ENZYMES AS BIOCATALYSTS

ENZYMES AS BIOCATALYSTSAnamika Chatterjee Guided by:12BCH002 Prof. Priya SaxenaCheck for conventional catalysts that can be replaced by enzymes1WHAT ARE ENZYMES ?Enzymes are biological molecules that act as catalysts . Every biochemical reaction in the cell is catalysed by specic enzymes.

Enzymes are in generalglobular proteinsand range from just 62 amino acid residues in size,to over 2,500 residues.

Like all proteins, enzymes are long, linear chains of amino acids thatfoldto produce athree-dimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties.

ENZYMES v/s CHEMICAL CATALYSTSgenerally more efcient (lower concentration of enzyme needed)

can be modied to increase selectivity, stability, and activity

highly selective

environment friendly and are completely degraded in the environment The most important advantage of a biocatalyst is its high selectivity.

Such high selectivity is very desirable in chemical synthesis as it may offer several benefits such as reduced or no use of protecting groups, minimized side reactions, easier separation, and fewer environmental problems.

Other advantages, like high catalytic efficiency and mild operational conditions, are also very attractive in commercial applications.LOCK & KEY MODEL

suggested byEmil Fischerin 1894

both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another

while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieveINDUCED FIT THEORYDaniel Koshlandsuggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts with the enzyme.

As a result, the substrate does not simply bind to a rigid active site; the amino acidside-chainsthat make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic functionENZYME KINETICS MICHAELIS MENTEN EQUATIONFree enzyme (E), Substrate (S), Enzymesubstrate complex (ES) and products (P)

v = k3 [ES]

v max = k 3 ([E] + [ES])

This function represents a typical saturation curve where Km equates to the substrate concentration when the velocity is half the maximum and serves to indicate the rate at which maximum velocity is reached (i.e. high Km means a slow rate and vice versa).

Experimentally determining Rate parameters

Lineweaver Burk plot

gives good estimates on Vm, but not necessarily on Km

Eadie Hostfee Plot

Eadie Hostfee plots can be subject to large errors since both coordinates contain but there is less bias on points at low [S].

Hanes Woolf Plot

This plot is used as the best possible approximation of Vm .

APPLICATIONSRange of potential applications

Advent of recombinant DNA technology has made possible to make formerly rare enzymes in large quantities and hence reduce cost.

Smaller quantities of ne high-purity enzyme preparations are also required for numerous applications.

These include roles as therapeutic agents, many of which are now recombinant products, components of food and medical diagnostic test kits, biosensors, as research tools and many other purposes.

There is an increasing demand for ne enzymes used in molecular biology, particularly restriction endonucleases and DNA polymerases,

IMMOBILIZATION

Enzymes that are more expensive to produce, including many intracellular microbial enzymes, and those employed in biosensors and analytical test systems, are often used in an immobilized form.

Benecial features:

reuse; suitability for application within continuous operations; their product is enzyme free, therefore further processing to remove or inactivate the enzyme is not required; improved enzyme stability; and reduced efuent disposal problems.

Enzymatic Fuel Cells

Advantages :Enzymes are easily mass manufactured and cheaper compared to the mining of precious metals which have an inelastic supply. Enzymes can also process organic compounds such as sugars and alcohols, which are exceedingly frequent in nature. Most organic compounds cannot be used as fuel with metal catalysts because the carbon monoxide formed during the fuel cells working will quickly poison the precious metals that the cell relies on, rendering it ineffective. Sugars and other biofuels can be harvested on a massive scale, and can be found in nearly any part of the world, thus making it an amazingly striking option from a logistics standpoint, and even more so for those concerned with the adoption ofrenewable energy sources.

Biomass is extensively used as fuels such as in production of bio- ethanol. However food security concerns prevent the large scale production of bio ethanol using sugars and starch. Hence, lignocellulose biomass is a possible alternative. Until recently, lignocellulose biomass was inert to enzymatic hydrolysis. However a patented method has been developed to produce biothanol from lignocelluloses biomass. The cellulose is separated from lignin and further processed to get sugars and finally ethanol. [8]Another patent has been extended to use sweet variety of sorghum for production of biofuels by increasing the sugar content and decrease lignocellulose content. [9]

Lignocellulosic biomass- dry plant matter composed of cellulose and lignin31CASE STUDY Two plant based enzymes with diverse applications were studied and an economic analysis was performed to evince that enzymes are cheaper to produce since the advent of recombinant technologies and hence a more viable option over industrial catalysts.

butyryl cholinesterase It is an enzyme that can act as a bio scavenger to offset the effects of cholinesterase inhibitors such as sarin . Also, it is already in use as bio defense countermeasures in several countries. At present, BuChE is extracted from outdated human blood supplies, but it can also be made recombinantly in cell culture, transgenic animals, and plant systemsSARINSarin, is used as achemical weaponowing to its extreme potency as anerve agentand it has been classified as aweapon of mass destruction inUN Resolution 687.

Production and stockpiling of sarin was outlawed as of April 1997 by theChemical Weapons Conventionof 1993, and it is classified as aSchedule 1 substance.

Sarin can be lethal even at very low concentrations, with death following within 1 to 10 minutes after direct inhalation due to suffocation from lung muscle paralysis .People who absorb a non-lethal dose, but do not receive immediate medical treatment, may suffer permanent neurological damage.

With the recent use of chemical nerve agents such as sarin, there is continued interest on the part of many governments in stockpiling BuChE as a countermeasure.

