Site Directed Mutagenesis of Subtilisin

Site Directed Mutagenesis of Subtilisin Introduction Subtilisin is a monomeric protein serine protease, which is produced by Gram positive bacteria and fungi.(1) Serine proteases are a subgroup of carbonyl hydrolase. These carbonyl hydrolases can be naturally occurring or recombinant. Naturally occurring carbonyl hydrolases consists of subtilisin matalloproteases, serine carboxypeptidase and thiol proteinase. In case of recombinant carbonyl hydrolase, the sequence coding for the wild type enzyme is altered to produce a mutant either by insertion, substitution or deletion of amino acid. Proteases are a diverse class of enzymes having several biological functions and specificities. The catalytic machinery of these enzymes is attributed to subtilisin and mammalian chymotrypsin related bacterial serine protease.(2) They are responsible for cleaving peptide bonds found in proteins. The proteases play an important role in cell wall turnover and are maximally expressed in the stationary phase. Serine proteases have molecular weights ranging f rom 25,000-30,000. They are inhibited by diidopropylfluorophosphate but are resistant to EDTA ( ethylene diamaine tetra acetic acid.) (3) The amino acid sequences of several subtilisins like subtilisin Carlsberg, subtilisin BPN, and subtilisin DY are known. Subtilisin BPN is a 275 amino acid serine protease, which is secreted by Bacillus amyloliquefaciens. This enzyme is industrially important and its gene has been cloned and expressed in Bacillus subtilis. This molecule has two enzyme binding sites- one of the site binds weakly to cations and the other one binds calcium with high affinity. These calcium binding sites are common characteristics of microbial proteases since they contribute towards kinetic and thermodynamic stability. Subtilisin is majorly used in industries wherein the environment contains high levels of metal chelators which remove the calcium from subtilisin , thus reducing its stability. It would be highly beneficial to create a stable subtilisin which would be independent of calcium. These mutated subtilisin enzymes will have an enhanced stability even in the presence of detergents and chaotropic ag ents. X-ray crystallographic analysis of mutants revealed that the conformational changes due to the mutations are localized, with very less distortion of the backbone structure. Thus a large increase in stability can be obtained with very minor changes in the protein structure. Mutants can be created by deletion, addition or substitution of amino acids at positions 75-83. The deletion of amino acids 75-83 has shown to eliminate the calcium binding site while still retaining its enzymatic activity. However, calcium free subtilisins are susceptible to proteolysis. This can be overcome by converting the active site serine 221 to cysteine (S221C). This allows the protein to fold without any auto-degradation by proteolysis. A recombinant DNA was created which encoded an active subtilisin protein without the ability to bind to calcium. The protein had no codons which specifies amino acids 75-83 and had certain additional stabilizing mutations at the N-terminal amino acids 1-8 or the ÃŽ ± helix amino acids 70-74 or the helix turn amino acids 84-89. Suitable host cells were transformed with an expression vector containing this DNA segment. Upon culturing the host cells the enzymatically active subtilisin mutant was expressed and recovered.(1) Fabric cleaning compositions containing subtilisin BPN mutants The ability to hydrolyze proteins can be exploited by incorporating these proteases as an additive to laundry detergents. Most of the stains on clothes are proteinaceous and these enzymes can efficiently aid in their removal. The amino acid sequence of the protease is responsible for its characteristics. The subtilisin BPN variants have modifications at 2-3 amino acid positions. This variant has an increased hydrolytic power and decreased adsorption to an insoluble substrate as compared to the original subtilisin. A decreased adsorption to the substrate results in a better cleaning performance. In subtilisin BPN, the amino acids ranging from position 199-220 form a large exterior loop on the molecule. This loop plays an important role in mediating the adsorption of enzyme to the surface bound peptide and mutations in this region will affect the adsorption. The substituting amino acids in case of a mutation are either neutral or negatively charged. The substituting amino acid for position 199 is mostly His, Ala, Cys, Pro, Glu, Asp, Gly, Asn or Gln and for position 207 is Glu or Asp. The cleaning compositions comprise of 0.001-1% of one or more enzyme variants. The enzyme variants can be used along with other conventional ingredients to produce efficient fabric laundry composition. These fabric cleaning compositions also contain various zwitterionic or anionic surfactants, hydrotopes, dyes or pigments, primary or secondary alcohols, chelating agents and brighteners. Fermentation: The Bacillus subtilis cells having the subtilisin mutant are allowed to grow till mid log phase in LB glucose broth and is then inoculated into a fermentor. The cells are grown overnight at 37ËÅ ¡C. Chloramphenicol is added for the antibiotic selection of mutagenized plasmid. The cells are removed from the broth by centrifugation The enzyme is purified by affinity adsorption or cation exchange chromatography. The active enzyme concentration is determined by the pNA assay. ( para nitro anilide) This assay measures the rate at which pNA is released as the enzyme hydrolyses the substrate. The rate at which the yellow colour is produced is measured at 410nm with the help of a spectrophotometer and is directly proportional to the enzyme concentration. The total protein concentration can also be estimated by determining the absorbance at 280nm. (4) Production of subtilisin variants Techniques used in microbiology, molecular biology, protein purification and recombinant DNA technology are all used in developing a subtilisin variant, which is a part of dish washing and fabric cleaning compositions. Cassette mutagenesis method for production of protease variants The gene encoding the protease is sequenced The sequence is screened for a point at which desired mutation can be made The sequences flanking this region are checked for the presence of restriction enzyme site so as to replace a short segment of the gene with an oligonucleotide which will then encode various mutants. The gene is mutated by primer extension The oligonucleotides are synthesized to have the same restriction sites, eliminating the need for synthetic linkers to create the restriction site. The genes coding for serine proteases of B.amyloliquefaciens, B.subtilis and B.licheniformis can be used as targets for mutagenesis. The gene can be inserted into a suitable vector and introduced into a host strain ( Bacillus PB92) for expression and production of mutant proteases. These mutations / substitutions enhance the performance and stability of subtilisin in detergent compositions. These serine proteases can be used in the form of granules and liquid composition both in laundry dishwashing and cosmetic applications. These enzymes are often in the form of encapsulated particles in order to protect it from the other components. Encapsulation also improves the enzyme performance and helps in regulating its availability. The encapsulating material can be derived from carbohydrates, silicates, polyvinyl alcohol, borates, PEG or paraffin waxes. Fabric cleaning performance by Blood Milk Ink microswatch assay. (BWI) This assay is performed on a microtitre plate. Samples of the subtilisin variants and reference subtilisin are obtained from filtered culture broths. 10ul samples of the enzyme are added to the BWI swatch plate along with 90ul of working detergent solution. The plates are incubated for 30 minutes. 100ul of the supernatant is transferred into a new microtiter plate and its absorbance is measured. Control wells contain the detergent solution without the protease sample. The measurement at 450nm tracks pigment removal and at 600nm tracks turbidity and cleaning. The performance index (PI) of the variant is calculated. PI compares the performance of variant and reference enzyme at the same protein concentration. PI>1 the variant is better than the wild type. PI=1 variant and standard have the same performance PI

