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CHAPTER 1
INTRODUCTION
1.1 ENZYMES FOR INDUSTRY
The nature’s only catalysts are considered to be the enzymes (Fariha
Hasan et al 2005). Enzymes offer the advantages of mild conditions of
reactions, specificity, and reduced wastage of chemicals. It is required in
0.1 % - 1.0 % of the substrate, minimal waste treatment cost and contribution
to the BOD, and the industries utilizing enzymes in their processes require
less cost to set up the facility. Most of the enzymes are from bacterial origin
and are very useful and stable than plant or animal origin. The advantages of
bacterial enzymes are consistent quality and availability throughout the year,
unlike the seasonal variation in plant sources. A total of only 2 % of the
microorganism in the world have been tested as enzyme sources. The
bacterial origin offer very high activities of enzymes, mostly are of neutral or
alkaline pH optimum, and thermostable. The catalytic properties of enzymes,
secreted by microorganisms, in the production of food products like cheese,
sourdough, vinegar, and in leather making, linen etc., are in use from 18th
century. The production of these microbial enzymes is cheaper, easier and
safer to produce by fermentation route and with the advent of recombinant
DNA technology it is produced with better or improved properties. The
recombinant gene technology offers many benefits to the enzyme industry.
This technology allows the use of safe, well documented organisms that are
easy to grow in fermentors, enhance the productivity, purity, activity and
stability. Genetic and environmental manipulation to increase the yield of
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cells (Demain 1971), to increase the enzyme activity of the cells by making
the enzyme of interest constitutive, or by inducing it, or to produce altered
enzymes (Betz et al 1974), may be employed easily using microbial cells
because of their short generation times, their relatively simple nutritional
requirements, and since screening procedures for the desired characteristic are
easier. The advent of recombinant DNA technology has led to
commercialization of many enzymes which could not be previously produced
in large quantities. The lowering of economic activity world-wide has not
crippled the pharmaceutical and biocatalyst enzymes industry. The increased
demand on industrial enzymes is focussed to reach $7 billion by the end of
2013. A new area of biocatalysis which is expanding dramatically is the
biotransformation for organic and fine chemical synthesis. Lipids form a
major composition of the earth’s biomass and to breakdown and transfer of
these lipids, lipolytic enzymes play a major role. Lipases are the principal
enzymes that are at the forefront of this development and are being used to
resolve the racemic mixtures, synthesis of chiral building blocks for
pharmaceuticals, agrochemicals and pesticides.
1.2 LIPASES - OCCURRENCE AND INDUSTRIAL
IMPORTANCE
Lipases (triacylglycerol hydrolases EC 3.1.1.3) are fat-splitting
‘ferments’ (Verger 1997) or lipid-hydrolysing enzymes that catalyse the
hydrolysis of water-insoluble esters of glycerol and long chain fatty acids.
The credit to the discovery of lipase goes to Claude Bernard, who in 1856 was
the first to report the existence of lipase in pancreatic juice which hydrolyzed
insoluble oil droplets into soluble products. The definition of lipase is just not
a carboxy - esterase that hydrolyses acyl glycerols but it is defined in terms of
kinetic terms based on interfacial phenomenon as a highly efficient enzyme
hydrolyzing substrates having a carboxylic ester group aggregated in water,
the property distinguishing lipases from esterases. These enzymes occur
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widely in nature and are found in most organisms from the microbial, plant
and animal kingdoms. The interest in microbial lipases was developed due to
a shortage of pancreas and the difficulties in the collection methods of other
sources of lipases. However lipases from the microbial flora comprising
bacteria, fungi and yeast have a wide-ranging commercial significance (Jaeger
and Reetz 1998) and the factors contributing to their industrial importance are
that they are i) stable in organic solvents, ii) do not require cofactors,
iii) possess a broad substrate specificity and iv) exhibit a high
enantioselectivity. The synthesis of biopolymers and biodiesel,
pharmaceutical products with enantiopurity, agrochemicals and flavour
compounds are the novel biotechnological applications that are carried out
with lipases. The Table 1.1 summarizes the sources of triacylglycerol lipases.
