Dextran is a corporate name given to a big category of homopolysaccharides composed of D-glucans with immediate a-1,6 glycosidic linkages (95%), with minor secondary linkages such as a-1,2, a-1,3, and a-1,4. It is produced by microorganisms such as Leuconostoc mesenteroides, Streptococcus sp., Acetobacter capsulatus, and Acetobacter viscosus.
Dextrans are soluble in water, have low toxicity, and comparative inertness. These properties make dextrans effective water-soluble carriers for dyes, indicators, and reactive groups in a broad assortment 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 and, by cross-linking, for the production of the chromatographic matrix Sephadex. They are also widely used as both anterograde and retrograde tracers in nerve cells. On the other hand, microbial synthesis of dextrans in damaged cane and Beta vulgaris or other products containing sucrose is a serious problem in the sugar and food industry. Dextran is also a structural component of dental plaque, which causes the development of dental cavities.
Dextranases are enzymes that cleave the a-1,6 glycosidic linkages of dextran to give 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 possibly some higher eukaryotes.
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 category is 1,4-alpha-D-glucan:1,6-alpha-D-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 and Lipomyces starkeyi dextranases have been characterized.
Dual-stimuli-responsive drug release, as in biodegradable polymer-structured hydrogels of gelatin and dextran. Hydrogels are used for a broad 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 belongingss appears assuring for applications in cell civilization and tissue technology.
Drug bringing device suitable for presenting drug to the colon. Brondsted et Al. studied the application glutaraldehyde dextran as a capsule stuff for colon-specific drug bringing. The dextran capsules were challenged with a dextranase solution, imitating the reaching of the drug bringing to the colon, so they broke and the drug was released as a dose pump. The result highlights the dextran capsules as promising campaigners for supplying a colon-specific drug bringing.
The betterment of brewing barm strain for beer industry. Due to the lifting demand for low-calorie drinks, including beer, recombinant strains of Saccharomyces cerevisiae have been produced by incorporating LSD1 cistron of Lipomyces starkeyi. Scerevisiae lacks the ability to bring forth extracellular depolymerising enzymes that can expeditiously emancipate fermentable sugar from abundant, polysaccharide rich substrates. By presenting the cistron mentioned above, adding an exogenic enzyme during beer agitation to accomplish amylum hydrolysis and oligosaccharide decrease can be avoided.
Categorization of dextranase based on amino acid sequence
Dextranases are dextran-degrading enzymes that form a diverse group of carbohydrases and transferases. The more recent categorization divides dextranases into two categories: endodextranases (α-1,6-glucan-6-glucosidase; 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 categorization depending on the type of reaction catalyzed and merchandise specificity.
Contrary to that system, the Carbohydrate Active Enzymes (CAZy) database describes the families on the basis of structural and mechanistic characteristics 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 categorization system, Henrissat and Bairoch have divided glucosylhydrolases and glycosyltransferases into five families based on the similarities in the amino acid sequences.
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 use starch as a sole source of carbon. Both species contain highly efficient amylolytic systems, allowing growth on starch with very high biomass outputs.
The family Lipomycetaceae is known to use certain heterocyclic compounds, such as imidazole, pyrimidine, and pyrazine and their derivatives, as sole nitrogen sources. 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 and it can use a variety of other compounds, like hexoses, pentoses, alcohols, and organic acids, as sole sources of carbon and energy. 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 usage of dextranase began in the 1940s, primarily 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.
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. Additionally, L. starkeyi is reported to have slow growth and difficulty avoiding contamination from other microorganisms during growth.
With that in mind, D. W. Koenig and D. F. Day (1989) undertook to set up conditions which would minimise the cost of the inducer for bring forthing an enzyme by utilizing a de-repressed mutation of L. starkeyi ATCC 12659 grown on glucose. Thus the mutant ATCC 20825 is capable of hyperproducing dextranase at low pH to supply biologically contaminant-free supernatant liquid incorporating dextranase.
