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Review Article Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts
Institute for Biotechnology and Bioengineering (IBB), Centre for Biological and Chemical Engineering, Instituto Superior T´ ecnico, Avenue Rovisco Pais, 1049-001 Lisboa, Portugal Correspondence should be addressed to Pedro Fernandes, email@example.com Received 7 July 2010; Accepted 1 September 2010 Academic Editor: Cristina M. Rosell Copyright © 2010 Pedro Fernandes. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Food and feed is possibly the area where processing anchored in biological agents has the deepest roots. Despite this, process improvement or design and implementation of novel approaches has been consistently performed, and more so in recent years, where signiﬁcant advances in enzyme engineering and biocatalyst design have fastened the pace of such developments. This paper aims to provide an updated and succinct overview on the applications of enzymes in the food sector, and of progresses made, namely, within the scope of tapping for more eﬃcient biocatalysts, through screening, structural modiﬁcation, and immobilization of enzymes. Targeted improvements aim at enzymes with enhanced thermal and operational stability, improved speciﬁc activity, modiﬁcation of pH-activity proﬁles, and increased product speciﬁcity, among others. This has been mostly achieved through protein engineering and enzyme immobilization, along with improvements in screening. The latter has been considerably improved due to the implementation of high-throughput techniques, and due to developments in protein expression and microbial cell culture. Expanding screening to relatively unexplored environments (marine, temperature extreme environments) has also contributed to the identiﬁcation and development of more eﬃcient biocatalysts. Technological aspects are considered, but economic aspects are also brieﬂy addressed.
Food processing through the use of biological agents is historically a well-established approach. The earliest applications go back to 6,000 BC or earlier, with the brewing of beer, bread baking, and cheese and wine making, whereas the ﬁrst purposeful microbial oxidation dates from 2,000 BC, with vinegar production [1–3]. Coming to modern days, in the late XIX, century Christian Hansen reported the use of rennet (a mixture of chymosin and pepsin) for cheese making, and production of bacterial amylases was started at Takamine (latter to become part of Genencor). Pectinases were used for juice clariﬁcation in the 1930s, and for a short period during World War II, invertase was also used for the production of invert sugar syrup in a process that pioneered the use of immobilized enzymes in the sugar industry . Still, the large-scale application of enzymes only became really established in the 1960s, when the traditional acid hydrolysis of starch was replaced by an approach based in
the use of amylases and amyloglucosidases (glucoamylases), a cocktail that some years latter would include glucose (xylose) isomerase [1, 2, 4, 5]. From then on, the trend for the design and implementation of processes and production of goods anchored in the use of enzymes has steadily increased. Enzymes are currently among the well established products in biotechnology , from US $1.3 billion in 2002 to US $4 billion in 2007; it is expected to have reached US $5.1 billion in a rough 2009 year, and is anticipated to reach $7 billion by 2013 [3, 5, 7–9]. In the overall, this pattern corresponds to a rise in global demand slightly exceeding 6% yearly [7, 9]. Part of this market is ascribed to enzymes used in large-scale applications, among them are those used in food and feed applications . These include enzymes used in baking, beverages and brewing, dairy, dietary supplements, as well as fats and oils, and they have typically been dominating one, only bested by the segment assigned to technical enzymes [11, 12]. The latter includes enzymes in the detergent, personal care, leather, textile and pulp, and
and high biodegradability. glucosidases and proteases from severalgenera [32. Enzymes are furthermore generally considered a natural product [18. These provide high-throughput capability for a speedy and detailed characterization of the performance of enzymes . marine environment has also been tapped as a source for useful enzymes from either microbial or higher organisms origin [57–60]. enzymes from plant or animal cells. When successfully implemented. 6% for feed enzymes and the remaining for technical enzymes . 68]. 45. moreover after the latter taking over Genencor (USA). which will be addressed latter in this paper. inulinases [46–49]. since operation at high temperatures (roughly above 45–50◦ C) minimizes the risk of microbial contamination. have been rebutted [21.2 paper industries [10. (b) eased implementation of enzyme cascade. which has been consistently undertaken. their high cost. The pace of development in emerging markets is suggestive that companies from India and China can join this restricted party in a very near future [15–17]. LCA is a methodology used to compare the environmental impact of alternative production technologies while providing the same user beneﬁts . ability to operate under mild conditions of pH. glucose (xylose) isomerases [42. A relatively large number of companies are involved in enzyme manufacture. Methodologies with a high level of parallelization. A recent survey on world sales of enzymes ascribes 31% for food enzymes. with DSM (The Netherlands) and BASF (Germany) lagging behind. Other examples of useful enzymes for food and feed. fructosyltransferases . 56]. and glycosyl hydrolases [67. From some years now. particularly thermophiles. but isolated from higher organisms [59. the extension of some reactions in relevant food applications is favored at relatively high temperatures (namely. Some of these enzymes are actually psychrophiles. Other examples of these enzymes. the implementation of enzyme-based technology has a positive impact on the environment . protein engineering. but hydrolases are possibly the prevalent one. The majority of enzymes used in food and feed processing is of terrestrial microbial origin. the undertaken approaches allow: (a) continuous operations at relatively high temperatures. 14]. another approach relies on screening eﬀorts. and xylanases [53. since there is a low amount of by-products. Roughly all classes of enzymes have an application within the food and feed area. and narrow range of substrates. Enzyme Research (namely. glucosidases [44. Relevant Enzymes: Tapping for Improved Biocatalysts 2. although care should be taken to avoid an operational environment that may lead by-product formation (namely. Along with these diﬀerent strategies focused on the enzyme molecule (namely. Life-cycle assessment (LCA) has conﬁrmed. Some examples of strategies taken to improve the performance of relevant enzymes for food and feed are given in Table 2. amylopullulanases . that within the range of given practical case studies. as summarized recently [26–31]. This latter environment has allowed the isolation of some promising biocatalysts. 44. temperature and pressure while displaying high activity and turnover numbers. such as the heat-stable invertase/inulinase from Thermotoga neapolitana DSM 4359 or inulinase from Cryptococcus aureus [61–63]. glucoamylases . a particularly delicate matter under continuous operation.1. as well as to overcoming the susceptibility to typical inhibitory molecules. 69]. preferably as high-concentrated solutions. anchored in computer-monitored microtiter plates equipped with optic ﬁbers and temperature control have also been developed. such as transglutaminase or even slow-growing microorganisms). are given in Table 3. and (c) the use of raw substrates. metal ion removal/addition) throughout the intermediate steps of a multistep biotransformation (namely. 65]. 23. levansucrases . USA and Japan. By allowing gene cloning in microorganisms compatible with industrial requirements. starch to high fructose syrup). esterase from Vibrio ﬁscheri . 52]. Some of the broad generalizations on the limitations of enzymes for application as biocatalysts in commercial scale. including food and feed processing. The widespread use of enzymes for food and feed processing is easily understandable. One of the methodologies to obtain improved biocatalyst relies on in-vitro modiﬁcations. isomerization of glucose to fructose). but major players are located in Europe. 64. Furthermore. General Aspects and the Screening Approach. 19]. namely. 13]. with some of which able to retain stability under temperatures of 90◦ C or higher. given the reduced need for processing the reaction media (pH adjustments. with some of them being calcium independent [32–38]. pullulanases [51. and screening-eﬀorts for isolation of promising enzymeproducing strains have accordingly been performed in such background [3. 24]. Some focus is given to extremophiles. particular care has been given to increasing thermal stability. 5. low productivity and stability. with Novozymes (45%) and Danisco (17%). the developments in recombinant DNA technology that occurred in the 1980s also had a huge impact on the application of enzymes in food and feed. Denmark is dominating. Particular focus was given to the prediction of the long-term stability of enzymes under moderate conditions using short-term runs (up to 3 hours). and the energy requirements are relatively low. Representative examples of the enzymes and their role in food and feed processing are given in Table 1. enzyme immobilization). hence performing best at low temperatures . 54]. enhancing the range of pH with catalytic activity and decreasing metal ions requirements. amylolytic enzymes. 2. Aiming at improving the performance of biocatalysts for food and feed applications. 43]. 22]. hence reducing the need for complex downstream process operations. 45]. this methodology enabled costfeasible production of enzymes that were naturally produced in conditions that prevented large-scale application . 11. with 5% and 4% [10. were reviewed by Gomes and Steiner . The whole contributes for developing sustainable and environmentally friendly processes. Maillard reactions). given their unsurpassed speciﬁcity. Examples of screened enzymes include the isolation of amylases. hence cutting back in costs related to upstream processing and increasing productivity [4.
