACT
INTRODUCTION: The study aimed to characterize breast imaging education and identify its strengths, difficulties and needs across five European countries according to student, radiographer and teaching staff perspectives. METHODS: An observation grid was used to collect data regarding breast imaging education and three questionnaires targeted to key-participants were developed/applied to collect data on strengths, difficulties and needs. Descriptive statistics and thematic analysis were performed according to the nature of the questions. RESULTS: Breast imaging curricula varied within and between countries. Response rate for questionnaires also varied (13-100%). More than one-third of the teaching staff (37.5%) was involved in breast research projects. This was identified as the main strength in breast imaging education followed by collaborations between hospitals and academia. Difficulties with their education program identified by the 97 students surveyed included teaching issues (45), breast positioning (18), variety of image evaluation strategies (10) and human interaction (6). The need to provide an explanation to the patient about the role of the student in the mammography setting, and performing exams and teaching at the same time (22.6%) was identified as difficult by radiographers. The need for education and training in communication, practice and technological developments was identified. CONCLUSIONS: A bridge between academia and clinical practice is extremely important in order to overcome recognized gaps between theory and practice. The development of a European education program covering the needs identified by the participants could be a possible solution to improve knowledge and access, and also to harmonize education and training across Europe.
Subject(s)
Attitude of Health Personnel , Breast/diagnostic imaging , Clinical Competence/statistics & numerical data , Mammography/methods , Radiology/education , Students, Medical/statistics & numerical data , Europe , Female , Humans , Surveys and QuestionnairesABSTRACT
INTRODUCTION: This study aims to compare motivations, expectations and work plans of students and teaching-staff from four different European radiography programs, it aims also to explore areas that could be included to advance post graduate studies. METHODS: Two different questionnaires (open- and closed-end questions) were applied to key-informants, students who had just completed their bachelor thesis and teaching-staff, to collect data regarding motivations, expectations, challenges and potentials for radiography education and, plans for further work. Descriptive statistics and thematic analysis were performed according to the nature of the questions. RESULTS: The response rates were 45% (students) and 68% (teaching-staff). The motivations to study radiography were similar between students: to work in a healthcare-service, helping people, manipulating high-end technologies, providing service while combining different knowledge (physics, patient-care, physiology, anatomy). 75% of the students did not reach all their expectations due to the lack of focused and updated content for some areas. The teaching-staff were expecting an extension of the radiographers' role. The development of advance studies in computed tomography and magnetic resonance was highlighted as important by students. Future work plans included: self-improvement, continuation of studies, specialization, research and collaborations. CONCLUSIONS: This study increased the understanding of radiography education and provides insights into future perspectives. Participants have similar motivations, expectations and future plans. Improvements in education should focus on technological developments and meeting job market demands. Further studies should be performed to identify approaches that acknowledge the specific needs of each country, while also providing strategies to harmonize radiography education in Europe.
Subject(s)
Career Choice , Motivation , Radiography , Radiology/education , Students, Medical/psychology , Adult , Faculty, Medical/psychology , Faculty, Medical/statistics & numerical data , Female , Humans , Male , Middle Aged , Norway , Portugal , Schools, Medical/organization & administration , Students, Medical/statistics & numerical data , Surveys and Questionnaires , Switzerland , United Kingdom , Young AdultABSTRACT
INTRODUCTION: The aim of this study was to compare radiography curricula, teaching/learning strategies, skill development, clinical practice outcomes and research development delivered by four European educational institutions. METHODS: This study was carried out in two phases: the first focused on curricula analysis; the second involved online questionnaires to ascertain data from two key-informants: students who had recently completed their bachelor thesis and teaching-staff. Questionnaires were designed to capture teaching and learning strategies, skill acquisition and outcomes of clinical practice and research. Descriptive statistics and thematic analysis were performed according to the nature of the questions. RESULTS: The European Credits Transfer System dedicated per core subject area (natural sciences, clinical practice, research, imaging technology, humanities) differed between institutions. Students classified technical, practical and communication skills as the most important, teaching-staff highlighted also critical thinking. The students defined as "very good" their experience in radiography (58.5%) and computed-tomography (45%). Magnetic resonance imaging practice was considered "Average" by 53% of the UK-students and "Good" by the other European students (40%). According to 71% (55/78) of the students, research work contributed to the development of critical/reflective thinking. CONCLUSIONS: The four radiography programs presented variations in curricula, contact-hours, clinical experience and outcomes. Research units allowed the participant-students to develop their critical thinking capabilities. The outcomes from clinical practice differ across the institutions, mainly due to differences in background and access to specialities. Further work is necessary to assess the real impact of different radiography programs on professional and academic mobility across Europe.