Lipomyces starkeyi (IGC 4047), when grown on dextran as an exclusive carbon source, produced a dextranase able to hydrolyze bluish dextran and Sephadex G-100. The molecular weight was 23 kDa, and the isoelectric point was 5.4. The dextranase of L. starkeyi (ATCC 20825) studied by Koenig and Day (1988, 1989a, 1989b) was analyzed by SDS-PAGE and produced four sets 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 signifiers.
L. starkeyi foods (YPDex/YPD)
The main ingredient in the chosen media is yeast extract. Yeast extract is a dried autolysate that facilitates rapid and elaborate growth when used in various media or agitation broth. It is a good source of amino-nitrogen and vitamins, particularly the water-soluble B-complex vitamins. However, yeast extract is reported to enhance glucose metabolism to lipids but inhibit lipolysis. The metabolic pathway consists of converting glycerin into pyruvate or glucose and then hydrolysis by a phosphatase gives glycerin again. The breakdown of this metabolic pathway could account for the apparently truncated numerous sets that SDS gives after prolonged storage of the yeast. Mycological peptone is incorporated into the media and discourages bacterial growth because of its acidity.
The environment that dextranases favor
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 10°C and 60°C at a pH of 6.0 . In the particular study, the effect of pH on enzyme activity was determined by changing the pH between 3.5 and 8.5 under the temperature of 30°C.
The pH of 3.4-4.5, 5.0-7.5, and 8.0-8.5 were maintained by Na acetate buffer (20 mM), citrate and phosphate buffer (20 mM), and sodium phosphate buffer (20 mM) respectively. The effects of metal ions (AlCl3, CaCl2, CoCl2, CuSO4, FeCl3, KCl, MgCl2, NaCl, NiSO4, MnCl2, and ZnCl2) and SDS on dextranase activity were assayed by incubation of dextranase with 1 mM metal ions or 1 mM SDS at pH 4.5 for 3 h at 37°C, and then the enzyme activity of dextranase was determined.
Ravi Kiran Purama and Arun Goyal (2008) in a survey for optimization of nutritional factors estimated dextransucrase activity in the cell-free extract of Leuconostoc mesenteroides. They analyzed 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 important. Sucrose was important at 99.99 % assurance degrees for dextransucrase production. K2HPO4 and yeast infusion were found important about 94 % degree for dextransucrase production. Beef infusion was important 91 % for dextransucrase production. Peptone and Tween 80 were found undistinguished with negative coeffficients for enzyme activities.
Methods used for enzyme activity measuring
Enzymatic activity is measured with the aid of research lab methods called enzyme checks. All enzyme checks step either the ingestion or production of merchandise over clip. Enzyme checks can be split into two groups harmonizing to their sampling method: uninterrupted checks, where the check gives a uninterrupted reading of activity, and discontinuous checks, where samples are taken, the reaction stopped and so the concentration of substrates/products determined.
Spectrophotometry is used to follow the course of the reaction by measuring a change in how much light the assay solution absorbs. Fluorimetric checks are used to make use of the difference in the fluorescence of the substrate from the product to measure enzyme reaction. These checks are generally much more sensitive than spectrophotometric checks, but can suffer from intervention caused by drosses and the instability of many fluorescent compounds when exposed to radiation.
Calorimetric checks measure the heat released or absorbed by chemical reactions. Chemiluminescence is used to measure the radiation emitted by some enzyme reactions to detect product formation. The detection of horseradish peroxidase by ECL is a common method of observing antibodies in western blotting.
Radiometry is used to measure the incorporation of radiation in substrates. Chromatographic assays measure product formation by dividing the reaction mixture into its constituents. 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 labeling the substrates/products with a radioactive or fluorescent label.
Methods and checks for dextranase activity measuring:
The large variability of available substrates makes it difficult to gauge enzyme activity because the reaction product is often an ambiguous mixture of sugar polymers. The existing checks try to compromise convenience, speed, and accuracy.