5. which allows for a high throughput. aiming at enhancing stability without compromising catalytic activity . . hence. and of a xylanase from Bacillus subtilis . The encouraging results obtained suggest the soundness of the approach to obtain a mutant glucose isomerase competitive with those currently used. The activation energy required by the triple 1F1 mutant (V185T/L282P/F186S) was roughly half of the wildtype. and computer-assisted design of proteins. 89]. Amylases and glucoamylases are enzymes used in starch processing. are actually produced by recombinant microorganisms. has been extensively applied. when compared with the parent T. clariﬁcation of fruit juices with pectinases to produce clear juice . improving thermal stability without decreasing enzyme activity is of 3. since it allows for reducing the amount of enzyme used in the process. 31]. milk processing with lactase for lactose-free milk [81– 83]. partly resulting from lack of uniformity in the US and EU legislation. The targeted improvements have not been restricted to thermostability. In order to control the pathway of the process. Large-scale glucose isomerization is carried out at 55–60◦ C and slightly alkaline pH [1. where the combination of each individual eﬀect is usually roughly additive . such as cheese maturing and milk coagulation with proteases [59. This pattern is often reported when glucose isomerases from hyperthermophilic strains operate in mesophilic environments. which involves temperatures typically in excess of 60◦ C. There is therefore interest in selecting an enzyme able to operate eﬃciently at temperatures close to those currently used but at a lower pH. Several enzymes (namely. Since extremophiles are often diﬃcult to grow under typical laboratory conditions if not nonculturable at all. in-vitro improvement of biocatalysts have been consistently implemented [90–93]. (1) The ﬁrst is the enhancement of the activity of the hyperthermostable glucose (xylose) isomerase from Thermotoga neapolitana at relatively low temperature and pH. Some relevant examples in the area of food and feed processing include the following. Improving Biocatalysts: Beyond Screening Taking advantage of the knowledge gathered on molecular biology. such as broadening the range of pH where the enzyme is active. The enzyme from the parent strain is highly active at 97◦ C. or production of oligosaccharides . This will be addressed in the following section. aiming for more eﬃcient biocatalysts [103–106]. but they have also addressed other features. These methods are also widely used when protein engineering is carried out. 108]. it can be improved by several substitutions of amino acids in a single mutant. which can be obtained from mixed microbial population from environmental samples. namely. 113]. where the environmental adaptation is reproduced in-vitro in a much hastened timescale. or lessening the temperature of operation while retaining high activity [91. IM6501 . The mutant glucose isomerase 1F1 obtained by Sriprapundh and coworkers displayed a roughly 5-fold higher activity at 60◦ C and pH 5.and long-range interactions. while being able to operate in a slightly acidic environment and 60◦ C. hence allowing for high activity at relatively low temperatures . The latter result from by-product and color formation occurring when the reaction is carried out at alkaline pH and high temperatures [31. of the glucoamylase from Aspergillus niger . Some of the research eﬀorts in this area has focused on the biochemical and molecular mechanisms underlying the stability of enzymes from extremophiles [31. Given that thermostability is determined by a series of short. These include protein processing. diﬀerent approaches have been developed in order to assess the potential of enzymes from such microorganisms.0 to 9. 94–96]. 107]. In order to screen the huge number of DNA-libraries typically generated for the intended property. through random mutagenesis and recombination. 99]. Recombinant microorganisms can then be obtained using mesophiles as hosts where the genes of interest from extremophiles have been expressed . Despite some complexity in the implementation of their use in large-scale applications. Two methodologies can be used for protein engineering . combined with process boundary conditions. Enhancing the stability of enzymes is of paramount importance when implementation of industrial processes is foreseen. Such knowledge is also particularly useful for protein engineering of known enzymes. of a phytase from Escherichia coli [112. enables some processes to be carried out with minimal deterioration of the raw material. 3 (i) The ﬁrst is directed evolution of enzymes.Enzyme Research Operation at low temperatures is also welcome since it also reduces the risk of microbial contamination. high-throughput methods have been implemented . α-amylases. This set of conditions results from the optimal range of pH (typically 7. without decay in thermostability . but it retains only 10% of its activity at 60◦ C. 80]. pullulanases) currently used in food processing. This methodology. and requires neutral pH for optimal activity.0) and temperature (60 to 80◦ C) for glucose isomerization displayed by most of the glucose isomerases used. (2) The second is the enhancement of the thermostability of the maltogenic amylase from Thermus sp. and was more thermostable than the wild type isomerase [104. of the amylosucrase from Neisseria polysaccharea . in starch hydrolysis. quite a few enzyme preparations have been accepted for industrial use [88. high-throughput processing. One approach relies on the generation and screening of target genes from DNA libraries. or selective pressure is applied [100– 102]. towards the optimization of the intended property. neapolitana isomerase. either a screening test for the assessed feature is performed after each round of modiﬁcation.
