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Review of Literature

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Lactic acid bacteria (LAB) are grouped under the phylum Firmicutes and consisting of a

number of Gram-positive bacterial genera. The recognized lactic acid bacteria belong to

the genera Aerococcus, Alloiococcus, Carnobacterium, Dolosigranulum, Enterococcus,

Globicatella, Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus,

Streptococcus, Tetragenococcus, Vagococcus and Weissella (Khalid, 2011). They are

Gram-positive, catalase-negative, aerotolerant, solely fermentative bacteria that gain their

energy from substrate-level phosphorylation. In general they grow well at pH 6.5. Most

strains lack several common metabolic pathways and thus require complex media

containing amino acids and vitamins for their growth. Many lactic acid bacteria are able

to grow in high sugar environments such as on the surface of fruits, vegetables, in milk,

and on the surface of skin.

There are two major physiological groups of lactic acid bacteria;

homofermentative lactic acid bacteria (such as Lactococcus and Streptococcus) and

heterofermentative lactic acid bacteria (Leuconostoc and Weissella) (Stackebrandt and

Teuber, 1988; Thompson, 1988). The homofermentative lactic acid bacteria produce

lactic acid as the sole fermentative product whereas the heterofermentative lactic acid

bacteria produce approximately equimolar amounts of lactic acid, ethanol and CO2

(Caplice and Fitzgerald, 1999; Jay, 2000; Kuipers et al, 2000).

LAB have several attractive key characteristics such as production of flavor,

antibacterial agent, perfuming the fermented product and imparting a texture to fermented

foods (Caplice and Fitzgerald, 1999). They are being used as starter in fermentation of

foods for centuries in food industry and having GRAS (Generally Recognized As Safe)

status. Some of the LAB species produce small heat-stable antibacterial compound

named as bacteriocin. Among these, nisin produced by certain strains of Lactococcus

lactis is mostly studied and widely applicable (Liang et al, 2010; Parada et al, 2007).

Lactococcus lactis

In the world literature the first bacterium that was isolated from pure culture by Josheph

Lister in 1873 was Streptococcus lactis which was reclassified further and named as

Lactococcus lactis (Suganthi et al, 2012). It is a microbe classified as a Lactic Acid

Bacterium because it ferments milk sugar (lactose) to lactic acid. Lactococci are typically

spherical or ovoid cells, about 1.2 µm by 1.5 µm, occurring in pairs and short chains.


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They are Gram-positive, non motile, and do not form spores. They have a

homofermentative metabolism and produced lactic acid as a sole fermentative product.

Based on its history in food fermentation L. lactis has the GRAS status (Generally

Regarded As Safe). L. lactis is one of the best characterized low G+C, Gram-positive

bacteria having detailed knowledge on genetics, metabolism and diversity.

L. lactis is a food-grade bacterium that is widely used as starter culture in milk

fermentation. L. lactis is vital for manufacturing cheeses such as Cheddar, Colby, cottage

cheese, cream cheese, Camembert, Roquefort and Brie, as well as other dairy products

like cultured butter, buttermilk, sour cream and kefir. It may also be used for vegetable

fermentations such as cucumber pickles and sauerkraut. The bacterium can be used in

single strain starter cultures, or in mixed strain cultures with other lactic acid bacteria

such as Lactobacillus and Streptococcus species. When L. lactis is added to milk, the

bacterium uses enzymes to produce energy molecules (ATP) from lactose. The byproduct

of ATP production is lactic acid. The lactic acid curdles the milk that then separates to

form curds, which are used to produce cheese and whey. L. lactis are widely distributed

in nature and are commonly found on plant surfaces and in milk (Salama et al, 1995).

L. lactis has two subspecies with few phenotype and genotype differences, L.

lactis subsp. lactis and subsp. cremoris. These organisms were originally classified under

the genus Streptococcus, but in 1985, it was assigned to the current genus. Both the

strains display high level of similarity in both phenotypic and genotypic properties. But

L. lactis subsp cremoris differ from L. lactis subsp lactis by a few phenotypic properties

like lack of growth at 400C, in 4% NaCl concentration, at pH 9.2 and also inability to

hydrolyze arginine (Mundit, 1986). Curdling of milk is not the bacterium's only role in

cheese production. The lactic acid produced by the bacterium lowers the pH of the

product and preserves it from the growth by unwanted bacteria and molds while other

metabolic products and enzymes produced by L. lactis contribute in the development of

subtle aromas and flavors. Besides its role in food fermentation and production of some

antibacterial compounds like lactic acid some of the L. lactis also produces small heat

stable bacteriocin, nisin which contribute the safety and self life of the fermented foods.

L. lactis are not only used in food fermentation and preservation but recently it also used

in molecular biology research as a host for the production of different proteins of

industrial importance. Engineered L. lactis are used in the production of membrane


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proteins and toxic proteins, in the delivery of gene, vaccine and therapeutic drugs

(Bahey-El-Din et al, 2010; Braat et al, 2006; Hanniffy et al, 2007). L. lactis is considered

an advantageous host for protein expression and delivery.

