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Invertase and its applications – A brief review
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 9 2 e7 9 7
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Review Article
Invertase and its applications e A brief review Samarth Kulshrestha a, Prasidhi Tyagi b, Vinita Sindhi a, Kameshwar Sharma Yadavilli c,* a
Department of Biological Sciences, Sri Venkateswara College, University of Delhi, Delhi 110021, India Department of Life Sciences, Sri Venkateswara College, University of Delhi, Delhi 110021, India c Department of Biochemistry, Sri Venkateswara College, University of Delhi, Delhi 110021, India b
article info
abstract
Article history:
Invertase, also called beta-fructofuranosidase cleaving the terminal non-reducing beta-
Received 9 January 2013
fructofuranoside residues, is a glycoprotein with an optimum pH 4.5 and stability at 50 C.
Accepted 16 July 2013
It is widely distributed in the biosphere especially in plants and microorganisms. Saccha-
Available online 25 September 2013
romyces cerevisiae commonly called baker’s yeast is the chief strain used for the production and purification of the enzyme. Invertase in nature exists in different isoforms. In yeasts, it
Keywords:
is present either as extracellular Invertase or intracellular Invertase. In plants, there are
Antiseptic
three isoforms each differing in biochemical properties and subcellular locations. Invertase
Baker’s yeast
in plants is essential not only for metabolism but also help in osmoregulation, develop-
Chromatography
ment and defence system. In humans, the enzyme acts as an immune booster, as an anti-
Invertase
oxidant, an antiseptic and helpful for bone cancer or stomach cancer patients in some
Purification
cases. The present study focuses upon the Invertase along with its application and purification from Saccharomyces cerevisiae. Invertase from baker’s yeast was purified by concentrating the crude extract with ammonium sulphate (70%), dialyzed using sample buffer (0.1 M Tris, pH 7.2) and followed by centrifugation. The resultant supernatant was then applied on DEAE-cellulose column equilibrated with Tris buffer. The enzyme was eluted with a step gradient of NaCl (0e0.5 M) in starting buffer. Fractions showing highest activity were pooled. The result contains the purification summary with the purification fold of 27.13 and recovery of 31.93%. For the better understanding the mechanism and structure of the purified enzyme characterization is essential. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Enzymes are complex globular proteins found in living cells, acting as a bio-catalyst facilitating metabolic reactions in an organism’s body. In 1878 Kuhne coined the term ‘enzyme’ from the Greek word, “enzumas”, which refers to the leavening of bread by yeast. Enzymes catalytic nature is
responsible for the functioning. It participates in a reaction without being consumed in the reaction, attaining a high rate of product formation by lowering down the Gibb’s free energy (DG ) required for the reaction to occur.1 Because of their specific nature enzymes can differentiate between chemicals with similar structures and can catalyze reactions over a wide range of temperatures (0e110 C) and in
* Corresponding author. Tel.: þ91 9910374426 (mobile). E-mail addresses: [email protected], [email protected] (K.S. Yadavilli). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.07.014
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 9 2 e7 9 7
the pH range 2e14. In industrial application, such qualities with an enzyme being non-toxic and biodegradable can result in high quality and quantity products, fewer byproducts and simpler purification procedures. Also enzymes can be obtained from different microorganisms and that too in large amount without using any chemical resistant approaches.2 In the West, the industrial understanding of enzymes revolved around yeast and malt where traditional baking and brewing industries were rapidly expanding. Much of the early development of biochemistry was centred on yeast fermentations and processes for conversion of starch to sugar.1 One such enzyme of our interest is “Invertase”. This article focuses on the extraction methods, purification approaches, catalytic nature and its application in today’s world. The primary source of energy in all living organisms is carbohydrates. Even non-reducing disaccharides like trehalose or sucrose also have other roles like acting as signalling molecule as well as stress protectants.3 Additionally monosaccharide like glucose or fructose plays regulatory functions in the central metabolic pathway of a cell’s metabolism.4 Thus, Invertase plays a central role as it is a sucrose hydrolyzing enzyme, named because of the inversion in the optical rotation during the hydrolysis of sucrose.
2.
Sources of Invertase
The official name for Invertase is beta-fructofuranosidase (EC.3.2.1.26), which implies that the reaction catalyzed by the enzyme, is the hydrolysis of the terminal non-reducing beta-fructofuranoside residues in beta-fructofuranosides.5 Invertase is widely distributed among the biosphere. It is mainly characterized in plants and microorganisms. Saccharomyces cerevisiae commonly called Baker’s yeast is the chief strain used for the production of Invertase commercially. They are found in wild growing, on the skin of grapes and other fruits.5 Though plants like Japanese Pear fruit (Pyrus pyrifolia), Pea (Pisum sativum), Oat (Avena sativa) can also be used, but generally microorganisms like S. _cerevisiae, Candida utilis, A. niger are considered ideal for their study.6
3.
Kinetics of Invertase enzyme
In contrary to most other enzymes, Invertase exhibits relatively high activity over a broad range of pH (3.5e4.5) with the optimum near pH of 4.5. The enzyme activity reaches a maximum at 55 C. The MichaeliseMenten (Km) value for the free enzyme is typically 30 mM (approx.).7 The enzyme is a glycoprotein, stable at 50 C. The cations 2 2 2 Hg þ, Agþ, Ca þ and Cu þ exhibit a marked inhibition of 8 the enzyme. Competitive inhibition was observed with the fructose analogue 2, 5-anhydro-D-mannitol suggesting that the enzyme was inhibited by the furanose form of fructose.9
4.
Isozymes of Invertase
4.1.
Isozymes in Baker’s yeast
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Invertase exists in more than one form in yeasts generally, either extracellular Invertase or intracellular Invertase.10 The external yeast Invertase is a glycoprotein containing about 50% carbohydrate, 5% mannose, 3% glucosamine, whereas internal Invertase contains no carbohydrate.9 The former one has a molecular weight of 135 KDa whereas the latter variety has a molecular weight of 270 KDa.8 It has been established that in depressed cells most of the Invertase is external whereas in fully repressed state all the Invertase is intracellular.7 Both differ in amino acid sequences particularly the internal Invertase does not contain cysteine. Both the enzymes are inhibited by Iodine and reactivated by mercaptoethanol. Both require an acid with pKa about 6.8 in its protonated form. Both are inhibited by cyanogen bromide in a biphasic reaction.11
4.2.