S30P. M375T. upon which the eﬄuent reaction stream has to be cooled to 60◦ C. Q411L. 12. P453L) which displayed an optimal reaction temperature 15◦ C higher than that of the wild-type and a half-life of roughly 170 min at 80◦ C.4 Table 1: An overview of enzymes used in food and feed processing (adapted from [10. in-situ emulsiﬁcation for dough conditioning. Phytases are added to animal feeds to improve phosphorus nutrition and . also led to a 23% decrease in speciﬁc activity. S119P. whey hydrolysis Cheese ﬂavor.12 kJ mol−1 increase in the free energy of thermal inactivation. A27C. the cooling step. However. ensuring uniform yeast fermentation Juice treatment. Starch liquefaction is performed at 105◦ C in the presence of α-amylase. ﬂour adjustment. dough conditioning Acetolactate decarboxylase Beer maturation Xylose (Glucose) isomerase Glucose isomerization to fructose relevance. The mutant thus allowed for a process with increased yield in amylose chains (31 g L−1 ). the mutant was claimed to be the only amylosucrase usable at 50◦ C. the mutant enabled the synthesis of amylose chains twice as long as those obtained by the wild-type enzyme at 30◦ C. ﬂavor improvement in milk and cheese. enhanced digestibility Glucoamylase Sacchariﬁcation Invertase Sucrose hydrolysis. a temperature at which the wild-type ThMA was fully inactivated in less than 1 minute. one of the mutations most accountable for enhanced thermal stability. T290A. 68]). low calorie beer Viscosity reduction in lupins and grain legumes used in animal feed. A398V. Wang and coworkers obtained a multiply-mutated enzyme (N20C. Furthermore speciﬁc activities and catalytic eﬃciencies remained unaltered. lower risk of contamination. cheese ripening Phospholipase In-situ emulsiﬁcation for dough conditioning Phytases Release of phosphate from phytate. Proteases (namely. production of invert sugar syrup Lactase Lactose hydrolysis. S169N. thermostable glucoamylases are aimed at. when mutant and wild type were compared . ﬂavor enhancer (beer) Dough strengthening. gluten) for savoury ﬂavors. juice clariﬁcation Peptidase Hydrolysis of proteins (namely. enhanced digestibility. The amylosucrase engineered by Emond and coworkers was a double mutant (R20C/A451T). At the latter temperature. 13. Class Oxidoreductases Enzyme Glucose oxidase Laccases Lipoxygenase Cyclodextrin Glycosyltransferase Fructosyltransferase Transglutaminase Role Dough strengthening Clariﬁcation of juices. Actually. In order to avoid. bread whitening Cyclodextrin production Enzyme Research Transferases Hydrolases Lyases Isomerases Synthesis of fructose oligomers Modiﬁcation of viscoelastic properties. milk clotting. thus resulting in the enhanced thermal stability of the mutant. support for lipid Lipase digestion in young animals. for sucrose concentrations of 600 mM. meat processing Starch liquefaction and sachcariﬁcation Increasing shelf life and improving quality by retaining moist. dough processing. prevention of chill haze formation in brewing Pectinase Mash treatment. meat tenderizer. as compared to the wild type . H391Y). displaying a 10-fold increase in the half-life at 50◦ C compared to the wild-type enzyme. so that glucoamylases can be used. Kim and coworkers obtained also a multiply-mutated amylase (R26Q. enhanced digestibility Pullulanase Sacchariﬁcation Xylanases Viscosity reduction. close to the active site. enhanced Galactosidase digestibility Glucanase Viscosity reduction in barley and oats used in animal feed. papain) enhanced digestibility and utilization. elastic and soft Amylases nature Bread softness and volume. synthesis of aromatic molecules Protein hydrolysis. T62A. chymosin. I333V. enhanced substrate and product solubility and overall productivity . or at least minimize. M375T. G137A. which displayed a 5. low-allergenic infant-food formulation. when compared to the wild type. soy.
As a result. found in North Atlantic). Directed evolution. since feed pelleting is carried out at high temperature (60 to 80◦ C). found mostly in Northeast Atlantic Ocean.0 and 7. mutants were obtained. botan shrimp (Pandalus nipponensis). Mangrove crab (Scylla serrata). Sardine Orange roughy (Hoplostethus atlanticus) Amylase Hydrolases Chymotrypsin Pepsin Protease to reduce phosphorus excretion.3. cerevisiae displayed a 20-fold increase in the speciﬁc activity when compared to the wild-type enzyme. Mutant displayed increased half-life from 15 min to about 70 min (100◦ C). found in Mediterranean sea and coastal North Atlantic Ocean. found in estuaries and mangroves of Africa. Atlantic cod (Gadus morhua). coli phytases. Protein engineering through site-directed mutagenesis. found in Caribbean sea and South Atlantic. with roughly 20% increased thermostability at 80◦ C . without compromising the kinetic parameters. Enzyme Role Starch liquefaction α-amylase Starch liquefaction Activity Targeted improvement Thermostability Strategy/comments Protein engineering through site-directed mutagenesis. Black and Mediterranean seas. found in Northeast Atlantic Ocean and Mediterranean sea.Enzyme Research Table 2: Some examples of strategies undertaken to improve the performance of enzymes with applications in food and feed. pentoses and tetroses pH-activity proﬁle  Substrate speciﬁcity  Table 3: Examples of enzymes isolated from various marine higher organisms with potential of application in food and feed (adapted from [68. E. The resulting mutant displayed a 3-fold increase in catalytic eﬃciency with L-arabinose as substrate. Enhanced activities are maintained between pH 6. Turbot (Scophthalmus maximus). Thermal stable enzymes are needed. since they have low activity at the physiological temperature of animals . close to Brazil. which are appealing to industrial application.5. and Southeast the Paciﬁc Ocean Deepwater redﬁsh (Sebastes mentella. white shrimp. due to the acidic pH optimum. thermostability and pH optimum Thermostability Activity pH-activity proﬁle       Xylose (glucose) isomerase Isomerization/epimerization of hexoses. and resistance to pepsin digestion. were thus engineered in order to improve their thermal stability. speciﬁcity phytate. carp (Cyprinus carpio). Baltic. rainbow trout (Oncorhynchus mykiss). The turnover number on D-glucose in some mutants was increased by 30%–40% when compared to the wild type at pH 7. Arctic capelin (Mallotus villosus). Marine sponges Spheciospongia vesperia. crayﬁsh. 69]). Protein engineering through site-directed mutagenesis Protein engineering through directed evolution Protein engineering through site-directed mutagenesis Immobilization Protein engineering through directed evolution Protein engineering through site-directed mutagenesis Protein engineering through directed evolution. After 3 rounds the mutant enzyme from S. Phytases produced by thermophiles do not provide a suitable approach. Class Transferases Enzyme Transglutaminase Source Muscles of atka mackerel (Pleurogrammus azonus). Asia and Australia. by promoting the hydrolysis of phytate into myoinositol and inorganic phosphate. Atlantic cod (Gadus morhua). and Geodia cydonium. 5 Reference   Baking l-arabinose isomerase Glucoamylase Lactase Pullulanase Phytase Tagatose production Starch sacchariﬁcation Lactose hydrolysis Starch debranching Animal feed pH-activity proﬁle pH-activity proﬁle Substrate speciﬁcity. scallop (Patinopecten yessoensis). Gilt-head (sea) bream (Sparus aurata).
