BLBS102-c08 BLBS102-Simpson March 21, 2012 12:8 Trim: 276mm X 219mm Printer Name: Yet to Come
168 Part 2: Biotechnology and EnzymologyThis word emphasizes the materials inside or secreted by the
organisms to enhance the fermentation (changes of raw mate-
rial). Buchner (1897) performed fermentation by using a cell-
free extract from yeast of significantly high light, and these
were molecules rather than lifeforms that did the work. In 1905,
Harden and Young found that fermentation was accelerated by
the addition of some small dialyzable molecules to the cellfree
extract. The results indicated that both macromolecular and mi-
cromolecular compounds are needed. However, the nature of
enzymes was not known at that time. A famous biochemist,
Willstatter, was studying peroxidase for its high catalytic ef- ́
ficiency; since he never got enough samples, even though the
reaction obviously happened, he hesitated (denied) to conclude
that the enzyme was a protein. Finally, in 1926, Summer pre-
pared crystalline urease from jack beans, analyzed the properties
of the pure compound, and drew the conclusion that enzymes are
proteins. In the following years, crystalline forms of some pro-
teases were also obtained by Northrop et al. The results agreed
with Summer’s conclusion.
The studies on enzyme behavior were progressing in paral-
lel. In 1894, Emil Fischer proposed a “lock and key” theory
to describe the specificity and stereo relationship between an
enzyme and its substrate. In 1902, Herri and Brown indepen-
dently reported a saturation-type curve for enzyme reactions.
They revealed an important concept in which the enzyme sub-
strate complex was an obligate intermediate formed during the
enzyme-catalyzed reaction. In 1913, Michaelis and Menten de-
rived an equation describing quantitatively the saturation-like
behavior. At the same time, Monod and others studied the kinet-
ics of regulatory enzymes and suggested a concerted model for
the enzyme reaction. In 1959, Fischer’s hypothesis was slightly
modified by Koshland. He proposed an induced-fit theory to
describe the moment the enzyme and substrate are attached,
and suggested a sequential model for the action of allosteric
enzymes.
For the studies on enzyme structure, Sanger et al. were the
first to announce the unveiling of the amino acid sequence of
a protein, insulin. After that, the primary sequences of some
hydrolases with comparatively small molecular weights, such
as ribonuclease, chymotrypsin, lysozyme, and others, were
defined. Twenty years later, Sanger won his second Nobel
Prize for the establishment of the chain-termination reaction
method for nucleotide sequencing of DNA. On the basis of this
method, the deduction of the primary sequences of enzymes
blossomed; to date, the primary structures of 55,410 enzymes
have been deduced. Combining genetic engineering technology
and modern computerized X-ray crystallography and/or Nucleai
magnetic resonance (NMR), about 15,000 proteins, including
9268 proteins and 2324 enzymes, have been analyzed for their
three-dimensional structures (Protein Data Bank (PDB): http://
http://www.rcsb.org/pdb). Computer software was created, and
protein engineering on enzymes with demanded properties
was carried on successfully (Fang and Ford 1998, Igarashi
et al. 1999, Pechkova et al. 2003, Shiau et al. 2003, Swiss-
PDBViewer(spdbv): http://www.expasy.ch/spdbv/mainpage
.htm, SWISSMODELserver: http://www.expasy.org/swissmod/
SWISS-MODEL.htm).FEATURES OF ENZYMES
Most of the Enzymes are ProteinsProteins are susceptible to heat, strong acids and bases, heavy
metals, and detergents. They are hydrolyzed to amino acids by
heating in acidic solution and by the proteolytic action of en-
zymes on peptide bonds. Enzymes give positive results on typi-
cal protein tests, such as the Biuret, Millions, Hopkins-Cole, and
Sakaguchireactions. X-ray crystallographic studies revealed that
there are peptide bonds between adjacent amino acid residues in
proteins. The majority of the enzymes fulfill the above criterion;
therefore, they are proteins in nature. However, the catalytic el-
ement of some well-known ribozymes is just RNAs in nature
(Steitz and Moore 2003, Raj and Liu 2003).Chemical Composition of EnzymesFor many enzymes, protein is not the only component required
for its full activity. On the basis of the chemical composi-
tion of enzymes, they are categorized into several groups, as
follows:- Polypeptide, the only component, for example, lysozyme,
trypsin, chymotrypsin, or papain. - Polypeptide plus one to two kinds of metal ions, for
example,α-amylase containing Ca^2 +, kinase contain-
ing Mg^2 +, and superoxide dismutase having Cu^2 +and/or
Zn^2 +. - Polypeptide plus a prosthetic group, for example, peroxi-
dase containing a heme group. - Polypeptide plus a prosthetic group and a metal ion, for
example, cytochrome oxidase (a+a 3 ) containing a heme
group and Cu^2 +. - Polypeptide plus a coenzyme, for example, many dehydro-
genases containing NAD+or NADP+. - Combination of polypeptide, coenzyme, and a metal ion,
for example, succinate dehydrogenase containing both the
FAD and nonheme iron.
Enzymes are SpecificIn the life cycle of a unicellular organism, thousands of reac-
tions are carried out. For the multicellular higher organisms
with tissues and organs, even more kinds of reactions are pro-
gressing in every moment. Less than 1% of errors that occur
in these reactions will cause accumulation of waste materials
(Drake 1999), and sooner or later the organism will not be able
to tolerate these accumulated waste materials. These phenomena
can be explained by examining genetic diseases; for example,
phenylketonuria (PKU) in humans, where the malfunction of
phenylalanine hydroxylase leads to an accumulation of metabo-
lites such as phenylalanine and phenylacetate, and others, fi-
nally causing death (Scriver 1995, Waters 2003). Therefore,
enzymes catalyzing the reactions bear the responsibility for pro-
ducing desired metabolites and keeping the metabolism going
smoothly.