Viscometric analysis was among the first to be used. This method measured the amount of enzyme that reduced the specific viscosity of the dextran solution by half in 10 minutes and is more suitable when dextranase hydrolyses the dextran molecule at random, producing long oligosaccharides.
Reducing-sugar checks or saccharogenic methods measure the rate of addition in reducing sugar as measured with the Somogyi check, the 3,5-dinitrosalicylic acid method (DNS), thiourea borax-modified O-toluidine coloring material reagent (35), and alkaline K ferricyanide solution (225). These methods test the presence of free carbonyl groups (C=O). It is a simple method normally used to analyze for reducing sugars produced from enzymatic hydrolysis of substrates such as starch and sucrose. The most common substrates applied are Dextran T2000,47 T-260,3 and T110.
A number of substances have been reported as interfering with DNS coloring material development, and citrate is one of them. Acetate and citrate are reported to enhance coloring material development, and the true adversary in this reaction is the proton (H+). This method is based on the release of short colored products from polymeric blue dextran and their selective colorimetric sensing at 610-650 nm after precipitation of the polymer.
DNS colorimetric checks reported in the literature are often alterations of the method of Webb and Spender-Martins (1983). E. F. Khalikova and N. G. Usanov (2001) developed a dextranase check utilizing an insoluble substrate, viz., Sephadex G-200 with Remazol Brilliant Blue dye.
The action form of dextranase was then studied by means of exclusion chromatography. Overall, this check was reported as convenient for quantitative dextranase sensing, relatively independent of the enzyme source, and is proposed as an inexpensive option to the known procedures using colored substrates.
The dextranase substrates can be either dye-releasing or fluorogenic. The check procedures based on these substrates are accurate, fast, and can be recommended for dextranase-producing microbial showing and enzyme purification.
Other check processs deserving adverting include a spectrophotometric method with the usage of Blue Dextran developed by Kauko K. Makinen and Illika K. Paunio ( 2004 ) who recommend it for column chromatography , and a method based on simple titration, developed by Eggleston and Gillian ( 2005 ) for easy usage at the sugar cane mill .
Fluorometric checks are based on mensurating the fluorescence of the samples and the consequences are frequently compared to a series of criterions of Penicillium sp. A really sensitive fluorometric check utilizing 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-labeled dextranase substrate is an amine-containing dextran-derived function that is labeled with the pH-insensitive, green fluorescent BODIPY FL dye, resulting in almost complete extinction of the conjugate’s fluorescence. The addition of the fluorescent degradation products of BODIPY FL dextran is relative to the amount of dextranase activity.
A suspension of Sephadex in a buffer is supplemented with agar, sterilized, and poured into Petri dishes. After the wells are filled with the test solution, they are left to incubate. The dextranase activity can be evaluated by the extent of the aura around the holes due to the iridescence 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. Molecular weight markers were used to build a standardization 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.
Slightly paradoxically, fermenter culture in industrial capacity often refers to highly oxygenated and aerobic growth conditions, whereas agitation in the biochemical context is a purely anaerobic procedure. Most practical industrial agitation procedures are based on complex media because of the cost and the choice of nutrients and the ease of nutrient preparation. For example, complex media for yeast agitation can be easily prepared in a lab by following the same formula 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 key studies of agitation dynamics because of the possibility of fluctuations 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 change from batch to batch. On the other hand, a defined medium can be reproduced time after time to ensure the replicability of biochemical experiments.
The disadvantage of a defined medium is that there is always the possibility of losing some important growth factors. The preparation 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 its own genetic and metabolic characteristics. These specificities will affect the property and activity of the final product. The yeast culture is stored in cryopreserved, 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 parent culture into a small flask (5 mL) of yeast culture medium: the starter culture. Then, the yeast culture is increasingly transferred to larger flasks until finally incubated in 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 foods for optimum yeast growth: molasses, N, phosphate, vitamins, and minerals. Their concentrations are continuously monitored, every bit good as the civilization physics-chemical parametric quantities.