ࡱ> ^`]g`bjbjss 9g\yffff  & & &8B&L&L &&&40'0'0'ezz z2444444$)hXe z^ezXff0'0'R333f f0' 0'23233, 0'& `={> &$ Ɲlӟ0~w,w$w ,z6|3]~D~zzzXXՒ^zzz d&% &% ffffff The novel technologies for the use of biocatalyst and biotransformations ura Vasi-Ra ki, University of Zagreb, Faculty of Chemical Engineering and Technology Savska c.16, 10000 Zagreb E-mail:dvracki@marie.fkit.hr Summary Throughout the history of mankind, microorganisms have been of tremendous social and economic importance. In the course of time, it was discovered that microorganisms could modify certain compounds by simple, chemically well-defined reactions, which were further catalyzed by enzymes. Nowadays, these processes are called biotransformations. Biotransformations are reactions in one or two-steps where the chemical structures of the substrate and product resemble one another. The reactions are catalyzed by isolated enzymes or enzymes in whole cells. These enzyme forms are named biocatalysts (Vasi-Ra ki, 2000) In comparison to fermentation processes fewer side-products are formed in enzymatic biotransformations, complex expensive fermentors are not required, aeration, agitation and sterility need not necessarily be maintained and substrate is not diverted into the formation of de novo cellular biomass. Isolated biocatalysts are especially useful if the reaction they catalyze is about to be completed, if they are resistant to product inhibition, and if they are active in the presence of low concentration of substrate (such as in detoxification reaction where pollutants are present in the waste stream). One-pot multi-enzyme reactions are much more feasible than a combined use of several chemical catalysts or reagents, especially as the latter often have be used in reactors made of special resistant materials to tolerate extreme conditions, such as the use of concentrated acids under elevated temperatures and pressures. Keywords: biocatalysts, biotransformations, malic acid, pyruvic acid, L-tert leucine Introduction Since early 1970s the use of biotransformations in industry for the fine as well as for the traditional chemicals, pharmaceutical and agrochemical intermediates production has steadily increased. Presently, approximately 100 different biotransformations are carried out in industry (Liese at al, 2000). Without doubt, there will be an increase in the industrial use of biocatalysts over the next 10 years because the biotransformations had led to a reduction in waste generation from 10 to 2 tones per ton of product. In the fine chemical sector, it would be in order of one million tones of waste per annum. Biotransformations can be carried out at ambient temperature and neutral pH without need for high pressure and extreme conditions thereby saving process energy. The use of biocatalysts has proven to be supplementary technology for the chemical industry. This technology allows in some case reactions, which are not easily conducted by classical reactions or in other cases it allows reactions, which can replace several chemical steps. Thus, highly chemo-, regio- and steroselective biotransformations can simplify manufacturing processes and make them even more economically attractive and environmentally acceptable. Recombinant DNA technology has dramatically changed enzyme production, because enzymes are synthesized in cells by the normal protein synthesis methods. A 5-10 year period required for classical enzyme development can be reduced to 1-2 years. Protein engineering, in combination with recombinant expression systems allows to plug in a new enzyme variant and to be very quick and cheaper at manufacturing levels. It is now well-known that enzymes do function in various forms, in organic solvents, and many in neat (pure) solvents or in supercritical fluids in the absence of added water, and finally in gas phases. Until now, mainly isolated hydrolases are used industrially with water as solvent (penicillin G splitting, hydrolysis of acrylonitrile, hydrolysis of methyl-p-methoxyphenyl-gycidate). On the another hand, oxidoreductases were used from whole cells in the industrial biotransformations (sorbit-sorbose oxidation, biocatalytic steroid hydroxylation). A considerable amount of time elapsed before the first isolated oxidoreductases were used in industry (synthesis of L-tert- leucine (Kragl et al.1996)). The development of the continuous coenzyme regenerating system by means of the isolated formate dehydrogenase had made it possible (Wichmann and Vasi-Ra ki, 2005). The biotransformations at mild conditions with high regio- and enantio-selective biocatalysts are green and economical alternative in chemical, pharmaceutical and