12/08/14

Enzimologia
Histrico, Conceitos e Mercado
Prof. Dr. Hermgenes David
Disciplina: Enzimologia

Interesse cientfico iniciado em meados do sculo passado.

Contribuies da Qumica Orgnica de Protenas e dos estudos com
metabolismo.
As enzimas do trado digestrio humano foram as primeiras a serem
identificadas
1783 Lazzaro Spallanzani observou a degradao enzimtica da
carne pelo suco gstrico;
1836 Theodor Schwann denominou de pepsina a substncia ativa
presente na secreo gstrica
Trabalhos de Wilheln Friedrich Kuhne
1876 apresentao dos resultados sobre o estudo com
"fermentos pancreticos
Publicao: Sobre Tripsina: Enzima do Pncreas

Enzimas - So catalisadores
biolgicos que tm por funo
viabilizar a atividade celular
permitindo que reaes qumicas
apropriadas aos seu funcionamento
ocorram em escalas de tempo
compatveis com as suas
necessidades.

B i o c a t l i s e - u s o d e
catalisadores biolgicos para
promover transformaes
qumicas em compostos
orgnicos. Ex.: qumica fina,
ingredientes farmacuticos.
Estereoseletividade
Environmentally friendly
reactions

Trabalhos de Wilheln Friedrich Kuhne

Sugesto da palavra enzima (do grego en: na; zymos: levedura)
Fermentos organizados e no organizados

1850 Louis Pasteur identificou os microrganismos como os

responsveis pelas fermentaes (20 anos antes de Kuhne) > a ao
dos fermentos dependia de clulas vivas
1897 Edward Buchner: extratos de leveduras eram capazes de
fermentar acar a lcool >> Nobel de Qumica de 1907
Incio dos trabalhos com isolamento e estudo de enzimas
Urease James Sumner em 1926 (Nobel de Qumica de 1946) natureza
protica das enzimas
1930 John Howard Northrop e colaboradores cristalizaram a pepsina,
tripsina e papana >>enzimas so proteicas

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1934 John Desmond Bernal: usou a pepsina cristalizada para obter a

primeira imagem por difrao de raios X de uma protena globular.
Tecnologia enzimtica

Enzimas
De natureza protica: intensamente exploradas biotecnologicamente
Ribozimas: entendimento de processos bioqumicos celulares

Tripsina foi usada em 1908 para o tratamento do couro e em 1913 para a

produo de detergente.
1874 Emil Hansen extraiu a renina de estmagos secos de bezerros
primeira preparao enzimtica minimamente purificada usada para (fins
industriais) a fabricao de queijos holandeses
Entre 1833 e 1876 (e at hoje) peptidases e amilases eram as enzimas
mais utilizadas.
Pepsina foi a primeira enzima cientificamente purificada, caracterizada
(cristalizada) estruturalmente e tambm foi uma das primeiras enzimas
imobilizadas.

Ribozimas bem caracterizadas: peptidil-transferase do RNAr, ntrons

do grupo I (auto-splicing), RNase P e ribozimas cabea-de-martelo.
Substratos frequentemente molculas de RNA que pode ser parte
da prpria ribozima.
Reaes fundamentais: transesterificao e hidrlise da ligao
fosfodister
Estrutura tridimensional importante para a atividade
Tamanho varivel
ntrons do grupo I pode ter mais de 400 nucleotdeos
Ribozima cabea-de-martelo: 2 fitas com 41 nucleotdeos

Especificidade >>> Quiralidade >>> produo especfica de

determinados ismeros.
Separao de subprodutos eliminada
Reduo qualitativa e quantitativa de efluentes industriais

Economia
Reduo da energia de ativao (economia celular)
Condies brandas de temperatura (economia de energia na
indstria)

Eficincia nmero de renovao

Exigem condies especficas de pH, polaridade do solvente e fora
inica para a sua atuao.

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STIO ATIVO - Regio da

reao com o substrato.

molcula

enzimtica

que participa da

Necessidade de cofatores em ensaios in vitro e nos processos onde a

catlise explorada industrialmente.

Pode possuir componentes no proticos: cofatores.

Possui aminocidos auxiliares e de contato.

Poro protica
APOENZIMA Cofator

Ativador:ons inorgnicos que

condicionam a ao cataltica das
enzimas.
Coenzima: molcula orgnica
complexa.

