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Enzymes are biological catalysts that accelerate chemical reactions by lowering the activation energy. Enzymes have an active site that provides a unique chemical environment, made up of certain amino acid R groups (residues). This unique environment is well-suited to convert particular chemical reactants for that enzyme, called substrates, into unstable intermediates called transition states. Enzymes and substrates are thought to bind with an induced fit, which means that enzymes and substrates undergo slight conformational adjustments upon substrate contact, leading to binding. Enzymes bind to substrates and catalyze reactions in four different ways: bringing substrates together in an optimal orientation, compromising the bond structures of substrates so that bonds can be more easily broken, providing optimal environmental conditions for a reaction to occur, or participating directly in their chemical reaction by forming transient covalent bonds with the substrates.
Enzyme action must be regulated so that in a given cell at a given time, the desired reactions are being catalyzed and the undesired reactions are not. Enzymes are regulated by cellular conditions, such as temperature and pH. They are also regulated through their location within a cell, sometimes being compartmentalized so that they can only catalyze reactions under certain circumstances. Inhibition and activation of enzymes via other molecules are other important ways that enzymes are regulated. Inhibitors can act competitively, noncompetitively, or allosterically; noncompetitive inhibitors are usually allosteric. Activators can also enhance the function of enzymes allosterically. The most common method by which cells regulate the enzymes in metabolic pathways is through feedback inhibition. During feedback inhibition, the products of a metabolic pathway serve as inhibitors (usually allosteric) of one or more of the enzymes (usually the first committed enzyme of the pathway) involved in the pathway that produces them.
A substance that helps a chemical reaction to occur is a catalyst, and the special molecules that catalyze biochemical reactions are called enzymes. Almost all enzymes are proteins, made up of chains of amino acids, and they perform the critical task of lowering the activation energies of chemical reactions inside the cell. Enzymes do this by binding to the reactant molecules, and holding them in such a way as to make the chemical bond-breaking and bond-forming processes take place more readily. It is important to remember that enzymes don’t change the ∆G of a reaction. In other words, they don’t change whether a reaction is exergonic (spontaneous) or endergonic. This is because they don’t change the free energy of the reactants or products. They only reduce the activation energy required to reach the transition state.
Figure 1: Enzymes lower the activation energy of the reaction but do not change the free energy of the reaction. Here the solid line in the graph shows the energy required for reactants to turn into products without a catalyst. The dotted line shows the energy required using a catalyst. This figure should say Gibbs Free Energy on the Y-axis and instead of noting deltaH should have deltaG. Attribution: Marc T. Facciotti (own work)
Enzyme Active Site and Substrate Specificity
The chemical reactants to which an enzyme binds are the enzyme’s substrates. There may be one or more substrates, depending on the particular chemical reaction. In some reactions, a single-reactant substrate is broken down into multiple products. In others, two substrates may come together to create one larger molecule. Two reactants might also enter a reaction, both become modified, and leave the reaction as two products. The location within the enzyme where the substrate binds is called the enzyme’s active site. The active site is where the “action” happens, so to speak. Since enzymes are proteins, there is a unique combination of amino acid residues (also called side chains, or R groups) within the active site. Each amino acid side-chain is characterized by different properties. Amino acids can be classified as large or small, weakly acidic or basic, hydrophilic or hydrophobic, positively or negatively charged, or neutral. The unique combination of amino acids, their positions, sequences, structures, and properties, creates a very specific chemical environment within the active site. This specific environment is suited to bind, albeit briefly, to a specific chemical substrate (or substrates). Due to this jigsaw puzzle-like match between an enzyme and its substrates (which adapts to find the best fit between the transition state and the active site), enzymes are known for their specificity. The “best fit” between an enzyme and its substrates results from the their respective shapes and the chemical complementarity of the functional groups on each binding partner.
Figure 2: This is an enzyme with two different substrates bound in the active site. The enzymes are represented as blobs, except for the active site which shows the three R-groups of each of the three amino acids located in the active site. These R groups are interacting with the substrates through hydrogen bonding (represented as dashed lines)
At this point in the class you should be familiar with all the types of bonds as well as the chemical characteristics of all the functional groups. For example, the R group of R180 in the enzyme depicted above is the amino acid Arginine (abbreviated as R) and has an R group that consists of several amino functional groups. Amino functional groups contain a nitrogen (N) and hydrogen (H) atoms. Nitrogen is more electronegative than hydrogen so the covalent bond between N-H is a polar covalent bond. The hydrogen atoms in this bond will have a positive dipole moment, and the nitrogen atom will have a negative dipole moment. This allows amino groups to form hydrogen bonds with other polar compounds. Likewise, the backbone carbonyl oxygens of Valine (V) 81 and Glycine (G) 121 the backbone amino hydrogen of V81 are depicted engaged in hydrogen bonds with the small molecule substrate.
Prepare for the Test
Look to see which atoms in the figure above are involved in the hydrogen bonds between the amino acid R groups and the substrate. You will need to be able to identify these on your own, hydrogen bonds may not be drawn in for you on the test.
If you changed the pH of the solution that this enzyme was located in, would the enzyme still be able to form hydrogen bonds with the substrate ?
Which substrate (the left or right one) do you think is more stable in the active site? Why? How?
Figure 3: This is an enzyme active site. Only the amino acids in the active site are drawn. The substrate is sitting directly in the center. Source: Created by Marc T. Facciotti (original work)
Prepare for the test: First, identify the type of macromolecule in the figure above. Second, draw in and label the appropriate interactions between the R groups and the substrate. Explain how these interactions might change if the pH of the solution changed.
Structural Instability of Enzymes
The fact that active sites are so well-suited to provide specific environmental conditions also means that they are subject to influences by the local environment. It is true that increasing the environmental temperature generally increases reaction rates, enzyme-catalyzed or otherwise. However, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the active site in such a way that they are less well suited to bind substrates. High temperatures will eventually cause enzymes, like other biological molecules, to denature, a process that changes the natural properties of a substance. Likewise, the pH of the local environment can also affect enzyme function. Active site amino acid residues have their own acidic or basic properties that are optimal for catalysis. These residues are sensitive to changes in pH that can impair the way substrate molecules bind. Enzymes are suited to function best within a certain pH range, and, as with temperature, extreme pH values (acidic or basic) of the environment can cause enzymes to denature.
