How is isomerase used

Sucrose and raffinose among pure carbon sources and corn cob and wheat husk among crude agro residues were found to yield high enzyme titers. Potassium nitrate among pure nitrogen sources and soy residues among crude sources gave maximum production. Quantitative effect of carbon, nitrogen, and inducer on GI was also determined. Plackett-Burman design was used to study the effect of different medium ingredients. Sucrose and xylose as carbon sources and peptone and soy residues as nitrogen sources proved to be beneficial for GI production.

GI EC 5. It is an equilibrium mixture of fructose and glucose. Fructose, also known as fruit sugar, is the sweetest natural sugar and is found in fruits, vegetables and honey.

HFCS has wide applications in pharmaceutical and food industries. It is added in medicated syrups, beverages, baking, canning and confectionary items as a sweetening agent [ 1 ]. Ge et al. Glucose isomerase from Streptomyces is a tetramer composed of four identical polypeptide chains of 43, daltons each [ 1 , 3 ].

Marshall and Kooi in [ 4 ] for the first time reported the production of glucose isomerase from Pseudomonas hydrophila. Since then many mesophilic, thermophilic, a few psychrophilic, aerobic, and anaerobic organisms have been reported to produce GI.

Streptomycetes have been the organisms of choice for GI production by numerous researchers. The thrust areas to work upon for medium formulation are independence of xylose and cobalt ions. Xylose works as an inducer for GI in majority of the cases excluding a few like Actinoplanes missouriensis [ 5 ]. Xylose-independent GI producer or an agro-residue rich in xylose which can substitute for pure and expensive xylose can be useful in making the production technology economic.

There are reports on Bacillus being used for GI production on xylan containing media also [ 6 ]. Most of the researchers have suggested the requirement of cobalt and magnesium in the production medium as cobalt provides thermostability and magnesium is required for optimum activity of the enzyme. The deleterious effects of cobalt on human health forces the elimination of cobalt from production medium [ 7 — 9 ]. Employing a suitable production medium for fermentative production of GI is a very crucial requirement of present time in order to develop an economically viable technology.

India being an agrobased country is a rich producer of agroresidues. These residues can be utilized for microbial production processes.

A collection of 75 actinomycetal isolates was developed from compost pit samples. The isolates exhibited cultural and morphological diversity [ 10 ]. All the isolates were screened for GI activity and the highest producer of the enzyme Streptomyces sp. SB-P1 was selected for medium optimisation studies for industrial purpose [ 11 ]. GI production is known to be enhanced by the presence of its inducer xylose.

The production is also influenced by the presence of magnesium and cobalt ions in some cases. Investigators have used various medium combinations for GI production according to the microorganism employed. The selected isolate Streptomyces sp. SB-P1 was grown in 11 different media combinations to screen the best combination supporting maximum GI production and biomass accumulation.

The medium combinations were Medium No. The centrifuged supernatant of fermented broth was used as crude enzyme extract for determination of GI activity by assay method described by Chen et al.

The growth response on different medium combinations was measured by estimating the dry weight. The biomass was separated from the nutrient medium by centrifugation and washed with distilled water. The highest GI production and highest biomass accumulation were achieved on different medium combinations.

These two media were used to screen pure and crude sources of carbon and nitrogen. The quantitative effect of best carbon, nitrogen source, and inducer xylose was also studied.

A set of 14 medium components were screened by Plackett Burman design. The ingredients studied by Placket Burman design were carbon sources glucose, sucrose, wheat husk, orange bagasse, peanut shell , nitrogen sources tryptone, peptone, yeast extract, soy flour, potassium nitrate , minerals magnesium sulphate, cobalt chloride , inducer xylose , and a buffering agent di-potassium hydrogen phosphate. The high and low concentrations of medium ingredients used are given in Table 1 and design of experiment is given in Table 2.

Various medium combinations were tried for the production of GI. The medium combinations containing xylose were found to produce good amount of enzyme as compared to the media without xylose. Media containing xylose as sole source of carbon exhibited poor growth but high enzyme yield. As also reported by Hasal et al. The highest enzyme production was observed in Medium No.

High biomass production may be due to the high protein content yeast extract in the medium as also reported by Givry and Duchiron [ 22 ]. The enzyme activity we observed for our isolate Streptomyces sp.

SB-P1 1. Similar case was observed in Medium No. The results of above comparison are graphically represented in Figure 1. Sucrose gave maximum enzymatic yield in both media Medium No. Among pure carbon sources enzyme yield in flasks containing xylose and lactose were comparable.