these enzymes can be used in dough making. When the latter contain cereals (namely.4 bonds in xylan polymers.4. as compared to the wild-type enzyme.5. rye or wheat). Enzyme Research as expressed by decreases in KM of roughly 35% and 25%. [126. when compared to the wild-type enzyme. Furthermore.5-fold increase in half-life. K41E and E121F substitutions allowed for increases in the speciﬁc activity of 2. 124] and of glucose (xylose) isomerase from Streptomyces diastaticus . which can be preferably obtained from crystallography or NMR.1-fold. to obtain mutants with enhanced catalytic properties . Amylosucrases can be used for the modiﬁcation or synthesis of amylose-type polymers from sucrose. but for some mutants. while they are to display high activity at about 40◦ C. which provides data on amino-acid propensities and on protein sequences. once implemented. 124]. and of glucoamylase from Aspergillus awamori . Other examples can be found elsewhere [120. Enzyme engineering through molecular simulations requires structural data from the native enzyme. or cereal by-products. These mostly aim to improve thermal stability and/or catalytic eﬃciency and/or to modify the range of pH/temperature where the enzyme is active—goals that were already referred to when examples of enzyme modiﬁcations using random mutagenesis were addressed. most xylanases are inactive at temperatures exceeding 60◦ C. As referred for phytases. of amylases from Bacillus spp. but their industrial application is somehow thwarted by the low catalytic eﬃciency on sucrose and by side reactions leading to the formation of sucrose isomers. (3) Third is the enhancement of the activity of the amylosucrase from Neisseria polysaccharea . as compared to the wild type. Van der Veen and co-works engineered mutant enzymes through error-prone PCR that displayed increases in activity up to 5-fold and in overall catalytic eﬃciency up to 2-fold. hence the need for enhancing thermal stability [114. Some relevant examples of this strategy in the area of food and feed processing are given. Xylanases added to the the formulations hence have to withstand these conditions. The mutation also led to a 10◦ C increase in the optimal temperature for reaction and enhanced activity at higher temperatures. 121]. unlike the wild-type enzyme . as compared with the original enzyme. which. Also used are molecular potential functions. with no changes in catalytic properties [123. containing a K46E substitution. G247D) displayed a 2. Computational methods are also welcome in directed evolution. Adequate processing of the data enable the output of generalized rules predicting the eﬀect of mutations on enzyme properties. Computational tools used for enzyme engineering have been recently reviewed . as compared to the wild-type enzyme . (ii) The second methodology underlines that rational pinpoint modiﬁcations in one or more amino acids are made. Miyazaki and coworkers obtained a triple-mutant xylanase (Q7H. and additionally a 45% increase in the speciﬁc activity. in brewing and in animal feed compositions.0 [112. the formulation of commercial feed often involves steps at high temperatures. 127]. and S179C) which retained full activity for 2 hours at 60◦ C. (1) The ﬁrst example underlines the enhancement of the thermostability of the recombinant glucose (xylose) isomerase from Actinoplanes missouriensis [123. missouriensis displayed an enhanced thermal stability. which is the temperature in the intestine of animals. Such features were ascribed to increased molecular .6 improved overall catalytic eﬃciency (kcat . when compared to the wild type. 117]. The mutant isomerase from A. 117]. alongside with improved stability at diﬀerent pH. barley. the overall catalytic eﬃciency of the mutants increased 1. N8F. xylanases improve the break-down of plant cell walls. Furthermore. The alterations promoted are performed based on the growing knowledge on the structure and functions of enzyme.6-fold as compared to the wild type. Another example was provided by Tian and coworkers who engineered a phytase from Aspergillus niger 113 through gene shuﬄing. in baking. maize. The double mutant isomerase (G138P.and 3. turnover number/KM . where selected amino acids are replaced with those suggested from the outcome of modeling. that displayed a decrease in activity at pH 5. which favors the ingestion of plant nutrients by the animals and consequently enhances feed consumption and growth rate. However. the mutants were able to produce amylose polymers from 10 mM sucrose on. and of aﬃnity for sodium phytate. Otherwise a model is built based on known enzyme structures with homologous sequences . Hence. albeit at the cost of decreased activity at lower temperatures. 113]. enable the prediction of the eﬀect of mutations in enzyme structure . the use of xylanases decreases the viscosity of xylancontaining feeds [116. where these changes are predicted to bring along the envisaged improvement in the targeted enzyme function. whereas the wild-type enzyme was inactivated within 5 minutes under the same conditions. No signiﬁcant changes in the pH activity proﬁle were observed. Information on this matter mostly comes from bioinformatics. as a tool to better lead the random mutagenesis . Xylanases catalyze the cleavage of β1. Their work provides an illustrative example on the use of random mutagenesis and recombination for the enhancement of the catalytic properties of enzymes with application on food and feed.and 1. Michaelis constant) within 50 to 150%. Ultimately this approach is put into practice by producing a site-directed mutant. Accordingly. Furthermore.
as compared to 83◦ C for the wild-type strain. harboring each N175H and Q268K mutations. respectively. engineered mutants R360S.0 and a temperature range within 50– 65◦ C. Thermostable enzymes were obtained through the introduction of disulﬁde bonds in highly ﬂexible region in the polypeptide chain of the enzyme. most of these display an alkaline pH optimum. (5) The ﬁfth example underlines the enhancement of the product proﬁle of cyclodextrin glycosyltransferases (CGTase) from diﬀerentgenera [137. Eﬃcient operation in the stomach of simple-stomached animals where phytate hydrolysis mostly occurs at a pH around 3. when compared with the wild type. The work demonstrates that cumulative improvements in pH activity and thermostability through mutation are compatible in this phytase. Such set of operational conditions matches the targeted goals and again shows that the basis for pHactivity proﬁle and thermostability in l-arabinose isomerase are quite independent and compatible. A similar pattern was also observed in the previous example dedicated to a mutant phytase. (2) The second example underlines the enhancement of the pH-activity proﬁle and of the thermostability of phytase from A.5. subtilis.5 exceeded in roughly 1. which allowed the synthesis of a signiﬁcant amount of polymer and a lower amount of oligosaccharide.Enzyme Research rigidity due to the introduction of a proline in the turn of a random coil . when compared to the wild type . niger. see . subtilis . These led to broader optimal temperature range within 50 to 65◦ C and to enhanced stability in acidic media. 132]. but in-vitro they also isomerize d-galactose into dtagatose . Furthermore. Although several thermostable l-arabinose isomerases have been isolated and characterized. Data gathered also showed that care had to be taken not to disrupt the hydrogen bond and salt linkage network in the catalytic center as a result of mutagenesis. These authors established the eﬀect of mutations on the reaction speciﬁcity (hydrolysis/transfructosylation).and pH activity proﬁle of the larabinose isomerase from Bacillus stearothermophilus US100 . which was the optimum of the latter. Multiply-mutated amylases obtained by Declerck and coworkers displayed considered enhanced thermal stability. this parameter went as high as 106◦ C. α(1 → 4) linked oligosaccharides form starch. 138]. Operation within the latter temperature range also rules away the use of divalent ions. Furthermore. For example. The latter keto-hexose is being used as a low-calorie bulk sweetener. . l-Arabinose isomerases catalyze the conversion of l-arabinose to l-ribulose in-vivo. Based on the temperature at which amylase initial activity is reduced by 50% for a 10-minute incubation. Rhimi and coworkers engineered two individual mutants. These enzymes promote the production of cyclodextrins. the hydrolytic activity of the mutants at pH 3. The work by Lin and coworkers on amylase mutants from Bacillus sp.5-fold that of the parent one at pH 5. mutants R423K and W271N were obtained. and the wild type was ineﬀective. displayed optimal activity within a pH range of 6. again arises the need for enzymes able to isomerize l-arabinose in an acidic environment and at relatively low temperature. which stabilize isomerases at high temperatures [133. 