Introduction to Enzymes: Production of Enzymes

In traditional practices, brown rice syrup is created by adding a small amount of sprouted barley grains (barley malt) to cooked, whole brown rice in a solution of heated water, similar to the production of beer wort. The enzymes supplied by the barley malt digest the carbohydrates, proteins and lipids to produce a sweet solution rich in simple carbohydrates with minor amounts of amino acid, peptides and lipids. The solution is strained off the grains and boiled to evaporate and concentrate the liquid to produce a low water syrup suitable for use as a sugar substitute. Such syrups are high in the simple sugar maltose and low in glucose and fructose, due to the enzymatic action of beta- and alpha amylase on starch supplied by the sprouted barley. These enzymes produce large amounts of maltose from starch digestion and generate very little glucose or fructose in the process.

The modern, commercial preparation of brown rice syrup differs slightly. The ingredients consist of 100% modified rice starch generated by processing brown rice to remove the protein, hemicellulose and lipid fractions. The modification usually involves heat-assisted liquefaction of brown rice with enzyme isolates to produce a solution full of solubilised dextrins (derived from the breakdown of starch) and heat coagulated protein-hemicellulose-lipid complexes. The undesirable components are easily separated and recovered as a separate food stuff or agro-residue, leaving a solution of nearly pure, rice dextrins. A similar product to the rice-dextrin (modified starch) produced by this step is often sold under the name of malto-dextrin, but this commercial product often employs corn or wheat flour as the ingredient rather than rice.

The rice-dextrin solution then undergoes a further heat-assisted saccharification step involving the addition of further enzyme isolates, which convert the complex carbohydrates (rice-dextrins) into a solution rich in the simple carbohydrate maltose. The solution is then partially evaporated by boiling, until the final desired water content of the syrup is achieved. Brown rice syrup generated by this process is protein, fibre (hemicellulose) and lipid free and usually consists of 6585% maltose, 1015% maltotriose, 520% dextrins and only 23% glucose. The final carbohydrate mix of brown rice syrups can be controlled and adjusted by the manufacturer.

The enzymes used in the liquefaction step are usually alpha-amylases derived from bacterial or fungal bioreactors (Bacillus species or Aspergillus species are the most commonly used microbe engines in the bioreactors). These convert starch into dextrins of various molecular sizes and the modified starch end product is usually given an appropriate DE (dextrose equivalent) rating to signify the degree of starch conversion and the amount of reducing sugars produced in the process. The enzymes used in the saccharification step are the amylolytic enzyme, beta-amylase (usually derived from Bacillus species) and the debranching enzyme, pullulanase (derived from Aerobacter species). These convert the dextrinised starch into simple carbohydrates (sugars) and lower molecular weight dextrins.

The modern industrial production of brown rice syrup does not involve the use of synthetic chemicals in the modification of flour and starch. The enzymes added in processing are naturally derived from organic bioreactors using methods similar to the creation of antibiotics.

Brown rice syrup is readily available in most western Chinese grocery stores as maltose or maltose syrup, in reference to the high maltose content of the sweetener. This product is almost always produced by the industrialized method.

Rice syrup has a shelf life of about a year, and once opened, should be stored in a cool, dry place.

Brown rice syrup is the sweetener found in some drinks, such as rice milk.

Brown rice syrup is produced on a commercial scale by several companies in the United States, Europe, and Asia.

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Glal hydrolysis and Ligation Adapter Dependent PCR assay (GLAD-PCR assay) is the novel method to determine R(5mC)GY sites produced in the course of de novo DNA methylation with DNMTA and DNMTB DNA methyltransferases. GLAD-PCR assay do not require bisulfite treatment of the DNA.

Method was specially designed to determine methylation of RCGY site of interest in human and mammalian genomes in excess of corresponding unmethylated sites. This is a typical situation for DNA preparations from clinical samples of blood and tissues.

GLAD-PCR assay is based on the new type of enzymes - site-specific methyl-directed DNA-endonucleases (MD DNA endonucleases). These enzymes are very similar to restriction enzymes in biochemical properties and cleave DNA completely, but act in opposite way: they cleave only methylated DNA and do not cleave unmethylated DNA at all. Mammalian DNA-methyltransferases DNMT1, DNMT3a and DNMT3b catalyze a reaction of DNA methylation.