The results of the effect of carbon sources checked in Medium No. Lactose also gave better enzyme yield which opens a way to use dairy industry wastes especially whey as a prospective crude and inexpensive carbon source. Hasal et al. They found all the sources except lactose suppressed the yield of GI. Lactose is also reported as an inducer for the enzyme produced by a recombinant E.

Among the crude sources wheat husk and corn cob yielded high enzyme titres in Medium No. Wheat husk is found to be common in both cases which contain good amount of xylose and xylan. Earlier investigators [ 13 , 17 , 24 ] have also reported high enzyme yield and good growth response of Streptomyces in presence of wheat bran. Straw hemicellulose also works as an inducer that can replace xylose and increase the GI yield [ 16 ].

Hemicelluloses containing substance is useful as a medium ingredient because of its low cost than xylan and xylose. Alkali-treated hemicelluloses are also used by Chen and Anderson, [ 25 ] to produce glucose isomerase. Various inorganic and organic nitrogen sources were tested for glucose isomerase production.

Among the pure nitrogen sources potassium nitrate was found to be more suitable for high yield. There are reports suggesting the inability of inorganic salts in giving higher yields of glucose isomerase but we observed a remarkable increase in the enzyme production in the flasks containing potassium nitrate.

Soy flour was pointed out as the best source among the crude components tested. The higher yields with agrobased and industrial residue are a good sign for its usage in industrial production media. Corn steep liquor is reported to increase the enzyme yield by Hasal et al. There are reports on ammonium salts been promoting GI production but soy residues are less preferred [ 1 ].

Givry and Duchiron [ 22 ] found Lactobacillus bifermentans could produce high amount of glucose isomerase only in presence of organic nitrogen sources like peptone, tryptone or yeast extract.

This is the first report to our knowledge stating high production of GI in presence of soy residues. This is again a beneficial result in the context that Madhya Pradesh, India is one of the major producers of soybean and its products so lots of soy residue are also generated.

The results of the effect of nitrogen sources checked in Medium No. Increasing concentrations of sucrose also increased the enzyme productivity but till a certain limit beyond which there was no substantial increase.

There was an increase in the productivity of glucose isomerase from 0. This must be due to the highest sucrose concentration reached which the organism can bear.

Sucrose is reported to give high yields of glucose isomerase by earlier researchers also. The results of quantitative effect of sucrose are shown in Figure 6 a. Production of enzyme increased from 4. The molecular mass of the enzyme was estimated by gel filtration on Sephacryl to be approximately kDa data not shown , and it consists of four identical subunits with a molecular mass of These values are slightly different from those of the glucose isomerases from Streptomyces sp.

EC10 kDa with 4 identical subunits of 42 kDa 2 and from Streptomyces sp. SK strain kDa with four kDa subunits 4. SDS-polyacrylamide gel electrophoresis of purified glucose isomerase.

CH7 1. Its optimal pH is 7. Regarding substrate specificity, under standard assay conditions the Michealis-Menten constant K m values of the enzyme for glucose and xylose were found to be The lower K m values for xylose, as compared to glucose, indicates that this glucose isomerase has a preference for xylose as a substrate over glucose, which is in agreement with glucose isomerases from other microorganisms.

However, its K m values for both substrates were found to be lower than those of Bacillus sp. Tx-3 K m values of and mM for glucose and xylose, respectively whereas its V max values are higher V max values of 1. Its K m value for glucose is lower than that of Streptomyces sp. PLC mM , but for xylose it is slightly higher 35 mM Due to its preference for xylose and its lower K m value for glucose when compared to those of the other strains mentioned above, glucose isomerase from CH7 has the potential for industrial applications such as high-fructose syrup production and xylose isomerization for substrate preparation for ethanol fermentation by S.

However, the enzyme from Streptomyces sp. CH7 was found to be capable of producing glucose xylose isomerase efficiently when grown in medium containing corn husks, simply prepared as milled particles, as a carbon source. This cheap and abundantly available carbon source will result in low enzyme production costs.

Therefore, it has the potential for industrial applications, especially for high-fructose syrup production and for substrate preparation for bioethanol fermentation from xylose a major component in hemicellulosic hydrolysates by Saccharomyces cerevisiae. This work was supported in part by CU. National Center for Biotechnology Information , U. Journal List Braz J Microbiol v.

Braz J Microbiol. Published online Jun 1. Author information Article notes Copyright and License information Disclaimer. Received Nov 3; Accepted Jun 7. All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License.