Table 1.1 Sources of triacylglycerol lipases
Sources Name
Mammalian Human Pancreatic Lipase
Horse Pancreatic Lipase
Pig Pancreatic Lipase
Guinea Pig Pancreatic Lipase
Fungal Rhizomucor meihei
Pencillium cambertii
Humicola lanuginose
Rhisopus oryzae
Candida rugosa
Candida antarctica lipase A
Candida antarctica lipase B
Aspergillus niger
Geotrichium candidum
Bacterial Chrombacterium viscosum
Pseudomonas cepacia
Pseudomonas aeruginosa
Pseudomonas fluorescens
Pseudomonas fragi
Bacillus thermocate nulatus
Staphylococcus hyicus
Staphylococcus aereus
Staphylococcus epidermidis
Courtesy: TIBTECH (1998)
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1.3 BIOCHEMICAL FEATURES OF LIPASES
The lipase family of enzymes exhibits a high degree of structural
and functional similarity. The three dimensional structure of lipases belong to
a characteristic folding pattern known as the hydrolase fold (Schrag and
Cygler 1993). The lipase structure is composed of a core of upto eight parallel
beta strands, connected and surrounded by alpha helices. The active site is
formed by a catalytic triad consisting of a serine residue as the nucleophile,
histidine as base and aspartic (or glutamic) acid as the acidic residue. The
active site residues are placed inside a hydrophobic pocket termed as
‘nucleophilic elbow’ and the pocket is covered by a lid like structure,
composed of one or two amphiphillic alpha helices. The activation of lipases
requires the opening up of the pocket by the displacement of the lid and this
process is known as interfacial activation
The catalytic mechanism of lipase hydrolysis consists of four
subsequent steps: i) the absorption and activation of the lipase at the interface
between aqueous and organic phase and then binding of the ester substrate
within the hydrophobic pocket; ii) in the second step, the nucleophilic oxygen
of the serine side chain attacks the carbonyl carbon atom of the ester bond
leading to the formation of a tetrahedral intermediate and this is stabilized by
one or two hydrogen bonding with amide nitrogen atom of the amino acid
residues located in the region called as ‘oxyanion hole’. iii) The ester bond is
then cleaved liberating an alcohol and leaves behind the acyl-enzyme
complex. In the last step, the acyl-enzyme is hydrolysed, when a water
molecule (sometimes another alcohol) enters the active site, thereby liberating
the free fatty acid (or a new trans-ester with the alcohol) and the enzyme is
regenerated. Example of lipase catalysis mechanism is given in Figure 1.1.
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Figure 1.1 Mechanism of lipase catalysis
Courtesy: Mats Holmquist (2000)
A wide range of assay protocols have been developed for the
estimation of lipase activity (Beisson et al 2000). Based on the general
triacylglycerol hydrolysis reaction, the lipase activity is assayed by the release
of either fatty acids or glycerol from triacylglycerols or fatty acid ester.
Various lipase assay methods and its principle are given in the Table 1.2.
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Table 1.2 Screening and analytical assays for lipase activity
Method and Materials Principle Remarks
i) Plate assay:Agar plates supplemented withTAGs such as tributyrin andtriolein (olive oil).
Lipolysis of TAGs producesa clear halo or colour
change of Phenol Red / Victoria Blue/ Nile Blue Sulphate, or fluorescencewith Rhodamine B under UV lightdue to dye-FFAs complexation.
Convenient for rapid screeningbut false-positives occur due tomedium acidification fromacidic metabolites
ii) Titrimetry:
pH stat (emulsified TAGs).
Simple titration using fats andoils, TAGs with / without pHindicators.
Potentiometric determination ofFFAs liberated upon hydrolysis.
Neutralization reaction of FFAs withNaOH to constant end-point pH.
Can generate kinetic constantsand needs expensive equipment.
Process is extremely timeconsuming and laborious.
iii) Spectrophotometry:Carboxylic esters of p-nitrophenol or 2, 4 dinitro- phenol.
Colorimetry: Fatty acidconjugates of -naphthol.
Turbidimetry: Tweens
Estimation of the hydrolysed yellow-coloured p-nitrophenol at 420 nm and2, 4-dinitrophenol at 360 nm.
Red colour of -naphthol monitoredat 505 nm, after complexation withdiazonium salt.
Precipitation of FFAs with calciumor copper and measurement ofturbidity at 500 nm.
Very simple procedure butassayed only for relatively purelipases.
The esters are not stable atextreme pH and are not lipasespecific substrates.
Simple, reproducible, sensitiveand generally used for plateassays
Synthetic dilauryl glycerolresourfin ester
Resourfin released by lipasehydrolysis, measured for absorbanceat 575 nm.
Not straightforward to use sinceit requires separation of reactantsand products.
iv) Fluorescence:Fluorogenic substrates such as
–linked pyrenic acyl-glycerolderivatives.
Non-flourescent 4-methylumbelliferyl oleate.
Lipolytic activity quantified in termsof increasing fluorescence intensitywith time.
Highly fluorescent 4 - methylumbelliferone released after lipaseaction and is therefore quantified.
Rapid assay but expensivesubstrates limit its usage.
Most sensitive assay butexpensive substrates.
v) Chromatographic procedures:TLC / GC / HPLC, and usingradiolabelled TAGs incase ofquantitative analysis
Analysis and quantification of theproduct or residual substrate throughspecific columns.