HOLOENZIMA

Sculo XIX - poucas enzimas identificadas

Adio do sufixo ASE ao nome do substrato:
gorduras (lipo - grego) LIPASE; amido (amylon - grego)
AMILASE
Nomes arbitrrios
Tripsina ( do grego, frico) e pepsina (do grego, digesto)
proteases
Inadequao da nomenclatura >> 1956 estabelecimento da Comisso
de Enzimas (International Comission on Enzymes)
Listar sistematicamente as enzimas
To consider the classification and nomenclature of enzymes and coenzymes,
their units of activity and standard methods of assay, together with the symbols
used in the description of enzyme kinetics.

12/08/14

NOMENCLATURA

VISITE E EXPLORE

Registro da Comisso de Enzimas 1961 (Encontro da IUB em Moscou)

http://www.chem.qmul.ac.uk/iubmb/enzyme/

1964 - Recommendations (1964) of the International Union of Biochemistry on

the Nomenclature and Classification of Enzymes.
1972 Reviso completa do documento Enzyme Nomenclature e publicao
de um novo documento similar ao de 1964.
Aps 1972 Uso de suplementos contendo novas entradas, delees e
correes.
Publicao inicial no jornal Biochimica et Biophysica Acta
Deste 1984: European Journal of Biochemistry
Em 1992 3196 entradas (1961 712 entradas)

CLASSIFICAO: quanto reao qumica catalisada

NOMENCLATURA

ATP + D-Glicose
ADP + D-Glicose-6-fosfato

IUBMB ATP : glicose fosfotransferase

1. Oxidoredutases (reaes de oxidao-reduo ou transferncia de eltrons)

1.1.atuando em CH-OH
1.2.atuando em C=O
1.3.atuando em C=O1.4.atuando em CH-NH2
1.5.atuando em CH-NH1.6.atuando em NADH, NADPH

Doador de eltrons: aceptor de eltrons oxidoredutase

Ex: -D-Glucose:Oxignio Oxidoredutase
Glicose oxidase
E.C. 1.1.3.4

2.Transferases (transferem grupos funcionais entre molculas)

2 - Transferase
7 - Fosfotransferases
1 - Fosfotransferase que utiliza grupo hidroxila como receptor
1 - D-glicose o receptor do grupo fosfato

2.1.grupos com um carbono

2.2.grupos aldedo ou cetona
2.3.grupos acil
2.4.grupos glicosil
2.7.grupos fosfatos
2.8.grupos contendo enxofre

Doador : aceptor-grupo transferido-transferase

ATP:D-hexose 6-fosfotransferase
Hexoquinase

3.Hidrolases (reaes de hidrlise)

Nome trivial: Hexoquinase

3.1.steres
3.2.ligaes glicosdicas
3.4.ligaes peptdicas
3.5.outras ligaes C-N
3.6.anidridos cidos

Substrato Hidrolase
Lactose Galactohidrolase
Lactase
E.C. 3.2.1.108

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CLASSIFICAO: quanto reao qumica catalisada

Quanto a origem
Animais, vegetais e microrganismos
Intracelulares tcnicas adicionais para obteno
Extracelulares

4.Liases (removem grupos dos substratos formando duplas ligaes ou convertem

grupos adicionados em duplas ligaes)
4.1. =C=C=
4.2. =C=O
4.3. =C=N-

Substrato prefixo (tipo de reao catalisada)-liase

Ex: Isocitrato glioxalato-liase
Isocitrato Liase
E.C. 4.1.3.1

Quanto ao modo de ao
Endoenzimas
Exoenzimas

5.Isomerases (transferncia de grupos dentro da mesma molcula para formar

ismeros)
Substrato prefixo (tipo de isomerizao)-isomerase
5.1.Racemases

Ex: D-G3P aldose-cetose -isomerase

Triose fosfato isomerase
E.C. 5.3.1.1

Quanto ao modo uso

Enzimas como insumos
A enzima catalisa a reao principal
As enzimas complementam caractersticas do produto
A enzima o produto

6.Ligases (catalisam reaes de formao de novas molculas a partir da ligao

entre duas pr-existentes, sempre s custas de energia)
6.1. C-O
6.2. C-S
6.3. C-N
6.4. C-C

Substrato X: substrato Y Ligase

Ex: acetil CoA: dixido de carbono ligase
Acetil-CoA carboxilase
E.C. 6.4.1.2

346

Limitado a um nmero relativamente pequeno, em relao ao total de

enzimas conhecidas
Uso anal-co disposi-vos e
reagentes para diagns-co e
anlise qumica