60 to 70◦ C. as compared to the wild type. (3) The third example underlines the modiﬁcation of the temperature. On the other hand these mutations reduced the aﬃnity for sucrose. Cumulative enhancements in both properties in the same enzyme were thus possible . strain TS-23 highlighted the relevance of E219 for the thermal stability of the enzyme . was thus enabled. For industrial application this presents the same drawbacks of by-product and color formation referred to when 7 the random mutation of glucose isomerases was addressed. reuteri has a key role in the determination of the size of the products obtained .5. In order to minimize the dispersion in the products obtained. for this could lead to a decrease in the speciﬁc activity and overall catalytic eﬃciency . and thermal stability. OrtizSoto and coworkers also showed that the product proﬁle of transfructosylation reactions could be adequately tuned through modiﬁcation of target residues of an inulosucrase from B. since the former are more hydrolytic. but the caloric value is only 30% of that of sucrose [131. The mutated glucoamylases engineered by Liu and Wang allowed to establish the role of several intermolecular interactions in thermal stability of these enzymes. Mutants also retained higher residual activity after incubation within 70 to 100◦ C.0 to 7. when compared to the wild type. molecular weight and acceptor speciﬁcity. since its taste and sweetness are roughly equivalent to sucrose. the thermal stabilization was not accompanied by a decrease in the catalytic activity . The transglycosylation catalyzed by the inulosucrase from L. 134]. Inulosucrases are used to synthesize fructooligosaccharides or fructan polymer from sucrose.5. harboring both modiﬁcations. (4) The fourth example underlines the modiﬁcation of the product proﬁle of inulosucrase from Lactobacillus reuteri  and from B. The data gathered showed that the −1 subsite in the inulosucrase from L. Hence. without signiﬁcantly aﬀecting the catalytic activity. This was achieved by combining several individual mutations that allowed for mutants that were quite active at pH 3. whereas the wild type synthesized levan. Y429N and R433A only synthesized oligosaccharides. reuteri leads to a wide range of fructooligosaccharides alongside with minor amounts of an inulin polymer. An engineered double mutant. as well as by the introduction of more hydrophobic residuesstabilized α-helices.
143]. and the same packing can work throughout some months to years. since these introduce 4. and a signiﬁcant (roughly 45%) decrease in the production of β-cyclodextrin when compared to the wild-type enzyme . isomaltulose synthase for the production of isomaltulose. Immobilization can be performed by several methods. since cleaning needs of the reactor are less frequent [1. loss of activity during immobilization procedures. and that changes in sugar-binding subsites −3 and −7 may result in mutant CGTases with altered product speciﬁcity . mass transfer limitations. although immobilization can prove critical for economic viability if costly enzymes are used. for the production of whey hydrolysates and for the production of tagatose. Wildtype CGTases typically produce a mixture of the three cyclodextrins when incubated with starch. or γ-cyclodextrin. which may prove restrictive for cyclodextrin applications involving human consumption [139. and β-fructofuranosidase for the production of fructooligosaccharides [144–146]. Increased thermal stability. aminoacylase for the production of amino acids. The two methods are not mutually exclusive and methodologies for engineering of enzymes can assemble both strategies . lipases for the interesteriﬁcation of edible oils. immobilization prevents denaturation by autolysis or organic solvents. risk of support deterioration under operational conditions. There is therefore a clear interest in obtaining a mutant CGTase capable of producing a particular type of cyclodextrin in a high rate. due to mechanical or chemical stress. lactase for lactose hydrolysis. they are of interest for both industrial and research applications. Van der Veen and coworkers engineered a double-mutant (Y89D/S146P) of CGTase from Bacillus circulans which displayed a 2fold increase in the production of α-cyclodextrin and a marked decrease in β-cyclodextrin when compared to the wild type. the largescale liquefaction of starch to dextrins by α-amylases is performed by free enzymes. up. hence leading to lower risks of microbial growth and to less demanding sanitation requirements. Along with this. 144]. are likely to play a key role in to the size of cyclodextrin products formed. The half-life of the bioreactor is also a critical issue when evaluating the economical feasibility of a bioconversion process. the possibility of enzyme leakage during operation. throughout the operational lifetime of the biocatalyst . it enables the control of the extension of the reaction. the cost of the support. In particular. ultimately targeted at the production of trans-free fat. β. particularly due to chemical interaction or steric blocking of the active site.5-fold increase in the production of α-cyclodextrin. the product stream is clear from biocatalyst. are immobilized enzyme reactors packed with glucose isomerase for the production of high-fructose corn syrup. Li and coworkers were also able to obtain CGTase mutants from Paenibacillus macerans strain JFB05-01 with increased speciﬁcity for α-cyclodextrin. double mutant D372K/Y89R displayed a 1. Upon identiﬁcation of the most adequate enzyme. which high speciﬁc productivity (massproduct inﬂuences biocatalyst-related production costs [1. despite the technical advantages of immobilization. and of modiﬁed triacylglycerols. Immobilization has some intrinsic drawbacks. namely. It allows for high-enzyme load with high activity within the bioreactor. which are accordingly termed α. including selective complexation with organic solvents. The puriﬁcation of a given cyclodextrin from the reaction mixture requires several additional steps. 140]. longer half-lives favoring process economics. Given their ability to form inclusion complexes with small hydrophobic molecules. Enzyme Research be minimized. the authors suggested that hydrogen bonds (S146) and hydrophobic interactions (Y89). Still. entrapment/microencapsulation. In the process. continuous operation (or batch operation on a drain-and-ﬁll basis) and process automation is possible. provided that immobilization is adequately designed [142.and downstream processing of the bioconversion systems.000–15. downstream process is simpliﬁed. 142]. namely. since biocatalyst is easily recovered and reused. operational and storage stabilization. of cocoa butter equivalents. Among this group. In the overall. invertase for the production of inverted sugar syrup. allowing for 12. immobilization procedure and processing the biocatalyst once exhausted. Economical issues are furthermore to be taken into consideration when commercial processes are envisaged. Examples of commercial bioreactors depict half-lives of several months to years. given the low cost of the enzyme . respectively. seven or eight. On the other hand. From the data gathered. through mutations at subsite −3. The resulting cyclodextrin may consist of six. and can bring along thermal. and cross-linking of enzyme aggregates.8 through an intramolecular transglycosylation reaction. The latter presents an alternative to carrier-bound enzymes. the enhanced stability allowing for consecutive reuse leads to −1 −1 massbiocatalyst ). which may hamper scale up. this can be formulated adequately for better process integration. and a (still) relative empirical methodology. hence leading to high-volumetric productivities. allowing for routine reactor operation above 60◦ C minimizes the risks of microbial growth. A typical example is the output of immobilized glucose isomerase. a starch oligosaccharide is cleaved and cleaved and the resulting reducing-end sugar is transferred to the non-reducing-end sugar of the same chain . and sanitation requirements have to be taken into consideration. Immobilization There are several issues that can be lined up to sustain enzyme immobilization. resulting in carrier-free macromolecules . A rule of thumb suggesting that the enzyme costs should be a few percent of the total production costs has been established .000 kg of dry-product high-fructose corn syrup (containing 42% fructose) per kilogram of biocatalyst. binding to a solid carrier. One of the most widely considered approaches for such formulation is enzyme immobilization. and substrate inhibition can .
