Experiments in the Purification and Characterization of Enzymes

Experiments in the Purification and Characterization of Enzymes PDF

Author: Thomas E. Crowley

Publisher: Academic Press

Published: 2014-01-11

Total Pages: 267

ISBN-13: 0124095933

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Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual provides students with a working knowledge of the fundamental and advanced techniques of experimental biochemistry. Included are instructions and experiments that involve purification and characterization of enzymes from various source materials, giving students excellent experience in kinetics analysis and data analysis. Additionally, this lab manual covers how to evaluate and effectively use scientific data. By focusing on the relationship between structure and function in enzymes, Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual provides a strong research foundation for students enrolled in a biochemistry lab course by outlining how to evaluate and effectively use scientific data in addition to offering students a more hands-on approach with exercises that encourage them to think deeply about the content and to design their own experiments. Instructors will find this book useful because the modular nature of the lab exercises allows them to apply the exercises to any set of proteins and incorporate the exercises into their courses as they see fit, allowing for greater flexibility in the use of the material. Written in a logical, easy-to-understand manner, Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual is an indispensable resource for both students and instructors in the fields of biochemistry, molecular biology, chemistry, pharmaceutical chemistry, and related molecular life sciences such as cell biology, neurosciences, and genetics. Offers project lab formats for students that closely simulate original research projects Provides instructional guidance for students to design their own experiments Includes advanced analytical techniques Contains adaptable modular exercises that allow for the study proteins other than FNR, LuxG and LDH Includes access to a website with additional resources for instructors

Biocatalysis for Practitioners

Biocatalysis for Practitioners PDF

Author: Gonzalo de Gonzalo

Publisher: John Wiley & Sons

Published: 2021-07-19

Total Pages: 54

ISBN-13: 352734683X

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This reference book originates from the interdisciplinary research cooperation between academia and industry. In three distinct parts, latest results from basic research on stable enzymes are explained and brought into context with possible industrial applications. Downstream processing technology as well as biocatalytic and biotechnological production processes from global players display the enormous potential of biocatalysts. Application of "extreme" reaction conditions (i.e. unconventional, such as high temperature, pressure, and pH value) - biocatalysts are normally used within a well defined process window - leads to novel synthetic effects. Both novel enzyme systems and the synthetic routes in which they can be applied are made accessible to the reader. In addition, the complementary innovative process technology under unconventional conditions is highlighted by latest examples from biotech industry.

Studies on Purification and Characterization of an Autolytic Enzyme from Cell Walls of Bacillus Subtilis

Studies on Purification and Characterization of an Autolytic Enzyme from Cell Walls of Bacillus Subtilis PDF

Author: Willie Claiborne Brown

Publisher:

Published: 1968

Total Pages: 122

ISBN-13:

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The purpose of this investigation was to purify and characterize the autolytic enzyme from cell walls of Bacillus subtilis 168. The crude enzyme was obtained by autolysis of purified cell walls in buffer at 37° C. Two purification methods were developed. The first involved fractional precipitation with ammonium sulfate. The enzyme activity was found in the 30-85% fraction. Purification by this method was 3.2 fold while the recovery was 35%. A second, more efficient, method was developed using ethanol as a precipitant in the presence of 0.1 M NaCl. Crude autolysates were precipitated with 75% cold ethanol. The precipitate was dissolved and exposed to 33% ethanol. This fraction was further purified by chromatography on Bio Gel A50m. Active fractions were pooled and concentrated. This scheme resulted in a purification of 14 fold and a yield of 31%. With heat-inactivated cell walls as substrate the partially purified autolytic enzyme was active at temperatures from 30°C to 62°C with maximum activity at 54°C. The pH optimum was broad (7-10); maximum activity occurred at pH 9-9.5. Divalent cations were required for activity. Activation occurred with Ba+, Ca++, Mg ++, and Mn ++. The reaction was inhibited by Fe++ and Cu++. Solutions of enzyme were stable for several hours at room temperature and for at least two months at -20°C. Activity was unaffected by freezing and thawing during this period. Lyophilization caused a 50% reduction in activity. No evidence for proteolytic activity was found. The partially purified enzyme contained 3% tightly bound organic phosphorus which was assumed to be in teichoic acid. This complex was not dissociated by several physical methods such as electrophoresis, ion-exchange chromatography, and gel filtration. These findings permitted tentative characterization of the enzyme as an acidic glycoprotein.

