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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
0003-2700/84/A351-1528$01.50/0 0 1984 American Chemical Society
Report Doris C. Warren Department 01 Chemistry HoustonBaptist University 7502 Fondren Rd. Houston, Tex. 77074-3298
IERS
in Membrane "bchnology and Chromatography: ADDkatiOnS for Bbtechnology
nnalytical chemistry and the analytical chemist will play an important role and participate more fully in the area of biotechnology and genetic engineering in the future. The analytical chemist will be working with the biochemist, the molecular biologist, and the process engineer to scale up bench-scale syntheses of biomolecules for processing large amounts of product. Analytical HPLC will have an increasingly important role in on-line monitoring of parameters in the synthesis process. Scaled-up HPLC will be important in the purification of the final product. The demands of modern biology will require the development of a breed of biotechnologically competent analytical chemist. This breed of analytical chemist will not only have to be well-grounded in traditional theory and skills, but must also be aware of the basic principles of biology, biochemistry, microbiology, molecular biology, biocatalysis, and the basic engineering processes associated with the production of biomolecules. Microorganisms, such as yeasts and bacteria, have been used by humans since they firat leavened bread, fermented wine, and aged cheese. Now bacteria are also being used in factories-producing drugs, foods, and chemicals through a process referred to as biotechnology. Biotechnology, which includes microbes used for industrial purposes, recombinant DNA, microbial biosynthesis, cloning, and gene splicing, is already being used in many areas including human health, agriculture, food processing, chemical production, energy, metals extraction, creation of forest uroducta, and environmental proteciion. Of all the forms of biotechnology, bioengineering holds the greatest POtential for solving the problems of industry. In bioengineering, also known
as genetic engineering, gene splicing, or recombinant DNA, a gene is taken from one organism and is slipped into the genetic material of another. The biochemist thus creates a hybrid without having to contend with the time delays and the role of chance encountered in cross-breeding. Bioengineering is being used in the health field for the synthesis of hormones, antibiotics, insulin, and interferons. Bipngineering also holds the promise of new food produds and the large-scale production of ethanol as an economical fuel. During World War I, when the Germans needed elrcerol to manufacture explosives, &man biochemist Carl Neuberg discovered that by adding sodium bisulfate M ferments1 fat (fermentation produces small amount8 of glycerol), the bacteria in the fat pro-
ANALYTICAL CHEMISTRY. VOL. 56. NO. 14, DECEMBER 1984
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duced greater amounts of glycerol and less ethanol. The Germans used this process to produce 1000 tons of glycerol each month. The British needed acetone to manufacture munitions and Chaim Weizmann developed an acetone-butanol fermentation process that was used for many years until displaced by a petroleum-based process. These were the first two practical applications of microorganisms in largemale industry other than food production. Microbial cells now have two main commercial applications: as a source of protein, mainly animal feed, and to carry out biological conversions, processes in which a compound changes into a structurally relaced compound by means of enzymes supplied by the cells. Reactions involving microorganisms have many advantages over chemical reagents. Chemical reactions require large amounts of energy to either heat or cool the reaction vessels, whereas microorganisms do not, and generally require solvents and inorganic catalysts, both of which may be pollutants. Chemical reactions, in addition, may leave unwanted by-products. There are four classes of industrially important microorganisms: yeasts, molds, single-cell bacteria, and actinomycetes. The commercially important products of these organisms fall into four major categories: the microbial cells themselves; the large molecules, such as enzymes, which they synthesize; their primary metabolic products. compounds essential to their growth; and secondary metabolic products, compounds not essential to their growth. Perhaps the most useful of the molecules manufactured by microorganisms are enzymes, biological catalysts important in the food and chemical industries for their efficiency, potency, and specificity under conditions of moderate temperature and acidity. When enzymes are produced by fermentation, their fermentation timesare short, their growth media inexpensive, and their screening procedures simple. Microbiologically produced enzymes are used in brewing, baking, textiles, meat tenderizers, 1530A
leather, detergents, and cheeses. The secondary metabolic products, those not essential to the microorganism’s growth, are the most important for the health industry. The best known are the antibiotics, hut alkaloids, toxins, and plant growth factors are also produced. The most important primary metabolic products produced in fermentation are amino acids, purine nucleotides, vitamins, and organics such as citric acid and riboflavin. Finally, organisms grown for the cells themselves me bakers’ yeast and Pruteen, a form of single-cell protein requiring methanol to grow and a possible re. placement for many animal feeds. indudrial production wHh microorganlsms The batch process and the continuous process may both be used in manufacturing with microorganisms. In the batch process, the fermentation vat or reaction vessel is filled with the starting materials, often including the microorganisms themselves. The biochemical conversion takes place in the containers over a period ranging from a few hours to several days. Ultimately the vessel is emptied, the product purified, and a new batch started. When a continuous process is used, the raw products are supplied and the finished products withdrawn, both a t a steady rate. In this process, all stages of the