Nisin

Nisin, a heat stable pentacyclic cationic peptide, produced by certain strains of

Lactococcus lactis, is one of the oldest known antibacterial compounds. It was first

described by Rogers (Rogers, 1928; Rogers and Whittier, 1928) and the proteinaceous

nature of this compound was first described by Hunter and Whitehead (1944). The

protein was characterized and called ‘nisin’ (Group N Inhibitory Substance) (Mattick and

Hirsch, 1947). This peptide was partially purified and analyzed by Mattick and Hirsch in

1944. However, the isolation and characterization of the biosynthetic gene of this

lanthionine ring-containing peptide was done in the last century (Buchman et al, 1988;

Kaletta and Entian, 1989; Rauch et al, 1990; Dodd et al, 1990; Horn et al, 1991; Engelk

et al, 1992 and Kuipers et al, 1993). The genes required for nisin production, maturation,

immunity and regulation are located on a conjugative transposon, Tn5276, which also

contains the determinants of sucrose metabolism (Dodd et al, 1990). Nisin biosynthetic

genes are transcriptionally organized in four operons, nisABTCIPRK, nisI, nisRK and

nisFEG (Qiao et al, 1996b, Ra et al, 1996; Li et al, 2006) (Fig. 2.1). Nisin is widely used

as a food-preservative in a broad range of products, including dairy products, liquid egg,

bakery products, vegetables, meat and fish (Delves-Broughton et al, 1996). Its food-grade

status, long history of safe use and high efficacy make it one of only a few commercially

applied bacteriocins. It effectively kills Gram-positive bacteria including spoilage and

pathogenic bacteria, such as Bacillus cereus, Listeria monocytogenes, Enterococci,

Staphylococci and Streptococci. Recently, nisin is being used in pharmaceutical and the

structural element of this peptide was exploited in the development novel antibiotics

(Breukink and de Kruijff, 2006).

The antimicrobial peptide, nisin, belongs to the class I bacteriocin called

lantibiotics (Klaenhammer, 1993; Nes et al, 1996), and to the Group A lantibiotics, a

class of related elongated post-translationally modified peptides. The members of this

class include subtilin, epidermin, gallidermin, Pep5, lacticin481 and the two-component

lantibiotic lacticin 3147 (Lubelski et al, 2008).


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Fig. 2.1. Transcriptional organization of nisin biosynthetic gene cluster (Lubelski et al, 2008).

Nisin structure and solubility

The primary structure of nisin was first proposed by Gross and Morell in 1971 (Fig. 2.2).

The 3-D structure of nisin has been investigated by high-resolution NMR spectroscopy

and the data showed that nisin has an elongated conformation with the termini of the

peptide pointing towards the middle of the molecule, which is enabled by a flexible hinge

region (van den Ven et al, 1991, and van den Hooven et al, 1996) (Fig. 2.4). It is a linear

amphiphilic, 34-residue polypeptide with a net positive charge of +4. It has five intra-

molecular sulfide bridges (ring A to E) with a molecular mass of 3510 daltons. Nisin

contains four unusual amino acids; lanthionine (Lan), β-methyllanthionine (MeLan), 2, 3-

dehydroalanine (Dha), and 2, 3-dehydrobutyrine (Dhb), which are the results of post-

translation modification (Fig. 2.3). During the post translational modification of nisin, as

proposed by Ingram (1970), serines and threonines are dehydrated to give Dha and Dhb,

respectively. Some of the dehydrated residues then react with the thiol (--SH) group of

nearby cysteine residues, forming Lan (from Dha) and MeLan (from Dhb) rings. Totally,

mature nisin has two Dha, one Dhb, one Lan, and four MeLan residues. The subsequent

dehydration reaction yielded five lanthionine rings of which two are located in the N-

terminal domain and three are situated in the C-terminal region of the peptide. These two

regions are separated by a flexible hinge region comprising of amino acid residues 20-22.

In solution this rigid ring structure imparts a screw-like helical conformation to nisin

(Slijper et al, 1989). In aqueous solution, nisin does not adopt a preferred conformation.

In a lipophilic environment, it adopts an amphiphilic α-helix shape with two domains.

The presence of lanthionine rings contribute in the hydrophobicity, rigid structure,

protease resistance and thermal resistance to the nisin. The N-terminal part of nisin is


10

hydrophobic and the C-terminal is hydrophilic (van den Ven et al, 1991). A high

proportion of basic amino acids give nisin a net positive charge (Breukink et al, 2000)

and it does not contain any aromatic amino acids (Bailey and Hurst, 1971).