Subject(s)
Clinical Competence , Curriculum , Technology, Radiologic/education , Adult , England , Female , Humans , Male , Norway , Portugal , Research , Surveys and Questionnaires , SwitzerlandABSTRACT
INTRODUCTION: This study aims to explore current challenges in mammography education from the perspectives of radiography teachers, mentors and students. METHODS: A qualitative study including two focus groups interviews, with radiography teachers/mentors (n = 5) and student radiographers (n = 5) exploring their perspectives on challenges in mammography education today. The content analysis methodology proposed by Graneheim and Lundman was applied to the interviews. RESULTS: Three main categories were identified, each with subcategories identified as: (1) Building Bridges; Applying Theoretical knowledge in Practice, Performing Mammograms, Communication and Quality Assessment (2) State of the Art in Mammography; Personal Attitudes and Skills, Quality Awareness and Patient Care (3) Exploring the Curriculum; Time Constraints, Capacity in Clinical Placement, Multidisciplinary Field and Elective Course. CONCLUSION: The short study period allocated to this discipline and lack of material resources were considered the main limitations in mammography education, both impacting on the development of students' skills. Breast positioning, patient communication and quality control were considered key factors affecting mammography performance, patient experience and diagnostic outcome and should therefore be the core focus in mammography education.
Subject(s)
Mammography , Mentors/psychology , Radiography , Radiology/education , Students/psychology , Teaching/psychology , Attitude of Health Personnel , Clinical Competence , Communication , Curriculum , Europe , Focus Groups , Humans , Mammography/standards , Patient Care/standards , Patient Positioning , Physician-Patient Relations , Qualitative Research , Quality ControlABSTRACT
The present study describes the one-step purification and biochemical characterization of an endo-1,4-ß-xylanase from Aspergillus tamarii Kita. Extracellular xylanase was purified to homogeneity 7.43-fold through CM-cellulose. Enzyme molecular weight and pI were estimated to be 19.5kDa and 8.5, respectively. The highest activity of the xylanase was obtained at 60°C and it was active over a broad pH range (4.0-9.0), with maximal activity at pH 5.5. The enzyme was thermostable at 50°C, retaining more than 70% of its initial activity for 480min. The K0.5 and Vmax values on beechwood xylan were 8.13mg/mL and 1,330.20µmol/min/mg of protein, respectively. The ions Ba2+ and Ni2+, and the compounds ß-mercaptoethanol and DTT enhanced xylanase activity, while the heavy metals (Co2+, Cu2+, Hg+, Pb2+ and Zn2+) strongly inhibited the enzyme, at 5mM. Enzymatic hydrolysis of xylooligosaccharides monitored in real-time by mass spectrometer showed that the shortest xylooligosaccharide more efficiently hydrolyzed by A. tamarii Kita xylanase corresponded to xylopentaose. In agreement, HPLC analyzes did not detect xylopentaose among the hydrolysis products of xylan. Therefore, this novel GH11 endo-xylanase displays a series of physicochemical properties favorable to its application in the food, feed, pharmaceutical and paper industries.
Subject(s)
Aspergillus/enzymology , Xylosidases/chemistry , Chromatography , Chromatography, High Pressure Liquid , Enzyme Activation , Enzyme Stability , Glucuronates , Hydrolysis , Kinetics , Mass Spectrometry , Models, Molecular , Molecular Weight , Oligosaccharides , Protein Conformation , Recombinant Proteins , Substrate Specificity , Xylosidases/isolation & purificationABSTRACT
BACKGROUND: Our aim was to characterize the influence of time-of-flight (TOF) and point spread function (PSF) recovery corrections, as well as ordered subset expectation maximization (OSEM) reconstruction parameters, in 82Rb PET/CT quantification of myocardial blood flow (MBF) and myocardial flow reserve (MFR). Rest and stress list-mode dynamic 82Rb PET acquisition data from 10 patients without myocardial flow defects and 10 patients with myocardial blood flow defects were reconstructed retrospectively. OSEM reconstructions were performed with Gaussian filters of 4, 6, and 8 mm, different iterations, and subset numbers (2 × 24; 2 × 16; 3 × 16; 4 × 16). Rest and stress global, regional, and segmental MBF and MFR were computed from time activity curves with FlowQuant© software. Left ventricular segmentation using the 17-segment American Heart Association model was obtained. RESULTS: Whole left ventricle (LV) MBF at rest and stress were 0.97 ± 0.30 and 2.30 ± 1.00 mL/min/g, respectively, and MFR was 2.40 ± 1.13. Concordance was excellent and all reconstruction parameters had no significant impact on MBF, except for the exclusion of TOF which led to significantly decreased concordance in rest and stress MBF in patients with or without perfusion defects on a coronary artery basis and in MFR in patients with perfusion defects. CONCLUSIONS: Changes in reconstruction parameters in perfusion 82Rb PET/CT studies influence quantitative MBF analysis. The inclusion of TOF information in the tomographic reconstructions had significant impact in MBF quantification.