Figure 4: Enzymes have an optimal pH. The pH at which the enzyme is most active will be the pH where the active site R groups are protonated/deprotonated such that the substrate can enter the active site and the initial step in the reaction can begin. Some enzymes require a very low pH (acidic) to be completely active. In the human body, these enzymes are most likely located in the lower stomach, or located in lysosomes (a cellular organelle used to digest large compounds inside the cell). Source: http://biowiki.ucdavis.edu/Biochemis..._pH_Inhibition
The process where enzymes denature usually starts with the unwinding of the tertiary structure through destabilization of the bonds holding the tertiary structure together. Hydrogen bonds, ionic bonds and covalent bonds (disulfide bridges and peptide bonds) can all be disrupted by large changes in temperate and pH. Using the chart of enzyme activity and temperature below, make an energy story for the red enzyme. Explain what might be happening from temperature 37C to 95C.
Figure 5: Enzymes have an optimal temperature. The temperature at which the enzyme is most active will usually be the temperature where the structure of the enzyme is stable or uncompromised. Some enzymes require a specific temperature to remain active and not denature. Source: http://academic.brooklyn.cuny.edu/bi...ge/enz_act.htm
Induced Fit and Enzyme Function
For many years, scientists thought that enzyme-substrate binding took place in a simple “lock-and-key” fashion. This model asserted that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view called induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild shift in the enzyme’s structure that confirms an more productive binding arrangement between the enzyme and the transition state of the substrate. This energetically favorable binding maximizes the enzyme’s ability to catalyze its reaction.
When an enzyme binds its substrate, an enzyme-substrate complex is formed. This complex lowers the activation energy of the reaction and promotes its rapid progression in one of many ways. On a basic level, enzymes promote chemical reactions that involve more than one substrate by bringing the substrates together in an optimal orientation. The appropriate region (atoms and bonds) of one molecule is juxtaposed to the appropriate region of the other molecule with which it must react. Another way in which enzymes promote the reaction of their substrates is by creating an energetically favorable environment within the active site for the reaction to occur. Certain chemical reactions might proceed best in a slightly acidic or non-polar environment. The chemical properties that emerge from the particular arrangement of amino acid residues within an active site create the energetically favorable environment for an enzyme’s specific substrates to react.
The activation energy required for many reactions includes the energy involved in slightly contorting chemical bonds so that they can more easily react. Enzymatic action can aid this process. The enzyme-substrate complex can lower the activation energy by contorting substrate molecules in such a way as to facilitate bond-breaking. Finally, enzymes can also lower activation energies by taking part in the chemical reaction itself. The amino acid residues can provide certain ions or chemical groups that actually form covalent bonds with substrate molecules as a necessary step of the reaction process. In these cases, it is important to remember that the enzyme will always return to its original state at the completion of the reaction. One of the hallmark properties of enzymes is that they remain ultimately unchanged by the reactions they catalyze. After an enzyme is done catalyzing a reaction, it releases its product(s).
Figure 6: According to the induced-fit model, both enzyme and substrate undergo dynamic conformational changes upon binding. The enzyme contorts the substrate into its transition state, thereby increasing the rate of the reaction.
Creating an Energy story for the reaction above
Using the figure above, answer the questions posed in the energy story.
1. What are the reactants? What are the products?
2. What work was accomplished by the enzyme?
3. What state is the energy in initially? What state is the energy transformed into in the final state? This one might be tricky still, but try to identify where the energy is in the initial state and the final state.
Why regulate enzymes?
Cellular needs and conditions vary from cell to cell, and change within individual cells over time. The required enzymes and energetic demands of stomach cells are different from those of fat storage cells, skin cells, blood cells, and nerve cells. Furthermore, a digestive cell works much harder to process and break down nutrients during the time that closely follows a meal compared with many hours after a meal. As these cellular demands and conditions vary, so do the needed amounts and functionality of different enzymes.
Regulation of Enzymes by Molecules
Enzymes can be regulated in ways that either promote or reduce their activity. There are many different kinds of molecules that inhibit or promote enzyme function, and various mechanisms exist for doing so. In some cases of enzyme inhibition, for example, an inhibitor molecule is similar enough to a substrate that it can bind to the active site and simply block the substrate from binding. When this happens, the enzyme is inhibited through competitive inhibition, because an inhibitor molecule competes with the substrate for active site binding. On the other hand, in noncompetitive inhibition, an inhibitor molecule binds to the enzyme in a location other than an allosteric site and still manages to block substrate binding to the active site.
Figure 7: Competitive and noncompetitive inhibition affect the rate of reaction differently. Competitive inhibitors affect the initial rate but do not affect the maximal rate, whereas noncompetitive inhibitors affect the maximal rate.
Some inhibitor molecules bind to enzymes in a location where their binding induces a conformational change that reduces the affinity of the enzyme for its substrate. This type of inhibition is called allosteric inhibition. Most allosterically regulated enzymes are made up of more than one polypeptide, meaning that they have more than one protein subunit. When an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits are changed slightly such that they bind their substrates with less efficiency. There are allosteric activators as well as inhibitors. Allosteric activators bind to locations on an enzyme away from the active site, inducing a conformational change that increases the affinity of the enzyme’s active site(s) for its substrate(s).
Figure 8: Allosteric inhibitors modify the active site of the enzyme so that substrate binding is reduced or prevented. In contrast, allosteric activators modify the active site of the enzyme so that the affinity for the substrate increases.
Many enzymes don’t work optimally, or even at all, unless bound to other specific non-protein helper molecules, either temporarily through ionic or hydrogen bonds or permanently through stronger covalent bonds. Two types of helper molecules are cofactors and coenzymes. Binding to these molecules promotes optimal conformation and function for their respective enzymes. Cofactors are inorganic ions such as iron (Fe2+) and magnesium (Mg2+). One example of an enzyme that requires a metal ion as a cofactor is the enzyme that builds DNA molecules, DNA polymerase, which requires bound zinc ion (Zn2+) to function. Coenzymes are organic helper molecules, with a basic atomic structure made up of carbon and hydrogen, which are required for enzyme action. The most common sources of coenzymes are dietary vitamins. Some vitamins are precursors to coenzymes and others act directly as coenzymes. Vitamin C is a coenzyme for multiple enzymes that take part in building the important connective tissue component, collagen. An important step in the breakdown of glucose to yield energy is catalysis by a multi-enzyme complex called pyruvate dehydrogenase. Pyruvate dehydrogenase is a complex of several enzymes that actually requires one cofactor (a magnesium ion) and five different organic coenzymes to catalyze its specific chemical reaction. Therefore, enzyme function is, in part, regulated by an abundance of various cofactors and coenzymes, which are supplied primarily by the diets of most organisms.
In eukaryotic cells, molecules such as enzymes are usually compartmentalized into different organelles. This allows for yet another level of regulation of enzyme activity. Enzymes required only for certain cellular processes can be housed separately along with their substrates, allowing for more efficient chemical reactions. Examples of this sort of enzyme regulation based on location and proximity include the enzymes involved in the latter stages of cellular respiration, which take place exclusively in the mitochondria, and the enzymes involved in the digestion of cellular debris and foreign materials, located within lysosomes.