Isoforms of Invertase in plants
Several isoforms of Invertase exist with different biochemical properties and subcellular locations in plants.10 On the basis of solubility, optimum pH, isoelectric point and subcellular localization, plant Invertase can be classified into three subgroups. Three biochemical subgroups of Invertase in plants: vacuolar (soluble acid), cytoplasmic (soluble alkaline) and cell wall bound Invertase. The presence of multiple isoform of Invertase in nature have functionally beneficial role to the plants.12
4.2.1.
Insoluble acid/cell wall bound Invertase
Insoluble acid Invertase (INAC-INV) is cell wall bound, glycosylated protein with a variable molecular weight ranging between 28 and 64 KDa. It has an optimum pH of 4.0, temperature optimum of 45 C and an isoelectric point of 9. Its activity is inhibited by 6.2 mM Copper sulphate. The Km and Vmax values for the above were found to be 4.41 mM and 8.41 U (mg/protein)/minute respectively.12 It is localized in the basal endosperm and pedicel tissue in maize kernels. Using immunological techniques, it was concluded that is involved in the normal development of the endosperm cells and maternal cells in pedicel tissues in maize. Using a bean as a plant material, in seed development, it was found in thin walls of the seed coat of the parenchyma cells. It is a true member of b-fructofuranosidases which can react with sucrose and raffinose as substrates.13
4.2.2.
Soluble acid/vacuolar Invertase
Vacuolar Invertase has an acidic pI with a pH range between 4.5 and 5.0. The enzyme has a Km for sucrose in the lowmillimoles range. Along with sucrose, it also hydrolyzes raffinose or stachiose being as a true member of bfructofuranoside family. The enzyme loses its activity when reacted by heavy metal ions like mercury or silver. Also, glucose acts as a non-competitive inhibitor for the enzyme and fructose being a competitive inhibitor. The mature polypeptide is N-glycosylated and has a molecular mass of approximately 70 KDa.14
794
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The first cloned plant acid Invertase was cell wall bound Invertase from carrot. This study revealed that each isoform of Invertase is encoded by a different gene. Although, the cDNA derived amino acid sequences share some common feature such as the pentapeptide Asn-Asp-Pro-Asn-Gly (bFmotif), which is close to the N-terminus of the mature protein, and a Cys residue and its neighbouring amino acids, which are located closed to the C-terminus. INAC-INV cDNA appear to have short C-terminus extensions which are not present in other Invertases having a critical role in vacuolar sorting signals.15 Soluble acid Invertase (AIV) have two or more isozymes, which can be purified and characterized from plants such as Japanese pear fruit, barley lectin or tobacco chitinase. In the process of purification, specific activities of purified Soluble acid Invertase I (AIV I) and Soluble acid Invertase II (AIV II) were found out to be 2670 and 2340 (nkat/mg protein), respectively. The Km values for sucrose of Soluble acid Invertase I (AIV I) and Soluble acid Invertase II (AIV II) were found to be 3.33 and 4.58 mM with an optimum pH of 4.5 for both the enzymes. With SDS-PAGE, AIV I and AIV II were found to be monomeric enzymes with molecular weight of 80 KDa and 86 KDa respectively.14 Soluble acid Invertase plays important biological functions related to sucrose metabolism and predominantly hydrolyzes sucrose for growth and developmental processes. Also, sucrose hydrolysis by soluble acid Invertase helps in regulation of osmotic pressure which is controlled by cell expansion which depends on size of vacuole.16
4.2.3.
Soluble alkaline/cytoplasmic Invertase
Soluble alkaline Invertase is a non-glycosylated polypeptide expressed at low levels. The two isoforms are encoded by the same gene and two transcripts originate from differential splicing of a hetero nuclear mRNA. The native polypeptides are homo tetramers with a molecular mass of 54e65 KDa. The enzyme has a Km of 10 mM for sucrose and is inhibited by glucose, fructose and Tris but not by heavy metal ions. Using cDNA expression, when the amino acid sequence of soluble alkaline Invertase was deduced, it lacks N-terminal signal peptide and has no similarity with other forms of Invertases. Soluble alkaline Invertase is not a member of b-fructofuranosidase family as it hydrolyzes sucrose only unlike other acid Invertases. It is found in all plant organs at different developmental stages, especially in the developing tissues implying it has growth related functions.3
5.
Role of Invertase in plants
5.1.
Plant osmoregulation and metabolism
To provide cell, fuel for respiration, carbon and energy for the synthesis of different compounds, Invertase cleave sucrose into corresponding monosaccharide. By generating the necessary sucrose concentration gradient between sites of phloem loading and unloading, Invertase also help in longdistance transport of sucrose. Hydrolysis of sucrose into glucose and fructose influences the osmotic pressure of cells and thus helps in cell elongation and plant growth. Developing
roots of carrot or elongating stems of bean are some of the organs of the plant which contain high activity of acid Invertase especially in rapidly growing tissues. High acid Invertase activity can also be correlated with the accumulation of hexoses in sugar storing sink organs such as fruit. Thus, indicating that a soluble acid Invertase also function as a regulator of sugar composition in the post harvest processes.15
5.2.
Sucrose allocation
In 1995, Weber et al studied the molecular physiology of photosynthetic unloading and portioning during seed development of fava bean and proposed that high level of hexoses exists in the cotyledons and the apoplastic endospermal space during the pre storage phase. The level of hexoses was found to be proportional to level of cell wall bound Invertase in the seed coat.17 It was also found that an early degeneration and withdraw of maternal cells from endosperm occurs when there is lack of Invertase activity resulting in an interruption of the transport of photo assimilates into the developing kernel.18
5.3.
Role of Invertase in plant development
In the early stages, by controlling sugar composition and metabolic fluxes, Invertase appears to play key role in plant development. Both isoenzymes i.e. cell wall Invertase and vacuolar Invertase performs functions in sucrose partitioning, when their activities have shifted development in favour of leaves.16 The higher levels of Invertase activity can be observed in oat internodes reflecting the increased energy and carbon requirements to sustain the biochemical reactions during growth period. Thus, suggesting that a close relationship exists between growth rate and level of Invertase activity. The degradation of carbohydrate in the tissue is also observed proportional to the enhancements in respiration, and protein and cell-wall biosynthesis during the growth period.14
5.4.
