Large Class Of Homopolysaccharides Biology Essay

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Dextranase definition and its uses

Dextran is a collective name given to a large class of homopolysaccharides composed of D-glucans with contiguous a-1, 6 glycosidic linkages (95%), with minor secondary linkages such as a-1, 2, a-1, 3 and a-1, 4 [74]. It is produced by microorganisms such as Leuconostoc mesenteroides, Streptococcus sp., Acetobacter capsulatus and Acetobacter viscus [44]. Dextrans are well soluble in water, have low toxicity, and relative inertness. These properties make dextrans effective water-soluble carriers for dyes, indicators, and reactive groups in a wide variety of applications. They are widely used in the pharmaceutical and biochemical fields. Dextrans of low molecular weight are used as an alternative to blood plasma. They are also used for clinical purposes such as drug delivery [82], and by cross-linking for the production of the chromatographic matrix Sephadex. They are also widely used as both anterograde and retrograde tracers in neurons [94]. On the other hand microbial synthesis of dextrans in damaged cane and beets or other products containing sucrose is a serious problem in sugar and food industry. Dextran is also a structural component of dental plaque which causes the development of dental caries [78], [85].

Dextranases are enzymes that cleave the a-1,6 glycosidic linkages of dextran to yield either glucose or isomaltose (exodextranases) or isomalto-oligosaccharides (endodextranases), and are only produced as extracellular enzymes by a small number of bacteria and fungi, including yeasts and perhaps some higher eukaryotes [44].

Enzymes in many groups can be classified as dextranases according to function: dextranhydrolases, glucodextranases, exoisomaltohydrolases, exoisomaltotriohydrases, and branched-dextran exo-1,2-alpha glucosidases. In particular the chemical reaction catalyzed is as follows:

(1,4-alpha-D-glucosyl)n + (1,4-alpha-D-glucosyl)m ↔ (1,4-alpha-D-glucosyl)n-1 + (1,6-alpha-D-glucosyl)m + 1

These enzymes belong to the family of glycosyltransferases, specifically the exosyltransferases. The systematic name of this enzyme class is: 1,4-alpha-D-glucan: 1,6-alphaD-glucan 6 alpha-d-glucosyltransferase. Other commonly used names include dextrin 6-glucosyltransferase and dextrin dextranase.

Many microorganisms are known to produce dextranase, including filamentous fungi belonging to the genera Penicillium, Aspergillus, Spicaria, Fusarium and Chaetomium, bacteria, e.g. Lactobacillus, Cellvibrio, Flavobacterium etc. The only yeasts reported to produce dextranases are members of the family Lipomycetaceae. Only Lipomyces kononenkoae [104] and Lipomyces starkeyi dextranases have been characterized [47].

Potential commercial uses of dextranases include:

The synthesis of potentially valuable oligosaccharides [30]

Potential mouthwash ingredients since isomaltose may be of significant importance for the prevention of dental caries [40], [41]

Clearance of dextran contamination in cane sugar processing [25]

Dual-stimuli-responsive drug release as in biodegradable polymer-structured hydrogels of gelatin and dextran [55]. Hydrogels are used for a wide range of biomaterials applications such as: contact lenses, drug delivery vehicles and tissue adhesives. Dextrans are polymers that mimic biological sugars found on tissue surfaces. The dextran hydrogel system with tunable mechanical and biochemical properties appears promising for applications in cell culture and tissue engineering [58]

Drug delivery device suitable for delivering drug to the colon [7], [8]. Brondsted et al. studied the application glutaraldehyde dextran as a capsule material for colon-specific drug delivery. The dextran capsules were challenged with a dextranase solution, simulating the arrival of the drug delivery to the colon, so they broke and the drug was released as a dose pump. The outcome highlights the dextran capsules as promising candidates for providing a colon-specific drug delivery

Also in site-specific drug delivery systems with the use of antibodies [69]

The improvement of brewing yeast strain for beer industry. Due to the rising demand for low-calorie beverages, including beer, recombinant strains of Saccharomyces cerevisiae have been produced by integrating LSD1 gene of Lipomyces starkeyi [101]. S. cerevisiae lacks the ability to produce extracellular depolymerising enzymes that can efficiently liberate fermentable sugar from abundant, polysaccharide rich substrates [75]. By introducing the gene mentioned above, adding an exogenous enzyme during beer fermentation to achieve starch hydrolysis and oligosaccharide reduction can be avoided

Carbohydrase activity produced can also be exploited in sensitive chromogenic bioassays for toxicity: a mycotoxin bioassay using the intracellular β-galactosidase activity of Kluyveromyces marxianus has been developed [20]

Fructose Dextran

Figure 1: Dextran formed from sucrose by Leuconostoc m. (reproduced from Sliman El-Sayed, 2007).