The following links will take you to a series of videos on kinetics. The first link contains 4 videos on reaction rates and the second link contains 9 videos related to the relationship between reaction rates and concentration. These videos are supplemental and are provided to give you an outside resource to further explore enzyme kinetics.
- Introduction to enzyme kineticst
- Reaction mechanism
- Allosteric regulation
Stability and Stabilization of Biocatalysts
R.M. de la Casa , . J.M. Sánchez-Montero , in Progress in Biotechnology , 1998
3.3 Esterification reaction
CRCL (from Sigma) and pure isoenzymes lipase A and lipase B are stereoselective in the recognition of the S(+) isomer of 2-arylpropionic acids  . Nevertheless, lipase B is more active and slightly more stereoselective than lipase A.
The stereopreference is not altered by fermentation conditions as we show in Figure 1 in the esterification (S) 2-phenyl propionic acid ( Figure 1 ) where we compare the commercial powder with one of the crude lipases.
Figure 1 . Esterification of (S) 2-phenyl propanoic acid with n-propanol:● LCC, ■ UAB.
We chose the lipase UAB for this experiment because these powder give us the best results in the hidrolysis reactions with long and short acid chain triglyceride (triolein, tributyrin).
Both in yield and in initial reaction rate UAB lipase is a better biocatalyst than commercial enzyme (CRCL).
It is well documented that the addition of water to the reaction mixture increases the yield and stereoselectivity of the esterification of (R,S) 2(4-isobutylphenyl) propionic acid [7,8] .
The esterification of (S) and (R)-ketoprofen without and with 200 μi H2O/ml media was carried out.
We can see the results of the esterification of (S)-ketoprofen without ( Figure 2a ) and with water ( Figure 2b ).
Figure 2a . Esterification of (S) ketoprofen without n-propanol with water: ● LCC, ■ UAB, ♦ UAB-1000, ╋ UAB-300.
Figure 2b . Estérification of (S)-ketoprofen with n-propanol with water: ● LCC, ■ UAB, ♦ UAB-1000, ╋ UAB-300.
In opposite of CRCL, UAB lipase is more active in water than when not present in water because better yield is achieved in Figure 2 a than in Figure 2 b.
In Table 3 we show the initial reaction rates of the esterification of (R) and (S)-ketoprofen with or without water. The influence of the water in the initial reaction rates is opposite in UAB and in CRCL. The enantioselectivity increases due to the effect of the water, in CRCL but decreases in UAB. This effect is different when observed at long reaction times (yield at 400 hours) where the addition of water is always positive.
Table 3 . Initial reaction rates of the esterification of (R) or (S) ketoprofen (mM ester/mg prot × hr)
|Lipases||with water||without water|
|UAB||2.18·10 −4||6.35·1O −5||343||2.41·10 −4||2.04·10 −5||118|
|UAB-1000||2.4·10 −5||1.94·10 −5||123||1.56·10 −5||1.62·10 −5||97|
|UAB-300||4.25·1 O −5||4.12·10 −5||104||3.33·I0 −5||2.55·10 −5||13|
|CRCL||3.72·10 −6||-||-||6.35·10 −7||9.23·1O −7||68|
The downstream process affects the catalytic activity in the synthesis ( Table 3 ) as in the hydrolysis ( Table 2 ). The lyophilization of crude lipase after equilibration in the presence of lactose (UAB) give us the best preparation because lactose acts as a water reservoir giving enough water to the lipase to be active  . When lactose is not present (UAB-1000 and UAB- 300), the water added is used to hydrate the external surface of the protein that slightly increases the esterification reaction rate ( Table 3 ) of both enantiomers. Thus, the enantioselectivity remains unaltered, although a slight increase of the yield is obtained. This behaviour is different to that observed with a UAB sample that has been preequilibrated with lactose.
Type IIS Restriction Enzymes
Type IIS restriction enzymes recognize asymmetric DNA sequences and cleave outside of their recognition sequence. They are useful for many applications, including Golden Gate Assembly. NEB currently offers over 50 Type IIS restriction enzymes. This table allows you to sort our enzymes by feature for easy comparison.
We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. Beginning April 2021, NEB will be switching our current BSA-containing reaction buffers (NEBuffer&trade 1.1, 2.1, 3.1 and CutSmart ® Buffer) to Recombinant Albumin (rAlbumin)-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart&trade Buffer). We anticipate that this switch may take as long as 6 months to complete. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Find more details at www.neb.com/BSA-free.
During this transition period, you may receive product with BSA or rAlbumin-containing buffers. NEB has rigorously tested both and has not seen any difference in enzyme performance when using either buffer. Either buffer can be used with your enzyme. All website content will be switched in April to reflect the changes, although you may not receive the new buffer with your product immediately.