Time-consuming and not suitedfor routine analysis.
vi) Interfacial tensio-metrymethods
Monomolecular film technique:Wilhelmy Plate method
Atomic forcemicroscopy:Infrared spectroscopy:
Lipolysis at lipid / water interfaceand so interfacial surface pressureregulated by substrate supply.
TAG bilayers supported on micahydrolyzed by lipases and regions ofthese indentations detected andstudied as function of time with AFMtip.
Lipase activity of TAGs in reversemicelles, monitored by recordingfourier-transform IR spectroscopyand products quantified using themolar extinction coefficients.
Extensive setup but providesprecise kinetic data.
Suited for kinetic modeling oflipase action but requiressophisticated instruments.
Expensive and sophis-ticatedinstruments involved.
Courtesy: Biotechnol. Appl. Biochem (2003)
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1.4 CANDIDA ANTARCTICA LIPASES
Among the widely used enzymes for biocatalytical purposes are the
lipases produced by different strains of genus Candida sp. In the late 1960’s
the yeast strain Candida antarctica was isolated from Lake Vanda in
Antarctica and was found to produce two different lipases (CALA and
CALB). The lipases produced by Candida antarctica are stable when
immobilized and the enzymes retain their activity at higher temperatures for
longer durations without significant loss in the enzyme activity. The two
lipases of C. antarctica were characterized and the amino acid and the DNA
sequences encoding these lipases were sequenced at Novo Nordisk A/S, by
Patkar, Hoegh and others. The lipase B was later crystallized and its structure
was also determined by Uppenberg et al (1994).
C. antarctica lipase B is found to exhibit varying physio-chemical
characteristics and is summarized in Table 1.3 (Kirk and Christensen 2002).
The lipase B has become one of the most prominent enzymes, in organic
synthesis, especially for the kinetic resolution of racemates. Currently, lipase
B is the widely targeted enzyme for protein engineering so as to improve and
optimize its substrate specificity and enantioselectivity (Lutz 2004).
Table 1.3 Characteristics of CALB
Characteristics CALB
Molecular weight (kDa)
Isoelectric point (pI)
pH optimum
Thermostability at 70°C
pH stability
33
6.0
7
15
7 - 10
Courtesy: Ole Kirk and Wurtz Kristensen (2002)
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As both C. antarctica lipases have gained significant commercial
importance and as the expression levels in the native organism are too low,
recombinant over-expression is needed for the large-scale production of these
biocatalysts.
1.5 PICHIA PASTORIS RECOMBINANT EXPRESSION SYSTEM
In the production of recombinant proteins, yeasts have offered
advantages over both bacterial and mammalian systems. The eukaryotic yeast
offers the capacity to post-translationally modify the secreted protein and to
glycosylate at lesser costs incurred in using mammalian system for
expression. The first yeast selected for production of heterologous eukaryotic
proteins was Saccharomyces cerevisiae, due to the accumulated knowledge of
its genetics and physiology. It is generally regarded as safe (GRAS) for
human use through experience with the organism in brewing and baking
industry. However, in the 1970s, the methylotrophic yeasts gained significant
importance, due to its rather unique ability to anabolise methanol to very high
cell mass and was used as a cattle feed as single cell protein. Later in the
following decade, Phillips Petroleum, together with Salk Institute
Biotechnology / Industrial Associates (SIBIA, CA) developed the
methylotrophic yeast Pichia pastoris into an efficient recombinant protein
production system. Pichia pastoris belonging to the yeast family are easy to
manipulate genetically as E. coli or Saccharomyces cervisiae. They have a
relatively high growth rate than higher eukaryotes and can be grown to higher
cell densities than bacteria, producing a 10 – 100 fold higher heterologous
protein.
Pichia pastoris has become an ideal host for the expression of
recombinant proteins (Cereghino and Cregg 2000) due to the contribution of
following factors: it can be easily manipulated at the molecular genetic level
(e.g. gene targeting, high-frequency DNA transformation, cloning by
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functional complementation); it can express proteins at high levels due to its
highly efficient and tightly regulated promoter from the alcohol oxidase I
gene (AOX1); and it can perform many ‘higher eukaryotic’ protein
modifications, such as glycosylation, disulfide-bond formation, and
proteolytic processing.
1.5.1 Genetics and Properties
P. pastoris, a methylotropic yeast is one of approximately a dozen
yeast species representing four different genera that are capable of
metabolizing methanol as its sole carbon source. The other genera include
Candida , Hansenula and Torulopsis. The methanol utilization pathway, of
these microbial genera is highly inducible and involves several unique
enzymes that take place initially in the peroxisomes where methanol is
oxidized to formaldehyde and hydrogen peroxide using molecular oxygen
from the AOX1 gene (Tschopp et al 1987b). The subsequent metabolic steps
take place in the cytoplasm. The first enzyme of the pathway, alcohol oxidase
(AOX1 and AOX2), can account for up to 35 % of the total protein in cells
when grown on methanol, whereas it is undetectable in cells grown on
glucose, glycerol or ethanol (Sreekrishna et al 1997). This highly inducible
and stringently regulated alcohol oxidase gene (AOX1) promoter has been
used to express heterologous proteins in P. pastoris.