164]. Among these are the synthesis of oligosaccharides with dextransucrase . cyprosin. have also been immobilized in liposomes. respectively. alginate and poly-l-lysine. while avoiding the problems associated with the use of free enzyme. known as dehydration-rehydration vesicles (DRVs). with up to 80% lactose conversion out of a substrate concentration of 250 gL−1 . This feature is enhanced along with the hydrophobicity of the membrane. was carried out in a perfectly mixed reactor and enzyme was recovered in a 10 kDa nominal molecular weight cutoﬀ. Methodologies for large scale production of these supports have been implemented [154. The resulting vesicle suspension is then dehydrated under freeze drying or equivalent method. the whole process of immobilization is performed in-situ. namely. The enzyme complex is released in a controlled manner due to pressure applied during cheese curd . as well as on the eﬀectiveness of the methodology for controlled release of enzymes [156. lactose hydrolysis with lactase . If liposome-based biocatalysts are used in a process under continuous operation. Within the same methodology. k-carragenan. alginate. are given hereafter. The resulting product presented some similarities to the commercially available Vivinal prebiotic . maltodextrin hydrolysis with glucoamylase . In these processes the biocatalyst could be eﬀectively reused or operated in a continuous manner. gellan. liposome-encapsulated lactase was incorporated in milk. regular replacement of the membrane may be required. Several enzymes. a polyvinyl-alcoholbased support in lens-shaped form. enzyme loading is relatively low. partly ascribed to excessive proteolysis and whey contamination. as well as some illustrative examples of their applications. enzyme leakage was minimized (a common drawback of hydrogels) while allowing for high activity and stability. chymosin. and leakage. and production of invert sugar syrup with invertase . a feature that will be addressed in (ii). Recent work in this particular application has used lactase as enzyme model and has focused on the optimization and characterization of the liposome-based immobilized system [163. glutaraldehyde) [148. mass transfer limitations may be relevant. Entrapment/(micro)encapsulation. allowing in-situ degradation of lactose . hence. A broad. Enzyme containment by a membrane has been used for the continuous production of galactooligosaccharides from lactose. biocatalyst separation has to be integrated (namely. The reaction. (a fungal protease/peptidase complex) entrapped in calcium alginate . 167]. This eﬀect was also observed in a previous work where alginate-entrapped inulinase was used 9 for sucrose hydrolysis . has been used for several applications in carbohydrate processing. the authors observed that increasing the concentration of CaCl2 and of sodium alginate to 4% and 3%. There is however some lack of consensus on the feasibility of its application on large scale. was eﬀectively used in cheese ripening. Flavourzyme. Details on the diﬀerent methods. in order to speed up the process. particularly of smaller enzymes from hydrogels (namely. providing high protection against proteolysis. the vesicles are disrupted in the stomach by the presence of bile salts. . a membrane device such as a hollow ﬁber or a microcapsule. namely. 155]. reduced yield and poor-quality cheese. and often leads to the loss of more than 50% native activity. After ingestion. Flavourzyme. the membrane still presents a boundary for overall mass transfer of substrate/products and enzyme molecules are prone to interact with the membrane material. This may be minimized by previously cross-linking the enzyme with multifunctional agent (namely. Cocktails of enzymes. the latter term could preferably be used when the core and the boundary layer(s) are made of diﬀerent materials. bacterial proteases and Palatase M (a commercial lipase preparation). In a diﬀerent concept. Besides. Still. trypsin. the resulting DRVs are multilamellar and larger (from 200 nm to a little above 1000 nm) than the original SUVs. as reﬂected by the use of the biocatalyst in 20 cycles of operation. In the latter work. which is particularly noticeable at high enzyme loadings . using an ultra-ﬁltration membrane). were immobilized in liposomes and successfully used to speed up cheddar cheese ripening . However. leading to supports that are often referred to as beads or capsules. gelatin). The polymeric network is formed in the presence of the enzyme. small (diameters usually below 50 nm) unilamellar vesicles (SUVs) is prepared in distilled water and mixed with an aqueous solution of the enzyme to be encapsulated. namely. The stability of an amylase immobilized biocatalyst was further enhanced with the addition of 1% silica gel to the alginate prior to gelation. 163. A conversion rate close to 95% in skim milk was observed for an initial substrate concentration close to 40 gL−1 . hence immobilization in membrane devices may have some adsorptive nature. Apart from the hollow ﬁber. resulting in low space-time yields and productivities. Although direct contact with an adverse environment is prevented. where the enzyme is contained within a given structure. with little loss (if any) of catalytic activity. in order to carry out lactose hydrolysis in continuous operation. 161. or a (reverse) micelle. Upon rehydration. 157. 149] or by promoting cross-linkage of the matrix after the entrapment . A typical example of the patterns suggested by data in Table 4 was observed by AbdelNaby when evaluating the immobilization of α-amylase through diﬀerent methods . generalized overview of the advantages and drawbacks of the diﬀerent immobilization approaches is given in Table 4. Encapsulation in lipid vesicles has been proved a mild method. 162]. In a particularly favored technique immobilization of enzymes in liposomes. while retaining more than 90% of the initial eﬃciency . based in batch mode. and higher melting-fat fraction of milk fat . lactase. and can capture solute molecules [161. This can be: a polymer network of an organic polymer or a sol-gel. Containment within an ultra-ﬁltration (UF) membrane allows the enzyme to perform in a fully ﬂuid environment.Enzyme Research a large portion of noncatalytic material. may occur. These include deﬁcient enzyme distribution. Neutrase. . This can account to about 90% to more than 99% of the total mass of the biocatalysts. a hollow-ﬁber module was used to contain lactase. Calcium alginate beads were also used to immobilize glucose isomerase  and α-amylase for starch hydrolysis to whey . The use of LentiKats.