|Enzyme||Heat Inact.||NEBuffer||Rxn Temp.||Activity at 37°C||Storage Temp.||Recognition Sequence||Recognition Sequence Length||Over- hang Length||Isoschizomers from NEB||Methylation Sensitivity**||Enz. Sub- type|
|AcuI|| ||Y||rCutSmart||37°C|| ||-20°C||CTGAAG(16/14)||6||2||IIC|
|AlwI|| ||N||rCutSmart||37°C|| ||-20°C||GGATC(4/5)||5||1||dam|| |
|BaeI|| ||Y||rCutSmart||25°C||20%||-20°C||(10/15)ACNNNNGTAYC(12/7)||7||5 & 5||IIC|
|BbsI *|| ||Y||NEBuffer r2.1||37°C|| ||-80°C||GAAGAC(2/6)||6||4|| ||IIT|
|BbsI-HF *|| ||Y||rCutSmart||37°C|| ||-20°C||GAAGAC(2/6)||6||4|| ||IIT|
|BbvI||Y||rCutSmart||37°C|| ||-20°C||GCAGC(8/12)||5||4|| |
|BccI|| ||Y||rCutSmart||37°C|| ||-20°C||CCATC(4/5)||5||1|| || || |
|BceAI|| ||Y||NEBuffer r3.1||37°C|| ||-20°C||ACGGC(12/14)||5||2|| ||CpG|| |
|BcgI||Y||NEBuffer r3.1||37°C|| ||-20°C||(10/12)CGANNNNNNTGC(12/10)||6||2 & 2||dam CpG||IIC|
|BciVI|| ||Y||rCutSmart||37°C|| ||-20°C||GTATCC(6/5)||6||1|| || |
|BcoDI|| ||N||rCutSmart||37°C|| ||-20°C||GTCTC(1/5)||5||4||BsmAI||CpG||IIT|
|BfuAI||Y||NEBuffer r3.1||50°C||50%||-20°C||ACCTGC(4/8)||6||4||BspMI||CpG|| |
|BmrI|| ||Y||NEBuffer r2.1||37°C|| ||-20°C||ACTGGG(5/4)||6||1|| || |
|BpmI||Y||NEBuffer r3.1||37°C|| ||-20°C||CTGGAG(16/14)||6||2|| ||IIC|
|BpuEI|| ||Y||rCutSmart||37°C|| ||-20°C||CTTGAG(16/14)||6||2|| ||IIC|
|BsaI-HF®v2 *|| ||Y||rCutSmart||37°C|| ||-20°C||GGTCTC(1/5)||6||4||dcm CpG||IIT|
|BsaXI|| ||N||rCutSmart||37°C|| ||-20°C||(9/12)ACNNNNNCTCC(10/7)||6||3 & 3|| ||IIC|
|BseRI|| ||Y||rCutSmart||37°C|| ||-20°C||GAGGAG(10/8)||6||2|| || ||IIC|
|BsgI||Y||rCutSmart||37°C|| ||-20°C||GTGCAG(16/14)||6||2|| ||IIC|
|BsmAI|| ||N||rCutSmart||55°C||50%||-20°C||GTCTC(1/5)||5||4||BcoDI||CpG|| |
|BsmBI-v2 *|| ||Y||NEBuffer r3.1||55°C||20%||-20°C||CGTCTC(1/5)||6||4||Esp3I||CpG||IIT|
|BsmFI|| ||Y||rCutSmart||65°C||50%||-20°C||GGGAC(10/14)||5||4||CpG dcm||IIC|
|BsmI|| ||Y||rCutSmart||65°C||20%||-20°C||GAATGC(1/-1)||6||2|| ||IIT|
|BspCNI|| ||Y||rCutSmart||25°C||75%||-20°C||CTCAG(9/7)||5||2|| ||IIC|
|BspMI||Y||NEBuffer r3.1||37°C|| ||-20°C||ACCTGC(4/8)||6||4||BfuAI|| || |
|BspQI *|| ||Y||NEBuffer r3.1||50°C||10%||-20°C||GCTCTTC(1/4)||7||3||SapI|| ||IIT|
|BsrDI|| ||Y||NEBuffer r2.1 ||65°C||30%||-20°C||GCAATG(2/0)||6||2|| ||IIT|
|BsrI|| ||Y||NEBuffer r3.1||65°C||20%||-20°C||ACTGG(1/-1)||5||2|| ||IIT|
|BtgZI *|| ||Y||rCutSmart||60°C||75%||-20°C||GCGATG(10/14)||6||4|| ||CpG||IIC|
|BtsCI|| ||Y||rCutSmart||50°C||50%||-20°C||GGATG(2/0)||5||2|| || |
|BtsI-v2|| ||Y||rCutSmart||55°C||75%||-20°C||GCAGTG(2/0)||6||2|| || ||IIT|
|BtsIMutI|| ||Y||rCutSmart||55°C||50%||-20°C||CAGTG(2/0)||5||2|| || ||IIT|
|CspCI||Y||rCutSmart||37°C|| ||-20°C||(11/13)CAANNNNNGTGG(12/10)||7||2 & 2||IIC|
|EarI|| ||Y||rCutSmart||37°C|| ||-20°C||CTCTTC(1/4)||6||3||CpG||IIT|
|EciI|| ||Y||rCutSmart||37°C|| ||-20°C||GGCGGA(11/9)||6||2|| ||CpG||IIC|
|Esp3I *|| ||Y||rCutSmart||37°C||-20°C||CGTCTC(1/5)||6||4||BsmBI-v2||CpG||IIT|
|FauI|| ||Y||rCutSmart||55°C||20%||-20°C||CCCGC(4/6)||5||2|| ||CpG|| |
|FokI||Y||rCutSmart||37°C|| ||-20°C||GGATG(9/13)||5||4|| ||dcm CpG|| |
|HgaI|| ||Y||NEBuffer r1.1||37°C|| ||-20°C||GACGC(5/10)||5||5||CpG|| |
|HphI|| ||Y||rCutSmart||37°C|| ||-20°C||GGTGA(8/7)||5||1||dam dcm|| |
|HpyAV|| ||Y||rCutSmart||37°C|| ||-20°C||CCTTC(6/5)||5||1|| ||CpG|| |
|MboII||Y||rCutSmart||37°C|| ||-20°C||GAAGA(8/7)||5||1|| ||dam|| |
|MlyI|| ||Y||rCutSmart||37°C|| ||-20°C||GAGTC(5/5)||5||0|| || |
|MmeI||Y||rCutSmart||37°C|| ||-20°C||TCCRAC(20/18)||6||2|| ||CpG||IIC|
|MnlI|| ||Y||rCutSmart||37°C|| ||-20°C||CCTC(7/6)||4||1|| || || |
|NmeAIII||Y||rCutSmart||37°C|| ||-20°C||GCCGAG(21/19)||6||2|| ||IIC|
|PaqCI|| ||Y||rCutSmart||37°C|| ||-20°C||CACCTGC(4/8)||7||4||CpG|| |
|PleI||Y||rCutSmart||37°C|| ||-20°C||GAGTC(4/5)||5||1||CpG|| |
|SapI *|| ||Y||rCutSmart||37°C|| ||-20°C||GCTCTTC(1/4)||7||3||BspQI|| ||IIT|
|SfaNI|| ||Y||NEBuffer r3.1||37°C|| ||-20°C||GCATC(5/9)||5||4||CpG|| |
* Cited for use in Golden Gate Assembly according to current literature
** Methylation sensitivity applies to the recognition motif only
Through suppression experiments and published reports, NEB has identified that these enzymes require more than one recognition site on the substrate to cleave optimally. For more information, visit Restriction Enzyme Cleavage: &lsquosingle-site&rsquo enzymes and &lsquomulti-site&rsquo enzymes.
Working Mechanism of Enzymes
As mentioned above, most of the enzymes are produced in the cells of living organisms. The production of enzymes is carried out by the cell, based on the instructions from the genes of that cell. So defects in the genes may result in defective enzymes, which do not work properly. The structure and function of each enzyme is different. They have to act upon different targets, that vary from one enzyme to another. Usually, a particular enzyme can act upon a specific target only. The course of action of enzymes are different and complex and so, there are various theories regarding this subject.