Cells And Cell Theory :: essays research papers

Cells and Cell Theory What advantages does small size give to a cell? Many cellular processes occur by diffusion, which is efficient over short distances, but less efficient over long distances. Since all materials going in and out of a cell must pass through the plasma membrane, the greater the surface area of this membrane, the faster a given quantity of molecules can pass through. Smaller cells have a much greater surface-to-volume ratio than larger cells and therefore can "feed" all areas of the cell in less time. What is "surface-to-volume ratio," and how does it affect cell size? The surface-to-volume ratio is a mathematical relationship between the volume of an object and the amount of surface area it has. This ratio often plays an important role in biological structures. Think of a cell as a sphere: The surface area of a sphere can be calculated by 4Ã ° r2 where r is the radius of the sphere. Volume of a sphere can be calculated by 4/3 Ã ° r3. An increase in r will increase the surface area by a power of two, but increase the volume by a power of three. This means that the volume will increase much faster than the surface area. This puts an upper limit on the size of a cell, because if the cell volume gets too big, there won't be enough membrane to transport the amount of food in and wastes out to support that large cell size. What is the difference between prokaryotic and eukaryotic cells? Prokaryotic cells are more simple: they are usually much smaller and don't have a nucleus or any other membrane-bound organelles. Bacteria are prokaryotes. Eukaryotic cells are much more complex, are usually larger, and have a nucleus and several other membrane-bound organelles that allow them to compartmentalize their functions. All multicellular plants and animals are eukaryotes. A helpful trick to remember is that "you" are a "eu"karyote. Are there any single-celled eukaryotes? Yes--yeast, for example. Yeast are single-celled organisms, but they do contain a membrane-bound nucleus, mitochondria, and other organelles. What are the advantages and disadvantages of prokaryotic compared to eukaryotic cells? Although prokaryotes may seem more primitive than eukaryotes, they are among the most successful species on our plant and comprise a very large percentage of the total mass of all living things on earth. Simple, small, and single-celled organisms can reproduce quickly and evolve quickly. Prokaryotes can generate millions of progeny in a short period of time.