Classification of dextranase based on amino acid sequence

Dextranases are dextran-degrading enzymes that form a diverse group of carbohydrases and transferases. The more recent classification divides dextranases into two classes: endodextranases (a-1,6-glucan-6-glucnohydrolase; also referred to as dextranase) and exodextranases ( glucan-1,6-α-glycosidase; also referred to as dextran glucosidases). The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB) provides a system of classification depending on the type of reaction catalyzed and product specificity (Table 1). Contrary to that system, the Carbohydrate Active Enzymes (CAZy) database describes the families on terms of structural and mechanical features of these enzymes; enzymes with different substrate specificities are placed in the same family and those that hydrolyze the same substrate are sometimes placed in different families. In another classification system, Henrissat and Bairoch [33] have divided glucosylhydrolases and glycosyltransferases into five families on the basis of the similarities in the amino acid sequences (Table 2).

Table 1: The IUB-MB classification system

EC3.2.1.11

Dextranases

EC3.2.1.70

Glucan-1,6-glucosidases

EC3.2.1.94

Glucan-1,6-isomaltosidases

EC3.2.1.95

Dextran-1,6-isomaltotriosidases

EC3.2.1.115

Branched-dextran exo-1,2-glucosidases

Table 2: Classification of dextran hydrolysing enzymes, based on amino acid sequences.

Dextran-glucosidases

Families 13 & 15

Isomaltodextranase

Family 27

Isomaltotriosidase

Family 49

Endodextranases

Families 49 & 66 (no sequence similarities between the two families)

Aoki and Sakano (1997) came up with 4 families [2]. They isolated and sequenced the isopullunase gene (ipuA) from Aspergillus niger ATCC 9642. The gene shows significant amino acid similarity to the dextranase produced by Penicillium minioluteum (PEMDEX) and Arthrobacter sp. (ARTDEX). Since ASNIPU shows great similarity to PEMDEX and ARTDEX, they can be classified as Family 1. In the same fashion, the researchers compared the amino acid sequences of dextranases and dextran-hydrolising enzymes, including ASNIPU.

Lipomyces species and Lipomyces starkeyi

Lipomyces starkeyi and Lipomyces kononenkoae belong to the Lipomycetaceae family and are the only yeasts reported to produce dextranases. The first Lipomyces species was identified by Robert Starkeyi in 1946 during a study of nitrogen-fixing bacteria: it was then that he discovered L. starkeyi, a fat-producing, ascosporogenous soil yeast. The family Lipomycetaceae was proposed later, in 1952 by Lodder and Kreger von Rij. Lipomyces species can utilize starch as a sole source of carbon. Both species contain highly efficient amylolytic systems, permitting growth on starch with very high biomass yields [97].

The family Lipomycetaceae is known to utilize certain heterocyclic compounds, such as imidazole, pyrimidine, and pyrazine and their derivatives, as sole nitrogen sources [92]. Information on the genome organization and molecular genetics of this group of yeasts is very limited.

The ascosporogenous soil yeast L. starkeyi has been reported to produce commercially useful extracellular dextranase activity [97], [52], [53], and it can utilize a variety of other compounds, like hexoses, pentoses, alcohols and organic acids, as sole sources of carbon and energy [46]. The strains of L. starkeyi currently used are NCYC 1436, IGC 4047, ATCC 12659 and its de-repressed mutant ATCC 20825.

L. starkeyi dextranases

Commercial use of dextranase began in 1940s, mainly by producing low-molecular-weight clinical dextran. Therefore, industrially practical mixed culture fermentation of L. starkeyi and Leuconostoc mesenteroides was capable of producing controlled-size dextrans in order to satisfy clinical use, in which dextranase produced by L. starkeyi hydrolyzed the high molecular weight dextran produced by L. mesenteroides to a controlled size [46]. The enzyme production system of L. starkeyi needs an inducer. Dextran is its normal inducer but it is a relatively expensive carbon source for large-scale fermentations. Also, L. starkeyi is reported to have slow growth and difficulty of avoiding contamination from other microorganisms during growth. With that in mind D. W. Koenig and D. F. Day (1989) undertook to establish conditions which would minimize the cost of the inducer for producing an enzyme by using a de-repressed mutant of L. starkeyi ATCC 12659 grown on glucose. Thus the mutant ATCC 20825 is capable of hyperproducing dextranase at low pH to provide biologically contaminant-free supernatant liquid containing dextranase.

Lipomyces starkeyi (IGC 4047), when grown on dextran as a sole carbon source produced a dextranase able to hydrolyse blue dextran and Sephadex G-100. The molecular weight was 23kDa and the isoelectric point was 5.4 [97]. The dextranase of L. starkeyi (ATCC 20825) studied by Koening and Day (1988, 1989a, 1989b) was analysed by SDS-PAGE and produced four bands, of molecular weights 65 kDa, 68 kDa, 71 kDa, and 78 kDa. Millson and Evans (2007) have isolated extracellular dextranase of L. starkeyi NCYC 1436 and have found that for their strain the enzyme occurs as three molecular weight species and seven isoelectric forms [68].

L. starkeyi nutrients (YPDex / YPD)

The main ingredient in the chosen media is yeast extract. Yeast extract is a dried autolysate which facilitates rapid and luxuriant growth when used in various media or fermentation broth. It is a good source of amino-nitrogen and vitamins, especially the water-soluble B-complex vitamins. However, yeast extract is reported to enhance glucose metabolism to lipids, but inhibit lipolysis [18]. The metabolic pathway consists of converting glycerol into pyruvate or glucose and then hydrolysis by a phosphatase gives glycerol again. The disruption of this metabolic pathway, could account for the seemingly truncated numerous bands that SDS gives after prolonged storage of the yeast. Mycological peptone is incorporated in the media and discourages bacterial growth because of its acidity.