Terapu-ca
e Frmacos
Inibidores

Enzimas

Protein technologies and commercial enzymes

Table 1
Enzymes used in various industrial segments and their applications.
Industry

Enzyme class

Application

Detergent (laundry and dish wash)

Protease
Amylase
Lipase
Cellulase
Mannanase
Amylase
Amyloglucosidase
Pullulanase
Glucose isomerase
Cyclodextrin-glycosyltransferase
Xylanase
Protease
Protease
Lipase
Lactase
Pectin methyl esterase
Pectinase
Transglutaminase
Amylase
Xylanase
Lipase
Phospholipase
Glucose oxidase
Lipoxygenase
Protease
Transglutaminase
Phytase
Xylanase
-Glucanase
Pectinase
Amylase
-Glucanase
Acetolactate decarboxylase
Laccase
Cellulase
Amylase
Pectate lyase
Catalase
Laccase
Peroxidase
Lipase
Protease
Amylase
Xylanase

Protein stain removal

Starch stain removal
Lipid stain removal
Cleaning, color clarification, anti-redeposition (cotton)
Mannanan stain removal (reappearing stains)
Starch liquefaction and saccharification
Saccharification
Saccharification
Glucose to fructose conversion
Cyclodextrin production
Viscosity reduction (fuel and starch)
effective
catalytic
Protease The
(yeast
nutrition
fuel) properties of enzymes have already
promoted
their introduction
into several
Milk clotting,
infant formulas
(low allergenic),
flavor industrial products and
Cheese flavor
processes. Recent developments in biotechnology, particularly in
Lactose removal
(milk)
areas such
as protein engineering and directed evolution, have
Firming fruit-based products
provided important tools for the efficient development of new
Fruit-based products
enzymes. This
has resulted in the development of enzymes with
Modify visco-elastic
properties
improved
foradjustment
established technical applications and in
Bread softness
andproperties
volume, flour
Dough conditioning
the production of new enzymes tailor-made for entirely new areas
Dough stability
and conditioning
(in situ emulsifier)
of application
where enzymes
have not previously been used.
Dough stability and conditioning (in situ emulsifier)
Dough strengthening
Addresses bread whitening
Dough strengthening,
Research and Development, Novozymes A/S, Krogshoejvej 36,
Biscuits, cookies
Bagsvaerd,
Laminated2880
dough
strengthsDenmark
*e-mail: oki@novozymes.com
Phytate digestibility
phosphorus release
Digestibility
Current Opinion in Biotechnology 2002, 13:345351
Digestibility

Starch and fuel

Indstria de
Alimentos e
Indstria
Qumica

Food (including dairy)

Baking

Tratamento de euentes
Indstria tx-l e produo de
papel e celulose

Animal feed
Beverage

Textile

Pulp and paper

Industrial enzyme applications

Ole Kirk*, Torben Vedel Borchert and Claus Crone Fuglsang

De-pectinization,
mashing see front matter
0958-1669/02/$
Juice treatment,
calorie
beer Ltd. All rights reserved.
2002low
Elsevier
Science
Mashing
DOI
10.1016/S0958-1669(02)00328-2
Maturation (beer)
Clarification (juice), flavor (beer), cork stopper treatment
Denim finishing, cotton softening
De-sizingIntroduction
Scouring The enzyme industry as we know it today is the result of a
Bleach termination
rapid development seen primarily over the past four decades
Bleachingthanks to the evolution of modern biotechnology. Enzymes
Excess dye removal
found in nature have been used since ancient times in the
Pitch control, contaminant control
production of food products, such as cheese, sourdough, beer,
Biofilm removal
wine and
vinegar,
and inimprovement
the manufacture of commodities such
Starch-coating,
de-inking,
drainage
Bleach boosting
as leather, indigo and linen. All of these processes relied on

century, aimed specifically at the productio

use of selected production strains, made it p
facture enzymes as purified, well-character
even on a large scale. This developme
introduction of enzymes into true industr
processes, for example, within the detergent,
industries. The use of recombinant gene
further improved manufacturing processes
commercialization of enzymes that could p
produced. Furthermore, the latest deve
modern biotechnology, introducing protein
directed evolution, have further revolutioni
ment of industrial enzymes (Figure 1). The
made it possible to provide tailor-made en
new activities and adapted to new process co
a further expansion of their industrial use.
Table 1, the result is a highly diversified in
growing both in terms of size and complexit

The majority of currently used industr

hydrolytic in action, being used for the
various natural substances. Proteases rema
enzyme type, because of their extensive
gent and dairy industries. Various carbohy
amylases and cellulases, used in indust
starch, textile, detergent and baking indu
the second largest group [1]. As illustrated
technical industries, dominated by the d