The NMR study of nisin was determined by Slijper et al, (1989), used aqueous

solution, by Chan et al, (1989), used both water and dimethyl sulfoxide (DMSO) as the

solvent and by Palmer et al, (1989), who studied the chemically synthesized individual

rings A and B. The NMR result suggest that nisin is quite flexible in solution and the

three small rings B, D and E are in β-turns which are fixed by the thioether bond formed

by the first and the fourth residue of the rings. The ring A and C are structurally variable

and are not so well defined. In aqueous solution nisin exist in two amphipathic structure

of which first domain contains Ala3 – Ala19 and the second domain contains Ala23 –

Ala28. The first domain contains A, B and C rings with the hydrophobic side chains of

Ile4, Dha5, Leu6, Ala15, Leu16 and Met17 on one face and lanthionines and hydrophilic

Lys12 on the opposite face. The second domain consists of D and E rings and

hydrophobic residues of Met21 and Ala24 and the hydrophilic side chains of Lys22 and

His27 protrudes from the opposite face. The hinge region is located around Met21 which

join the ABC and DE rings. The C- terminal domain contains hydrophilic and charged

residues whereas N- terminal domain contains hydrophobic residues and only a single

charged residue, Lys 12.

Nisin normally occurs in the more stable dimer (Jarvis et al, 1968) conformation.

The solubility and stability of nisin are highly dependent on the pH of the solution. In

aqueous solution, it is most soluble and stable at pH 3.2-3.3 (Davies et al, 1998; Kelly et

al, 2000) and both solubility and stability of nisin decrease at neutrality, whereas an alkali

environment inactivates nisin (Hurst, 1978). Nisin Z has improved solubility at high pH

values, compared to nisin A, due to more hydrophilic nature of asparagine compared to

the deprotonated histidine and nisin Z is widely distributed than nisin A (de Vos et al,

1993). Nisin remains stable after autoclaving at 115.6°C at pH 2.0, but loses 40% of its

activity at pH 5.0 and more than 90% at pH 6.8 (Tramer, 1966). Stability of nisin also

depends on several other factors such as presence of other chemicals and the protective

effect of proteins. Storage at refrigerated temperature gives no detectable chemical or

biological changes to this peptide (Motlagh et al, 1991).


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Fig. 2.2. Structure of natural nisin variants; (a) Nisin A (Gross and Morell, 1971), (b) Niisn Z (Mulders et

al, 1991) and (c) Nisin Q (Zendo et al, 2003).

Fig. 2.3. Dehydration of serine and threonine and formation of lanthionine and β-methyllanthionine by

thioether linkage with cysteine (Jack et al, 1995)

Natural variants of Nisin

Six natural nisin variants have been described so far and are nisin A (Gross and Morell,

1971), nisin Z (Mulders et al, 1991), nisin Q (Zendo et al, 2003), nisin F (de

Kwaadsteniet et al, 2008) and two variants of nisin U (nisin U and nisin U2) (Wirawan et

al, 2006). Nisin A, Z, Q and F are produced by some strains of L. lactis and nisin U and


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U2 are produced by Streptococcus uberis. Premature nisin contains 57 amino acid of

which 23 amino acids are in the leader peptide and 34 amino acids in the propeptide.

Nisin Z differs from nisin A by having a glutamine instead of a histidine in position 27.

Nisin Q differs from nisin A in the positions: – 8 (K→T), – 2 (P→T), 15 (A→V), 21

(M→L), 27 (H→N) and 30 (I→V) (Fig. 2.5). Nisin F differ from nisin A in the position

27 (H→N) and 30 (I→V). The leader peptide of nisin U and U2 is one amino acid longer

than the leader of nisin A, Z and Q. An additional Glu seems inserted between positions –

8 and – 9 of nisin A, Z and Q. After alignment of the leader peptides 9 (nisin U) or 10

(nisin U2) other positions of the leader peptide differ from the leader peptide of nisin A

(Fig. 2.6). Nisin U and U2 propeptides are three amino acids shorter than nisin A, Z and

Q and differ from nisin A in positions 15 (A→I), 18 (G→ Dhb), 20 (N→P), 21 (M→L),

27 (H→G), 29 (S→H) 30 (I→F) and 31 (H→G). Nisin U2 additionally differs from nisin

A in position 1 (I→V) (Lubelski et al, 2008). A comparative analysis of natural nisin

variants was shown in Table 2.1.

Fig. 2.4. 3D-structure of nisin molecule. Grey balls demonstrate hydrophobic and black ones hydrophilic

amino acids. It can be seen that hydrophobic amino acids form one face of the molecule and hydrophilic

another on the opposite side of the molecule. Structured domains I and II with the thioether rings and the

flexible hinge region of the molecule are signed (Koponen, 2004).


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The two nisin variants (nisin A and nisin Z) showed 83% similarity in their

activity (Morency et al, 2001). But nisZ gene is widely distributed among nisin producing

L. lactis strains (de Vos et al, 1993). Nisin Z having high diffusion rate (de Vos et al,

1993) and less solubility at low pH than nisin A (Rollema et al, 1995). Nisin A does not

kill Listeria sp. in presence of fat, but Mitra et al, (2011) showed that nisin Z have the

capability to kill Listeria sp. in presence of fat due to high diffusion rate of nisin Z. The

detailed properties of nisin Q, nisin F, nisin U and U2 are yet to discover.