Guide to Protein Purification

Guide to Protein Purification PDF

Author: Richard R Burgess

Publisher: Academic Press

Published: 2009-11-03

Total Pages: 915

ISBN-13: 0080923178

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Guide to Protein Purification, Second Edition provides a complete update to existing methods in the field, reflecting the enormous advances made in the last two decades. In particular, proteomics, mass spectrometry, and DNA technology have revolutionized the field since the first edition’s publication but through all of the advancements, the purification of proteins is still an indispensable first step in understanding their function. This volume examines the most reliable, robust methods for researchers in biochemistry, molecular and cell biology, genetics, pharmacology and biotechnology and sets a standard for best practices in the field. It relates how these traditional and new cutting-edge methods connect to the explosive advancements in the field. This "Guide to" gives imminently practical advice to avoid costly mistakes in choosing a method and brings in perspective from the premier researchers while presents a comprehensive overview of the field today. Gathers top global authors from industry, medicine, and research fields across a wide variety of disciplines, including biochemistry, genetics, oncology, pharmacology, dermatology and immunology Assembles chapters on both common and less common relevant techniques Provides robust methods as well as an analysis of the advancements in the field that, for an individual investigator, can be a demanding and time-consuming process

Undergraduate Research in the Sciences

Undergraduate Research in the Sciences PDF

Author: Sandra Laursen

Publisher: Jossey-Bass

Published: 2010-06-10

Total Pages: 352

ISBN-13: 0470625619

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Undergraduate research (UR) is widely believed to enhance the learning experience of students in science, technology, engineering, and mathematics programs. This is the first comprehensive, practical, research-based book on undergraduate research. It addresses how the benefits to UR participants arise; compares the benefits of UR with other types of educational activities or experience; the long-term value of UR; and more. Intended to assist both existing and new UR practitioners with program design and evaluation needs, the book will also be useful to the wider community of academics, policy-makers, and funders of UR programs.

Microbial Enzymes and Biotechnology

Microbial Enzymes and Biotechnology PDF

Author: W.M. Fogarty

Publisher: Springer Science & Business Media

Published: 2012-12-06

Total Pages: 482

ISBN-13: 9400907656

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Biotechnology is now one of the major growth areas in science and engineering and within this broad discipline enzyme technology is one of the areas earmarked for special and significant developments. This publication is the second edition of Microbial Enzymes and Biotechnol ogy which was originally published in 1983. In this edition the editors have attempted to bring together accounts (by the relevant experts) of the current status of the major areas of enzyme technology and specifically those areas of actual and/or potential commercial importance. Although the use of microbial enzymes may not have expanded at quite the rate expected a decade ago, there is nevertheless intense activity and considerable interest in the whole area of enzyme technology. Microbial enzymes have been used in industry for many centuries although it is only comparatively recently that detailed knowledge relating to their nature, properties and function has become more evident. Developments in the 1960s gave a major thrust to the use of microbial enzymes in industry. The commercial success of alkaline proteases and amyloglucosidases formed a bed-rock for subsequent research and development in the area.

Production, Purification and Characterization of Thermostable Protease from Alkaliphilic and Thermophilic

Production, Purification and Characterization of Thermostable Protease from Alkaliphilic and Thermophilic PDF

Author: Seden Güracar

Publisher:

Published: 2011

Total Pages: 152

ISBN-13:

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Proteases are the hydrolase enzymes that catalyze the hydrolysis of the peptide bonds in the primary structure of proteins and peptids. They are used to cleave the proteins specifically to produce useful peptides in the processes. Proteases are present in a wide variety of living organisms and they also show different physicological, physicochemical, biological, chemical functions on the earth. They are the most important enzymes in the industry, accounting for 60% of the total enzyme scales in the world. The microorganisms that were previously isolated and characterized as a Bacillus sp. from Balçova Geotermal region in İzmir were used in the experiments. The aim of this study was to produce the protease enzyme from alkaliphilic and thermophilic Bacillus sp., purify and determine the properties of the enzyme with the characterization steps. When the screening studies and growth conditions were investigated, it was understood that the alkaliphilic and thermophilic Bacillus sp. produced extracellular protease enzyme. This extracellular protease enzyme was purified by ammonium sulphate precipitation and ion exchange chromatography chromatograpy. The yield and purification fold after purification of the enzyme were 33% and 1.41, respectively. In the characterization studies, the results indicated that the protease enzyme had highest activity at pH 8.0 and 55 C. The protease enzyme lost 20% of its activity at pH 4.0 and it lost 10% of its activity at pH 10.0. The protease enzyme at temperatures below 55 C lost 15% of its activity and also the protease enzyme at temperatures above 55 C lost 25% of its activity. The protease enzyme was stable at different pH values during 3 hours and at different temperature values during 6 hours. When compared the substrates, casein showed higher activity. The effect of organic solvents and surfactants on protease activity was investigated and the results indicated that the protease enzyme was stable in the presence of 10% of the organic solvents and 1% of the surfactants. PMSF and the protease inhibitor coctail decrease the activity of the protease.

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