There are three phenotypes of P. pastoris strains with regard to
methanol utilization and these phenotypic characteristics are an important
factor during P. pastoris cultivation process and protein production
(Macauley-Patrick et al 2005). The methanol utilization plus phenotype, or
Mut+ grows on methanol at the wild-type rate and require high feeding rates
of methanol in large-scale fermentations. A maximum of 3 % methanol is
used to express protein cloned in Mut+ strains. The MutS, or methanol
utilization slow phenotype, have a disruption in the AOX1 gene. The cells
then rely on the weaker secondary alcohol oxidase gene AOX2 for methanol
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metabolism and slower methanol utilization strain is produced. A maximum
of 1 % methanol is used to express protein cloned in MutS strains. The Mut ,
or methanol utilization minus phenotype, are unable to grow on methanol,
since these strains have both AOX genes deleted. Deletion of AOX genes does
not affect the strains ability to induce expression at high levels from the AOX1
promoter (Chiruvolu et al 1997). The different Mut phenotypes have
exhibited varied effects for the production of recombinant proteins. The lower
growth and protein production of MutS on methanol is particularly preferable
for production of recombinant proteins where the folding is rate-limited.
Though Mut+ expression is higher than that with cells of the MutS phenotype
when higher methanol concentrations were used, the former strains may get
oxygen limited at high methanol concentrations, leading to cell lysis.
Recombinant P. pastoris strains are obtained by transforming the
host strains with the constructed plasmid, which on electroporation gets
integrated into the chromosome at a specific locus and generates a genetically
stable transformants / clone. Chromosomal integration is highly desirable than
the use of episomal plasmid expression systems as episomal plasmids tend to
have low copy number, which affects the amount of product expressed.
Integration into the genome can occur via homologous recombination when
the vector / expression cassette contains regions that are homologous to the
P. pastoris genome and the integration can occur via gene insertion or gene
replacement. Integration by gene insertion can result in tandem multiple
integration events due to repeated recombination events at a rate of 1 – 10 %
of transformants. Integrations are usually site-directed at the HIS4 gene or at
the primary alcohol oxidase (AOX1) locus. Transformations that target gene
replacement generally result in single copy transformants; however, gene
replacement transformants are usually more genetically stable. Figure 1.2 and
Figure 1.3 illustrate the gene integration and gene replacement event that
occur in Pichia genome by homologous recombination.
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Figure 1.2 Integration of expression cassette by gene replacement in
Pichia genome
Figure 1.3 Integration of expression cassette by gene insertion in Pichia
genome
(Courtesy: Invitrogen life technologies, Multi-Copy Pichia Expression Kit, Manual,
p. 63, p. 66)
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1.5.2 P. pastoris AOX system
Methylotrophic yeast, Pichia pastoris can utilize methanol as the
sole source of carbon and energy (Figure 1.4). Methanol induces a specific
methanol utilization pathway leading to expression of key enzymes under the
control of tightly regulated promoters. One of these key enzymes, alcohol
oxidase (AOX), catalyses the oxidation of methanol to formaldehyde and
hydrogen peroxide. Since hydrogen peroxide is very toxic to the cells, the
reaction takes place in specialized organelles called peroxisomes, where
hydrogen peroxide is degraded into oxygen and water by the activity of the
enzyme catalase (Cereghino et al 2000). AOX is encoded by two genes in
P. pastoris namely, AOX1 and AOX2, former being responsible for the
majority of alcohol oxidase activity in the cell (Cregg et al 1989). The AOX1
gene expression is controlled at the level of transcription and the presence of
methanol is essential to induce high levels of transciption. 5 % of RNA
isolated from methanol grown cells is from the AOX1 gene, whereas AOX1
message is undetectable in cells grown on any other carbon source (Tschopp
et al 1987a). In a bioreactor, cultures with methanol feeding at growth
limiting rates, AOX1 transcription levels can be as high as 30 % of total
soluble protein. Like the S. cerevisiae GAL1 gene, AOX1 gene is under the
control of two mechanisms, repression / derepression and induction. The
repressing carbon source can be any other carbohydrate other than methanol,
does not result in transcription of AOX1 (Higgins and Cregg 1998).