biochemical conversion must occur a t essentially the same rate. The batch process is like the operation of a steel mill and the continuous process like the workings of a refinery. Continuous methods work best with large volumes, but until now most products of microbiology have been made in batches. In both processes, the organisms need a medium to grow in, usually water, which generally must contain carbon, nitrogen, phosphorus, and oxygen for the organism to feed on. The medium must be well mixed so all the microorganisms can reach the nutrients and substrate. In theory, continuous industrial process has several advantages over the batch process. The continuous process can produce a potentially higher volume of substances for a plant of a given size. The continuous process also saves catalysts. Although the production process does not consume them, they are thrown out with the medium when used in the batch process. Losses can be prevented by recycling the catalysts even though it is difficult to take living cells from a stream of fluid and return them to the reactor. Another technique would be to keep the catalysts inside the reactor on a packed bed or solid support on which the cells are encouraged to grow.
ANALYTICAL CHEMISTRY, VOL. 56,
NO. 14, DECEMBER 1984
Presently the batch method is used more frequently in industrial microbiology because of the flexibility of production offered. A fermentation vat can produce one batch of a product, be emptied, and then produce another batch of a totally different product in higher demand. Raw materials must be treated before being added to the vat, and the final product must be treated carefully when it is later removed from the vat. Many of the products are fragile chemically, so when the desired product is removed from the spent medium, the temperature and pH of the solution must be carefully controlled. Since the product is usually dissolved or suspended in a large amount of water, the water must be removed from the product or the product removed from the water. This is where various separation techniques are required. Evaporation, distillation, absorbing the product on a solid material, and dissolving the product in a solution with greater solubility are the techniques generally used to remove the product from the solution. Newer separation methods that are not nearly so energy intensive and that are capable of more precise separationsmembrane techniques including reverse osmosis and ultrafiltration and various types of chromatography-are becoming more and more important. Of the industries that rely on the microbiological process, the manufacture of cultured dairy products, i.e., cheese-making, is second in sales only to that of alcoholic beverages. Breadmaking and the large-scale cultivation of bakers’ yeast is one of the most sophisticated branches of industrial microbiology. In 1979, the wholesale value of prescription drugs sold in the US. totaled nearly $7.5 billion. About $1.5 billion came from the sale of drugs produced using microorganisms. Most of the pharmaceuticals made this way used fermentation vats with a maximum volume of 100,000 L. Culturing of the industrial strains of fungi or bacteria is started in smaller tanks; then they are transferred to the larger fermenters. The temperature, pH, oxygen supply, and nutrients in the medium are strictly controlled, and the mixture is stirred by blades inside the fermenter. Biotechnology Instrumentation The “gene machines,” the companies producing them, and the different chemical processes and reagents licensed for these machines have received a great deal of publicity. However, this type of equipment is only one of three major types of instrumentation that can be identified as playing a very significant role in the fast-growing genetic engineering-biotechnology sector. These three major areas of
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rdure 1. Typical protein purifichuw and Sequence method biotechnology instrumentation can he identified as: protein-amino acid analysis and sequencing; nucleic acid-nucleotide synthesis; and purification of producta, which is an important component in the first two areas as well. Protein-amino acid analyzers Amino acid analyzers or the more sophisticated protein sequencers fall into this category. The amino acid analyzer, which is actually a specialized high-performance liquid chromatograph (HPLC), can provide a complete analysis of a mixture of the amino acids derived from a particular protein. A protein sequencer chemi-
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cally hydrolyzes a protein one amino acid at a time. As each amino acid comes off the protein molecule in sequence, it is identified by HPLC. There are three basic steps in amino acid sequencing: complete chemical hydrolysis, site-selective hydrolysis, and terminal-site derivative hydrolysis. In complete chemical hydrolysis, the first step is to hydrolyze all the peptide bonds of pure polypeptide. The amino acid mixture is analyzed by ion exchange chromatography to de- , termine not only which amino acids are present in the molecule, but bow many of each type are present. In site-selective hydrolysis, partial enzymatic hydrolysis of the peptide chain using the digestive enzyme trypsin cleaves the polypeptide chain at known specific bond sites. The hydrolysis is specific in that only those peptide bonds are broken in which the carboxyl group is contributed by either lysine or arginine. The number of smaller peptides produced is dependent on the number of lysine and arginine residues present in the original polypeptide. Fragments produced by trypsin hydrolysis are then separated using various chromatographic methods. Terminal-site-selective hydrolysis involves a chemical method called Edman degradation, which labels and removes only the NHa-terminal amino acid fragment of a protein, leaving all other peptide bonds intact. The phenylthiohydantoin (PTH) derivative generated in each step is identified chromatographically upon removal. The peptide residue can he treated again and again until all amino acids making up the fragment have been identified in order, or sequenced. A typical protein purification and sequence method is shown in Figure 1.