ABSTRACT
From cultures of thermophilic soil fungus Humicola grisea var thermoidea, a δ-lactam derivative (3-(2-(4-hydroxyphenyl)-2-oxoethyl)-5,6-dihydropyridin-2(1H)-one) that displayed anti-allergic activity was isolated, which was predicted by in silico computational chemistry approaches. The in vitro anti-allergic activity was investigated by ß-hexosaminidase release assay in rat basophilic leukaemia RBL-2H3 cells. The δ-lactam derivative exhibited similar anti-allergic activity (IC(50) = 18.7 ± 6.7 µM) in comparison with ketotifen fumarate (IC(50) = 15.0 ± 1.3 µM) and stronger anti-allergic activity than azelastine (IC(50) = 32.0 µM). Also, the MTT cytotoxicity assay with RBL-2H3 cells showed that δ-lactam does not display cytotoxicity at concentrations lower than 50 µM. This study suggests that the δ-lactam derivative has the potential to be used as a lead compound in the development of anti-allergic drugs for clinical use in humans.
Subject(s)
Anti-Allergic Agents/chemistry , Anti-Allergic Agents/pharmacology , Ascomycota/chemistry , Lactams/chemistry , Pyridones/chemistry , Pyridones/pharmacology , Animals , Cell Degranulation/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Computer Simulation , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Inhibitory Concentration 50 , Ketotifen/pharmacology , Magnetic Resonance Spectroscopy , Molecular Structure , Phthalazines/pharmacology , Rats , Soil Microbiology , beta-N-Acetylhexosaminidases/metabolismABSTRACT
Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 degrees C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 degrees C and pH 6.5 for A. terricola, and 65 degrees C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 degrees C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t(50) of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4-3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and beta-xylosidase were detected which might act synergistically with xylanase.
Subject(s)
Aspergillus ochraceus/classification , Aspergillus ochraceus/enzymology , Cellulose/metabolism , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/metabolism , Wood/chemistry , Endo-1,4-beta Xylanases/isolation & purification , Enzyme Activation , Enzyme Stability , Species SpecificityABSTRACT
This study describes the production of xylanases from Aspergillus niveus, A. niger, and A. ochraceus under solid-state fermentation using agro-industrial residues as substrates. Enzyme production was improved using a mixture of wheat bran and yeast extract or peptone. When a mixture of corncob and wheat bran was used, xylanase production from A. niger and A. ochraceus increased by 18%. All cultures were incubated at 30 degrees C at 70-80% relative humidity for 96 h. For biobleaching assays, 10 or 35 U of xylanase/g dry cellulose pulp were incubated at pH 5.5 for 1 or 2 h, at 55 degrees C. The delignification efficiency was 20%, the brightness (percentage of ISO) increased two to three points and the viscosity was maintained confirming the absence of cellulolytic activity. These results indicated that the use of xylanases could help to reduce the amount of chlorine compounds used in cellulose pulp treatment.