Food (including dairy)

Baking

Animal feed
Beverage

Textile

Pulp and paper

Fats and oils

Organic synthesis
Leather
Personal care

Amyloglucosidase
Pullulanase
Glucose isomerase
Cyclodextrin-glycosyltransferase
Xylanase
Protease
Protease
Lipase
Lactase
Pectin methyl esterase
Pectinase
Transglutaminase
Amylase
Xylanase
Lipase
Phospholipase
Glucose oxidase
Lipoxygenase
Protease
Transglutaminase
Phytase
Xylanase
-Glucanase
Pectinase
Amylase
-Glucanase
Acetolactate decarboxylase
Laccase
Cellulase
Amylase
Pectate lyase
Catalase
Laccase
Peroxidase
Lipase
Protease
Amylase
Xylanase
Cellulase
Lipase
Phospholipase
Lipase
Acylase
Nitrilase
Protease
Lipase
Amyloglucosidase
Glucose oxidase
Peroxidase

technical industries, first of all the detergent industry [2].

The fastest growth over the past decade has been seen in
the baking and animal feed industries, but growth is also
being generated from applications established in a wealth
of other industries spanning from organic synthesis to
paper and pulp and personal care. This review will, segment
by segment, discuss the most important recent developments
in the technical use of enzymes and will consider the

MERCADO MUNDIAL
Segundo dados do estudo World

Saccharification
Saccharification
Glucose to fructose conversion
Cyclodextrin production
Viscosity reduction (fuel and starch)
Protease (yeast nutrition fuel)
Milk clotting, infant formulas (low allergenic), flavor
Cheese flavor
Lactose removal (milk)
Firming fruit-based products
Fruit-based products
Modify visco-elastic properties
Bread softness and volume, flour adjustment
Dough conditioning
Dough stability and conditioning (in situ emulsifier)
Dough stability and conditioning (in situ emulsifier)
Dough strengthening
Dough strengthening, bread whitening
Biscuits, cookies
Laminated dough strengths
Phytate digestibility phosphorus release
Digestibility
Digestibility
De-pectinization, mashing
Juice treatment, low calorie beer
Mashing
Maturation (beer)
Clarification (juice), flavor (beer), cork stopper treatment
Denim finishing, cotton softening
De-sizing
Scouring
Bleach termination
Bleaching
Excess dye removal
Pitch control, contaminant control
The effective catalytic properties of enzymes have already
Biofilm removal
promoted
their introduction
into several industrial products and
Starch-coating,
de-inking,
drainage improvement
Bleach boosting
processes. Recent developments in biotechnology, particularly in
De-inking,
drainage
fiber modification
areas
such improvement,
as protein engineering
and directed evolution, have
Transesterification
provided important tools for the efficient development of new
De-gumming, lyso-lecithin production
enzymes.
has resulted
in the development of enzymes with
Resolution
of chiralThis
alcohols
and amides
improved
properties
for established technical applications and in
Synthesis
of semisynthetic
penicillin
Synthesis
of production
enantiopureofcarboxylic
acids tailor-made for entirely new areas
the
new enzymes
Unhearing,
bating
of application
where enzymes have not previously been used.
De-pickling
Antimicrobial (combined with glucose oxidase)
Addresses
Bleaching,
antimicrobial
Research and Development, Novozymes A/S, Krogshoejvej 36,
Antimicrobial
2880 Bagsvaerd, Denmark
*e-mail: oki@novozymes.com

12/08/14

PERFIL
A maioria das enzimas industriais obtida de microrganismos
345
Ferramentas como
metagenmica, protemica e bioinformtica so usadas para
prospeco, estudo e uso das enzimas.

Industrial enzyme applications

A produo em escala industrial pode ser feita usando tcnicas de
Ole Kirk*, Torben Vedel Borchert and Claus CroneFuglsang

most recent Current

technological
that2002,
have13:345351
facilitated
Opinion inadvances
Biotechnology
these developments.
0958-1669/02/$ see front matter
2002 Elsevier Science Ltd. All rights reserved.