Nisin A ITSISLCTPG CKTGALMGCN MKTATCHCSI HVSK

Nisin Z ITSISLCTPG CKTGALMGCN MKTATCNCSI HVSK

Nisin Q ITSISLCTPG CKTGVLMGCN LKTATCNCSV HVSK

Nisin F ITSISLCTPG CKTGALMGCN MKTATCNCSV HVSK

Nisin U ITSKSLCTPG CKTGILMTCP LKTATCGCHF G

Fig. 2.5. Amino acid sequences of mature nisin variants.

Fig. 2.6. Sequence of leader peptide (above) and structure (below) of natural nisin variants (Lubleski et al,

2008).

Nisin Biosynthesis

Nisin A, encoded by nisA, is a lanthionine ring containing peptide that is ribosomally

synthesized as a prepeptide of 57 amino acid residues. The unmodified precursor of nisin

is processed by specific maturation machinery that is responsible for dehydration


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reactions and ring formation (NisBC), transport across the cytoplasmic membrane (NisT)

and cleavage of the leader peptide (NisP), which liberates biologically active nisin,

consisting of 1 lanthionine, 4 methyllanthionines, 1 dehydrobutyrine, 2 dehydroalanines

and 21 unmodified amino acids (Kuipers et al, 1993a) (Fig. 2.7; Fig. 2.8). Unmodified

prenisin, contains 57 amino acid residues, which after translation are targeted to a

modification at the cytoplasmic membrane (Siegers et al, 1996). The N-terminal leader

peptide containing 23 amino acid residues are important in recognition of unmodified

prenisin by the modification and transport proteins (Kuipers et al, 1993a, Siegers et al,

1996, Li et al, 2006; Kuipers et al, 2006). The first step of nisin maturation is performed

by the NisB dehydratase, which, after interaction with the leader peptide, dehydrates

serines and threonines in the nisin pro-peptide (Kuipers et al, 2006; Koponen et al, 2002).

Dehydrated residues may then participate in regioselective cyclization with the help of

NisC (Li et al, 2006, and Koponen et al, 2002). Modified nisin is subsequently

transported via the ABC-transporter NisT (Kuipers et al, 1993, Qiao et al, 1996a; Kuipers

et al, 2004). (Methyl) lanthionine-containing nisin that still contains a leader sequence

remains biologically inactive. Only after the proteolytic cleavage of the N-terminal leader

sequence, which is mediated by a protease called NisP (Kuipers et al, 1993; Qiao et al,

1996b), nisin becomes active and able to induce NisRK, a two-component system that

regulates its biosynthetic and immunity genes.

NisB is a membrane-associated enzyme that converts serines and threonines to

dehydroalanines and dehydrobutyrines, respectively, in the nisin prepeptide. NisB is

117.5-kDa protein that, according to the UniProt database prediction, contains one

potential transmembrane segment, ranging from residues 838 to 851. Cellular localization

of NisB has been studied and it was suggested that NisB is primarily associated with the

cytoplasmic membrane (Engelke et al, 1992). For a long time it was believed that the

lantibiotic transport and modification machinery is highly specific for a dedicated

substrate but recent reports clearly demonstrated that nisin-modifying enzymes possess

broad substrate specificity (Kuipers et al, 2004; Kluskens et al, 2005). These findings

opened the possibility for the biotechnological application of the nisin modifying

enzymes as novel tools to introduce dehydrated amino acids and lanthionine rings into a

variety of non-lantibiotic peptides. The presence of these unusual amino acids can modify


15

biological activity of the peptides and protect them against proteolysis (Kluskens et al,

2005).

NisC, besides the cyclization reaction, also perform the dehydration reaction.

NisC has been shown to be membrane associated and to interact physically with other

members of the nisin transport and modification machinery as well as with nisin itself

(Siegers et al, 1996). Kuipers et al, (2004) compared plasmid-based overexpression of

nisABT with nisABTC genes and concluded that NisC catalyzes (methyl) lanthionine

formation.

NisT is an ABC transporter that consists of putative α-helices that transverse the

cytoplasmic membrane five times and a hydrophilic nucleotide-binding domain that binds

ATP and is needed to energize the transport. A typical ABC transporter includes four

modules, i.e., two transmembrane segments and two nucleotide-binding domains. Thus,

NisT, which is a half-transporter and contains only two out of four modules commonly

found in ABC transporters. NisT was shown to interact with NisC and was implicated to

be a part of a putative membrane modification and transport complex (Siegers et al,

1996). Deletion/disruption of nisT abolishes secretion of nisin, and as a result of the

inability of the cell to secrete it, nisin accumulates in the cytoplasm (Qiao et al, 1996a;

Ra et al, 1999). It cannot only transport fully modified nisin but also partially modified or

completely unmodified peptides. Moreover, various non-lantibiotic peptides were

successfully transported by NisT provided that they were fused to the leader sequence of

nisin (Kuipers et al, 2004).