Subject(s)
Aspergillus niger/enzymology , Aspergillus ochraceus/enzymology , Aspergillus/enzymology , Cellulose/metabolism , Endo-1,4-beta Xylanases/biosynthesis , Endo-1,4-beta Xylanases/isolation & purification , Endo-1,4-beta Xylanases/metabolism , Fermentation , Hydrogen-Ion Concentration , Industrial Waste , Nitrogen/metabolism , Substrate Specificity , TemperatureABSTRACT
Two strains (15.1 and 15.8) of the thermophilic fungus Scytalidium thermophilum produced high levels of intracellular glucoamylases, with potential for industrial applications. The isoform I of the glucoamylase produced by 15.1 strain was sequentially submitted to DEAE-Cellulose and CM-Cellulose chromatography, and purified 141-fold, with 5.45 percent recovery. The glucoamylase of strain 15.8 was purified 71-fold by CM-Cellulose and Concanavalin A-Sepharose chromatography, with 7.38 percent recovery. Temperature and pH optima were in the range of 50-60ºC and 5.0-6.0, respectively, using starch and maltose as substrates. The glucoamylase of S. thermophilum 15.8 was more stable (t50 > 60 min) than that of S. thermophilum 15.1 (t50= 11-15 min), at 60ºC. The glucoamylase activities were enhanced by several ions (e.g. Mn2+ and Ca2+) and inhibited by β-mercaptoethanol. The glucoamylase from 15.1 strain showed a Km of 0.094 mg/ml and 0.029 mg/ml and Vmax of 202 U/mg prot and 109 U/mg prot, for starch and maltose, respectively. The hydrolysis products of starch and maltose, analyzed by TLC, demonstrated glucose as end product and confirming the character of the enzyme as glucoamylase. Differences were observed in relation to the products formed with maltose as substrate between the two strains studied. S. thermophilum 15.8 formed maltotriose in contrast with S. thermophilum 15.1.
Duas linhagens (15.1 e 15.8) do fungo termofílico Scytalidium thermophilum se mostraram produtoras de grandes quantidades de glucoamilases, com potencial aplicação industrial. A isoforma I de glucoamilase produzida pela linhagem 15.1 foi submetida seqüencialmente a cromatografia em colunas de DEAE-celulose e CM-celulose, sendo purificada 141 vezes com porcentagem de recuperação de 5,45 por cento. A glucoamilase da linhagem 15.8 foi purificada 71 vezes através do uso de colunas de cromatografia de CM-celulose e Concanavalina A-sepharose com porcentagem de recuperação de 7,38 por cento. Temperatura e pH ótimo foram de 50-60ºC e 5,0-6,0 respectivamente, utilizando-se amido e maltose como substratos. A glucoamilase de S. thermophilum 15.8 se mostrou mais estável (t50 > 60 min) que a de S. thermophilum 15.1 (t50 =11-15min) a 60ºC. As glucoamilases tiveram suas atividades enzimáticas aumentadas na presença de vários íons (ex: Mn2+, e Ca2+) e inibidas por β-mercaptoetanol. A glucoamilase da linhagem 15.1 apresentou um Km de 0,094 mg/ml e 0,029 mg/ml and Vmax de 202U/mg prot e 109U/mg prot, para amido e maltose respectivamente. A análise do produto da hidrólise de amido e maltose por TLC, demonstrou que o produto final era glucose, confirmando as características da enzima como glucoamilase. Diferenças entre as duas linhagens foram observadas com relação aos produtos formados tendo maltose como susbstrato, a linhagem 15.8 de S. thermophilum produziu maltotriose como produto final em contrate com a linhagem 15.1.
Subject(s)
Clinical Enzyme Tests , Enzymes/analysis , Fungi , /analysis , In Vitro Techniques , Industrial Microbiology , Chromatography , Culture Media , Hydrolysis , MethodsABSTRACT
Rhizopus microsporus var. rhizopodiformis produced high levels of alpha-amylase and glucoamylase under solid state fermentation, with several agricultural residues, such as wheat bran, cassava flour, sugar cane bagasse, rice straw, corncob and crushed corncob as carbon sources. These materials were humidified with distilled water, tap water, or saline solutions--Segato Rizzatti (SR), Khanna or Vogel. The best substrate for amylase production was wheat bran with SR saline solution (1:2 v/v). Amylolytic activity was still improved (14.3%) with a mixture of wheat bran, corncob, starch and SR saline solution (1:1:0.3:4.6 w/w/w/v). The optimized culture conditions were initial pH 5, at 45 degrees C during 6 days and relative humidity around 76%. The crude extract exhibited temperature and pH optima around 65 degrees C and 4-5, respectively. Amylase activity was fully stable for 1 h at temperatures up to 75 degrees C, and at pH values between 2.5 and 7.5.