Environment that dextranases favour

Dextranase activity is affected by temperature, pH, metal ions and nutrients. According to Lin Chen et al (2007), dextranase activity is optimized between temperatures of 10oC and 60oC at pH of 6.0 [12]. In the particular study, the effect of pH on enzyme activity was determined by varying the pH between 3.5 and 8.5 under the temperature of 30oC. The pH of 3.4-4.5, 5.0-7.5, and 8.0-8.5 were maintained by sodium acetate buffer (20mM), citrate and phosphate buffer (20mM) and sodium phosphate buffer (20mM) respectively. The effects of metal ions (AlCl3, CaCl 2, CoCl2, CuSO4, FeCl3, KCl, MgCl2, NaCl, NiSO4, MnCl2 and ZnCl2) and SDS on dextranase activity were assayed by incubation of dextranase with 1mM metal ions or 1 mM SDS at pH 4.5 for 3h at 37oC, and then the enzyme activity of dextranase was determined.

Ravi Kiran Purama and Arun Goyal (2008) in a study for optimization of nutritional factors, estimated dextransucrase activity in the cell free extract of Leuconostoc mesenteroides. They analysed the regression coefficients and t-values of six ingredients: yeast extract, sucrose, intercept, K2HPO4, beef extract, peptone and Tween 80. Yeast extract, sucrose, beef extract, and K2HPO4 displayed a positive effect for enzyme production whereas, peptone and Tween 80 had a negative effect on enzyme production. The variables with confidence levels greater than 90% were considered as significant. Sucrose was significant at 99.99% confidence levels for dextransucrase production. K2HPO4 and yeast extract were found significant about 94% level for dextransucrase production. Beef extract was significant 91% for dextransucrase production. Peptone and Tween 80 were found insignificant with negative coeffficients for enzyme activities.

Methods used for enzyme activity measurement

Enzymatic activity is measured with the help of laboratory methods called enzyme assays. All enzyme assays measure either the consumption or production of product over time. Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined [11], [20].

Continuous assays:

Spectrophotometry in which you follow the course of the reaction by measuring a change in how much light the assay solution absorbs

Fluorimetric assay in which we make use of the difference in the fluorescence of substrate from product to measure enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light

Calorimetric assay in which the heat released or absorbed by chemical reactions is measured

Chemiluminescence in which the light emitted by some enzyme reactions is measured so as to detect product formation. The detection of horseradish peroxidase by ECL is a common method of detecting antibodies in western blotting

Discontinuous assays:

Radiometry in which the incorporation of radioactivity in substrates is measured

Chromatographic assays measuring product formation by separating the reaction mixture into its components. This is usually done by high-performance liquid chromatography (HPLC), but thin layer chromatography can also be used. Although this approach needs a lot of consumables its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag

Methods and assays for dextranase activity measurement

The large variability of available substrates makes it difficult to estimate the enzyme activity, because the reaction product is often an undefined mixture of sugar polymers. The existing assays try to compromise convenience, speed and accuracy [44]

Viscosimetric analysis was among the first to be used [31], [35], [36]. This method measured the amount of enzyme which reduced the specific viscosity of the dextran solution by half in 10min. and it is more suitable when dextranase hydrolyses the dextran molecule at random, producing long oligosaccharides.

Reducing-sugar assay or saccharogenic methods measure the rate of increase in reducing sugar as measured with the Somogyi assay, the 3,5-dinitrosalicylicacid method (DNS) [102], thiourea borax-modified O-toluidine colour reagent (35) and alkaline potassium ferricyanide solution (225). These methods test the presence of free carbonyl group (C=O). It is a simple method commonly used to analyze for reducing sugars produced from enzymatic hydrolysis of substrates such as starch and sucrose [67].The most common substrates applied are Dextran T2000,47 T-260,3 and T110 [54], [72]. A number of substances have been reported as interfering with DNS colour development and citrate is one of them. Acetate and citrate are reported to enhance colour development and the true antagonist in this reaction is the proton (H+) [96]. This method is based on the release of short coloured products from polymeric blue dextran and their selective colorimetric detection at 610-650nm after precipitation of the polymer. DNS colorimetric assays reported in literature are often modifications of the method of Webb and Spender-Martins (1983). E. F. Khalikova and N. G. Usanov (2001) developed a dextranase assay using an isoluble substrate, namely, Sephadex G-200 with Remazol Brilliant Blue dye [45]. The action pattern of dextranase was then, studied by means of exclusion chromatography. Overall, this assay was reported as convenient for quantitative dextranase detection, relatively independent of the enzyme source, and is proposed as an inexpensive alternative to the known procedures utilizing coloured substrates.

The dextranase substrates can be either dye-releasing or fluorogenic. The assay procedures based on these substrates are accurate, fast and can be recommended for dextranase-producing microbial screening and enzyme purification.