NisP belongs to the subtilisin family of serine proteases (Pfam entry:

Peptidase_S8). It contains an N-terminally located Sec-signal sequence (residues 1 – 22)

that is likely responsible for targeting and transport of NisP out of the cell via the Sec

pathway (Lubelski et al, 2008). The nisin prepeptide with the leader sequence attached

does not show significant antimicrobial activity (Qiao et al, 1996b; Kuipers et al, 1993).

NisP helps in the proteolytic processing of nisin (van der Meer et al, 1993). Neither

unmodified prenisin nor dehydrated prenisin could be cleaved by NisP, indicating that

one or more thioether rings are required for NisP activity (Kuipers et al, 2004).

Strains of L. lactis that produce nisin have developed immunity against the

bactericidal activity of nisin. Immunity is conferred by two different systems: lipoprotein

NisI and ABC transporter NisFEG (Siegers et al, 1995).


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NisI was first described in an early work on the characterization of the nisin gene

cluster (Kuipers et al, 1993). It is a 245 amino acid lipoprotein with a consensus

lipoprotein signal sequence, which is post-translationally removed. Subsequently, the

protein is anchored to the extracellular side of the cell membrane via lipid modification of

the N-terminal cysteine residue (Qiao et al, 1995). Previously it was suggested that nisI

function as a nisin intercepting molecule and was shown to exist in two forms: a lipid-

free form secreted into the growth medium and a membrane-associated lipoprotein, a

situation not uncommon for lipoproteins (Qiao et al, 1996b, Takala et al, 2004, Stein et

al, 2003; Koponen et al, 2004). Both of the forms were shown to bind with nisin (Stein et

al, 2003) and, interestingly, lipid-free NisI enhanced immunity of L. lactis more

efficiently in the strain expressing nisEFG as compared to the strain lacking these genes

(Takala et al, 2004).

NisFEG immunity proteins were first described by Siegers and Entian (1995).

NisFEG proteins form an ABC transporter complex, where NisE and NisF are

homologous to the ABC transporters of the HisP family, NisF is a cytoplasmic ATP

binding protein and NisG together with NisE are integral membrane proteins (Siegers and

Entian, 1995). Since many ABC transporters consist of four domains, two of which are

hydrophobic and two are ATPases, it is hypothesized that a NisF2EG complex is formed

(Peschel and Gotz, 1996). According to Stein et al, (2003) NisFEG contain nisin

expelling properties.

Immunity and production of nisin in L. lactis is regulated by the NisRK mediated

two-component system (Engelke et al, 1994). NisK phosphorylates itself in the presence

of nisin and transfers a phosphoryl group to an aspartate of NisR, which triggers binding

of the response regulator to nisA and nisF promoters (Engelke et al, 1994; Kuipers et al,

1995). This initiates transcription of nisABTCIPRK operon as well as nisFEG (Qiao et al,

1996b, Kuipers et al, 1995; de Ruyter et al, 1996). Until recently it was believed that

transcription of nisI is controlled only by the nisA operator in nisABTCIPRK operon but

nisI has its own constitutive promoter (Li et al, 2006).

NisK is a histidine sensor kinase that is localized in the cytoplasmic membrane

and serves as a receptor of fully maturated nisin (Kuipers et al, 1995). Extracellularly

present and modified nisin binds to NisK and initiates a signal transduction cascade,

which starts with autophosphorylation of histidine of NisK (van der Meer et al, 1993,


17

Engelke et al, 1994; Kuipers et al, 1995). Subsequently, phosphate is transferred to NisR,

which is a transcriptional activator that binds to promoter regions of nisABTCPRK and

nisFEG inducing transcription of genes that are required for nisin biosynthesis and

immunity (Qiao et al, 1996b, Kuipers et al, 1995; de Ruyter et al, 1996). The promoter of

the nisRK operon was shown to be independent of nisin regulation and the nisRK genes

are constitutively expressed (de Ruyter et al, 1996). Recently, it has been reported that

prenisin, which is produced by a translocator deficient strain and accumulates

intracellularly, can also induce NisK and initiate a signal transduction pathway. It was

suggested that prenisin is cleaved by an unidentified intracellular protease(s) which can

activate extracellularly located NisK (Hilmi et al, 2006).

Fig. 2.7. Model for nisin biosynthesis (Entian et al, 1996).

Fig. 2.8. Post Translational processing of Nisin (Lubelski et al, 2008).