Subject(s)
Amylases/chemistry , Amylases/metabolism , Bioreactors/microbiology , Cell Culture Techniques/methods , Dietary Fiber/microbiology , Triticum/microbiology , Zea mays/microbiology , Enzyme Activation , Enzyme Stability , Species SpecificityABSTRACT
Two strains (15.1 and 15.8) of the thermophilic fungus Scytalidium thermophilum produced high levels of intracellular glucoamylases, with potential for industrial applications. The isoform I of the glucoamylase produced by 15.1 strain was sequentially submitted to DEAE-Cellulose and CM-Cellulose chromatography, and purified 141-fold, with 5.45% recovery. The glucoamylase of strain 15.8 was purified 71-fold by CM- Cellulose and Concanavalin A-Sepharose chromatography, with 7.38% recovery. Temperature and pH optima were in the range of 50-60°C and 5.0-6.0, respectively, using starch and maltose as substrates. The glucoamylase of S. thermophilum 15.8 was more stable (t50 > 60 min) than that of S. thermophilum 15.1 (t50= 11-15 min), at 60°C. The glucoamylase activities were enhanced by several ions (e.g. Mn(2+) and Ca(2+)) and inhibited by ß- mercaptoethanol. The glucoamylase from 15.1 strain showed a Km of 0.094 mg/ml and 0.029 mg/ml and Vmax of 202 U/mg prot and 109 U/mg prot, for starch and maltose, respectively. The hydrolysis products of starch and maltose, analyzed by TLC, demonstrated glucose as end product and confirming the character of the enzyme as glucoamylase. Differences were observed in relation to the products formed with maltose as substrate between the two strains studied. S. thermophilum 15.8 formed maltotriose in contrast with S. thermophilum 15.1.
ABSTRACT
The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K (m) 0.28 and 0.22 mmol/L, with upsilon (lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.
Subject(s)
Alkaline Phosphatase/isolation & purification , Fungal Proteins/isolation & purification , Rhizopus/enzymology , Alkaline Phosphatase/metabolism , Cations/pharmacology , Chromatography, Agarose , Chromatography, DEAE-Cellulose , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Fungal Proteins/metabolism , Hot Temperature , Hydrogen-Ion Concentration , Molecular WeightABSTRACT
Biochemical properties of a termostable alkaline phosphatase obtained from the mycelium extract of A. caespitosus were described. The enzyme was purified 42-fold with 32% recovery by DEAE-cellulose and concanavalin A-Sepharose chromatography. The molar mass estimated by Sephacryl S-200 or by 7% SDS-PAGE was 138 kDa and 71 kDa, respectively, indicating a homodimer. Temperature and pH optima were 80 degrees C and pH 9.0. This enzyme was highly glycosylated (approximately 74% saccharide content). The activity was enhanced by Mg2+ (19-139%), NH4+ (64%), Na+ (51%) and Mn2+ (38%). 4-Nitrophenyl phosphate (4-NPP) was preferentially hydrolyzed, but glucose 1-phosphate (93%), UTP (67%) and O-phosphoamino acids also acted as substrates. V(lim) and K(m) were 3.78 nkat per mg protein and 270 micromol/L in the absence of Mg2+ and 7.35 nkat per mg protein and 410 micromol/L in the presence of Mg2+, using 4-NPP as substrate. The purified alkaline phosphatase removed the 5'-phosphate group of a linearized plasmid without showing DNAase activity, indicating its potential for recombinant DNA technology.
Subject(s)
Alkaline Phosphatase/isolation & purification , Alkaline Phosphatase/metabolism , Aspergillus/enzymology , Mycelium/enzymology , Alkaline Phosphatase/chemistry , Deoxyribonucleases/metabolism , Kinetics , Substrate SpecificityABSTRACT
The osmotically-sensitive os-1 mutant of Neurospora crassa overproduced conidial alkaline phosphatase. The enzyme was purified by Phenyl-Sepharose CL-4B chromatography and Sephadex G-200 gel filtration. PAGE analysis of the purified enzyme suggested the occurrence of aggregation and/or disaggregation phenomena. The enzyme is a glycoprotein containing 16% saccharide, with apparent molar mass of 137 kDa. Two protein bands (36 and 62 kDa) were observed in SDS-PAGE, suggesting that the native enzyme was a trimer. The pI was estimated to be 2.7, and optima of pH and temperature were 9.5 and 65 degrees C, respectively. The enzyme showed broad substrate specificity, hydrolyzing preferentially 4-nitrophenyl phosphate, O-phosphoamino-acids and 2-phosphoglycerate. The hydrolysis of 4-nitrophenyl phosphate was stimulated by Co(II) (26%), Ni(II) (23%) and Mg(II) ions (80%). The enzyme was stable for up to 6 months at 4 degrees C in 5 mmol/L Tris-HCl buffer and also upon storage at 25 degrees C for 10 d. The kinetic and structural properties of the conidial enzyme purified from the os-1 mutant were quite different from those of the wild type strain. The enzyme overproduction observed in the mutant may be related to cell wall alterations that affect the process of enzyme secretion.