1.5.3 P. pastoris expression strains
Pichia pastoris strains are now available with a wide variety of
genotypes. All P. pastoris expression strains are derived from NRRL - Y
11430 (Northern Regional Research Laboratories, Peoria, IL). The strains
have one or many auxotrophic mutations which help in selection of
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Figure 1.4 The methanol pathway in P. pastoris
Courtesy: Michael M. Meagher (2000)
1:alcohol oxidase, 2: catalase, 3: formaldeyde dehydrogenase, 4:
formate dehydrogenase, 5:dihydroxyacetone synthase,
6:dihydroxyacetone kinase, 7: fructose 1,6-biphosphate
aldolase, 8: fructose 1,6-biphosphatase (Cereghino et al 2000).
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expression vectors having the selectable marker gene when transformed. All
of these strains with the mutations grow on complex media but require
supplementation with the appropriate nutrient(s) like histidine for growth on
minimal media after transformation.
P. pastoris strains KM71, GS115 and SMD1168 are defective in the
histidine dehydrogenase gene (his4). These strains allow the transformants to
be selected based on their ability to grow in non - histidine containing agar
media. The Pichia strains X–33 and GS115 are differentiated from other
strains based on their ability to utilize methanol as they have a functional
copy of the alcohol oxidase 1 gene (AOX1) responsible for approximately
85 % of the utilization of methanol by the alcohol oxidase enzyme and are
designated as Mut+ phenotype. P. pastoris strains KM71 and KM71H have a
partial insertion in AOX1 gene and thus rely only on AOX2 for methanol
utilization. This strain grows slower than wild type strains on methanol
showing MutS phenotype (methanol utilization slow phenotype).
The Pichia strains such as SMD1163 (his4 pep4prb1), SMD1165
(his4 prb1) SMD1168 (his4 pep4) are protease deficient strains, and they have
been shown to be effective in reducing degradation of some foreign proteins
(Brierley et al 1998, White et al 1995). These strains have a disruption in the
genes encoding proteinase A (PEP4) and / or proteinase B (PRB1)
(Sreekrishna et al 1997). Proteinase A is a vacuolar aspartyl protease
necessary for the activation of vacuolar proteases, such as carboxypeptidase Y
and proteinase B. Proteinase B has about half the activity of the processed
enzyme before being activated by proteinase A. Therefore, pep4 mutants
eliminate the activity of proteinase A and carboxypeptidase Y, and partially
reduce proteinase B activity. The prb1 mutants eliminated activity of
proteinase B, whereas pep4 prb1 double mutants showed a significant
reduction or elimination of all three of these protease activities (Jahic et al
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2003). Various P. pastoris strains used for heterologus protein production are
given in Table 1.4 with their features.
Table 1.4 Genotype and Phenotype of P. pastoris strains
Strain Genotype Phenotype Reference
GS115 his4 Mut+, His- (Cregg et al 1985)
KM71his4, aox1:
ARG4;arg4Muts, His-
X-33 Wild type — (Li et al 2001)
SMD1168 His4, pep4 Mut+, His-, pep4- (White et al 1995)
SMD1165 his4, prb1 Mut+, His-, prb1- (Abdulaev et al 1997)
SMD1163his4, prb1,
pep4
Mut+, His-,
pep4, prb1- (Sreekrishna et al 1997)
(Courtesy: Invitrogen life technologies, Multi-Copy Pichia Expression Kit, Manual)
1.5.4 Alternative promoters
Although the AOX1 promoter has been successfully used to express
numerous foreign genes, there are circumstances in which this promoter may
not be suitable. Alternative promoters to the AOX1 promoter are the
P. pastoris GAP, FLD1, PEX8, and YPT1 promoters. P. pastoris
glyceraldehyde 3 - phosphate dehydrogenase (GAP) gene promoter provides
strong constitutive expression on glucose at significant level (Waterham et al
1997). Since the GAP promoter is constitutively expressed, it is not a good
choice for the production of proteins that are toxic to the yeast. The FLD1
gene encodes a glutathione - dependent formaldehyde dehydrogenase, a key
enzyme required for the metabolism of certain methylated amines as nitrogen
sources and methanol as a carbon source (Shen et al 1998). The FLD1
promoter can be induced with either methanol as a sole carbon source (and
ammonium sulfate as a nitrogen source) or methylamine as a sole nitrogen
source (and glucose as a carbon source). The FLD1 promoter offers the
flexibility to induce high levels of expression using either methanol or
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methylamine, an inexpensive nontoxic nitrogen source. For proteins where
there are limitations in the post translational machinery, the P. pastor is PEX8
and YPT1 promoters may be of use. The PEX8 gene encodes a peroxisomal
matrix protein that is essential for peroxisome biogenesis (Sears et al 1998). It
is expressed at a low but significant level on glucose and is induced modestly
when cells are shifted to methanol. The YPT1 gene encodes a GTPase
involved in secretion, and its promoter provides a low but constitutive level of
expression in media containing glucose, methanol, or mannitol as carbon
sources
1.5.5 Glycosylation
Glycosylation is the most common post - translational modification
of proteins prior to protein secretion. Approximately 0.5 – 1.0 % of the
translated proteins in eukaryotic genomes are glycoproteins. Glycosylation
occurs in the lumen of the endoplasmic reticulum after protein translation. It
is thought that since many mammalian native proteins are glycosylated, it
must be necessary to have the correct glycosylation patterns on recombinant
proteins to ensure their biological activity. In yeast the glycosylation pattern is
different that of mammalian systems. In N - linked glycosylation of proteins
the mannose units added are in excess compared to mammalian systems.