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Organization of nisin gene cluster and Regulation of nisin Biosynthesis

The biosynthesis of lantibiotic nisin by a number of L. lactis is generally encoded by

cluster of 11 genes responsible for the synthesis, maturation, immunity, and regulation

(Siezen et al, 1996, Kleerebezem and Quadri 2001) (Fig. 2.9). The genes are

transcriptionally organized as nisA/ZBTCIPRK, nisRK, nisI, and nisFEG, and is located

on a large conjugative nisin-sucrose transposon (Buchman et al, 1988, Engelke et al,

1992, Engelke et al,1994, Kuipers et al,1993a, Kuipers et al,1995, de Vos et al,1995,

Siegers and Entian, 1995, Siezen et al, 1996; Ra et al, 1996). Of these genes, the nisA/Z

gene encodes nisin A/Z precursor peptide consisting of 57-amino acid residues,

containing a 23-amino acid residues N-terminal leader peptide that is involved in

directing the modification and targeting process of nisin precursor (van der Meer et

al,1993, Kleerebezem and Quadri 2001). nisB and nisC encode membrane-associated

proteins involved in the intracellular post-translational modification reaction such as

serine and threonine residues are dehydrated to become dehydroalanine and

dehydrobutyrine. Subsequently, five of the dehydrated residues are coupled to upstream

cysteines, thus form the thioether bonds that produce the characteristic (β-methyl)

lanthionine rings (Engelke et al, 1992, Kuipers et al, 1993, 1995; Siegers et al, 1996).

nisT encodes a putative transporter protein of ABC translocator family that is involved in

the translocation of the fully modified nisin precursor across the cytoplasmic membrane

(Qiao and Saris, 1996a). nisP encodes a subtilisin-like protease involved in extracellular

proteolytic activation. During or shortly after translocation of the nisin precursor, the

leader peptide is removed by the subtilisin-like protease to form an extracellular mature

nisin (van der Meer et al, 1993; Qiao, 1996). Two systems that are involved in immunity

to nisin of the producing cell are derived from nisI and nisFEG. nisI encodes a

lipoprotein involved in the self-protection of the producing bacterium against nisin

(Kuipers et al, 1993a; Qiao et al, 1995) and nisFEG encodes a putative ABC exporter

involved in nisin extrusion (Siegers and Entian 1995; Dodd et al, 1996). nisR and nisK

encode a response regulator (van der Meer et al, 1993) and a sensor kinase of the

histidine protein kinase family (Engelke et al, 1992, de Vos et al, 1995, Immonen et al,

1995; Siegers and Entian 1995), respectively, that belong to a class of two-component

regulatory systems (Stock et al, 1989). It has been shown that both genes are related to

the regulation of nisin biosynthesis (van der Meer et al, 1993, Kuipers et al, 1993,


19

Kuipers et al, 1995, Engelke et al, 1994, de Ruyter et al, 1996, Kleerebezem et al, 1997;

Kleerebezem et al,1999).

The regulation of nisin biosynthesis is a complex process involving the cascade of 11

genes. Biosynthesis of nisin appears to be under the control of NisRK mediated two-

component response regulatory proteins, a large family of proteins involved in regulation

of a variety of physiologically important processes (Kleerebezem et al, 1997). Nisin play

an important role in the regulation of its own biosynthesis. Nisin can be regarded as a

peptide pheromone, which induces the biosynthesis of nisin via NisK by direct protein-

peptide interaction. NisK is a two domain structured protein consisting of membrane

anchoring transmembrane domain and cytosolic ATPase domain. The extracellularly

produced nisin, act as signal, is sensed by the transmembrane domain of NisK by binding

of nisin to that domain. Upon receiving the signal, the cytoplasmic ATPase domain is

phosphorylated at His-residue by converting ATP → ADP using the kinase activity. This

domain has dual function as kinase and phosphatase. The phosphatase activity of this

domain donates its bound phosphate to the response regulator NisR. The NisR is also

consists of two domain, response and regulatory domain. The response domain of NisR

received the phosphate and phosphorylated at specific Asp-residue and becomes

activated. The activated NisR then binds to the inducible promoters (nisA/Z and nisF) of

nisin operons and transcribed the nisin biosynthetic genes (Fig. 2.10). Nisin biosynthesis

is thus solely regulated by nisin (de Ruyter et al, 1996; Kuipers et al, 1995). Both mature

modified and premature nisin can induce the nisin genes. The regulatory genes nisRK

themselves are transcribed from their own promoter which is assumed to be not

dependent on nisin induction (de Ruyter et al, 1996). The transcription from the nisA and

nisF promoter in the nisin gene cluster is directly related to the concentration of nisin in

the medium. This property is extremely useful for the development of a controlled gene

expression system.


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Fig. 2.9. Model for the biosynthesis of nisin. The nisin precursor is modified by the putative enzymes

NisB and NisC and translocated across the membrane by the exporter NisT. The precursor is

extracellularly processed by NisP, resulting in the release of mature nisin. NisK senses the presence of

nisin in the medium and autophosphorylates. The phosphate-group is transferred to NisR, which

activates transcription of the genes nisABTCIP and nisFEG. NisI, F, E, and G protect the cell from the

bacteriocidal activity of nisin. P: promoter region, P*: nisin-regulated promoters. (van Kraaij et al, 1999).

Fig. 2.10. Model for nisin mediated signal transduction involving the sensor kinase, NisK, and the response

regulator, NisR (de Ruyter, 1998).