agrochemical industry. The range of customers considering the utilization of enzymes, as a replacement to conventional chemical methods, appears to be growing. It appears that enzyme-based processes are gradually replacing conventional chemical-based catalyst e.g. the use of enzymes as catalyst provides a very new way of polymer synthesis; most of these polymers are otherwise very difficult to synthesize by conventional chemical catalysts (ESAB, 2005). It is no longer the case that biotransformations are relevant only to high added-value products such as pharmaceuticals. Bulk chemicals including polymers may have biotransformations such as conversion of methane to methanol (Chevron Research and Technology and Maxygen) or conversion of sugars to 3-hydroxypropionic acid (Cargill Inc. USA) or dehalogenation step in Dow2 s alkene oxide process. The next generation of biotransformations based process will target large volume chemicals and polymers and will compete directly with petroleum-based products. Biotransformations are becoming competitive with conventional routes, but industry expert believe that further improvements in enzymatic catalysis and fermentation engineering may be required before many companies are prepared to announce world-scale bioprocess plants. Bioprocessing proponents see a future in which micro-organisms are replaced by purified enzymes, synthetic cells or crop plants. Today, both the academic and the industrial community see biocatalysis as a highly promising area of research, especially for the development of sustainable technologies for the production of chemicals and more selective and complex active ingredients in pharmaceuticals and agrochemicals. Production of pyruvate Pyruvic acid and its salts serve as an effective starting material for the synthesis of drugs, agrochemicals and fat burners (Zeli, 2003). It is also a valuable substrate for the enzymatic production of amino acids such as L-dihydroxyphenylalanine (L-DOPA) (Li et al, 2001). There are two different approaches for the production of pyruvate: a) the classical chemical routes and b) biotechnological routes. The classical chemical routes: There are several chemical syntheses for pyruvate production. In the oldest one, which is described, pyruvate is produced by the dehydration and decarboxylation of tartaric acid in the presence of potassium hydrogen sulfates at 220 oC (Howard and Fraser, 1932). Decarboxylation of diethyltartarate (Sugiyama et al, 1992), oxidation of propylen glycol (Tsujino et al, 1992) and oxidative dehydrogenation of lactic (Ai and Ohdan, 1995), in the presence of heavy metals as catalysts and high temperature are also recently described. These chemical processes have in common that they are energy-intensive and that they use a heavy metals. Accordingly, these processes are not environmentally friendly. Therefore, more sustainable green process alternatives have been developed in the last two decades. Biotechnological routes: These process alternatives can be divided into three different approaches using: 1) isolated enzymes (Burdick and Schaeffer, 1987; Eisenberg et al, 1997); 2) resting cells (Izumi et al, 1982; Ogawa et al, 2001; Schinschel and Simon, 1993); and 3) fermentation (Li et al, 2001; Yokota et al, 1994). The bioconversion of glucose to pyruvate with non-growing, acetate auxotrophic cells of Escherichia coli YYC202 ldhA::Kan (Gerharz et al, 2002; Zeli, 2003; Zeli et al, 2003a; Zeli et al, 2004; Zeli et al, 2004a,), which is presented in Figure 1., offers the opportunity to produce pyruvate from sustainable, low cost substrate, glucose, with high product/substrate yield (YP/G=1.78 mol/mol), high volumetric productivity (QP=145 gpyruvate/L/d and high product titers of about 65 g/L. C6H12O6 ! 2 C3H4O3 + 2 H2O Glucose Pyruvate  Figure 1. Non-growing, acetate auxotrophic cells of Escherichia coli YYC202 ldhA::Kan (Gerharz et al, 2002, Zeli, 2003) Production of L-malic acid The production of malic acid has received great interest because this four carbon dicarboxylic acid is commonly used as a food and beverage acidulant, mainly as the D, L-racemate mixture, in food industry (Giacobbe et al, 1980). Moreover, to a lesser extent, the L-isomer of the acid, which holds about 10 % of malic acid market (Bressler et al, 2002), is used as a component of amino acid infusions and in the treatment of hyperammonemia and liver dysfunction (Goldberg et al, 1991). L-malic acid is incorporated in powdered soft drinks in conjunction with aspartame, as a flavor fixative and is used as an emulsifier for the manufacture of margarine and mayonnaise. L-malic acid as a monomer is used in synthesis of