Nucleic acid-nucleotide synthesis Amino acid analysis and protein sequencing are used in tandem with the oligonucleotide synthesizer. Once amino acid analysis and protein se-
ANALYTICAL CHEMISTRY, VOL. 56. NO. 14, DECEMBER 1984
quencing reveal which amino acids are present and in what order, the oligonucleotide synthesizer can be programmed to synthesize a gene that, when placed into a bacterium, will manufacture the protein. Simply stated, the oligonucleotide synthesizer is an automatic, computer-controlled instrument that uses programmed chemical reactions to automatically couple nucleotides to produce short stretches of DNA to specification. Nucleic acids are long-chained polymers composed of nucleotides. The sequence of nucleotides is the repository of all genetic information carried by chromosomes. There are two chemically different types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Both DNA and RNA are composed of four different nucleotides. Each nucleotide contains a nitrogenous base known as a purine or a pyrimidine; a sugar, ribose in RNA, deoxyribose in DNA; and a phosphoryl group (Figure 2). Nucleotides are the phosphate esters of nucleosides. The four commonly occurring deoxyribonucleotides found in DNA are deoxyadenylate, deoxythymidylate, deoxyguanylate, and deoxycytidylate. The compositional variation in the nucleotide is accounted for solely by the purine (adenine or guanine) or pyrimidine (thymine or cytosine) base attached to the C-1' position of the sugar (deoxyribwe). Each amino acid in a protein chain is represented by a coding triplet that comprises three successive nucleotides in DNA. For example, a triplet of three consecutive adenines (A-A-A) in DNA is translated as phenylalanine in a growing protein chain. Each of the 20 amino acids, of which all proteins are made, has ita own genetic code. The nucleotides in a polynucleotide chain are joined by a bond between the phosphate group of one nucleotide and the sugar of another nucleotide. The bond is generally formed between the phosphate group (located on carbon 5' of ita sugar) and carbon 3' of
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The inheren& high yields of the Applied Biosystems phosphoramldite DNA synthesis method have been increased by a new solid support, Controlled Pore Glass! CPG. with a long alkyl chain attachment to the nucleoside, lmproves stepwise yields by 2-4%. With coupling yields now routinely 98-100%. probes can frequently be used without purification. In addition, whole genes may be constructed with longer fragments requiring fewer Putfficationsand ligations. Model 380A DNA Synthesizers,operating In over 180 laboratoriesworldwide. produce more than 4.000 oligonucleotides every month. And the numbers are hcreashg rapidly. New developments, such as CPG and the large-scale lO-dcromole synthesis columns, continue to T d the range of apPUcationsas -11 as b p ~ w the e mults obtained. Applied Bksystems chemistsand 38OA users have d i n e d procedures for the analysk and mi.ficauon of oligonucleolides. Their work includes techniques for preparative and analytical gel electrophoresis, "LC. 5'-end labeling and Maxam-CiIbert sequendng. We've complled this data h the latest 38OA Users BulleUn. For a complhentary copy, contact your local Applied Blosystems representatbe, one of the omces below.or &de reader service
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APPLIED BIOSYSTEMS. INC.. 850 Lincoln Centre Drive. Foster City. CA 94404. (800)874-9868 In California (800)831-3582 Telex: 470052 IU CI I O ~ D C A . D D , Icn nlnCVCTCLIC C L m U nnmehrc0 r n A n C I A l D b , n n d o r l t I 1 I P c 9 C..erm~nu.nf