Other assay procedures worth mentioning include a spectrophotometric method with the use of Blue Dextran developed by Kauko K. Makinen and Illika K. Paunio (2004) who recommend it for column chromatography [62], and a method based on simple titration, developed by Eggleston and Gillian (2005) for easy use at the sugar cane factory [19].

Fluorometric assays are based on measuring the fluorescence of the samples and the results are often compared to a series of standards of Penicillium sp. A very sensitive fluorometric assay using amino-dextran-70 coupled with fluorescent dye BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid, succinimidyl ester) as the substrate was described by M. Zhou et al. (1998). The BODIPY FL dye-labelled dextranase substrate is an amine-containing dextran derivative that is labelled with the pH-insensitive, green fluorescent BODIPY FL dye, resulting in almost total quenching of the conjugate's fluorescence. The increase of the fluorescent degradation products of BODIPY FL dextran is proportional to the amount of dextranase activity [102].

A suspension of Sephadex in a buffer is supplemented with agar, sterilized, and poured in Petri dishes, and after the wells are filled with the test solution, they are left to incubate. The dextranase activity can be evaluated by the extent of halos around the holes due to the opalescence of Sephadex. Milson and Evans (2007), measured dextranase activity using SDS PAGE as described by Laemmli (1970), using both mini-gel and Protean II electrophoresis systems, and stained using Coomassie Blue [68], [56]. Molecular weight markers were used to construct a calibration curve, from which molecular weights of dextranase were determined. Native gel electrophoresis was performed, but the loading buffer and the gel lacked SDS and β-mercaptoethanol and the samples were not heated prior to loading on the gel. In the same study, dextranase activity was estimated in SDS gels, without extraction, by a plate modified from the method of Lawman and Bleiweis (1991) [57].

FL versus DNS assay method

The classic method (DNS) for measuring glycosidases through release of reducing activity is simple and inexpensive and, as cited above, has been modified in several studies so as to suit the researchers' needs. It may, however, have some pitfalls. The reaction taking place is the following:

aldheyde group -----------oxidation------------> carboxyl group

3,5-dinitrisalicylic acid ------------reduction-------------> 3-amino,5-nitrosalycilic acid

(Nam Sun Wang, University of Maryland)

The above reaction scheme shows that 1 mole of sugar reacts with 1 mole of 3,5-dinitrisalicylic acid. However, it is suspected that there are many side reactions, and the actual stoichiometry is more complicated than that previously described. Different reduced sugars yield different colour intensities; thus it is necessary to calibrate for each sugar. Apart from the oxidation, other side reactions may compete for the availability of 3,5-dinitrisalicylic acid. Consequently, the calibration curve may be affected and the intensity of the developed colour may be enhanced. Therefore, the method has low specificity and one must run blanks diligently if the colorimetric results are to be interpreted correctly and accurately [96].

Another obstacle to be dealt with when using DNS is non-linearity. One cause of non-linearity could be the common practice of diluting reaction products before quantification of reducing compounds and another is the insufficiency of substrates.

The fluorometric assay (FL), seems to gain ground in the most recent studies as faster and more accurate and it seems to leave space for modifications and combined use with other methods (see §1.3.1). A standard curve is constructed from Penicillium sp. and then compared with the one derived from Lipomyces starkeyi.

As described in the previous paragraph dextranase activity is estimated by the increase of the fluorescent products of dextran degradation. However, if too many fluoro are conjugated to the dextran molecule undesired may come up.

Molecular Probes TM seems to overcome this problem by removing as much of the free dye as possible and then assaying the fluorescent dextran by (TLC) to ensure that it is free of low molecular weight dyes. So, in general, FL seems to yield accurate curves. Millson and Evans (2007), used an assay of dextranase activity which was a variation on that reported by Zhou et al. (1998). In that study, fluorescence vs. dextranase activity produced a linear log [68], [102].

Purification of L. starkeyi dextranase

Dialysis tubing

Dialysis tubing is typically used for changing the buffering solution of a protein and is also a method for concentrating protein solutions by dialysis against a hygroscopic environment (e.g. PEG, Sephadex). The protein solution is contained within a membrane which permits solute exchange with a surrounding solution and whose pore size prevents the protein from escaping. Except for small volumes, this method is time-consuming [11].

Filtration - Ultrafiltration

Ultrafiltration (UF) is a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semi-permeable membrane. Suspended solids and solutes of high molecular weight are retained, while low molecular weight solutes pass through the membrane. UF is not fundamentally different from microfiltration or nanofiltration, except in terms of the size of the molecules it retains. [11], [77].

SDS-PAGE

Purification of Lipomyces starkeyi dextranase is carried out mainly by running a SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) analysis. The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non-disulfide-linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. SDS binds in a ratio of approximately 1.4g SDS per 1.0g protein. The size of the protein is directly related to the distance it migrates through the gel. Dextranase molecules migrate as bands based on size. Each band can be detected using stains such as Coomassie blue dye [77].

Modifications to the polypeptide backbone, such as N- or O- linked glycolylisation, however have a significant impact on the apparent molecular weight. Thus, the apparent molecular weight is not a true reflection of the mass of the polypeptide chain.