The advantages of speed of prototyping, manufacturing flexibility, and ease of indoor scale-up are clearly differentiating features of transient systems and explain why this approach has been widely adopted in the manufacture of many PMP products.

CELLULASESCellulase complex, a mixture of 46 enzymes used to saccharify cellulosic feedstocks for the manufacture of ethanol as a bio fuel.

This target was chosen for study because, for more than 30 years, the price of cellulases has been a major hindrance to the economic feasibility of cellulosic ethanol programs. Cellulases provide a good example of a tremendously cost sensitive product class and hence serve as a novel enzyme to perform studies. Cellulases currently under evaluation in bioethanol programs are all produced by microbial fermentation.

Despite decades of research on lowering cellulase manufacturing costs, these enzymes still account for 2040% of cellulosic ethanol production costs.

Hence, lowering the cost of the biocatalyst is critical to the eventual adoption of bio fuel processes that utilize renewable plant biomass feedstocks without competing with food or feed supplies. An alternative to fermentation produced cellulases is the production of these enzymes in crop plants, with the ultimate goal of producing cellulases at commodity agricultural prices.METHODS SuperPro Designer ,Version 9.0 (Intelligen, Inc., Scotch Plains, NJ; http://www. intelligen.com/), was the software used for the modelling of both case studies. It is generally used for techno economic modeling and is utilized for process simulation and flowsheet development. It is capable of performing mass and energy balances, equipment sizing, batch scheduling/debottlenecking, capital investment and operating cost analysis, and profitability analysis.

Nicotina host plants were the source for obtaining the active ingredient of both the enzyme classes . Nicotiana species, notably N. tabacum, N. excelciana, and N.benthamiana, are preferred hosts for PMB manufacture due to their metabolic versatility, permissiveness to the propagation of various viral replicons, and high expression yields achievable with a wide range of targets. Use of these hosts for production of clinical trial materials is also familiar to FDA and other regulatory agencies, thus facilitating Nicotianas acceptance in regulation-compliant manufacturing.

Plants are also free of adventitious agents that can infect humans and animals (a concern in cell-based systems and transgenic animals) and this inherent safety feature pays dividends by enabling the streamlined purification of the final product without the need for adventitious agent removal steps.

Plants eukaryotic protein processing enable them to synthesize complex classes of biomolecules, such as monoclonal antibodies and therapeutic enzymes.

Adventitious agent- foreign susbtance that is added inadvertently not natural or hereditary ( basically biochemical contamination)41 Recent advances in glycoengineering of host plants have enabled the production of human- and mammalian-identical (or at least mammalian similar) molecules that exhibit comparable or even superior pharmacology to their cell culture-derived counterparts.

Inescapably, the growth of the population in developing world regions, the aging of the population in industrialized countries, population displacement due to political turmoil, degradation of environmental quality, and depletion of non renewable resources are serious challenges that have not been and likely cannot be readily met only by the existing product manufacturing platforms. This creates new opportunities for plant-based systems to yield lower cost and more widely accessible biopharmaceuticals, food, feed, fuels, and industrial materials.

RESULTS BuChE could be manufactured in plants using transient expression for approximately $234 per 400-mg dose if an existing toll-manufacturing facility were available to accommodate production of 25kg/year of purified enzyme (equivalent to 62,500 doses/yr).

If a new facility with that capacity needs to be built, the cost per dose is projected to increase to approximately $474.

Further economic gains could be possible if capacity were to be increased to 100 kg of enzyme per year or more, which, in a toll manufacturing scenario, could reduce the cost of BuChE to below $200/dose. Even with conservative assumptions, these costs are dramatically below the costs obtainable with blood extraction processes for this enzyme and may be substantially lower than those for transgenic approaches.

In addition, the combination of speed of product prototyping enabled by transient expression, the superior quality and functionality of the BuChE obtained, lack of adventitious agents, and the rapid scalability of plant systems should make plants the preferred platform for the rapid and cost effective production of this and similar products.Atransgeneis a gene or genetic material that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another. The introduction of atransgenehas the potential to change the phenotype of an organism.

44 With the assumptions and process parameters adopted for the cellulose case study , our results show that high density field cultivation of tobacco induced to synthesize several enzymes of the cellulase complex could be competitive with fungal cellulases produced by fermentation for the saccharification of biomass in the production of cellulosic ethanol

REFERENCES[1] Tyler Johannes, Michael R. Simurdiak , Huimin Zhao, Biocatalysis, Encyclopedia of Chemical Processing, 2006 , pp 102 [2] http://en.wikipedia.org/wiki/Enzyme, 28/01/15, 20:20[3] Saha B. et al., Applied Biocatalysis in Specialty Chemicals and Pharmaceuticals; ACS Symposium Series; American Chemical Society: Washington, DC, 2001[4] Kim Gail Clarke, Bioprocess Engineering, Woodhead Publishing , 2013

[5] http://www.kyrobio.eu/Anton_Glieder.html, 8/4/2015, 20:34[6] Michael J. Waites, Industrial Microbiology: An Introduction, Blackwell Science, 2001[7] http://en.wikipedia.org/wiki/Enzymatic_biofuel_cell, 8/4/2015, 21:00[8]K. Ties, Method for producing ethanol by fermentation from lignocellulosic biomass, 2011[9] M.Joachim, C.T. Martin, B.Remy, Compositions and Methods for biofuel crops, 2011[10] Daniel Tus, Tiffany Tu, and Karen A. McDonald, Manufacturing Economics of Plant-Made Biologics: Case Studies in Therapeutic and Industrial Enzymes, Hindawi Publishing Corporation, BioMed Research International Volume 2014, Article ID 256135,