Invertase in defence mechanism
Invertase results in a link reaction between carbohydrate degradation and pathogen responses. This includes the phenomenon of high sugar resistance in which key pathogenesis related genes which are sugar inducible get over expressed in the plant apoplast. This results in an increased expression of Pathogen Related (PR) proteins and thus increased resistance against viral infections. The regulation of extracellular Invertase by phytohormones could also contribute to plant pathogen responses involving in expression of various defences related genes. In this process the extracellular Invertase induced by sugars provides a mechanism in which the sink strength will elevate increasing the sugar concentration. This induces PR genes and represses photosynthetic genes in addition to signals derived from the pathogen.19
6.
Mechanism of yeast Invertase
An imidazolium cation protonates the glycosidic oxygen atom. Departure of the natural alcohol group will leave behind
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 9 2 e7 9 7
an unstable intermediate carbonium ion in which the electron deficiency is spread over the C-2 atom as well as the ring oxygen atom. The active-site carboxylate anion will function during this and the previous stage by stabilizing the electrondeficient species [Fig. 1]. The next stage is the attack on the C-2 cation by a nucleophilic oxygen atom of an alcohol or water to yield a fructoside or fructose.11
7.
Gene regulation of yeast Invertase
The SUC2 is responsible for two forms of Invertase: a secreted invertase which is responsible for hydrolysis of sucrose and raffinose and an intracellular invertase having no significant physiological use.20 The SNF1 (sucrose nonfermenting) gene encodes a protein kinase. The SNF3 gene is needed for glucose transport. Hex2 probably allelic to regl is responsible for glucose insensitive expression of galactokinase and Invertase. Mutations in cid1, reg1 and hxk2 lead to high invertase activity under glucose under expressing conditions and produce wildtype levels under derepression conditions. Reg1 (encodes a regulatory subunit of a protein phosphatase) and hxk2 (structural gene for hexokinase P II) are responsible for making other glucose responsible genes glucose insensitive. They along with cid1 (constitutive invertase derepression) have a sensory role in monitoring the availability of glucose and regulating the activity of protein kinase encoded by SNF1. SSN6 directly affects the gene expression. The SSN6 gene product is a substrate of the SNF1 protein kinase and a regulator of SUC2. It can also have other functions.21
8.
Regulation of Invertase in plants
8.1.
By phytohormones
Gibberellic acid plays a central role in regulating Invertase levels (GA3) promoting cell elongation essential for flower induction. High Invertase activity can be seen in several
795
plant organs such as sugarcane stem, Jerusalem artichoke tubers, beet roots, lentil epicotyls, internodes of beans and oat, etc. Cytokinins promote cell and thus an enhanced demand for carbohydrate is needed for active growth. This phenomenon is bolded by the fact that tissues with higher activity of extracellular Invertase (rapidly growing tissues), also contain elevate concentration of cytokinin phytohormone. Ethylene is the only phytohormone which stimulates the down-regulation the expression of extracellular Invertase by repressing its mRNA transcripts.3
8.2.
Under abiotic stress
Under salt stress, plants produce photo assimilates which support crucial processes such as growth, maintenance and osmotic adjustment. An increase in sucrose in source leaves occurs with a decrease in photosynthesis rate due to feedback inhibition under saline condition. The extracellular Invertase plays a key role in those species in which the step of phloem unloading of sucrose is apoplasmic and also in the control of assimilate allocation. In the above process, when the extracellular Invertase is impaired or the phloem unloading pathway is symplasmic, the vacuolar acid Invertase and neutral Invertase play the major role.19 A decrease in export of assimilates and a decrease in crop production occurs under water stress due to inductions of large alterations in sourceelink reactions. Under such conditions, the elevated activities of soluble and insoluble Invertase get blocked during pollination and early kernel development in maize.19 Under drought conditions, in mature maize leaves, cell wall Invertase activity does not get affected but an increase in vacuolar Invertase activity can be seen leading to accumulation of hexoses in the leaves.18 Low oxygen stress in maize root tips decreases Invertase expression and therefore, decreasing the Invertase/sucrose ratio. Thus, by conserving sucrose and ATP and reduction of the hexose-based sugar signalling system, plants acclimatized to low oxygen condition.22
Fig. 1 e Mechanism of Invertase.
796
9.
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Applications of Invertase enzyme
An equimolar mixture of fructose and glucose (invert syrup) obtained by sucrose hydrolysis is sweeter than sucrose due to high degree of sweetness of fructose, as a result the sugar content can be increased without crystallization of the material.6 The production of non-crystallizable sugar syrup from sucrose is one of the major applications of Invertase enzyme. Invert syrup has hygroscopic properties which makes it useful in the manufacturing of soft- centred candies and fondants as ahumectants.23 Alcoholic beverages, lactic acid, glycerol etc. produced by fermentation of sucrose containing substrates requires the use of Invertase. It is also associated with insulinase for the hydrolysis of inulin (poly-fructose) to fructose.15 Other application of the enzyme is seen in drug and pharmaceutical industries. Also it is used in the manufacture of artificial honey and plasticizing agents which are used in cosmetics. Enzyme electrodes are used for the detection of sucrose. Formation of undesirable flavouring agents as well as coloured impurities do not take place on enzymatic hydrolysis of sucrose instead of acid hydrolysis.24 Immobilized Invertase is used for continuous hydrolysis of sucrose as the resulting shifts in the pH can be used to prevent the formation of oligosaccharides by the transferase activity associated with the soluble enzyme.23 Invertase being a powerful anti-microbial agent and an anti-oxidant aids in the prevention of bacterial infestations and gut fermentation due to oxidation.25 Raw honey was used in ancient India in killing bacteria, reducing intestinal ailments and was given to patients having a weak heart. It can also be used in subsiding bacterial infections because of its ability to extract moisture from the body of the patient. According to a European study on 18000 patients, honey has been proved effective in treating respiratory tract infection such as bronchitis, asthma and allergies. Invertase along with other enzymes has also been shown to help cure colds, flu and other respiratory problems.26
10.