In most cases, SDS-polyacrylamide gel electrophoresis is carried out with a discontinuous buffer system in which the buffer in the reservoirs is of a different pH and ionic strength from the buffer used to cast the gel. After migrating through a stacking gel of high porosity the SDS-polypeptide complexes are deposited in a very thin zone (or stack) on the surface of the resolving gel. The discontinuous buffer system that is most widely used was originally devised by Orstein (1964) and Dvis (1964) [77]. The sample and the stacking gel contain Tris Cl (pH 6.8), the upper and lower buffer reservoirs contain Tris-glycine (pH 8.3) and the resolving gel contains Tris Cl (pH 8.8). All components of the system contain 0.1% SDS [56].

Precipitation methods of proteins

Precipitation is widely used in downstream processing of biological products, especially proteins. It serves to concentrate and fractionate the target product from various contaminants, as in biotechnology industry where precipitation helps to eliminate contaminants commonly contained in blood. The underlying mechanism of precipitation is to alter the solvation potential of the solvent and thus lower the solubility of the solute by addition of a reagent.

Precipitation is usually induced by any of the following methods [11]:

Salting out

Isoelectric point precipitation

Precipitation with organic solvents

Non-ionic hydrophilic polymers

Flocculation by polyelectrolytes

Polyvalent metallic ions

Salting out

This the most common type of precipitation. Normally a neutral salt is added, such as ammonium sulphate, which compresses the solvation layer and increases protein - protein interactions. As the salt concentration of a solution is increased, more of the bulk water is associated with the ions. Consequently, less water is available to partake in the solvation layer around the protein, which exposes hydrophobic interactions, aggregate and precipitate from solution.

Isoelectric point precipitation

The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein becomes zero. At a solution pH that is above the pΙ the surface of the protein is primarily negatively charged and therefore like-charged molecules will exhibit repulsive forces. At a solution pH that is below the pI, the surface of the protein is primarily positively charged and repulsion between proteins occurs. At the pI, the negative and positive charges cancel, repulsive electrostatic forces are reduced and the dispersive forces predominate, and will, therefore, cause aggregation and precipitation. The pI of most proteins lies in the pH range of 4-6. Mineral acids, such as hydrochloric and sulphuric acid are used as precipitants. The greatest disadvantage to isoelectric point precipitation is the irreversible denaturation caused by the mineral acids. For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins, rather than target protein.

Precipitation with organic solvents

Ethanol or methanol, if added to a solution may cause the proteins of the solution to precipitate. As the organic solvent gradually displaces water from the surface of the protein and binds it in layers around the organic solvent molecules, the solvation layer around the protein decreases. In that state, the protein can aggregate by attractive electrostatic and dipole forces. Parameters to consider are temperature (should be less than 0°C to avoid denaturation), pH and protein concentration of the solution. Miscible organic solvents decrease the dielectric constant of water, which in effect allows two proteins to come together. At the pI the relationship between the dielectric constant and protein solubility is given by:

log S = k/e2 + log S0

S0 is an extrapolated value of S, e is the dielectric constant of the mixture and k is a constant that relates to the dielectric constant of water [98].

Non- ionic hydrophilic polymers

Dextrans, polyethylene glycols and other polymers are used in precipitation of proteins due to their low flammability and are less likely to denature biomaterials compared to pI precipitation. These polymers attract water molecules away from the salvation layer around the protein, which enforces protein-protein interactions and induces precipitation. For the case of polyethylene glycol, the following equation models precipitation:

ln(S) +pS = X - αC

C is the polymer concentration, P is a protein-protein interaction coefficient, α is protein- polymer interaction coefficient and

X = ( μi - μi0 )RT

μ is the chemical potential of component I, R is the universal gas constant and T is the absolute temperature [98].

Flocculation by polyelectrolytes

Polyelectrolytes form extended networks between protein molecules in solution. These include alginate, carboxylmethylcellulose, polyacrylic acid, tannic acid and polyphosphates. The pH of the solution determines the effectiveness of these polyelectrolytes. Anionic polyelectrolytes are used at pH above the pI. Cationic polyelectrolytes are used at pH above the pI. The precipitate may dissolve back into the solution if an excess of polyelectrolytes is used.

Polyvalent metallic ions

Enzymes and nucleic acids are precipitated with the use of metal salts at low concentrations. Most frequently polyvalent metallic ions used are Ca+, Mg+, Mn+ or Fe+.

Precipitation reactors

Industrial scaled reactors that are used to precipitate large amounts of proteins, such as recombinant DNA polymerases from a solution include:

Batch reactors

The agent is slowly added to the protein solution under mixing, so the aggregating particles tend to be regular in shape. The protein particles are exposed to a wide range of shear stresses for a log period of time and become mechanically stable.

Tubular reactors

The precipitating reagent and the feed protein solution are contacted in an area of mixing and then added into enlongeted tubes where precipitation occurs. Plug flow is approached by the elements as they move along the tubes. The tubular reactor is inexpensive to be constructed but can become long and slow in case that aggregation of the particles occur slowly.