An Overview: In general, the working mechanism of an enzyme can be described as follows. Each enzyme acts upon a specific target called substrate, which is transformed into usable products through the action of the enzyme. In other words, the enzyme reacts with the substrate forming an enzyme-substrate complex. Once the reaction is complete, the enzyme remains the same, but the substrate transforms to products. For example, the enzyme sucrase acts upon the substrate sucrose to form products – fructose and glucose.
Lock and Key Theory: This is one of the theories that explain the working mechanism of enzymes. As per this theory, each enzyme has a specific area (called active site) that is meant for a particular substrate to get attached. The active site of the enzyme is complementary to a specific part of the substrate, as far as the shapes are concerned. The substrate will fit into the active site perfectly, and the reaction between them takes place.
The right substrate will fit into the active site of the enzyme and form an enzyme-substrate complex. It is at this active site that the substrate is transformed to usable products. Once the reaction is complete, and the products are released, the active site remains the same and is ready to react with new substrates. This theory was postulated by Emil Fischer in 1894. This theory provides a basic overview about the action of enzymes on the substrate. However, there are certain factors that remain unexplained. As per this theory, the amino acids (in unbound state) at the active site are responsible for its specific shape. There are certain enzymes that do not form any shape in the unbound form. The lock and key theory fails to explain the action of such enzymes.
Induced-fit Theory: This theory was formulated by Daniel E. Koshland, Jr. in 1958. This theory too supports the lock and key hypothesis that the active site and substrate fits perfectly and their shapes are complementary. According to the induced-fit theory, the shape of the active site is not rigid. It is flexible and changes, as the substrate comes into contact with the enzyme.
To be more precise, once the enzyme identifies the right substrate, the shape of its active site changes so as to fit the latter exactly. This results in formation of the enzyme-substrate complex and further reactions. As this theory explains the working mechanism of numerous enzymes, it is widely accepted than the lock and key hypothesis.
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Factors that Affect Enzymes’ Action: The activities of enzymes are affected by various factors, like the temperature, pH, and concentration. Usually, high temperatures boost the rate of reactions involving enzymes. The optimal temperature for such reactions are said to be around 37 ºC to 40 ºC. Once the temperature rises above this level, the enzymes get denatured and they are no longer fit for reaction with substrates. Variations in pH may also affect the working mechanism of enzymes. The optimum pH level may vary from one enzyme to another, as per the site of their action. Variations from that pH level may slow down the activity of enzymes and very high or low pH results in denatured enzymes that cannot hold the substrate properly. The rate of enzymatic activities may increase with the concentration of enzymes and substrates.
This is only a brief overview about the working mechanism of enzymes. Human body produces numerous enzymes that are responsible for a wide range of chemical reactions, which are necessary for our survival. Right from respiration and digestion, enzymes are involved in so many functions. Some of these enzymes are used for industrial purposes too. Enzymes in laundry detergents are responsible for removing the stains and make the clothes clean. Some are used in preparing foods and beverages.
For every enzyme, there is an optimum pH value, at which the specific enzyme functions most actively. Any change in this pH significantly affects the enzyme activity and/or the rate&hellip
In a chemical reaction, the step wherein a substrate binds to the active site of an enzyme is called an enzyme-substrate complex. The activity of an enzyme is influenced by&hellip
Rennin is an enzyme that is essential for the digestion of proteins. It helps digest milk in young mammals. This BiologyWise article lists out the function of rennin enzyme.
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I had the bacteria in my gut analysed. And this may be the future of medicine
W e are all familiar with "gut feelings", "gut reactions" and "gut instincts", but how much do we really know or care about our guts? As we become increasingly more aware of what we put in our stomachs, it's striking how ignorant we remain of what takes place in our intestines. And it turns out there is an awful lot going on down there.
Microbiologists have made some startling advances in revealing our innermost secrets. It turns out that there is a complex ecosystem deep within us that is home to a fantastic diversity of life – of which very little belongs to our species.
For most of us, suspicious of foreign bodies, it's a struggle to comprehend that at our very core we are less than – or rather much more than – human. But, the fact is, there are about 100 trillion organisms living in the gut. If you put them all together they would be about the size of a football. In terms of cells, the microbial kind outnumber their human counterparts by about three to one. And in terms of genes, the microbial advantage is more like 300 to one.
That means there is a tremendous amount of us that is not, so to speak, us. This raises a whole range of interesting philosophical and anatomical questions, of which the most urgent might be: should we be worried?
Well, I wasn't much concerned about bacteria before I got the contents of my gut tested. I took a fairly relaxed view that as long as the lavatory was regularly bleached, I brushed my teeth and kept the kitchen surfaces reasonably clean then I didn't have to think too much about what goes on at the microbial level. But there's nothing like spooning your own faecal matter into a Perspex container to make you stop and contemplate just what it is that we're full of. That unpleasant task is precisely what I found myself doing last October, as I gathered a stool sample to send off, cold-packed to the BioSciences Institute at University College Cork in Ireland.
The institute is one of Europe's leading centres for the study of what is now referred to as the microbiome – that is all the bacteria, viruses, fungi, archaea and eukaryotes that inhabit the human body, inside and out. The simplistic view of these guests has traditionally centred on their parasitic or pathogenic aspects. Either they were fairly harmlessly hitching a free ride or were a direct threat to their host.
But the latest thinking presents this vast army of microbes as a vital component in furnishing and maintaining human health. Such is the microbiome's importance that it is now viewed by scientists as a separate organ with its own dynamic metabolic activity. But what precisely is that activity and is it all going to plan with me?
Paul O'Toole is a professor at the Alimentary Pharmabiotic Centre, which is part of the BioSciences Institute at Cork. A keen marathon runner, he looks like he knows a thing or two about intestinal fortitude. He co-ordinated a government-funded study – fortuitously launched just before the Irish economy collapsed – entitled Eldermet, which was aimed at helping the Irish food industry develop food products for old people. To do that, they needed a knowledge base of the gut microbiota. So O'Toole began examining how diet affects the microbiota of Ireland's elderly population.
There is an element of poacher-turned-gamekeeper to his career because he started out as something of a bacterial enemy. "I spent about 15 years working on pathogens where you're trying to kill them," he tells me in his office. "I did my PhD in staphylococcus. One organism, one gene. I worked on a condition called scalded skin syndrome syndrome where staphylococci infect the umbilical stump and if they produce a toxin all the baby's skin peels off."
From combatting staphylococci, he moved into probiotics – the organisms that are supposed to be good for us – which in commercial form have been decanted into capsules and yoghurts and advertised to the public as "friendly bacteria". But he discovered that he couldn't effectively study probiotics in isolation because their benefits were often indirect.