since it allows for support regeneration. Examples include the immobilization of α-amylase  and of levansucrase  on glutaraldehyde-treated chitosan beads. and it was used in a ﬂuidized-bed reactor. This methodology has been used for lactase immobilization in magnetic poly(GMAMMA). formed from monomers of glycidylmethacrylate and ethylmethacrylate. Ion-exchanger resins are typical supports for ionic binding. among others. It involves chemically reactive sites of the protein such as amino groups. These are of weak nature and easily allow for enzyme leakage from the support. sulfhydryl groups. due to support binding to critical residues for enzyme activity. The latter comprehends hydrophobic and van der Waals interactions. derivatives of synthetic polymers. quaternary aminoethyl anion exchange. previously activated with glutaraldehyde and using PEI as spacer [185. QAE-dextran. typically with the ε-amino group of lysine. carboxyl groups. Curiously. When wood shavings were used as support. Dowex. or UGI reaction. namely. carboxymethyl. Amberlite. temperature or even as a result of ﬂow rate or abrasion. namely. polyethylenimine and glutaraldehyde . Sepabeads) with a high density of epoxide functional groups aimed at multipoint attachment. CMSepharose.(QAE) cellulose. 184]. and phenol residues of tyrosine. the ﬁrst reported application of enzyme immobilization was of invertase onto activated charcoal . among them are derivatives of cross-linked polysaccharides. using α-amylase as model enzyme . Ionic binding to a carrier involves interaction of negatively or positively charged groups of the carrier with charged amino-acid residues on the enzyme molecules . A recent paper by Gopinath andSugunan illustrates the increased trend for leakage when adsorption is compared with covalent binding. since it enables the expedite removal of spent enzyme and its replacement with fresh enzyme . alkylation and arylation.10 Table 4: A generalized characterization of immobilization methods. 191]. or physical nature. ionic strength. among them are amide bond formation. Within this methodology for immobilization. and cross-linked with ethyleneglycol dimethacrylate . through the glutaraldehyde reaction between the free amino groups of chitosan and the enzyme molecule. Recent examples include the immobilization of invertase in Dowex . in Duolite . On the other hand. glucoamylase onto dried oxidized bagasse . QAE-Sephadex. or the imidazole group of histidine. aiming at its application for sucrose hydrolysis. Anther example is the immobilization of pectinase in egg shell for the preparation of low-methoxyl pectin. each under consecutive batch operation. Recently invertase was immobilized in diﬀerent types of sawdust. namely. However. Eupergit. and invertase immobilized on nylon6 microbeads. hence enhancing stabilization [190. desorption can be turned into an advantage if performed under a controlled manner. The binding can be carried out by several methods. where enzyme-support interaction can be of covalent. Duolite. CLECs Intermediate/High Low Intermediate Intermediate Intermediate Cannot be changed Impossible Enzyme Research Entrapment High Intermediate/High Intermediate Low Intermediate/Diﬃcult Can be changed Impossible Binding to a solid carrier. and it retained 65% of the original activity after 10 hours of continuous operational regime in a column reactor . Covalent binding is the strongest form of enzyme linking to a solid support. unlike immobilization by covalent binding. for the immobilization of cyclodextrin glycosyltransferases to glyoxylagarose supports for the production of cyclodextrins . or on poly(hydroxyethyl methacrylate)/glycidyl methacrylate ﬁlms . The immobilized biocatalyst could be reused for 32 times at 30◦ C. glucoamylase or invertase immobilized onto montmorillonite K-10 activated with aminopropyltriethoxysilane and glutaraldehyde [183. polyethylenimine (PEI) .(DEAE-) cellulose. 186]. or for the immobilization of dextransucrase on Eupergit C .(CM-) cellulose. onto polyglutaraldehydeactivated gelatin . Immobilization method Parameter Covalent Activity High Range of application Low Immobilization eﬃciency Low Cost Low Preparation Easy Substrate speciﬁcity Cannot be changed Regeneration Possible Carrier binding Ionic High Intermediate Intermediate Low Easy Cannot be changed Possible Adsorption Low Intermediate High High Diﬃcult Can be changed Impossible CLEAs. and steric hindrance. the immobilized invertase retained 90% of the original activity after 20 cycles of 15 minutes. operated at an optimum ﬂow rate of 5 mL h−1 and 35◦ C . ionic. on polyurethane treated with hydrochloric acid. highlight should be given to the introduction of commercial supports (namely. after environmental shifts in pH. to confer high rigidity to the enzyme molecule. Ionic interaction may be favored if enzyme leakage is not an issue. namely. Other examples are the surface immobilizations of α-amylase on alumina  and in zirconia . Diaion. and in epoxy(amino) . the immobilization of pectinase onto Amberlite IRA900 Cl through glutaraldehyde cross-linking . diethylaminoethyl. and resins coated with ionic polymers. or onto macroporous copolymer of ethylene glycol dimethacrylate and glycidyl methacrylate through the carbohydrate moiety of the enzyme . DEAESephadex. in poly(glycidyl methacrylate-co-methyl methacrylate beads grafted with PEI . on poly(styrene-2-hydroxyethyl methacrylate) microbeads activated with epichlorohydrin . this often brings along loss of activity during the process of immobilization.