Analytical approach
In the commenced study, an attempt was made to purify Invertase from Baker’s yeast, common form of S. cerevisiae. The present study deals with the appliance of various biochemical techniques like ammonium sulphate precipitation, dialysis and ion-exchange chromatography.
10.1.
Experimentation summary
Invertase is used for the inversion of sucrose in the preparation of invert sugar and high fructose syrup (HFS). It is one of the most widely used enzymes in food industry where fructose is preferred than sucrose especially in the preparation of jams and candies, because it is sweeter and does not crystallize easily. A wide range of microorganisms produce Invertase and thus can utilize sucrose as a nutrient. Commercially Invertase is biosynthesized chiefly by yeast strains of S. cerevisiae. In the following analysis, active dried yeast was taken and enzyme extract was prepared. The extract was subjected for
ammonium sulphate precipitation. The resultant pellet after centrifugation was dialyzed using Tris-Phosphate buffer. The supernatant obtained after centrifugation was subjected onto ion-exchange chromatography using DEAE-cellulose and TriseHCl.27,28 Step gradient technique is used for elution of the sample with NaCl concentration ranging from 0 to 0.5 M. The purification fold of the enzyme comes out to be 27.13 with a recovery of 31.93%.
11.
Conclusion
Invertase is a key metabolic enzyme hydrolyzing betafructofuranoside residues, existing in various forms of life and even found as different isoforms. These isoforms provide an extra edge to the organism’s survival capability. These isoforms appear to regulate the entry of sucrose into different utilization pathways. Invertase is of high importance in plants developmental processes, carbohydrate partitioning and in abiotic as well as biotic interaction. Multiple genes encode for above proteins responsible for Invertase action. With immobilized enzyme technology, Invertase demand has increased for its vital role in food industry. The above article provides a practical hand on introduction of many general considerations and corresponding strategies encountered during the course of isolating a specific protein from its initial biological source. With the advent of technology and modern gadgets, our knowledge for the subject has increased tremendously. Despite these accomplishments, some questions still need to be answered/such as why Invertase is so specific for its function, why nature has selected these isoforms and their role in Invertase evolution. Solution to these questions may also arise from multidisciplinary approaches. The knowledge gained will help to understand one of the most fundamental processes of carbohydrate metabolism and its utilization pathway according to an organism’s need. With biotechnological manipulation, Invertase can be transformed into a billion dollar solution for yield improvement in plants and crops, support for cancer patients and high quality anti-oxidant product.
Conflicts of interest All authors have none to declare.
references
1. Maciel Ma´rcia Jaqueline Mendonc¸a, Silva Ademir Castro e, Ribeiro Helena Camara˜o Telles. Industrial and biotechnological applications of ligninolytic enzymes of the basidiomycota: a review. Electron J Biotechnol. 2010;13(6):14e15. 2. Enzymatic Production of Invert Sugar. Du¨sseldorf, Germany: Enzyme Company; 2007:1e10. 3. Plant invertases: structure, function and regulation of a diverse enzyme family Vasileiosfotopoulus. J Biol Res. 2005;4:127e137. 4. Molnapatsongpim, Vaithanomsat Pilanee, Vanichsriratana Wirat, Sirisansaneeyakul Sarote.
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Enhancement of Inulinase and Invertase Production from a Newly Isolated Candida Guilliermondii. TISTR 5844.45. 2011:675e685. Romero-Gomez S, Augur C, Viniegra-Gonzalez G. Invertase production by Aspergillus nı´ger in submerged and solid-state fermentation. Biotechnol Lett. 2000;22:1255e1258. Uma C, Gomathi D, Multhulakshmi C, Gopalkrishana VK. Production, purification and characterization of invertase by Aspergillus flavus using fruit peel waste as substrate. Advan Biol Res. 2010;4(1):31e36. Vu TKH, Le VVM. Biochemical studies on the immobilization of the enzyme invertase (EC.3.2.1.26) in alginate gel and its kinetics. ASEAN Food J. 2008;15(1):73e78. Ali S, Haq I. Kinetics of improved extracellular bD-fructofuranosidase fructohydrolase production by a derepressed Saccharomyces cerevisiae. Lett Appl Microbiol. 2007;45:160e167. Workman Wesley E, Way Donal F. Purification and properties of the b-fructofuranosidase from Kluyveromyces fragilis. FEBS Letters. 1983 August;160(1-2):16e20. Lahiri Sagar, Basu Avghya, Sengupta Shinjinee, et al. Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis. Arch Biochem Biophys. 2012;522:90e99. Shall S, Baseer A, Waheed A. The Mechanism of Action of Yeast Invertase. Biochem J. 1971 March;122(1):19e20. Kim Donggiun, Gunsuplee, Chang Man, et al. Purification and biochemical characterization of insoluble acid invertase from pea seedlings. J Agric Food Chem. 2011;59:11228e11233. Belcarz A, Ginalska G, Penel C. The novel non glycosylated invertase from Candida utilis. J Biochem Biophys Acta. 2002;1594:40e53. Hashizume Hiroshi, Tanase Koji, Shiratake Katsuhiro, Mori Hitoshi, Yamaki Shohee. Purification and characterization of two soluble acid invertase isozymes from Japanese peer fruit. Phytochemistry. 2003;63:125e129. Friedrich Micscher Institute, Switzerland. Invertases, primary structure, function and roles in plant development and sucrose partitioning. Plant Physiol. September, 1999;121.