"I realised I needed to study the whole canvas," he says. And that was how he came to find himself involved with the microbiome, just when it was starting to become the subject of intensive biomedical research.
There are two labs, O'Toole explains, that processed my sample. The first was the wet lab, where, through various molecular assaults, DNA was extracted, 95% of which was bacterial. This was then sent to an external company to be sequenced – there were over 30,000 sequences – and then a huge file of data was crunched by what O'Toole called "a bunch of computer nerds who sit around all day generating stats" in the institute's data lab.
Just a year ago, that process cost upwards of £400. Now it can done for as little as £15. What you get are a couple of pie charts that list the microbiota found in the gut at different phylogenetic levels and a narrative explanation as to what their significance is. Phylogentic levels in this instance simply refer to different levels of resolution.
At the broadest level, the phylum level, my microbiota, in common with everyone else's, was dominated by two types: firmicutes and bacteroidetes. The western diet, by which we tend to mean the North American diet, is high in fat and protein. In this diet bacteroidetes usually make up more than 55% of the gut microbiota, and sometimes, in North America itself, as much as 80%. In Europe, the average numbers vary from country to country. In my case I had 34%.
The opposite to a North American diet is what O'Toole calls a "natural diet". "Our antecedents on the plains of Africa weren't chewing on burgers," he explains. "They were running around eating plant foods and leaves and occasionally eating a squirrel if they were lucky."
On a plant-based diet, the microbiota is tipped in favour of the other major phylum, firmicutes. Some of the complex carbohydrates in plants cannot be digested by our bodies alone. They have to be broken down by the gut microbiota, which produce enzymes to chop up the long chains and ferment them into short-chain fatty acids such as butyrate – which is made exclusively by bacteria – acetate and propionate.
These fatty acids are beneficial to the body. Butyrate, for example, provides an energy source that the cells lining our intestines can directly access. It also controls the proliferation of cells in the intestine and is thought to possess anti-carcinogenic properties. All of which meant that my score of 51% firmicutes was a healthy sign.
Zooming into the genus level, which offers a more detailed look at my microbial composition, the good news continued. I had three times as much of the butyrate-producing roseburia than the healthy cohort used in O'Toole's study. Many more lachnospira than normal but many fewer bacteroides (not to be confused with bacteroidetes) and alistipes – as O'Toole put it, in more scientific terms, "bugger all".
Again these were positive results. Lachnospira degrade pectins and ferment dietary fibres and I have three times more than typical. And bacteroides are often associated with meat-based, high-protein, high-fat diets, just as alistipes tend to be more present in people who eat less plant-based food. In sum that meant my gut – the lack of six-pack notwithstanding – was probably in good shape. Of course, it's not the sort of thing you can boast about at dinner parties. "I've got significantly higher than average amounts of lachnospira," is unlikely to be a conversational gambit that will impress non-microbiologists, even if you do manage to pronounce the word correctly. But just as we now know that high cholesterol is something to be avoided, so too might we soon begin to become aware of the sorts of bacteria counts that are markers for good health, especially as the price of testing comes down.
There were, however, one or two results that O'Toole struggled to make sense of. In particular my high levels of natranaerobius, a genus of bacteria that thrive in high-salt, highly alkaline environments. Did I eat a lot of sushi? No, while I love fish, I tend to prefer it cooked. Did I prepare a lot of fish? No more than once a week.
Although he found nothing sinister in the natranaerobius, it perturbed him that he couldn't quite put his finger on the cause of its abundance in my gut. But by then he had managed to make a blind prediction of my diet that was uncannily accurate. He saw very little evidence of meat-eating – I haven't eaten meat for 30 years. But there was plenty of evidence of high fibre, which is good because bacteria feed on fibre. If we don't feed bacteria, they feed off us – specifically the mucus lining in our large intestine. There was also evidence of lots of fish and a large range of vegetables. All of which exactly represents my diet.
I suggest that it must be satisfying to get his prediction so right.
"It's a bit spooky all right," he agrees. "But it made me think about the utility of it. I mean, it's not particularly useful to tell people what they eat."
O'Toole is interested in the diagnostic potential of the microbiome. "We could probably guess what your inflammatory parameters are," he says, fixing me with one of those expressions in which GP's specialise when looking up from studying your medical notes: neutral, unyielding, and anxiety-inducing. Not only do I not know what my inflammatory parameters are, I don't know what inflammatory parameters means.
O'Toole explains that significant links have been established between gut microbiota and inflammation, sarcopenia and cognitive function.
"Inflammation," he says, "is not a swollen thumb. Inflammation means how activated your immune system is. I would guess that your inflammatory markers are baseline. Flat. In old people they're not. In old people, the immune system is typically turned on and that's not good, because if it's turned on, when they get a winter flu all their energies are expended chasing ghosts. So you want to turn down the inflammation."
Sarcopenia means loss of muscle mass. It happens as we get older because the body becomes less efficient at turning protein into muscle, which is why older people need to have more protein. "We think that the narrowing of gut bacteria in old people is making the intestine less efficient at absorbing proteins," says O'Toole.
Cognitive function is partly related to what's known as the brain-gut axis. As all those phrases like "gut wrenching" and "gut feeling" suggest, there is indeed an intimate link between the brain and the gut. Our intestines are acutely responsive to shifts in our emotions and mental states. But it's a two-way street: studies suggest that our brains and emotions are also sensitive to what's going on in our guts.
T ypically, cognitive function is only slowly diminished as we get older, but in some cases it can quickly accelerate.
"There are physiological reasons like Alzheimer's and senile dementia that explain rapid cognitive impairment," O'Toole says. "But the rate of loss could also be affected by compounds made by bacteria, and that's what we're targeting. Bacteria produce chemicals which are analogues – in other words they look identical to normal human transmitters. What we hope is that we can improve the ability of old people to process data."
Common to all these issues, particularly among the aged, is the narrowing of the gut microbiota which, in turn, is usually the result of a narrowing of diet. This is a point that O'Toole repeatedly emphasises.
"Diversity is the key. What we see with people on narrow diversity diets is that the microbiota collapses. A good analogy would be an ecosystem like a rainforest, where you've got loads of plants and animals interacting. It's evolved over tens of thousands of years, then one of the key species, a tree, gets cut down and you get ecological collapse.
"And if you had a gentleman whose wife died and she had done all the cooking, and then he's suddenly eating toast and marmalade, the diversity of gut microbiota will collapse – because diversity of diet correlates with diversity of microbiota – and you will get a range of health problems associated with that."