Continuous stirred tank reactors

CSTR reactors also known as vat or back mix reactors, run at steady state with a continuous flow of reactants and products in a well-mixed tank. It is a type of reactor mainly used in chemical engineering. A CSTR often refers to a mathematical model which is used to estimate the key unit operation variables when using a continuous agitated-tank reactor to reach a specified output. Perfect mixing is demanded.

Precipitation of L. starkeyi

The most common precipitation methods in the case of L. starkeyi cited in literature are:

Isoelectric focusing

Koening and Day (1988) used precast IsoGel agarose isoelectric focusing plates, pH 5.0-8.5. A standard mixture of proteins was applied in the lane next to each sample and the protein profile was quantified by densitometer scans. The enzyme activity in the gel was determined by slicing an unstained gel into 0.9 mm sections. Each section was placrd in a test tube with 1.0 ml 0.05 M citrate/phosphate (pH 5.5) buffer, allowed to elute overnight at 4oC and assayed for enzyme activity. This method separated the protein mixture into five isoelectric bands. All five forms were found to have dextranase activity and exhibited the same Km values.

Organic solvents

Polyethylene glycol precipitation is often used. Nishimura et al. (2002) used this method in an effort to prepare total DNA from L. starkeyi for taxonomy analysis. They added phenol solution (phenol: chloroform: isoamyl alcohol=25:24:1) to a test tube of Tris-SDS. The solution was stirred and centrifuged. Then the aqueous phase was transferred to a fresh polypropylene tube and TE buffer was added to the organic phase and centrifuged again. The second aqueous phase was combined with the first. A phenol extraction as above was repeated.

Preservation methods of proteins

It seems that innovation has found a realm of expansion when it comes to preserving proteins. The maintenance of protein stability through every phase of laboratory research, such as homogenization and freeze-drying, is critical for success Proteins need to be preserved and stabilized for several reasons. Running industrial processes at an elevated temperature, making mutations in normal meso proteins, and storing them for long periods are some of them. Stability is usually defined as the midpoint of the F ↔ U transition, in which F is the folded protein and U is unfolded. Stability can be determined by using denaturing agents. For instance, the equilibrium can be determined as function of the urea concentration. In that case, a mutation that increases the ureum concentration at which U and F are in 50 - 50 equilibrium is called stabilizing mutation. Calcium binding proteins, for example, normally are more stable at higher calcium concentrations. The stability of a protein can be improved by methods as diverse as mutagenesis, increased or decreased ion concentrations, inorganic solvents, using higher or lower protein concentrations, adding helper proteins or removing proteases from the medium [96], [73].

Freeze drying

Freeze drying prevents stability problems that may be posed during storage. While working with proteins in the lab, they should be kept on ice.  Since proteins are generally more stable at colder temperatures, maintenance at low temperatures even for short duration is recommended.  Typically, proteins are stored freeze-dried (lyophilized), frozen inappropriate buffers, or refrigerated at 4°C.  For short-term storage of proteins (hours to days), a standard laboratory refrigerator at 4°C is satisfactory providing the buffer used to solvate the protein provides all the necessary components necessary to stabilize the protein of interest.  These components can include reducing agents, hydrophobic additives, and protease inhibitors added to buffers.  Along with the use of gloves mentioned previously, protease inhibitors prevent denaturation due to contamination from these lytic agents potentially present in the protein source.  Additionally, antibacterial agents such as sodium azide can be added to inhibit bacterial growth.  Care must be taken, however, since antibacterial agents and protease inhibitors represent deliberate contamination of the sample.  Proper controls must be evaluated to insure no deleterious interaction with the protein of interest will occur [34].

Quick freezing

Quick freezing the sample at -20o C is used for long term storage (days to weeks). Addition of stabilizers such as glycerol helps prevent damage to the protein during freezing and thawing.  Typical concentrations for glycerol are 10% to 50%.  Again, care must be exercised since glycerol may negatively affect any chromatography methods subsequently used for sample handling or further purification after thawing of the sample.  Although stable while frozen, repeated thawing and freezing of a sample can lead to degradation and loss of activity.  During the freezing process proteins are exposed to extremes of salt concentration and pH.  Along with the use of stabilizers such as glycerol, rapid freezing of the protein solution limits the time the protein is exposed to these extreme conditions.  The rapid freezing process is typically performed by immersing the protein solution in a dry ice bath containing either acetone or ethanol followed by frozen storage at -20°C.  Along with rapid freezing, the thawing process should also be rapid for the same rationale as when freezing.  This can be accomplished by immersion in running lukewarm water.   Even when performed rapidly, repeated freezing and thawing of protein samples is considered ill advised.  It is advised to divide the original protein sample into several individual aliquots.  As sample is needed, a lone aliquot is thawed.  In this way the entire sample is not exposed to the perils of repeated phase changes.

Lyophilisation

Lyophilisation or desiccation is a freeze drying process in which the protein will eventually be reduced to a dehydrated powder for convenient storage in a laboratory freezer.  Although theoretically ideal, there are several hazards along the way.  As before, the protein must be rapidly frozen to avoid the pitfalls previously mentioned.  The protein must be dissolved in either deionized water or buffer containing lyophilisable salts.  If not, buffer salts will remain with the protein after the lyophilisation process is complete.  After the protein solution is frozen, it is attached to a lyophiliser where the frozen solution sublimes leaving the protein behind, usually as a fluffy white powder.  A major problem that occurs quite frequently with lyophilisation is the inability to redissolve the lyophilized protein, which indicates denaturation during the process.  Prior to lyophilizing the entire protein sample, it's advantageous to lyophilize a small aliquot to determine if the protein can be properly recovered.