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16. Ji X, Vander Ende W, Van Laaere A, Cheng S, Bunnett J. Structure, evolution and expression of the two invertase gene families of rice. J Mol Evol. 2005 May;60(5):615e634. 17. Li Z, PalmerW M. High invertase activity in tomato reproductive organs correlates with the enhanced sucrose import into and heat tolerance of young fruit. J Exp Bot. 2012 Feb;63(3):1155e1166. 18. Kaur H, Gupta AK, Kaur N, Sandhu JS. High invertase activity for a prolonged period in developing seeds/podwell of wild chick pea is detrimental to seed filling. Indian J Exp Biol. 2012 Oct;50(10):735e743. 19. Roitsch T, Balibrea ME, Hofman M, Proels R, Sinha AK. Extracellular invertase: key enzyme and PR protein. J Exp Bot. 2003;54(382). 20. Brandao RL, Etchebehere L, Queiroz CC, Tropia MJ. Evidence for involvement of Saccharomyces cerevisiae protein kinase C in glucose induction of HXT genes and derepression of SUC2. FEMS Yeast Res. 2002;2:93e102. 21. Lenore Neigeborn, Marian Carlson. Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations. Genetics. 1987 Feb;115(2):247e253. 22. Rioig P, Gozalbo D. Candida albicans UB13 and UB14 promoter regions confer differential regulation of invertase production to Saccharomyces cerevisiae in response to stress. Int Microbiol. 2002 Mar;5(1):33e36. 23. Kotwal SM, Shankar V. Immobilized invertase. Biotechnol Adv. 2009;27:311e322. 24. Young Linda, Cauvain Stanley P. Technology of Bread MakingIn: The Scientific Name for Baker’s Yeast Is Saccharomyces Cerevisiae. Berlin: Springer; 2007, ISBN 0-387-38563-0; 2007:79. 25. MAXINVERT. Invertase for food processing. 26. Edward F. The Health Benefits Of Invertase. http://www. globalhealingcenter.com/natural-health/invertase/ [Published on July 1, 2011, Last Updated on August 7, 2013]. 27. Muriel Mercier-Bonin, Christian Fonade. Enzyme recovery during gas/liquid two-phase flow micro filtration of enzyme/yeast mixtures. Biotechnol Bioeng. 2002 Dec;80(6):610e621. 28. Gjerde Douglas T, Fritzx James S. Ion Chromatography. Weinheim: Wiley-VCH; 2000, ISBN 3-527-29914-9; 2000.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Review Article
Invertase and its applications e A brief review Samarth Kulshrestha a, Prasidhi Tyagi b, Vinita Sindhi a, Kameshwar Sharma Yadavilli c,* a
Department of Biological Sciences, Sri Venkateswara College, University of Delhi, Delhi 110021, India Department of Life Sciences, Sri Venkateswara College, University of Delhi, Delhi 110021, India c Department of Biochemistry, Sri Venkateswara College, University of Delhi, Delhi 110021, India b
article info
abstract
Article history:
Invertase, also called beta-fructofuranosidase cleaving the terminal non-reducing beta-
Received 9 January 2013
fructofuranoside residues, is a glycoprotein with an optimum pH 4.5 and stability at 50 C.
Accepted 16 July 2013
It is widely distributed in the biosphere especially in plants and microorganisms. Saccha-
Available online 25 September 2013
romyces cerevisiae commonly called baker’s yeast is the chief strain used for the production and purification of the enzyme. Invertase in nature exists in different isoforms. In yeasts, it
Keywords:
is present either as extracellular Invertase or intracellular Invertase. In plants, there are
Antiseptic
three isoforms each differing in biochemical properties and subcellular locations. Invertase
Baker’s yeast
in plants is essential not only for metabolism but also help in osmoregulation, develop-
Chromatography
ment and defence system. In humans, the enzyme acts as an immune booster, as an anti-
Invertase
oxidant, an antiseptic and helpful for bone cancer or stomach cancer patients in some
Purification
cases. The present study focuses upon the Invertase along with its application and purification from Saccharomyces cerevisiae. Invertase from baker’s yeast was purified by concentrating the crude extract with ammonium sulphate (70%), dialyzed using sample buffer (0.1 M Tris, pH 7.2) and followed by centrifugation. The resultant supernatant was then applied on DEAE-cellulose column equilibrated with Tris buffer. The enzyme was eluted with a step gradient of NaCl (0e0.5 M) in starting buffer. Fractions showing highest activity were pooled. The result contains the purification summary with the purification fold of 27.13 and recovery of 31.93%. For the better understanding the mechanism and structure of the purified enzyme characterization is essential. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Enzymes are complex globular proteins found in living cells, acting as a bio-catalyst facilitating metabolic reactions in an organism’s body. In 1878 Kuhne coined the term ‘enzyme’ from the Greek word, “enzumas”, which refers to the leavening of bread by yeast. Enzymes catalytic nature is
responsible for the functioning. It participates in a reaction without being consumed in the reaction, attaining a high rate of product formation by lowering down the Gibb’s free energy (DG ) required for the reaction to occur.1 Because of their specific nature enzymes can differentiate between chemicals with similar structures and can catalyze reactions over a wide range of temperatures (0e110 C) and in
* Corresponding author. Tel.: þ91 9910374426 (mobile). E-mail addresses: [email protected], [email protected] (K.S. Yadavilli). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.07.014
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the pH range 2e14. In industrial application, such qualities with an enzyme being non-toxic and biodegradable can result in high quality and quantity products, fewer byproducts and simpler purification procedures. Also enzymes can be obtained from different microorganisms and that too in large amount without using any chemical resistant approaches.2 In the West, the industrial understanding of enzymes revolved around yeast and malt where traditional baking and brewing industries were rapidly expanding. Much of the early development of biochemistry was centred on yeast fermentations and processes for conversion of starch to sugar.1 One such enzyme of our interest is “Invertase”. This article focuses on the extraction methods, purification approaches, catalytic nature and its application in today’s world. The primary source of energy in all living organisms is carbohydrates. Even non-reducing disaccharides like trehalose or sucrose also have other roles like acting as signalling molecule as well as stress protectants.3 Additionally monosaccharide like glucose or fructose plays regulatory functions in the central metabolic pathway of a cell’s metabolism.4 Thus, Invertase plays a central role as it is a sucrose hydrolyzing enzyme, named because of the inversion in the optical rotation during the hydrolysis of sucrose.
2.
Sources of Invertase
The official name for Invertase is beta-fructofuranosidase (EC.3.2.1.26), which implies that the reaction catalyzed by the enzyme, is the hydrolysis of the terminal non-reducing beta-fructofuranoside residues in beta-fructofuranosides.5 Invertase is widely distributed among the biosphere. It is mainly characterized in plants and microorganisms. Saccharomyces cerevisiae commonly called Baker’s yeast is the chief strain used for the production of Invertase commercially. They are found in wild growing, on the skin of grapes and other fruits.5 Though plants like Japanese Pear fruit (Pyrus pyrifolia), Pea (Pisum sativum), Oat (Avena sativa) can also be used, but generally microorganisms like S. _cerevisiae, Candida utilis, A. niger are considered ideal for their study.6
3.