What I hope to gain from a Bachelor’s Degree in Business Administration

Management studies has always interested me and I am currently pursuing a Bachelor’s degree in business administration to work towards my aims and objectives which are described below and to pursue my interest in management studies and at the same time obtain a full fledged degree in Business Administration.By working towards this degree I hope to become a more well rounded personality alongside gaining more knowledge, skills and expertise in the field of business and management which I can wield towards my other long term goals, to evolve into a dynamic and capable leader in the field of business.Through this Business Administration program I hope to gain maximum and up to date knowledge and skills including technical skills to be successful in any area of business administration. I want to be able to understand the theoretical aspects of the knowledge and skills that I gain as well as apply them practically. I want to be able to explore and gain a thorough knowledge of conce pts, principles, theories, practices of modern business writers as well as the classical business writers.I hope that the program will enable me to build a strong foundation to pursue and be successful in any area of business administration by helping me understand and find the relation between the various interrelated areas of business administration. I hope that it would give me a good head start in analytical and strategic business studies. The skills that I wish to gain include soft skills, technical skills, analytical, mental skills, problem solving skills, negotiation skills and leadership skills. I want this program to help me improve my written and verbal communication.I want to ensure that my analytical skills are honed well to be able to analyze business conditions and decide the appropriate strategy to handle those conditions. I want to be able to hone my skills to be able to conduct research if necessary in various media and analyze the data that is gathered to reach con clusions about the business scenario. I want to be able to make presentations as and when required, to analyze the data collected and to reach business goals and to present a proposal to somebody and to negotiate and convince people in various business scenarios.I want to be able to make use of my skills and knowledge in the job market to play a good leadership and management role. I want this program to help me transform into an efficient and effective practitioner of ethical business practices. I want to be able to nurture my skills and knowledge in a rich interactive and intellectual environment by exchanging ides with other students, professors and eminent business personalities.I want to be able to take part in cultural activities and other extracurricular activities that will help me become a more balanced person and to contribute more wholesomely towards my community and my profession. I also want to go through various disciplines and understand the relationship between them and how they are relevant to Business management. I view this Business Administration program as a significant stepping-stone towards my long term goals and professional development in the field of business to enable me to turn into a competent business professional.

Critical Infrastructure Protection - 1106 Words

Introduction When it comes to protecting an infrastructure, careful planning and coordination needs to take place. Protecting an infrastructure takes an important security initiative called Critical Infrastructure Protection (CIP). The United States critical infrastructure is protected by the Department of Homeland Security. Examine three areas of the Department of Homeland Security: mission, operations and responsibilities The first area is the Department of Homeland Security’s mission. The Department of Homeland Security (DHS) was created in 2002 by the Homeland Security Act. In 2003 the DHS started their operations. The DHS primary responsibilities and objectives includes prevention, protection, and response to issues that threaten†¦show more content†¦No matter how hard you try or the efforts placed on prevention, no one can stop or prevent a natural disaster from causing damage to computer systems, downtime, and damage or disruption to other essential services. The only thing that ca help you with a natural disaster is to have recovery and contingency plans in place. The most unpredictable and dangerous attack comes from human threats. This can especially be damaging when it comes from someone in the inside, because they know what the security measures are and other protective measures that might be in place. An attack from an insider is hard to prevent because they have been given legitimate access to systems, they are most familiar with the computers and applications, and they will know how to cause damage. Evaluate the effectiveness of IS professionals in regard to protecting the U.S.’s critical infrastructure When evaluating the effectiveness of an IS professional in relation to protecting our nations critical infrastructure, issue arise from the outdated government regulation. Most of the efforts are non-effective because the government is trying to use 19th century solutions for 21st century problems. There are many regulations that are put into place but the difficulty comes when trying to change or remove the regulation that is currently in place. The direct result of slow regulation change is that cybersecurity regulations are already outdated before they are releases. The federalShow MoreRelatedEssay about Assignment 2: Critical Infrastructure Protection1280 Words   |  6 Pages Assignment 2: Critical Infrastructure Protection Eustace LangleyCIS 502 May-23-2013 Facilitator: Dr. Gideon U. Nwatu Strayer University Critical Infrastructure Protection Introduction The Presidential Policy Directive (PPD) on Critical Infrastructure Security and Resilience advances a national unity of effort to strengthen and maintain secure, functioning, and resilient critical infrastructure (The White House Office of the Press Secretary, February 2013). 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