Mode of action of Nisin

Nisin exhibits antimicrobial activity towards closely related Gram-positive bacteria,

including Streptococci, Staphylococci, Lactobacilli, Micrococci, Listeria, and most

spore-forming species of Clostridium and Bacillus. However, it shows little or no activity

against Gram-negative bacteria, yeasts or molds (O’Keeffe and Hill, 2000). Generally,


21

Gram-negative cells are protected from nisin because of the presence of an outer

membrane in cell wall, but when the outer membrane is weakened by any treatment that

make their cell walls permeable to nisin makes them susceptible to nisin. Such

treatments include exposure to chelating agents (20 mM EDTA), detergents (Tween 80),

sub-lethal heat, osmotic shock and freezing (Delvis-Broughton, 2005; Stevens et al,

1991). Interestingly, nisin effectively kill bacteria in nanomolar concentrations and it

works in a concentration dependent fashion; thus the more bacteria present in a food the

more nisin may be required. Nisin has dual killing mechanism; targeted pore formation

and inhibition of cell wall synthesis (Brotz and Sahl, 2000; Wiedemann et al, 2001) (Fig.

2.11).

The specific target of nisin for pore formation is lipid II (Fig. 2.12). According to

the model proposed by Hasper et al, (2004), nisin first form a 1:1 complex with lipid II

and then one more nisin molecule binds to lipid II, giving a 2:1 nisin: lipid II complex.

Finally, a staple pore complex is formed by the insertion of nisin molecules into a

perpendicular orientation with respect to the membrane surface, giving final pore

complex of 8 nisin and 4 lipid II molecules with a diameter of 2-2.5 nm (Wiedemann et

al, 2004). Binding of nisin onto lipid II not only causes collapses of the proton motive

force and membrane integrity via pore formation, but also interferes with cell-wall

synthesis by blocking lipid II from incorporation into peptidoglycan (Wiedemann et al,

2001). Thus nisin kills bacteria by formation of pores in the cytoplasmic membrane and

inhibition of peptidoglycan by binding with lipid II.

High resolution NMR spectroscopy studies of nisin-lipid II interactions in model

membrane systems (Hsu et al, 2002) and site-directed tryptophan spectroscopy studies of

nisin “topology” in lipid II-containing membranes (van Heusden, 2002) demonstrated

that the N-terminal domain is the key structural element involved in the binding of nisin

to lipid II, whereas the hinge region between ring clusters A-B-C and D-E is most

important for pore formation. The C-terminal domain of nisin is important for initial

binding as well as antimicrobial activity when pores are formed in a target-independent

fashion, i.e., without lipid II (Breukink et al, 1997; van Kraaij et al, 1997). Such activity

is also observed at relatively high nisin concentrations (µM range). Further studies have

revealed that an intact N-terminal of nisin binds to the lipid II by hydrogen bonds as a

result pyrophosphate cage is formed (Hsu et al, 2004).


22

In case of bacterial spore, effect of nisin is known to be different from that of

vegetative cell and is mostly sporostatic. Nisin affects the post-germination stages of

spore development by inhibiting pre-emergent swelling, the out-growth and formation of

vegetative cells (Hitchins et al, 1963; Gould, 1964).

Fig. 2.11. Wedge models of pore formation by nisin (Moll et al, 1997).

Fig. 2.12. Lipid II-mediated model of pore formation by nisin (Wiedeman et al, 2001).


23

Application of Nisin

In 1969, nisin was approved for use as an antimicrobial in food by the Joint FAO/WHO

Expert Committee on Food Additives. Since then nisin has been given the food additive

number 234 and is permitted currently for use in over 80 countries in the World. The

suitability of nisin as a food preservative arises from the following characteristics: it is

non-toxic, the producer strains of L. lactis are regarded as safe (food-grade); it is not used

clinically; there is no apparent cross-resistance in bacteria that may effect antibiotic

therapeutics; it is quickly digested and stability at high temperature at low pH.

Nisin shows increased solubility in an acid environment and becomes less soluble

as the pH increases. However, owing to the low level of nisin used in food preservation,

solubility does not present a problem. Nisin solutions are most stable to autoclaving

(121°C for 15 min) in the pH range 3.0–3.5 (<10% activity loss). At pH values

below and above this range, there is marked decrease in activity (>90% loss at pH 1

or 7). Losses of activity at pasteurisation temperatures are significantly less

(approximately 20% during s tandard processed cheese manufacturer at pH 5.6–5.8).

Food components can also protect nisin during heat processing as compared to a buffer

system.

The stability of nisin in a food system during storage is dependent upon three

factors: incubation temperature, length of storage and pH. In cold processed foods,

proteolyt ic enzymes can affect nisin stability. The food additives, titanium dioxide and

sodium metabisulphite can also adversely affect nisin stability (Delvis-Boughton, 2005).

Since 1953, nisin has been sold under the trade name of Nisaplin® marketed by Aplin

and Barrett, Ltd (Thomas et al, 2000).