Kinetics of Invertase enzyme
In contrary to most other enzymes, Invertase exhibits relatively high activity over a broad range of pH (3.5e4.5) with the optimum near pH of 4.5. The enzyme activity reaches a maximum at 55 C. The MichaeliseMenten (Km) value for the free enzyme is typically 30 mM (approx.).7 The enzyme is a glycoprotein, stable at 50 C. The cations 2 2 2 Hg þ, Agþ, Ca þ and Cu þ exhibit a marked inhibition of 8 the enzyme. Competitive inhibition was observed with the fructose analogue 2, 5-anhydro-D-mannitol suggesting that the enzyme was inhibited by the furanose form of fructose.9
4.
Isozymes of Invertase
4.1.
Isozymes in Baker’s yeast
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Invertase exists in more than one form in yeasts generally, either extracellular Invertase or intracellular Invertase.10 The external yeast Invertase is a glycoprotein containing about 50% carbohydrate, 5% mannose, 3% glucosamine, whereas internal Invertase contains no carbohydrate.9 The former one has a molecular weight of 135 KDa whereas the latter variety has a molecular weight of 270 KDa.8 It has been established that in depressed cells most of the Invertase is external whereas in fully repressed state all the Invertase is intracellular.7 Both differ in amino acid sequences particularly the internal Invertase does not contain cysteine. Both the enzymes are inhibited by Iodine and reactivated by mercaptoethanol. Both require an acid with pKa about 6.8 in its protonated form. Both are inhibited by cyanogen bromide in a biphasic reaction.11
4.2.
Isoforms of Invertase in plants
Several isoforms of Invertase exist with different biochemical properties and subcellular locations in plants.10 On the basis of solubility, optimum pH, isoelectric point and subcellular localization, plant Invertase can be classified into three subgroups. Three biochemical subgroups of Invertase in plants: vacuolar (soluble acid), cytoplasmic (soluble alkaline) and cell wall bound Invertase. The presence of multiple isoform of Invertase in nature have functionally beneficial role to the plants.12
4.2.1.
Insoluble acid/cell wall bound Invertase
Insoluble acid Invertase (INAC-INV) is cell wall bound, glycosylated protein with a variable molecular weight ranging between 28 and 64 KDa. It has an optimum pH of 4.0, temperature optimum of 45 C and an isoelectric point of 9. Its activity is inhibited by 6.2 mM Copper sulphate. The Km and Vmax values for the above were found to be 4.41 mM and 8.41 U (mg/protein)/minute respectively.12 It is localized in the basal endosperm and pedicel tissue in maize kernels. Using immunological techniques, it was concluded that is involved in the normal development of the endosperm cells and maternal cells in pedicel tissues in maize. Using a bean as a plant material, in seed development, it was found in thin walls of the seed coat of the parenchyma cells. It is a true member of b-fructofuranosidases which can react with sucrose and raffinose as substrates.13
4.2.2.
Soluble acid/vacuolar Invertase
Vacuolar Invertase has an acidic pI with a pH range between 4.5 and 5.0. The enzyme has a Km for sucrose in the lowmillimoles range. Along with sucrose, it also hydrolyzes raffinose or stachiose being as a true member of bfructofuranoside family. The enzyme loses its activity when reacted by heavy metal ions like mercury or silver. Also, glucose acts as a non-competitive inhibitor for the enzyme and fructose being a competitive inhibitor. The mature polypeptide is N-glycosylated and has a molecular mass of approximately 70 KDa.14
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The first cloned plant acid Invertase was cell wall bound Invertase from carrot. This study revealed that each isoform of Invertase is encoded by a different gene. Although, the cDNA derived amino acid sequences share some common feature such as the pentapeptide Asn-Asp-Pro-Asn-Gly (bFmotif), which is close to the N-terminus of the mature protein, and a Cys residue and its neighbouring amino acids, which are located closed to the C-terminus. INAC-INV cDNA appear to have short C-terminus extensions which are not present in other Invertases having a critical role in vacuolar sorting signals.15 Soluble acid Invertase (AIV) have two or more isozymes, which can be purified and characterized from plants such as Japanese pear fruit, barley lectin or tobacco chitinase. In the process of purification, specific activities of purified Soluble acid Invertase I (AIV I) and Soluble acid Invertase II (AIV II) were found out to be 2670 and 2340 (nkat/mg protein), respectively. The Km values for sucrose of Soluble acid Invertase I (AIV I) and Soluble acid Invertase II (AIV II) were found to be 3.33 and 4.58 mM with an optimum pH of 4.5 for both the enzymes. With SDS-PAGE, AIV I and AIV II were found to be monomeric enzymes with molecular weight of 80 KDa and 86 KDa respectively.14 Soluble acid Invertase plays important biological functions related to sucrose metabolism and predominantly hydrolyzes sucrose for growth and developmental processes. Also, sucrose hydrolysis by soluble acid Invertase helps in regulation of osmotic pressure which is controlled by cell expansion which depends on size of vacuole.16
4.2.3.
Soluble alkaline/cytoplasmic Invertase
Soluble alkaline Invertase is a non-glycosylated polypeptide expressed at low levels. The two isoforms are encoded by the same gene and two transcripts originate from differential splicing of a hetero nuclear mRNA. The native polypeptides are homo tetramers with a molecular mass of 54e65 KDa. The enzyme has a Km of 10 mM for sucrose and is inhibited by glucose, fructose and Tris but not by heavy metal ions. Using cDNA expression, when the amino acid sequence of soluble alkaline Invertase was deduced, it lacks N-terminal signal peptide and has no similarity with other forms of Invertases. Soluble alkaline Invertase is not a member of b-fructofuranosidase family as it hydrolyzes sucrose only unlike other acid Invertases. It is found in all plant organs at different developmental stages, especially in the developing tissues implying it has growth related functions.3
5.
Role of Invertase in plants
5.1.