He goes on to tell me that my microbial diversity is impressively wide and that, by way of summary, he would suggest that my diet is "pretty bloody good". Forget the 5-2 diet, I suddenly feel like writing a bestselling diet book entitled Guts: The Microbial Guide to Healthy Eating. In one sense, of course, it's no great achievement. Studies show that it only takes a short time of a changed diet to dramatically change the microbiota, although it changes back just as quickly as soon as the diet is dropped.
But this apparently superficial relationship between food and microbes is in reality rather profound because first it speaks of a co-evolution with the human body over tens of thousands of years. Like all organisms and species, humans have evolved to have a particular relationship with a particular set of microbes.
There are hundreds of thousands of kinds of microbes on Earth but only about a thousand enjoy an association with humans. Thus, secondly it suggests that we need to stop thinking of ourselves as separate entities from the microbes that have colonised our bodies.
"We came through the period of medicine in which we developed antibiotics," says O'Toole. "Until the second world war we were dying from stupid things like pneumonia and galloping septicemia from a small wound. So antibiotics were a major success. Then we've had the backlash where we've prescribed them too much and can't control the pathogens. But now we have a more intelligent understanding of humans as chimeras."
A germ-free existence would be an unhappy one. Tests have shown that a mouse raised in a lab devoid of bacteria fails to develop a proper immune system or an effective digestive system. It has to consume a lot more food to extract calories. Humans are first colonised by microbes during birth. Then through breast milk, which contains both probiotics (beneficial microbes) and prebiotics (compounds that foster the growth of probiotics).
"There is strengthening evidence," says O'Toole, "that the explosion of auto-immune diseases and immune disregulation diseases in western society may be due to suppression of gut bacteria from infancy onwards.
"The immune system in babies is probably taught to distinguish between self and non-self in the context of bacteria. There are two recent papers in the publication Nature showing that butyrate is important in enlisting regulatory T-cells, a branch of immune cells that control the processes involved in inflammatory bowel disease and irritable bowel syndrome."
It takes about two years from birth through a process of selection for a child to attain a mature microbiome. There are several phenomena that may contribute to childhood microbial diminishment. One is the increase in caesarian sections.
"Babies who were previously colonised in the birth canal with their mother's microbiota now have a gut microbiota that is more like the walls of the hospital than it is mum's vaginal microbiota."
Another is lack of breast milk, and a third is the increased use of antibiotics. O'Toole says that one study suggests that repeated use of antibiotics tips the microbiota towards one that promotes obesity. In fact there are many studies around the globe that are still in their infancy but which point up connections between the microbiota and diseases and complaints as diverse as irritable bowel syndrome, inflammatory bowel disease, type-two diabetes, Parkinson's, Alzheimer's, autism, depression, cardiovascular disease and colon cancer.
But so far none of it is conclusive and much is highly speculative. After the initial claims about the potential health benefits of microbiome research – the kind that tend to help funding – there has been a bit of a sceptical backlash.
Several articles have pointed out that there has been plenty of hyperbole but not enough substance. And as yet the medical profession isn't rushing to produce microbiome specialists.
"Medicine is notoriously slow to adopt new ideas," says O'Toole. He cites the case of Barry Marshall, an Australian doctor whose claim to have established a bacterial cause of peptic ulcers and gastric cancer was comprehensively ridiculed by the medical establishment in the 1980s. "About 20 years later he got the Nobel prize."
The problem, he says, is that microbiologists have been very good at discovering gut bacteria and identifying what roles they might play, but they have been slow to develop mechanisms to establish firm causal links and practical applications.
"I personally hope it doesn't become the solution for everything because it's not going to be credible, it's simply not true. There's plenty of evidence that most human major diseases have a physiological or lifestyle basis, but it's probable in some of those that the gut microbiota is a modulating factor that contributes to the overall risk."
Right now, O'Toole would like to like to reduce the lower diversity microbiota in the elderly by means of dietary supplements. "But we worry that, as the World Wildlife Fund says, extinction may be forever. That if a particularly good bacterium is missing from an elderly person, we may not be able to get it back by diet alone."
The solution in that case might be fecal microbiota transplantation, which O'Toole helpfully clarifies, "is the idea of transplanting someone else's poo into a recipient". Which neatly brings us back to where I started. If collecting your own excrement is counter-intuitive, then injecting it into someone else runs against every decent human instinct.
But it's already happening in North America and O'Toole suggests that such transplants may help prevent ulceration of the colon – a condition that nearly killed my father some years back.In the end, it's all comes back to what you put in and take out. And in that tireless cycle of life, we shouldn't be appalled if not even our waste need go to waste.
Please note: the BioSciences Institute is not able to offer individual analysis, and did so for the purposes of this piece only.
Molecular Biology Enzymes Market By Key Players (Becton Dickinson, Agilent Technologies, Thermo Fisher Scientific, Merck) Based on 2020 COVID-19 Worldwide Spread
The report on the Global Molecular Biology Enzymes Market has published by the Market Research Store. The report provides the client the latest trending insights about the Molecular Biology Enzymes market. You will find in the report include market value and growth rate, size, production consumption and gross margin, pricings, and other influential factors. Along with these you will get detailed information about all the distributors, suppliers and retailers of the Molecular Biology Enzymes market in the report. The competitive scenario of all the industry players are mentioned in-detail in the report. Due to the pandemic the market players have strategically changed their business plans.
Some of the key industry players that are operating in the Molecular Biology Enzymes market are:
- F. Hoffmann-la Roche
- QIAGEN N.V.
- Agilent Technologies
- Takara Bio
- Thermo Fisher Scientific
- Becton Dickinson
- New England Biolabs
Through the month of the analysis, research analysts predicted that the Molecular Biology Enzymes market reached XX million dollars in 2019 and the market demand will reach XX million dollars by 2026. During the forecast period 2020 to 2026 the expected CAGR is XX%. The increasing investments in the research and development activities and the rising technological advancements in the Molecular Biology Enzymes market, increasing the market growth.
Due to the increase of pandemic world-wide several market issues has generated around the world. Such as, economic crisis in various regions along with loss of employment.
The questions that are answered in the report:
- What are the challenges for the Molecular Biology Enzymes market created by the outbreak of the global pandemic?
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- What are the developing regions in the Molecular Biology Enzymes market?
Overall industries are struggling on the global platform to revive the markets. It has been observed that through the pandemic almost every market domain has been impacted.
The Molecular Biology Enzymes market regional presence is showcased in five major regions Europe, North America, Latin America, Asia Pacific, and the Middle East and Africa. In the report, the country-level analysis is also provided.
The Molecular Biology Enzymes market is segmented into Product Types:
The Molecular Biology Enzymes market is segmented into By End User/Application:
- Academic & Research Institutes
- Hospitals & Diagnostic Centers
- Pharmaceutical & Biotechnology Companies
The major points that are covered in the report:
Overview: In this section, the global Molecular Biology Enzymes Market definition is given, with an overview of the report in order to provide a board outlook about the nature and contents of the research study.