Sorbic acid

Sorbic acid or 2,4-hexadienoic acid, is a natural organic compound used as food preservative. Its chemical formula is C6H8O2. It was first isolated from the unripe berries of the Rowan (Sorbus aucuparia), hence its name.Sorbic acid and its mineral salts, such as sodium sorbate and calcium sorbate, are antimicrobial agents often used as preservatives in food and drinks to prevent the growth of mold, yeast and fungi. In general the salts are preferred over the acid form because they are more soluble in water. The optimal pH for the antimicrobial activity is below pH 6.5 and sorbates are generally used at concentrations of 0.025% to 0.10%. Adding sorbate salts to food will however raise the pH of the food slightly so the pH may need to be adjusted to assure safety.

Protein crystals

The crystallization of proteins makes them ready for use in dry or slurry formulations. Protein crystals are encapsulated within a matrix comprising a polymeric carrier to form a composition. This enhances preservation of the native biologically active tertiary structure of the proteins and create a reservoir which can slowly release active protein where and when it is needed.

Glutaraldehyde

Glutaraldehyde is also used to prevent leakage of proteins immobilized on Sepharose without destroying their biological functions [54]. In particular, glutaraldehyde was used by Kowal and Parsons (1980) at concentrations ranging from 0.015 to 0.25% (v/v) to crosslink proteins, which had been coupled to Sepharose by conventional methods. As a result, glutaraldehyde crosslinking reduced immuno-globulin G leakage from Sepharose-immunoadsorbents to undetectable levels without noticeably affecting antigen-binding activity. It also reduced leakage of lactoperoxidase from solid-phase lactoperoxidase with only a moderate reduction of enzymatic activity.

Preservation

Safe storage is of vital importance for efficient research in mycology. Repeated subculturing, which is frequently used as a routine method for preservation of fungi and yeasts, is not very practical for storing large numbers of cultures. In order to eliminate these disadvantages, various methods have been developed. These include: lyophilisation (freeze-drying), storage in liquid nitrogen (LN) and L-drying. Also, even though many yeast strains may be stored at temperatures between 4oC and 12oC after sub-culturing for intervals of 6 to 8 months, the teleomorphic members of ascomycetous yeasts, like L. starkeyi, lose the ability to sporulate on successive cultivation on laboratory media [92].

Freeze drying

Freeze drying is a generally accepted method for yeast storage, having the advantages of conferring longevity and genetic stability, as well as being suitable for easy worldwide postal distribution of the cultures in glass ampules. However, preservation by freeze-drying tends to be much more labour intensive than storage in liquid nitrogen and requires a higher level of skill to produce an acceptable product. Strain viabilities are generally low, typically being between 1 and 30%, as compared to >30% for those of yeast preserved frozen in liquid nitrogen. There are also several yeast genera, including Lipomyces, Leucosporidium, and Rhodosporidium which have particularly low survival levels and frequently cannot be successfully freeze-dried by the standard method [5].

Liquid nitrogen

Liquid nitrogen seems to surpass all others in preserving genomic and phenotypic features. It is a safe and reliable method for long-term maintainance of most yeast species, especially those not amendable to freeze-drying. The cryopreservation process includes freezing and thawing and the protocol in these procedures plays an important part. The boiling point of nitrogen (-196oC) is suitable for storage of a variety of cells (bacteria, yeasts and other fungi, tissue cultures, viruses etc.) but temperatures of -60oC to -135oC are the most suitable for yeasts [92]. The cryoprotective additives (CPAs) used in the frozen storage of microorganisms (viruses, bacteria, fungi, algae, and protozoa) include a variety of simple and more complex chemical compounds, but only a few of them have been used widely and with satisfactory results: these include dimethylsulfoxide (Me2SO), glycerol, blood serum or serum albumin, skimmed milk, peptone, yeast extract, saccharose, glucose, methanol, polyvinylpyrrolidone (PVP), sorbitol, and malt extract. Glycerol ensures high rates of survival as well as genetic stability [92].

Liquid drying

Liquid drying (L- Drying) involves vacuum-drying of samples from the liquid state without freezing. As a long-term preservation method, it is more effective than freeze-drying, but it demands specialized equipment. (UNESCO) Stability of L-dried cultures during storage is of vital importance. A high level of residual moisture content or exposure to oxygen have detrimental effects on the dried product. Liquid dried material is hygroscopic and its exposure to moisture during storage may destabilize the product. The higher the storage temperature, the faster the product will degrade. Thus, low temperatures ensure long shelf life. The unsealed L-dried ampoules can safely be stored for several years at about -30oC. The use of consumable solid desiccant materials for organic liquid dehydration is very current in refinery or chemistry processes, especially when the water concentration is very low (<1000 ppm). In spite of its common use, the kinetics of liquid drying by solid desiccants is not well known, and scale-up of industrial drying processes using such materials is always problematic.