an approach followed by Kweon and coworkers. CLECs of glucose isomerase packed in a column were also used for the concentration/puriﬁcation of xylitol from dilute or impure solutions. where. leading to the ﬂuidized-bed reactor. and accordingly are pumping costs. Therefore. carrier free. (5) Fifth are CLECs of glucose isomerase. the range of ﬂow rates used must be such as to provide a compromise between reasonable pressure drop. despite the potential of immobilization to improve enzyme performance by enhancing activity. when the pectinase activity of the immobilized biocatalyst was compared with the free form. as compared with entrapment and binding to a solid carrier. CLEAs of wild type and two mutant levansucrases were assayed for oligosaccharides/levan and for fructosyl-xyloside synthesis. stability. hence. When placed in a packed-bed. fructosyltransferase in DEAE-cellulose for the production of fructosyl disaccharides .to 3fold higher than the corresponding CLEAs. (4) Fourth.1 mm . led to biocatalysts with improved thermal stability as compared to the free form (over 2-fold increase in half-lives) . The most common form of enzymatic reactors for continuous operation is the packed-bed setup. formed by either glutaraldehyde or diimidates. Furthermore. in order to keep the pressure drop within reasonable limits. whereas the product (and unconverted substrate) freely permeates. a basket reactor was developed. but operated in upﬂow mode.1. which can be signiﬁcantly fastened by high-throughput approaches . This is basically a variation of the packed-bed reactor. they are restricted to batch mode. these displayed a 40. Each method for enzyme immobilization has a unique nature. a commercial enzyme preparation containing pectinase. but is seldom . Minimization of external mass-transfer resistances with enhanced ﬂow rates can be considered. the feasibility of its application to reactor conﬁguration and mode of operation has also to be considered in the selection process of the most adequate immobilized biocatalyst for a given bioconversion. (1) First is the immobilization of Pectinex Ultra SPL.05 mm.9% of the free form . namely. under similar operational conditions as for the free enzyme. but when coupled to a membrane setup with suitable cutoﬀ. glucoamylase onto SBA-15 silica  and in epoxy(amino) Sepabeads . Although the speciﬁc activity of the three free enzymes was 1. Typical Bioreactors. leading to a biocatalyst displaying highly concentrated enzyme activity. dimethylmalonimidate. The residence time allowed by the ﬂow rates required for ﬂuidization may however result in low conversion yields. allowing its separation from other sugars. The CLEA biocatalyst displayed a slight (30%) in the Vmax . where the biocatalyst particles are not in close contact which each other. This can be overcome by operating a battery of reactor or by operation in recycle mode . and cellulose activities . xylose and glucose. 207]. Commercially available carriers such as Eupergit C have particle sizes of roughly 0. (6) Sixth. particularly when hydrogels are considered.Enzyme Research Sepabeads . or speciﬁcity. A careful evaluation and characterization of the methodology addressed is thus required. including the natural substrates. These should not have sizes below 0. aimed at the conversion of glucose into fructose for the production of high fructose corn syrup. while achieving the same 45% yield in fructose. all CLEA preparations displayed enhanced thermal stability when compared with the corresponding free enzymes . 4. maximal reaction rate/KM ratio. CLEAs of glucoamylase. or carrierbound). Enzyme molecules can be modiﬁed chemically or genetically modiﬁed to enhance immobilization eﬃciency. The use of CLEAs is favored given the lower complexity of the process. with Ca2+ being acknowledged to inactivate glucose isomerase . the resulting enzyme preparation allowed for ﬂow rates that matched or even exceeded 11 those processed by commercially available enzyme preparations (either free. Carrier-free macroparticles. Bioconversions with free enzymes are carried out in stirred tanks. (2) Second is the immobilization of lactase for the hydrolysis of lactose. which acts as an immobilization device. under similar operational condition . but a signiﬁcant enhancement in thermal stability (a roughly 10fold increase in half-life). Ionic binding to Sepabeads-like supports has acknowledged multipoint attachment nature. who obtained a cyclodextrin glycosyltransferase fused with 10 lysine residues to improve ionic binding to SP-Sepharose . lactase immobilization in PEI-grafted Sepabeads . minimal diﬀusion layer and high conversion yield. pressure drop is low. and there are still relatively few examples of its application to enzymes used in the area of food processing. Again. Commonly operated in down-ﬂow mode. is used to cross-link enzyme aggregates (CLEAs) or crystals (CLECs). the CLEA yielded 78% monosaccharides in 12 h as compared to 3. glucose isomerase in DEAE-cellulose  or in Indion 48-R . Among those are following. When on their own. glutaraldehyde). dimethylsuccinimidate.25. Shear stress induced by stirring creates a hazardous environment for immobilized biocatalysts. basically a cylindrical column holding a ﬁxed bed of catalyst particles (Figure 1). (3) Third. where a bifunctional reagent (namely. In order to overcome this. they can be integrated in a continuous process. no speciﬁc approach tackles simultaneously these diﬀerent features.to 200-fold higher speciﬁc activity than the equivalent Eupergit-C-immobilized enzyme preparations. since the enzymes are rejected by the membrane. and dimethylglutarimidate. xylanase. Recovery of the adsorbed xylitol was achieved by elution with CaCl2 solutions. high stability and low production costs [142. This approach is recent. The approach was based on the high speciﬁcity of the enzyme crystals towards xylitol. since they are prone to abrasion.
which can be produced under mild condition. “History of industrial biotransformations— dreams and realities. the accumulated evolutionary enzyme engineering expertise. and D. WileyVCH. Such progresses have been made through the ever-continuing developments in molecular biology. 2006. more stable (to temperature and pH). R. S. while maintaining the targeted activity or evolving novel activities.” in Industrial Biotransformations. J. This is of particular relevance for application in the food and feed sector. A. The latter can be particularly restrictive in the food and feed sector. less dependent on metal ions and less susceptible to inhibitory agents and to aggressive environmental conditions. which furthermore encompass both technical and economic requirements. and the immobilized biocatalyst ﬁt for a given process and product may be totally unsuitable for another. All these strategies either isolated or preferably suitably integrated have been put into practice in food and feed. Alongside with these strategies. 5. G. . Liese. it can be foreseen that eﬀorts will be towards the development of immobilized biocatalyst with suitable chemical. Voragen. 1–35. It also favors process integration. 2nd edition. Poulsen and H.  P. and the implementation of high-throughput methodologies. B. Whitaker. with the latter often combined with the output of new goods. Conclusions and Future Perspectives The integration of enzymes in food and feed processes is a well-established approach. Seelbach. K. Wandrey. Hence. with high level of parallelization. by allowing the concerted use of enzymes that naturally have diverse requirements for eﬀective application. Weinheim. to improve existing processes or to implement new ones. and despite the developments made in this particular ﬁeld. since most products are of relatively low added value. A. the immobilization of enzymes has also been a key supporting tool for rendering these proteins ﬁt for industrial application.. physical. Again. that can be used in diﬀerent reactor conﬁgurations and that comply with the economic requirements for large-scale application. Given the diversity of enzyme nature and applications this pattern is unlikely to be reversed. for it allows enhanced performance under operational conditions that minimize the risk of microbial contamination. the (bio)computational tools. Acknowledgment ˜o para a Ciˆ Pedro Fernandes acknowledges Fundac ¸a encia e a Tecnologia (Portugal) for ﬁnancial support under program Ciˆ encia 2007. and geometric characteristics. “History of enzymology with emphasis on food production. Klaus Buchholz. Therefore. Reactors (a) to (d) are depicted under continuous mode of operation. there is no universal support and method for enzyme immobilization aimed at application in food and feed (let alone the overall range of possible ﬁelds of use). Germany. Vasic-Racki.” in Handbook of Food Enzymology. J. there is still the lack of a set of unanimously applicable rules for the selection of carrier and method of enzyme immobilization. resulting from novel enzymatic activities. used.12 Fluid in Fluid out Fluid in Fluid out Enzyme Research Fluid in Fluid out (product rich) Biocatalyst recycle Fluid out (a) Packedbed reactor Fluid in (b) Fluidizedbed reactor Ultraﬁltration unit Free enzyme Immobilized enzyme (d) Stirred basket reactor Free enzyme Immobilized enzyme (e) Stirred batch reactor (c) Perfectly mixed reactor with recycle Figure 1: Examples of bioreactor conﬁgurations commonly used in bioconversion processed involving free or immobilized enzymes. These endeavors have been anchoring in innovative approaches for the design of new/improved biocatalysts. Eds. but evidence clearly shows that dedicated research eﬀorts are consistently being made as to make this application of biological agents more eﬀective and/or diversiﬁed. whereas reactor (e) is depicted. this trend is foreseen to be further implemented. W. pp. and C. possibly due to mass transfer resistances associated . References  D. Given the recent developments in this ﬁeld. enabling the eﬃcient and timely screening/characterization of the biocatalysts. while simultaneously enabling the improvement of their catalytic features.
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