Since the glycosylated gene products from P. pastoris generally have much
shorter of 10 to 12 mannose subunits than those expressed in S. cerevisiae
which will have 20 – 50 mannose units, it is much more attractive host for the
expression of recombinant proteins (Bretthauer et al 1999). P. pastoris is
capable of forming both O - and N - linked glycosylation to the secreted
proteins (Goochee et al 1991). Pichia - derived glycosylated proteins have the
potential to trigger inappropriate immune responses if used as
pharmaceuticals. The immunogenicity of Pichia - derived proteins is an issue
that has attracted interest in the literature with regard to humanizing the
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glycosylation patterns (Asami et al 2000, Choi et al 2003). Candida
antarctica lipase B gene was cloned in E. coli and in Pichia pastoris and the
function of non glycosylated lipase B produced is similar to that of wild type
Pichia pastoris, indicating glycosylation is not required for the activity (Gaub
et al 2006, Hult et al 2008).
1.5.6 Gene dosage
Gene copy number has been identified as a ‘rate - limiting’ step in
the production of recombinant proteins from P. pastoris (Clare et al 1991).
Increasing the number of copies of the expression cassette generally has the
effect of increasing the amount of protein expressed (Clare et al 1991,
Romanos et al 1995, Vassileva et al 2001).
A number of researchers have had significant success with
increasing gene dosage utilizing both the AOX1 and GAP promoters (Clare
et al 1991, McGrew et al 1997, Vassileva et al 2001), therefore, before
considering increasing the gene copy number as an optimization strategy for
recombinant protein production from P. pastoris, the identity of the promoter
must be considered in advance. Depending on the type of protein, upto certain
gene copy number there is proportional effect in the recombinant protein
production after which there is no increase in productivity. Increasing the
gene copy number might reasonably be expected to exert a knock - on effect
on transcription and translation, both of which may become rate - limiting due
to a lack of resources, such as precursors and energy (Hohenblum et al 2004).
However, in Pichia it has been proposed that it is more likely that any
limitations are due to post - translational events, such as folding within the
endoplasmic reticulum, membrane translocation and signal sequence
processing.
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1.5.7 Codon Usage
In most of the expression studies in P. pastoris the gene containing
higher percentage of AT rich regions are poorly expressed due to improper
transcription termination of mRNA. For better expression, these genes have to
be modified with respect to Pichia preferred codon. The frequency of codon
usage from highly expressed proteins is given in Table 1.5. The optimal GC
content for good expression of protein is around 40 - 45 % and this is
achieved by altering the codon to most Pichia preferable one.
1.5.8 Protein secretion
One of the most valuable option in recombinant Pichia pastoris
expression system is the availability of secretion signals that can be flanked to
the protein of interest, causing the protein to be secreted out of the cell into
the medium used for growth.