New technologies for enzyme discovery

DOI 10.1016/S0958-1669(02)00328-2
Natural microorganisms
have over the years been a great
source of enzyme diversity. The developments in bioinforIntroduction
matics and the availability of sequence data have increased
Theefficiency
enzyme industry
as wean
know
it today is
the result of a
immensely the
of isolating
interesting
gene
rapid development seen primarily over the past four decades
thanks to the evolution of modern biotechnology. Enzymes
found in nature have been used since ancient times in the
production of food products, such as cheese, sourdough, beer,
wine and vinegar, and in the manufacture of commodities such
as leather, indigo and linen. All of these processes relied on
either enzymes produced by spontaneously growing microorganisms or enzymes present in added preparations such as
calves rumen or papaya fruit. The enzymes were, accordingly,
not used in any pure or well-characterized form. The developEnzymes (2009)
ment of fermentation processes during the later part of the last

fermentao submersa ou semi-slida.

century, aimed specifically at the production of enzymes by

Asmade
enzimas
so
obtidas do sobrenadante das culturas ou so extradas
use of selected production strains,
it possible
to manufacture enzymes as purified, well-characterized preparations
even on a large scale. This development allowed the
introduction of enzymes
into true industrial
and
Exemplos
deproducts
preparaes
disponveis comercialmente
processes, for example, within the detergent, textile and starch
industries. The use of recombinant
gene
technology
has
Preparaes de baixo custo e grandes volumes: malte como fonte de
further improved manufacturing processes and enabled the
amilases
e o not
sobrenadante
concentrado de fermentaes.
commercialization of enzymes that
could previously
be
produced. Furthermore, the latest developments within
Preparaes
purificadas: finalidade mdica, analtica e
modern biotechnology, introducing
protein engineering altamente
and
directed evolution, have further cientfica
revolutionized the development of industrial enzymes (Figure 1). These advances have
made it possible to provide tailor-made enzymes displaying
new activities and adapted to new process conditions, enabling
a further expansion of their industrial use. As illustrated in
Table 1, the result is a highly diversified industry that is still
growing both in terms of size and complexity.
The majority of currently used industrial enzymes are
hydrolytic in action, being used for the degradation of
various natural substances. Proteases remain the dominant
enzyme type, because of their extensive use in the detergent and dairy industries. Various carbohydrases, primarily
amylases and cellulases, used in industries such as the
starch, textile, detergent and baking industries, represent
the second largest group [1]. As illustrated in Figure 2, the
technical industries, dominated by the detergent, starch,
textile and fuel alcohol industries, account for the major
consumption of industrial
enzymes. Overall,
the estimated
MERCADO
MUNDIAL
value of the worldwide use of industrial enzymes has
grown from $1 billion [1] in
1995
to
$1.5
billion
in 2000 [2].
Segmentos:
This growth, however, has stagnated in some of the major

Enzimas especiais: >> 42,6% em 2014.

Custo elevado de algumas formulaes
Enzimas teraputicas: anticoagulantes, antitumorais, antibiticas,
Classical enzyme development
antiinflamatrias
Nature's diversity
Enzimas usadas no diagnstico
Molecular evolution
Enzimas usadas na pesquisa cientfica
Enzimas usadas em qumica fina

Crescimento da demanda mundial de 6,5%/ano.

Figure 1
China, ndia e Coria do Sul, com maior oportunidade
deversus
crescimento
The steps involved in classical
state-ofthe-art development of enzymes.
Present enzyme development
EUA como maiores consumidores
Creating biological
diversity

Segmentos:

Primary screening

Enzimas industriais: >> 60% do mercado (Biotecnologia Branca)

Secondary screening
Enzimas Tcnicas: setores de limpeza, txtil, couros, lcool
combustvel, papel e celulose. Exemplos: proteases, amilases,
Creating expression
system
lipases e celulases
Up-scaling process
Enzimas para alimentos (laticnios e pes) e bebidas: Amilases,
peptidases, pectinases, glicose isomerase
Production
Enzimas para rao animal aumento de digestibilidade e do
valor nutricional das raes. Ex.: fitases, xilanases, proteases e
amilases

Classical mutagenesis

Fermentation
Purification
Formulation

Up-scaling process
Production
Current Opinion in Biotechnology

12/08/14

Novas possibilidades >> ao longo do semestre

Melhoria nas caractersticas organolpticas dos alimentos

Controle mais efetivo do processo de fabricao
Menores gastos operacionais
Melhoria na funo enzimtica
Criao de novas enzimas abzimas (anticorpos catalticos,
p.ex.)

Exemplos:
Adio de enzimas ao queijos com o objetivo de alterar o
aroma e sabor
Uso da glucoamilase de A. niger na produo de cerveja
light
Melhoria na estabilidade e atividade enzimtica utilizando
tcnicas de engenharia gentica.