DNMT1 maintains DNA methylation pattern in vivo modifying a new strand after replication.

DNMT3a and DNMT3b are responsible for DNA methylation de novo including abnormal hypermethylation in cancer cells.It is well known that hypermethylation of CpG-islands in regulatory regions of promoter and/or first exon in a variety of genes often occurs at early stages of sporadic carcinogenesis. This leads to downregulation of the genes expression in tumor cells, whereas in a healthy tissue the corresponding genes remain to be active. Thus, the detection of such epigenetic biomarkers is one of the most promising diagnostic and prognostic tools

Study of DNMT3a and DNMT3b substrate specificity has shown that both enzymes predominantly recognize RCGY site and modify internal CG-dinucleotide to form 5-R(5mC)GY-3/3-YG(5mC) R-5 sequence. One of new enzymes GlaI recognizes and cleaves site R(5mC)GY. Due to this unique substrate specificity, GlaI is a convenient tool for identification of de novo methylated sites in the human and mammalian DNA.GLAD-PCR assay includes 3 simple steps:

GlaI hydrolysis of the studied DNA. At this step only R(5mC)GY sites are hydrolyzed. Unmethylated RCGY sites remain uncut.

The universal adapter ligation. As an adapter an oligonucleotide duplex 5-CCTGCTCTTTCATCG-3/3-pGGACGAGAAAGTAGCp-5 is used, where p means phosphate.

Subsequent real-time PCR with Taqman probe. Genome primer and TaqMan probe are designed for DNA region of interest, another hybrid primer consist of two parts: one part is complementary to the universal adapter and another one - to the DNA at the point of GlaI hydrolysis. GLAD-PCR assayAssay is performed in one tube, takes about 23 hours and determines even several copies of DNA with R(5mC)GY site of interest.

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Structural DifferencesThe primary differentiating feature between the MWC model and KNF model lies in the scale of conformational changes. While both suggest that a protein's affinity for a given ligand changes upon binding of the ligand, the MWC model suggests that this occurs by a quaternary conformational change that involves the entire protein, moving from T state to favoring the R state. On the other hand, the KNF model suggests these conformational changes occur on the level of tertiary structure within the protein, as neighboring subunits change conformation with successive ligand binding.

Unlike the MWC model, the KNF model offers the possibility of "negative cooperativity". This term describes a reduction in the affinity of the other binding sites of a protein for a ligand after the binding of one or more of the ligand to its subunits. The MWC model only allows for positive cooperativity, where a single conformational switch from the T to R states results in an increase in affinity for the ligand at unligated binding sites. Ligand binding to the T state thus cannot increase the amount of the protein in the T, or low-affinity, state.

Negative cooperativity is observed in a number of biologically significant molecules, including tyrosyl-tRNA synthetase and glyceraldehyde-3-phosphate dehydrogenase. In fact, in a systematic literature review performed in 2002 by Koshland and Hamadani, the same literature review that coined i3 cooperativity, negatively cooperating proteins are seen to compose slightly less than 50% of scientifically studied proteins that exhibit cooperativity, while positively cooperating proteins compose the other, slightly greater than 50%.

Functional Differences in HemoglobinHemoglobin, a tetrameric protein that transports four molecules of oxygen, is a highly biologically relevant protein that has been a subject of debate in allostery. It exhibits a sigmoidal binding curve, indicating cooperativity. While most scientific evidence points to concerted cooperativity, research into the affinities of specific heme subunits for oxygen has revealed that under certain physiological conditions, the subunits may display properties of sequential allostery.Nuclear magnetic resonance (NMR) studies show that in the presence of phosphate, deoxygenated human adult hemolglobin's alpha heme subunits display increased affinity for molecular oxygen, when compared to beta subunits. The results suggest either a modified concerted model, in which alpha subunits have a greater affinity for oxygen in the quaternary low-affinity T state, or a sequential model, in which phosphate binding creates a partially oligomerized state that stabilizes a low affinity form of the beta subunits, distinct from a T or R state. Thus, depending on physiological conditions, a combination of the MWC and KNF models appears to most comprehensively describe hemoglobin's binding characteristics.