A variety of secretory signal sequences are used such as the
P. pastoris acid phosphatase signal (PHO), yeast invertase signal (SUC2)
(Chang et al 1986; Payne et al 1995), factor secretion signal etc, (including
native signals sequence present in the parent organism to secrete the protein)
have been used with success. During the recombinant protein expression, the
endopeptidase sequence (Glu-Lys-Arg*) separates the signal sequence from
the protein coding gene and the yeast Kex2 signal peptidase cleaves the signal
peptide at this sequence and the mature protein is secreted in the culture
medium. Each signal has its particular advantage and there is no common rule
by which the most effective sequence can be used. The S. cerevisiae MAT
prepro signal peptide has found most success and is the one of most
frequently incorporated into P. pastoris expression vectors (Sreekrishna et al
1997). The factor, a yeast peptide pheromone consisting of 13 amino acid
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Table 1.5 Frequency of codon usage in highly expressed Pichia pastoris
genes
Amino acid Codon Number Fraction Amino acid Codon Number Fraction
Gly GGG 0.00 0.00 Trp TGG 39.00 1.00
Gly GGA 59.00 0.22 End TGA 0.00 0.00
Gly GGT 197.00 0.74 Cys TGT 35.00 0.83
Gly GGC 9.00 0.03 Cys TGC 7.00 0.17
Glu GAG 112.00 0.58 End TAG 1.00 0.20
Glu GAA 80.00 0.42 End TAA 4.00 0.80
Asp GAT 56.00 0.32 Tyr TAT 18.00 0.12
Asp GAC 118.00 0.68 Tyr TAC 128.00 0.38
Val GTG 10.00 0.05 Leu TTG 120.00 0.52
Val GTA 8.00 0.04 Leu TTA 21.00 0.09
Val GTT 107.00 0.50 Phe TTT 24.00 0.19
Val GTC 87.00 0.41 Phe TTC 104.00 0.81
Ala GCG 1.00 0.00 Ser TCG 6.00 0.03
Ala GCA 25.00 0.10 Ser TCA 14.00 0.07
Ala GCT 147.00 0.60 Ser TCT 89.00 0.47
Ala GCC 71.00 0.29 Ser TCC 71.00 0.37
Arg AGG 2.00 0.01 Arg CGG 2.00 0.01
Arg AGA 111.00 0.79 Arg CGA 0.00 0.00
Ser AGT 8.00 0.04 Arg CGT 26.00 0.18
Ser AGC 3.00 0.02 Arg CGC 0.00 0.00
Lys AAG 145.00 0.79 Gln CAG 31.00 0.34
Lys AAA 38.00 0.21 Gln CAA 59.00 0.66
Asn AAT 18.00 0.13 His CAT 11.00 0.13
Asn AAC 119.00 0.87 His CAC 77.00 0.88
Met ATG 60.00 1.00 Leu CTG 35.00 0.15
Ile ATA 0.00 0.00 Leu CTA 7.00 0.03
Ile ATT 93.00 0.56 Leu CTT 43.00 0.18
Ile ATC 72.00 0.44 Leu CTC 7.00 0.03
Thr ACG 5.00 0.03 Pro CCG 0.00 0.00
Thr ACA 8.00 0.05 Pro CCA 97.00 0.57
Thr ACT 86.00 0.50 Pro CCT 66.00 0.39
Thr ACC 74.00 0.43 Pro CCC 7.00 0.04
Courtesy: http://www.kazusa.or.jp/codon/
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residues plays an important role in the process of mating initiation. It is
known that factor is initially synthesized as a larger precursor,
prepro- -factor (Brake et al 1984). Prepro factor consists of a signal
sequence, prosegment and the repeats of spacer peptides followed by mature
factor sequences. This leader sequence is popular for its high level of
expression in the yeast system (Brake et al 1984, Hashimoto et al 1998a, b).
In certain cases the recombinant protein expression have been tried with its
native signal sequence of the protein and found to produce more stable
recombinant protein than using factor signal sequence (Koganesawa et al
2001). The Saccharomyces cerevisiae secretion signal peptide ( -factor) was
used with AOX1 promoter for the production of Yarrowia lipolytica and very
high activity was produced in fed – batch cultivation (Tan et al 2007). This
showed the possibility that there is some influence in the secretion of
recombinant protein with its secretion signal sequence.
1.5.9 Fermentation of Pichia pastoris
A property of the P. pastoris system is the ease with which
expression strains can be scaled - up from shake flask to high cell density
fermenter cultures. Efforts have gone into the optimization for the production
of heterologous proteins which have led to availability of detailed fed-batch
and continuous culture protocols (Stratton et al. 1998). The advantage of
P. pastoris relative to S. cerevisiae is that it prefers a respiratory pathway
rather than a fermentative mode of growth. In high cell density cultures of
S. cereviseae, ethanol (the product of S. cerevisiae anaerobic fermentation)
rapidly builds to toxic levels which limit further growth and foreign protein
production. Since Pichia pastoris prefers respiratory growth, it can be
cultured to high cell densities (500 - 600 OD600) in the controlled environment
of the fermenter with little risk of `pickling' itself. Most importantly, it is only
21
in fermenter, where parameters such as pH, aeration and carbon source feed
rate can be controlled, that it is possible to achieve high expression of foreign
protein.
The high salts / high cell density fermentation protocol was first
reported by Brierley et al (1990) using P. pastoris which expressed bovine
lysozyme C2 as a model system and resulted in an expression of up to
600 mg/L of the recombinant lysozyme. Currently, the three-stage process is
typically utilized for the production of foreign proteins in fermenter cultures
of P. pastoris. In the first stage, the recombinant strain is batch-cultured in a
simple defined medium with a non-fermentable carbon source such as
glycerol to accumulate biomass. The second stage is a fed - batch transition
phase in which glycerol is fed to the culture at a growth - limiting rate to
further increase the biomass concentration and to prepare (derepress) the cells
for induction. The third stage, induction phase, is started by adding methanol
to the culture at a slow rate, which facilitates the culture’s acclimation to
methanol and initiates the synthesis of the recombinant protein. The methanol
feed rate is then adjusted upwards periodically until the desired growth rate is
reached.