Strategies Analysis of Industry Players: This Strategic Analysis will help to gain competitive advantage over their competitors to the market players.
Essential Market Trends: Depth analysis of the market&rsquos latest and future trends is provided in this section.
Market Forecasts: In this segment, accurate and validated values of the total market size in terms of value and volume have provided by the research analyst. Also the report include production, consumption, sales, and other forecasts for the global Molecular Biology Enzymes Market.
Regional Analysis: In the global Molecular Biology Enzymes market report major five regions and its countries have been covered. Market players will have estimates about the untapped regional markets and other benefits with the help of this analysis.
Segment Analysis: Accurate and reliable foretell about the market share of the essential sections of the Molecular Biology Enzymes market is provided.
- North America
- Latin America
- Asia Pacific
- Middle East and Africa
Frequently Asked Questions
Which are the dominant player engaged in the Molecular Biology Enzymes industry?
- F. Hoffmann-la Roche
- QIAGEN N.V.
- Agilent Technologies
- Takara Bio
- Thermo Fisher Scientific
- Becton Dickinson
- New England Biolabs
Which sectors are forecasted to occupied the highest share in the Molecular Biology Enzymes business?
As per Molecular Biology Enzymes market analysis, North America is forecasted to occupied major share in the Molecular Biology Enzymes market.
How can I get analytical data of dominant industry player of Molecular Biology Enzymes market?
The statistical data of the dominant industry player of Molecular Biology Enzymes market can be acquired from the company profile segment described in the report. This segment come up with analysis of major player’s in the Molecular Biology Enzymes market, also their last five-year revenue, segmental, product offerings, key strategies adopted and geographical revenue produced.
Which segment are offer in this report?
The report come up with a segment of the Molecular Biology Enzymes market based on Type, Region, and Application, Also offer a determined view on the Molecular Biology Enzymes market.
Which market fluctuations have an effect on the business most?
The report offers a nitty-gritty estimation of the market by providing data on various viewpoints that incorporate, restraints, drivers, and opportunities threats. This data can help in making suitable decisions for stakeholders before investing.
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The sample report for Molecular Biology Enzymes market can be received after the apply from the website.
INHERITANCE OF TWO GENES
Dihybrid Cross: When two pairs of characters are studied in the cross, it is called dihybrid cross.
Mendel selected yellow colour (YY) and green colour (yy) as seed colour. He further selected round seeds (RR) and wrinkled seeds (rr) for seed texture. In this case, yellow colour is dominant over green colour, while round texture is dominant over wrinkled texture.
F1 Generation: When gametes RY and ry were crossed, all plants in F1 generation produced yellow and wrinkled seeds (RrYy). The genotype was heterozygous in these plants. Yellow colour and round texture showed dominance.
When plants of F1 generation were allowed to self pollinate, the result could be shown by following Punette Square.
The plants of F2 generation produced 3 types of seeds, i.e. round yellow, wrinkled yellow, round green and wrinkled green in ratio 9:3:3:1. Based on this observation, Mendel proposed the Law of Independent Assortment.
Law of Independent Assortment: When two pairs of traits are combined in a hybrid, segregation of one pair of characters is independent of the other pair of characters.
Chromosomal Theory of Inheritance: Chromosomes as well as genes occur in pairs. The two alleles of a gene pair are located on homologous sites on homologous chromosomes. Sutton and Boveri argued that the pairing and separation of a pair of chromosomes would lead to the segregation of a pair of factors they carried. Sutton united the knowledge of chromosomal segregation and Mendelian principles and termed it the Chromosomal Theory of Inheritance.
Linkage: The physical association of genes on a chromosome is called linkage.
Recombination: Combination of non-parental genes is called recombination.
Morgan carried out several dihybrid crosses in Drosophila to study genes that were sex-linked. Morgan hybridized yellow-bodied, white-eyed females to brown-bodied, red-eyed males. He intercrossed the F1 progeny. He observed that the two genes did not segregate independently of each other, and the F2 ratio deviated very significantly from the 9:3:3:1 ratio. Morgan was aware that the genes were located on the X chromosome. He could see that when the two genes in a dihybrid cross were situated on the same chromosome, the proportion of parental gene combinations were much higher than the non-parental gene combinations. This was attributed to the physical association or linkage of the two genes. Morgan also found that even when genes were grouped on the same chromosome, some genes were tightly linked, while others were loosely linked. The tightly linked genes showed very low recombination, while the loosely linked genes showed higher recombination. For example genes for white and yellow colours were tightly linked and showed only 1.3% recombination. On the other hand, genes for white and miniature wing showed 37.2% recombination because they were loosely linked.
What does elevated ALT level means? And when is treatment needed?
ALT (Alanine Aminotransferase / SGPT) is a type of enzyme found in liver cells. When the liver cells are functioning normally, the ALT enzymes should be contained within the liver cells.
You can imagine each liver cells as a balloon, and the ALT enzymes are the air inside the balloon. When the balloon is damaged, the air will be released. And when the liver cells is damaged, ALT enzymes are released into the bloodstream, therefore we are able to find out the level of liver function from a liver function blood test (LFTs).
The level of ALT is the primary indicator of liver health as this type of enzyme are only mainly found in liver cells. Normal ALT values are around 10-40 units per litre. This range might vary according to different countries or laboratories, but the upper limit is usually between 35-40. 
Therefore an elevated ALT level simply means liver damage, the higher ALT number indicates more severe damage to the liver.
When is treatment needed for elevated ALT level?
Different substances can causes damages to liver cells and elevated ALT level, i.e. Alcohol, medication, fat, heavy metals, or even excessive amount of meat intake. And it is normal for our body to have a small amount of ALT in the bloodstream, therefore the normal range of ALT is 10-40 units per litre.
Our liver is very forgiving and has a great self-repairing ability. If the ALT level is elevated in short term (less than 1 month), it is not considered to be a problem as the liver will be able to heal back itself.
But if the elevated ALT is longer than 1 month, this indicates a problem in the liver, which could be fatty liver, hepatitis, alcohol liver disease, etc. If ALT level is above normal range for longer than 3 months, it is considered to be chronic liver disease, where the liver is under constant injury, and treatment is required in order to prevent more serious liver diseases like: fibrosis, cirrhosis or liver cancer.
The types of treatment required for elevated ALT is different depending on the causes, some might require treatment for the cause of liver damage, and some treatment might focus on enhancing the ability of liver function and simply by protecting the liver cells, like fatty liver.