This article has been cited by other articles in PMC. Abstract Streptomyces sp. Keywords: agricultural residues, glucose xylose isomerase, production, purification, Streptomyces. Protein content Protein content was determined by the Lowry method 17 using bovine serum albumin as a standard. Enzyme assays D-Glucose isomerase was assayed in a reaction mixture that contained 0. Purification of glucose isomerase The mycelial extract referred to as the crude enzyme from Streptomyces sp. Estimation of molecular mass The apparent molecular mass of the purified enzyme in native form was determined by gel filtration on Sephacryl S using ferritin kDa , catalase kDa and globulin kDa as molecular mass standards.

Open in a separate window. Figure 1. Figure 2. Figure 3. Purification, molecular characteristics and properties of glucose isomerase Glucose isomerase from Streptomyces sp. Table 2 Summary of purification of glucose isomerase from Streptomyces sp. Figure 4. Figure 5. Figure 6. Metal Relative activity fold No metal added 1. Bangrak P. Continuous ethanol production using immobilized yeast cells entrapped in loofa-reinforced alginate carriers. Belfaquih N.

Enzyme Microl. Bhosale S. Molecular and industrial aspects of glucose isomerase. Borgi M. Glucose isomerase of the Streptomyces sp. SK strain : purification, sequence analysis and implication of alanine residue in the enzyme thermostability and acidotolerance.

Enrichment of phenylalanine ammonia lyase of Rhodotolura yeast. Enzyme and Microb. Deshmukh S. Medium optimization for the production of glucose isomerase from thermophilic Streptomyces thermonitrificans. Even though the product and the substrate have the same molecular formula, they have different bond connectivity and spatial arrangement.

The isomerases are known to catalyse many different biological reactions. Glycolysis and carbohydrate metabolism are a couple of examples for the same. The isomerases function by catalysing changes within just one molecule. Since they change one isomer to another, the end product has the same molecular formula but an alternate physical structure. Even though there are many different existing varieties of isomers they can be classified into two different groups-structural isomers and stereoisomers.

In structural isomers, the bonds are ordered differently or they differ in their bond connectivity from one other. Structural isomers can be catalysed by intramolecular lyases, oxidoreductases and transferases. On the other hand, stereoisomers have the same order of individual bonds and even the same connectivity but there is a difference in three-dimensional arrangement of the bonds.

The stereoisomers are catalysed using racemases, epimerases and cis-trans isomers. Enzyme catalysed reactions are known to have their own unique classification number. The enzyme classification category for isomerase-catalysed reactions is EC 5. The isomerase can then be further categorised into six sub-classes:. The racemases and epimerases function by inverting stereochemistry at the target chiral carbon.

While racemases act on molecules with one chiral carbon, the epimerases act on molecules with multiple chiral carbons but act only one of them. This category is then further broken down depending on what the enzyme acts upon, for example, action on amino acids or carbohydrates. This class is for the isomerases that catalyse the isomerization of the cis-trans isomers. Certain alkenes and cycloalkanes could have cis-trans stereoisomers. These isomers are distinguished through the positioning of the substituent groups relative to that of the plane of reference instead of absolute configuration.

The difference between cis-trans isomers is that cis isomers have their substituent groups on the same side while the trans isomers have their groups on opposite sides.

This class is not broken down further into sub-classes. These isomerases function by catalysing the transfer of electrons from one molecule to the other. Simply put, they catalyse the reaction that oxidises one part of the molecule while reducing the other part.

Depending on their processes intramolecular oxidoreductases can be divided into further sib-classes. Intramolecular transferases mutases are used to catalyse the movement of functional groups from one part of the molecule to another.

The intramolecular transferases can be sub-classified down depending on which functional group the enzyme moves. Intramolecular lyases function in reactions where a group is considered to be removed from one part of the molecule that forms a double bond while still being covalently attached to the molecule. Some of the reactions catalysed by intramolecular lyases involve the breaking of the ring structure. This class cannot be further classified.

An example of this kind of mechanism, i. The ring is closed after the formation of a ketose. A classic example of epimerization is the Calvin cycle when D-ribulosephosphate is converted into D-xylulosephosphate by ribulose-phosphate3-epimerase. The difference between the substrate and the product is in the stereochemistry at the third carbon in the chain. The process involves the deprotonation of the third carbon to make a reactive enolate intermediate.

An example of an intramolecular transferase is chorismate mutase. Chorismate mutase catalyses the change of chorismate to prephenate. The latter is used as a precursor for L-tyrosine and L-phenylalanine in some plants and bacteria.

This reaction is a Claisen modification that can continue with or without the isomerase, however, the rate increments 10 6 fold due to the chorismate mutase.