The methanol feeding strategy during the induction phase is one of
the most important factors for maximizing heterologous protein production
(Cos et al 2006). Monitoring and control of methanol concentration is a key
parameter in P. pastoris expression system and involves different strategies
such as using on-line methanol sensors either for liquid or gas phase, by
dissolved oxygen level, or by constant feeding rate to optimize the induction
phase. Moreover the methanol induction phase also depends on the optimum
conditions (eg: temperature, pH and culture medium), phenotype and specific
characteristics of the heterologous protein product.
22
The methanol feeding strategy differs with regard to the methanol -
utilising ability of P. pastoris strains. For the Mut+ strains, strategies based on
the pO2 of the culture have led to high yields of recombinant proteins with
respect to the amount of methanol fed and to the biomass formed (Invitrogen
2002). This method is based on modifying the methanol feeding rates in order
to avoid methanol exhaustion as indicated by a sharp increase in dissolved
oxygen. A rational feeding strategy proposed by Zang et al (2000) which is
based on the predetermined exponential rate to maintain a constant desired
growth rate has also shown to be more efficient in the recombinant protein
production. Generally it has been observed that when the cells are fed with
methanol at a growth-limiting rate, the induction of protein expression is
almost 3 to 5 times higher than in cells growing in excess of methanol (Trinh
et al 2003).
The fermentation strategy for MutS KM71 strain was the same as
with Mut+ GS115 strains. The methanol feed rate was lesser in order to
maintain methanol at a concentration between 0.2 % and 0.8 % in the
fermentor. The methylotrophic yeast strains when grown under limiting
concentrations of methanol leads to the formation of alcohol oxidase and
catalase in their peroxisomes. The breakdown product of methanol by alcohol
oxidase, hydrogen peroxide is degraded by catalase. The supplementation of
the media with hydrogen peroxide during induction phase along with
methanol increases oxidation of methanol (Duff 1990). However, in some
reports it was suggested that feeding multicarbon substrates in addition to
methanol show increased productivities (Zang et al 2003). The most used
co-substrate has been glycerol, but it is important to optimize the ratio of
methanol to glycerol in the substrate feeding rate, as excess glycerol represses
the heterologous protein production. Other carbon sources such as sorbitol,
mannitol, alanine and lactic acid have also been used as co-feeding substrates
(Xie et al 2005). These substrates have the advantage that they are non AOX1
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repressive carbon sources and have shown increased protein productivities but
their biomass yields are lower than glycerol.
A major problem that has hampered the protein productivities
during Pichia pastoris fermentations has been with proteolysis. The secreted
recombinant proteins are proteolytically degraded in the culture medium by
the proteases, secreted or released from the lysed cells (Sinha et al 2004). The
use of protease-deficient strains has been found to enhance the yield and the
quality of various heterologous proteins but these strains are not as vigorous
as wild strains and have lower viability (Cereghino and Cregg 2000).
Nitrogen limitations have also been found to increase protease activity
(Kobayashi et al 2000) and maintaining the ammonium requirements as well
as addition of amino-acid rich supplements (eg. peptone, casamino acids) to
the culture medium (Ohya et al 2002) has been observed to reduce
proteolysis.
Another major problem associated with the production of
recombinant proteins using Brierleys medium composition is that a lipid layer
is formed during fermentation, may be due to the increased lysis of cells. So
the fermentation strategy was shifted to reduced concentration of the medium
composition suggested by Brierley et al to half (Brady et al 1998). The
reduction of salt concentration reduced the lipid layer formation which
hindered in downstream processing.
The yields of some recombinant protein have also been influenced
by lower cultivation temperature (Hong et al 2002) and lowering of pH values
(Jahic et al 2002) possibly due to poor stability of these proteins, folding
problems at higher temperatures, and release of more proteases from dead
cells at higher pH. Therefore, achieving good expression levels using Pichia
pastoris recombinant expression systems involves an optimum combination
of the cultivation strategies.
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1.6 OBJECTIVES
The current work is an attempt to clone and express Candida
antarctica lipase B using recombinant DNA technology. The CALB has a
very broad range of substrate specificity and the main factor which limits the
industrial use of CALB is the high price of the immobilized enzyme product.
In an attempt to reduce the cost factor of the immobilized enzyme, different
strains of Pichia pastoris have been used for transformation. To attain this
goal the objectives of the current work was designed as follows:
Cloning of CALB in pPICZ B vector, transforming into Pichia
pastoris GS115 and KM71 strains and checking the level of
lipase activity.
Increasing the copy number of CALB in pPICZ B (Multicopy)
and pPIC9k (High copy), transforming into Pichia pastoris
GS115 strain and checking the level of lipase activity.
High cell density fermentation of all the Pichia clones to
maximize productivity of lipase B.