Fermentation

Fermentation is the process of deriving energy from the oxidation of organic compounds, such as carbohydrates, using an endogenous electron acceptor, which is usually an organic compound. Fermentation does not necessarily have to be carried out in an anaerobic environment, however. For example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to oxidative phosphorylation, as long as sugars are readily available for consumption. Sugars are the most common substrate of fermentation, and typical examples of fermentation products are ethanol, lactic acid, and hydrogen. However, more exotic compounds can be produced by fermentation, such as butyric acid and acetone. Yeast carries out fermentation in the production of ethanol in beers, wines and other alcoholic drinks, along with the production of large quantities of carbon dioxide . Therefore, sugars are metabolized to volatile fatty acids (VFAs). Glycolysis (via the traditional Embden-Meyerhoff pathway) is the enzymatic breakdown of glucose into pyruvate. This process requires ADP and NAD+ as co-factors , and produces ATP and NADH inside the cell. A cell can always find a way to hydrolyze ATP back into ADP when needed (no oxidation/reduction is involved in ATP hydrolysis), but NAD+ is a more difficult problem, as its formation involves oxidation of NADH. Under aerobic conditions, the electrons from NADH can ultimately be passed to oxygen, but under anaerobic conditions, another electron acceptor is needed. (bioewsonline) Although the final step of fermentation (conversion of pyruvate to fermentation end-products) does not produce energy, it is critical for an anaerobic cell since it regenerates nicotinamide adenine dinucleotide (NAD+), which is required for glycolysis. This is important for normal cellular function, as glycolysis is the only source of ATP in anaerobic conditions [11].

Fermentation Procedure

The proper procedure for a batch fermentation is first to inoculate a small flask of nutrient broth with a pure culture from a Petri dish, a culture tube (containing liquid nutrient), or a slant tube (containing solid gel). The inoculated flask is constantly agitated in a temperature controlled flask shaker. A small amount of the culture in the original flask is pipetted out during the exponential growth phase, or log phase, and is used to inoculate the next flask. This process is repeated a few times to ensure that the culture is acclimated before it is employed to study the fermentation kinetics. A similar process of repeated inoculation is carried out in the fermentation industry to build up enough inoculum needed to seed a larger fermentor. To reduce the shock resulting from a drastic change in the growth environment, the composition of the media used in preparing the inoculum should optimally be identical as that used in the main process. Fermentation produces two by-products: ethanol and CO2. Production of ethanol is measured but often the CO2 released is easier to measure as an indicator of fermentation.

The common procedure in designing lab fermentation is as follows:

Nutrient (YPDex or a mixed stock of minerals and proteins) is autoclaved

Cotton gauze plugs are placed on the flasks and then they are autoclaved

Flasks are then cooled to room temperature

Sterile nutrient is poured into the flasks aseptically

Yeast suspension is transferred to the flask

The inoculum is left to incubate from 2 to 24 hours

Glucose concentration is measured by DNS agent, sucrose and ethanol

Industrial scale fermentation

Somewhat paradoxically, fermenter culture in industrial capacity often refers to highly oxygenated and aerobic growth conditions, whereas fermentation in the biochemical context is a strictly anaerobic process. Most practical industrial fermentation processes are based on complex media because of the cost and the choice of the nutrients and the ease of nutrient preparation. For example, complex media for yeast fermentation can be easily prepared in a lab by following the same recipe as that used in the YPG agar, minus the agar: 5g/l yeast extract, 10g/l Peptone, and 5g/l glucose. However, the use of complex media is discouraged in the fundamental studies of fermentation kinetics because of the possibility of variations in the nutrient composition from run to run. For example, the exact content of a yeast extract preparation is not known, and its nutritional quality may vary from batch to batch. On the other hand, a defined medium can be reproduced time after time to ensure the reproducibility of biochemical experiments. The disadvantage of a defined medium is that there is always the possibility of missing some important growth factors. The formulation of a defined medium is often a tedious process of trial and error. However, a well formulated defined medium can support the healthy growth and maintenance of cells as effectively as, or sometimes superior to, a complex one.

Strain selection

There are thousands of different yeast strains, each with own genetic and metabolic characteristics. These specificities will impact the property and activity of the end product. The yeast culture is stored in cryopreserver, in liquid nitrogen. The identity of the culture may be confirmed using genetic and biochemical techniques, such as DNA fingerprinting with PCR or RFPL.

Bioconversion

At Grenaa, Denmark, in 2007, Dr Ildar Nisamedtinov at Lallemand's International Selenium Yeast seminar presented that the first step of yeast culture consists of inoculating an extract of the mother culture into a small flask (5mL) of yeast culture medium: the starter culture. Then the yeast culture is progressively transferred to larger flasks until being finally incubated into the industrial fermentor. Nutrients are added incrementally into the fermentor, according to the yeast culture density, to optimize yeast growth and conversion. The industrial culture medium contains all the nutrients for optimal yeast growth: molasses, nitrogen, phosphate, vitamins and minerals. Their concentrations are continuously monitored, as well as the culture physics-chemical parameters.

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