biodegradable polymers (Rossignol at al, 1999; Wada et al, 1996). It is presumed, that about 40 000 t of malic acid are used worldwide annually. The traditional method for preparing L-malic acid was by extraction from apple juice which has 0.4-0.7 % of L-malic acid and therefore it is not economical. Hence, today malic acid is produced by two additional processes: 1) chemical synthesis via hydratation of maleic or fumaric acid at high temperature and high pressure, yielding the racemic mixture, and 2) enzymatic synthesis, whereby fumaric acid is transformed to L-malic acid. Direct fermentation of carbohydrates to L-malic acid by Aspergillus flavus, though well-know, is not used in industry because of moderate productivity and the potential toxicity of the producing organism (Peleg et al, 1988). In the current industrial enzymatic process, fumaric acid is continuously converted to L-malic acid by immobilized whole cells of Brevibacterium ammoniagenes or Brevibacterium flavum (Takata and Tosa, 1993), containing the enzyme fumarase with high activity. The yield of L-malic acid reaches about 70 % of theoretical. The unconverted fumarate is recycled. The enzymatic reaction is carried out at neutral pH and results in L-malic acid salts. Thus, downstream processing involves separation of the un-reacted substrate as well as isolation of free acid. The enzymatic batch process using Corynebacterium glutamicum (Daneel and Faurie, 1994) also exists, but continuous process is more economical. The reaction catalyzed by fumarase is as follows:  EMBED ISISServer  Figure 2. Enzymatic process for L-malic acid synthesis. Fumaric acid is transformed into L-malic acid by addition of water molecule to the double bond. The process is a typical equilibrium reaction. The enzyme is highly stereospecific. Fumaric acid is obtained using byproducts resulting from the production of phthalic anhydride. The application of fumaric acid in the industrial field is limited by its low solubility therefore the conversion to L-malic acid is a solution. Even though the enzymatic process is environmentally more acceptable, the cost of L-malic acid produced by fumarase reaction from fumaric acid is higher than chemically synthesized acid. Therefore, the attempt to make it more economically is needed. Thus, a strain Saccharomyces cerevisiae was engineered to overproduce fumarase (Bressler et al, 2002). The advantage of this microorganism lies in increased productivity; lack of succinate accumulation and of course, compatibility with the requirements of food product safety. The yeast was immobilized in small glasslike beads of the composite alginate-silicate matrix ensuring the long-time stability. It was found (Vrsalovi Prese ki and Vasi-Ra ki, 2005) that Saccharomyces bayanus (UVAFERM BC) had the high activity of fumarase. Cells was permeabilized with 0.2 % (w/v) CTAB for 5 min. The average achieved conversion of fumaric acid of up to 82 % gives 21, 40, 83 and 175 mM of l-malic acid respectively from 25, 50, 100 and 210 mM fumaric acid. Production of L-tert-leucine The synthesis of L-tert-leucine is an example of the application of oxidoreductases with cofactor regeneration in an industrial continuous process (Kragl et al, 1992; 1993; Kragl et al, 1996; Bommarius et al., 1998). This chiral amino acid is important building block for drug synthesis (Fig. 3). Its chemical synthesis is not known.  Figure 3. L-tert-leucine a building block for drug synthesis (Liese et al, 2000). Therefore, only the enzymatic synthesis by oxidoreductase is available.  Figure 4. The enzymatic synthesis of L-tert-leucine from trimethyl-pyruvate catalyzed by leucine dehydrogenase with simultaneous regeneration of cofactor by formate dehydrogenase (Kragl et al, 1996). The kinetics of the substrate and the cofactor of this reaction can be described by Michaelis-Menten double substrate kinetics (Kragl et al, 1996, Vasi-Ra ki et al, 2003), taking into consideration a competitive product inhibition. The calculations for this synthesis (Kragl et al., 1992) indicated the way in which the total turnover number depends on the cofactor concentration. The dependence shown is valid for a conversion of 90 % adjusted by the corresponding variation of the residence period. Whereas the space-time yield rises with an increasing cofactor concentration, the total turnover number achievable simultaneously drops. This behaviour is found both for the polymer-enlarged and for the native cofactor. The space-time yield is somewhat higher for the native cofactor since a higher reaction rate is achieved due to favourable kinetic parameters. From the economic point of view, a compromise must be