Plant osmoregulation and metabolism
To provide cell, fuel for respiration, carbon and energy for the synthesis of different compounds, Invertase cleave sucrose into corresponding monosaccharide. By generating the necessary sucrose concentration gradient between sites of phloem loading and unloading, Invertase also help in longdistance transport of sucrose. Hydrolysis of sucrose into glucose and fructose influences the osmotic pressure of cells and thus helps in cell elongation and plant growth. Developing
roots of carrot or elongating stems of bean are some of the organs of the plant which contain high activity of acid Invertase especially in rapidly growing tissues. High acid Invertase activity can also be correlated with the accumulation of hexoses in sugar storing sink organs such as fruit. Thus, indicating that a soluble acid Invertase also function as a regulator of sugar composition in the post harvest processes.15
5.2.
Sucrose allocation
In 1995, Weber et al studied the molecular physiology of photosynthetic unloading and portioning during seed development of fava bean and proposed that high level of hexoses exists in the cotyledons and the apoplastic endospermal space during the pre storage phase. The level of hexoses was found to be proportional to level of cell wall bound Invertase in the seed coat.17 It was also found that an early degeneration and withdraw of maternal cells from endosperm occurs when there is lack of Invertase activity resulting in an interruption of the transport of photo assimilates into the developing kernel.18
5.3.
Role of Invertase in plant development
In the early stages, by controlling sugar composition and metabolic fluxes, Invertase appears to play key role in plant development. Both isoenzymes i.e. cell wall Invertase and vacuolar Invertase performs functions in sucrose partitioning, when their activities have shifted development in favour of leaves.16 The higher levels of Invertase activity can be observed in oat internodes reflecting the increased energy and carbon requirements to sustain the biochemical reactions during growth period. Thus, suggesting that a close relationship exists between growth rate and level of Invertase activity. The degradation of carbohydrate in the tissue is also observed proportional to the enhancements in respiration, and protein and cell-wall biosynthesis during the growth period.14
5.4.
Invertase in defence mechanism
Invertase results in a link reaction between carbohydrate degradation and pathogen responses. This includes the phenomenon of high sugar resistance in which key pathogenesis related genes which are sugar inducible get over expressed in the plant apoplast. This results in an increased expression of Pathogen Related (PR) proteins and thus increased resistance against viral infections. The regulation of extracellular Invertase by phytohormones could also contribute to plant pathogen responses involving in expression of various defences related genes. In this process the extracellular Invertase induced by sugars provides a mechanism in which the sink strength will elevate increasing the sugar concentration. This induces PR genes and represses photosynthetic genes in addition to signals derived from the pathogen.19
6.
Mechanism of yeast Invertase
An imidazolium cation protonates the glycosidic oxygen atom. Departure of the natural alcohol group will leave behind
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an unstable intermediate carbonium ion in which the electron deficiency is spread over the C-2 atom as well as the ring oxygen atom. The active-site carboxylate anion will function during this and the previous stage by stabilizing the electrondeficient species [Fig. 1]. The next stage is the attack on the C-2 cation by a nucleophilic oxygen atom of an alcohol or water to yield a fructoside or fructose.11
7.
Gene regulation of yeast Invertase
The SUC2 is responsible for two forms of Invertase: a secreted invertase which is responsible for hydrolysis of sucrose and raffinose and an intracellular invertase having no significant physiological use.20 The SNF1 (sucrose nonfermenting) gene encodes a protein kinase. The SNF3 gene is needed for glucose transport. Hex2 probably allelic to regl is responsible for glucose insensitive expression of galactokinase and Invertase. Mutations in cid1, reg1 and hxk2 lead to high invertase activity under glucose under expressing conditions and produce wildtype levels under derepression conditions. Reg1 (encodes a regulatory subunit of a protein phosphatase) and hxk2 (structural gene for hexokinase P II) are responsible for making other glucose responsible genes glucose insensitive. They along with cid1 (constitutive invertase derepression) have a sensory role in monitoring the availability of glucose and regulating the activity of protein kinase encoded by SNF1. SSN6 directly affects the gene expression. The SSN6 gene product is a substrate of the SNF1 protein kinase and a regulator of SUC2. It can also have other functions.21
8.
Regulation of Invertase in plants
8.1.
By phytohormones
Gibberellic acid plays a central role in regulating Invertase levels (GA3) promoting cell elongation essential for flower induction. High Invertase activity can be seen in several
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plant organs such as sugarcane stem, Jerusalem artichoke tubers, beet roots, lentil epicotyls, internodes of beans and oat, etc. Cytokinins promote cell and thus an enhanced demand for carbohydrate is needed for active growth. This phenomenon is bolded by the fact that tissues with higher activity of extracellular Invertase (rapidly growing tissues), also contain elevate concentration of cytokinin phytohormone. Ethylene is the only phytohormone which stimulates the down-regulation the expression of extracellular Invertase by repressing its mRNA transcripts.3
8.2.
Under abiotic stress
Under salt stress, plants produce photo assimilates which support crucial processes such as growth, maintenance and osmotic adjustment. An increase in sucrose in source leaves occurs with a decrease in photosynthesis rate due to feedback inhibition under saline condition. The extracellular Invertase plays a key role in those species in which the step of phloem unloading of sucrose is apoplasmic and also in the control of assimilate allocation. In the above process, when the extracellular Invertase is impaired or the phloem unloading pathway is symplasmic, the vacuolar acid Invertase and neutral Invertase play the major role.19 A decrease in export of assimilates and a decrease in crop production occurs under water stress due to inductions of large alterations in sourceelink reactions. Under such conditions, the elevated activities of soluble and insoluble Invertase get blocked during pollination and early kernel development in maize.19 Under drought conditions, in mature maize leaves, cell wall Invertase activity does not get affected but an increase in vacuolar Invertase activity can be seen leading to accumulation of hexoses in the leaves.18 Low oxygen stress in maize root tips decreases Invertase expression and therefore, decreasing the Invertase/sucrose ratio. Thus, by conserving sucrose and ATP and reduction of the hexose-based sugar signalling system, plants acclimatized to low oxygen condition.22
Fig. 1 e Mechanism of Invertase.