made between space-time-yield and total turnover number, which directly determines the cost of the cofactor. Due to the low price now achieved, the native cofactor can be used economically in the continuous process for the smaller achievable total turnover numbers. The understanding of the enzyme reaction engineering of such systems is useful in determining the optimum reaction conditions. The overall reaction rate for the formation of L-tert-leucine as a function of the concentration of trimethylpyruvate and the cofactor concentration for 90 % substrate conversion rises with the increasing cofactor concentration whereas it drops with the rising substrate concentration. At the given conversion of 90 %, this drop can be attributed to increasing product inhibition. The activity ratio of the two enzymes applied also influences the achievable space-time yield. Different conversions are achieved at a constant enzyme ratio by varying the residence time. In calculating the enzyme molar fraction, the enzyme activities determined under the initial reaction rate conditions are used. At low conversions, the maximum space-time yield must therefore be found at the molar fraction of 0.5. As the conversion increases, the maximum of the achievable space-time yield is shifted towards smaller enzyme molar fractions since the production enzyme and the regeneration enzyme are influenced to a different extent by the concentrations of the reactants, which are changed with increasing conversion. To achieve the same reaction rates for both enzymes under the concentration conditions prevailing in the reactor, the production enzyme must be applied in larger quantities due to the very strong product inhibition. The CSTR with the ultra filtration membrane is by far one of the simplest reactor configurations for continuous process realisation and is widely used in many biochemical reactions. While being advantageous for reactions with substrate inhibition, this reactor is disadvantageous in cases of severe product inhibition as in the synthesis of L-tert-leucine from trimethylpyruvic acid. A system of two membrane reactors in series can be proposed as a solution for the reaction in question (Kragl et al, 1996). From the economic point of view, a compromise has to be made between the space-time yield, the conversion and the activity of two enzymes that are used. Different conversions at a constant enzyme ratio are achieved by varying the residence time. A comparison among the three types of reactors shows that the maximum space-time yield is found in the batch reactor at the enzyme activity fraction of 0.5. The maximum of the achievable space-time yield in the batch reactor is shifted towards smaller values in the CSTRs in series. The smallest enzyme activity fraction is found for the single CSTR. This can be explained by the fact that the production enzyme (LeuDH) and the regenerating enzyme (FDH) are influenced to a different extent by the concentrations of the reactant and the product. These concentrations are changed according to the different increase of conversion in each reactor type. The main reason responsible is the product inhibition by L-tert-leucine. Therefore, with increasing conversion the maximum of the space-time yield is shifted to lower enzyme activity fractions for all three reactors. It is therefore possible to minimise the biocatalyst cost in the reactor by maximising the space-time yield if other destabilising effects are excluded. The minimal biocatalyst cost is achieved in the batch reactor. 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Mi ) = > _f7;DESv{+.34CG[_*&&&) ))3*++f+l+++ꢗꏆ{{h6OJQJ]hH*OJQJhOJQJh6]nH tH hnH tH hmHnHuh_*)h| h6] h5\ h5CJh56CJ] h6hmHnHtH uh h\ h5 h5CJ0F z DERS*b#@%&&&&)dh`h$a$)**3*S-T-m-.222222J3L3N3P3T3V3b3d3$d5$7$8$9DH$a$$a$ & Fd5$7$8$9DH$d5$7$8$9DH$$ & Fa$$a$+-- .%.3.8.u.z....//// /&000N0X0t0~0111426282D2T222222222222222222222F3H3J3L3P3лаЪ h<aJ h<aJ haJ h5CJ\aJ h5CJ$\aJ h5H*\aJ h5\aJ h5CJ \aJ hH*OJQJh6OJQJ]hOJQJ7P3R333 4404:4f44'6,666'7,7@8E8R8W88::6;;;;;;;==4>5>6>I>J>L>O>Q>1@,A;ATAAACDDĹĹĹĹĹĵxĵhOJQJnH tH j;F hCJUVjhUjhCJUmHnHu h6] h6hh6]nH tH hnH tH h5OJQJ\h6OJQJ]hOJQJ*jhCJOJQJUmHnHtH u/d3f3h3j3l3n3p3r3d4f4448D;>>3>4>M>N>$a$$a$$d5$7$8$9DH$a$$d5$7$8$9DH$`a$$d5$7$8$9DH$a$d5$7$8$9DH$N>O>Q>R>>>@?1@,AEIJJMNQNTT,U3U:U?UXYs[[[ݸݸ hCJj[chUjhCJUmHnHu h6]h5CJ\jhUh6]mH sH h6mH sH hmH sH h56\] h5\h h:8GIMNESSAUs[[[[[\\z]{]*^+^^^p_q_"`$`aabb$`a$$a$[[[[~\\\\]1][]k]m]p] ^^ ^K_d_g_q_"`}bbIckccdddddddd$e2eCeZefff!f|ffffIg`gcgggghрҀՀ-5>MP RU h6CJhh5CJ\mH sH h6CJ]mH sH hCJmH sH  hCJh5CJ\h6CJ]Hbccddddgehe+f,fffmgng !Z[؂قЃуz$a$crobiol. 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