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Applications of Invertase enzyme
An equimolar mixture of fructose and glucose (invert syrup) obtained by sucrose hydrolysis is sweeter than sucrose due to high degree of sweetness of fructose, as a result the sugar content can be increased without crystallization of the material.6 The production of non-crystallizable sugar syrup from sucrose is one of the major applications of Invertase enzyme. Invert syrup has hygroscopic properties which makes it useful in the manufacturing of soft- centred candies and fondants as ahumectants.23 Alcoholic beverages, lactic acid, glycerol etc. produced by fermentation of sucrose containing substrates requires the use of Invertase. It is also associated with insulinase for the hydrolysis of inulin (poly-fructose) to fructose.15 Other application of the enzyme is seen in drug and pharmaceutical industries. Also it is used in the manufacture of artificial honey and plasticizing agents which are used in cosmetics. Enzyme electrodes are used for the detection of sucrose. Formation of undesirable flavouring agents as well as coloured impurities do not take place on enzymatic hydrolysis of sucrose instead of acid hydrolysis.24 Immobilized Invertase is used for continuous hydrolysis of sucrose as the resulting shifts in the pH can be used to prevent the formation of oligosaccharides by the transferase activity associated with the soluble enzyme.23 Invertase being a powerful anti-microbial agent and an anti-oxidant aids in the prevention of bacterial infestations and gut fermentation due to oxidation.25 Raw honey was used in ancient India in killing bacteria, reducing intestinal ailments and was given to patients having a weak heart. It can also be used in subsiding bacterial infections because of its ability to extract moisture from the body of the patient. According to a European study on 18000 patients, honey has been proved effective in treating respiratory tract infection such as bronchitis, asthma and allergies. Invertase along with other enzymes has also been shown to help cure colds, flu and other respiratory problems.26
10.
Analytical approach
In the commenced study, an attempt was made to purify Invertase from Baker’s yeast, common form of S. cerevisiae. The present study deals with the appliance of various biochemical techniques like ammonium sulphate precipitation, dialysis and ion-exchange chromatography.
10.1.
Experimentation summary
Invertase is used for the inversion of sucrose in the preparation of invert sugar and high fructose syrup (HFS). It is one of the most widely used enzymes in food industry where fructose is preferred than sucrose especially in the preparation of jams and candies, because it is sweeter and does not crystallize easily. A wide range of microorganisms produce Invertase and thus can utilize sucrose as a nutrient. Commercially Invertase is biosynthesized chiefly by yeast strains of S. cerevisiae. In the following analysis, active dried yeast was taken and enzyme extract was prepared. The extract was subjected for
ammonium sulphate precipitation. The resultant pellet after centrifugation was dialyzed using Tris-Phosphate buffer. The supernatant obtained after centrifugation was subjected onto ion-exchange chromatography using DEAE-cellulose and TriseHCl.27,28 Step gradient technique is used for elution of the sample with NaCl concentration ranging from 0 to 0.5 M. The purification fold of the enzyme comes out to be 27.13 with a recovery of 31.93%.
11.
Conclusion
Invertase is a key metabolic enzyme hydrolyzing betafructofuranoside residues, existing in various forms of life and even found as different isoforms. These isoforms provide an extra edge to the organism’s survival capability. These isoforms appear to regulate the entry of sucrose into different utilization pathways. Invertase is of high importance in plants developmental processes, carbohydrate partitioning and in abiotic as well as biotic interaction. Multiple genes encode for above proteins responsible for Invertase action. With immobilized enzyme technology, Invertase demand has increased for its vital role in food industry. The above article provides a practical hand on introduction of many general considerations and corresponding strategies encountered during the course of isolating a specific protein from its initial biological source. With the advent of technology and modern gadgets, our knowledge for the subject has increased tremendously. Despite these accomplishments, some questions still need to be answered/such as why Invertase is so specific for its function, why nature has selected these isoforms and their role in Invertase evolution. Solution to these questions may also arise from multidisciplinary approaches. The knowledge gained will help to understand one of the most fundamental processes of carbohydrate metabolism and its utilization pathway according to an organism’s need. With biotechnological manipulation, Invertase can be transformed into a billion dollar solution for yield improvement in plants and crops, support for cancer patients and high quality anti-oxidant product.
Conflicts of interest All authors have none to declare.
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16. Ji X, Vander Ende W, Van Laaere A, Cheng S, Bunnett J. Structure, evolution and expression of the two invertase gene families of rice. J Mol Evol. 2005 May;60(5):615e634. 17. Li Z, PalmerW M. High invertase activity in tomato reproductive organs correlates with the enhanced sucrose import into and heat tolerance of young fruit. J Exp Bot. 2012 Feb;63(3):1155e1166. 18. Kaur H, Gupta AK, Kaur N, Sandhu JS. High invertase activity for a prolonged period in developing seeds/podwell of wild chick pea is detrimental to seed filling. Indian J Exp Biol. 2012 Oct;50(10):735e743. 19. Roitsch T, Balibrea ME, Hofman M, Proels R, Sinha AK. Extracellular invertase: key enzyme and PR protein. J Exp Bot. 2003;54(382). 20. Brandao RL, Etchebehere L, Queiroz CC, Tropia MJ. Evidence for involvement of Saccharomyces cerevisiae protein kinase C in glucose induction of HXT genes and derepression of SUC2. FEMS Yeast Res. 2002;2:93e102. 21. Lenore Neigeborn, Marian Carlson. Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations. Genetics. 1987 Feb;115(2):247e253. 22. Rioig P, Gozalbo D. Candida albicans UB13 and UB14 promoter regions confer differential regulation of invertase production to Saccharomyces cerevisiae in response to stress. Int Microbiol. 2002 Mar;5(1):33e36. 23. Kotwal SM, Shankar V. Immobilized invertase. Biotechnol Adv. 2009;27:311e322. 24. Young Linda, Cauvain Stanley P. Technology of Bread MakingIn: The Scientific Name for Baker’s Yeast Is Saccharomyces Cerevisiae. Berlin: Springer; 2007, ISBN 0-387-38563-0; 2007:79. 25. MAXINVERT. Invertase for food processing. 26. Edward F. The Health Benefits Of Invertase. http://www. globalhealingcenter.com/natural-health/invertase/ [Published on July 1, 2011, Last Updated on August 7, 2013]. 27. Muriel Mercier-Bonin, Christian Fonade. Enzyme recovery during gas/liquid two-phase flow micro filtration of enzyme/yeast mixtures. Biotechnol Bioeng. 2002 Dec;80(6):610e621. 28. Gjerde Douglas T, Fritzx James S. Ion Chromatography. Weinheim: Wiley-VCH; 2000, ISBN 3-527-29914-9; 2000.