Dairy products

Processed cheese products: - In processed cheese spreads, nisin at levels 12.5

mg/kg and above is effective in delaying or preventing growth and subsequent toxin

production by facultative aerobic Bacillus spp and the spores of C. botulinum types A

and B (Delvis-Broughton, 2005).

Other pasteurised dairy products:- Other pasteurised dairy products, such as dairy

desserts, cream, clotted cream and mascarpone cheese, often cannot be subjected to full

sterilisation without damaging quality and are thus sometimes preserved with nisin


24

which increase in shelf life at 7oC with 3.75 mg/kg nisin for 20 days (Delvis-

Broughton, 2005). The addition of nisin to pasteurized milk is permitted in some

countries. Nisin was added before pasteurization resulted in significant shelf-life

extension of the milk at 80C and in 370C (Mitra et al, 2011).

Natural cheese:- Cheeses such as Emmenthal and Gouda have been made with

sufficient nisin content to provide protection against growth of Clostridium spp.,

Staphylococcus aureus and Listeria monocytogenes (Delvis-Broughton, 2005).

Yoghurt:- Nisin (at the levels of 0.5–1.25 mg/kg) has the potentiality to increase the

shelf life of yoghurt.

Dahi:- Fermentation of milk with nisin producing L. lactis strain produced dahi which

displayed antibacterial property against spoilage and pathogenic bacteria including

Listeria monocytogenes. When L. monocytogenes was mixed with dahi at 5.2 logCFU/ml

and stored at 4°C, the number of L. monocytogenes gradually decreased.

(Mitra et al, 2010).

Egg products

Nisin at 2.5–5 mg/L gives significant increases in shelf life and protection against

growth of the psychroduric food poisoning bacteria B. cereus and L. monocytogenes in

Pasteurised liquid egg products (whole, yellow and white) and value-added egg

products (eg omelettes, scrambled eggs, pancake mixes).

Pasteurised soups

Nisin at levels of 2.5–5 mg/L is effective at preventing or delaying outgrowth of

psychroduric spoilage Bacillus spp. during prolonged storage.

Flour based products

Addition of nisin to the batter mix at 3.75 mg/kg to prevent the growth of B. cereus.

Canned foods

Nisin is used in canned foods mainly for the control of thermophilic spoilage. Examples

of use are canned peas, carrots, peppers, potatoes, mushrooms, okra, baby sweet corn,

and asparagus. Nisin is also used in canned dairy puddings containing semolina and

tapioca. Bacterial spoilage of canned high acid foods (pH below 4.5) is restricted to

non-pathogenic spoilage species such as C. pasteurianum, B. macerans and B.

coagulans. Nisin addition levels of 1.25–2.50 mg/kg are used in high acid tomato-based

products.


25

Meat products

Concern regarding the high levels of nitrite in cured meat has resulted in research

investigating the use of nisin as a partial replacement for nitrite. Only high (and

uneconomic) levels of nisin, achieved good control of C. botulinum.

Seafoods

Nisin at 25 mg/kg in combination with a reduced heat process, that does not cause

product damage of lobster meat, achieved a Listeria kill significantly better than either

heat or nisin alone. Washing crabmeat with nisin reduced levels of L. monocytogenes

Natural sausage

To prevent subsequent outgrowth nisin was evaluated to reduce outgrowth of spores in

desalinated casings (Wijnker et al, 2011).

Salad dressings

Reduced acidity may improve the flavour of cold blended salad dressings but using reduced

levels of acetic acid and raising the pH from 3.8 to 4.2 can make salad dressings prone to

lactic acid bacterial spoilage. Such growth has been successfully controlled by nisin at

2.5–5.0 mg/L.

Alcoholic beverages

Acid tolerant lactic acid bacteria of the genera Lactobacillus, Pediococcus, and Leuconostoc can

spoil beer and wine and nisin, at levels of 0.25–2.5 mg/L, is effective in preventing such

spoilage. Yeasts are unaffected by nisin, thus the preservative can be added d u r i n g

the fermentation. Nisin can be added to fermenter to prevent or control contamination

and can also be used to increase the shelf-life of unpasteurised and bottle-conditioned

beers.

Medical Application of Nisin

There is a gradual demand of nisin in food preservation and health due to a relative broad

killing spectrum, stability at high pH and temperature, cost effective production process

and is safe for human consumption. Nisin is the only bacteriocin that has been approved

by FDA and WHO as food preservative and being used for more than 80 countries in the

world. In pharmaceutical, nisin has been used to treat peptic ulcer caused by Helicobacter

pylori as it is stable at stomach pH and resistant to stomach protease pepsin, to inhibit the

growth of multi-drug resistant pathogens like Staphylococcus and Streptococcus and it

can be used as antimicrobial barrier in implanted medical devices like catheters and


26

tracheotomy tubes (Pag and Sahl, 2002, van Kraaij et al, 1999, Post, 1996, Delves-

Broughton et al, 1996, Severina et al, 1998; Bower et al, 2002).

Nisin have a role in the treatment of systemic diseases caused by antibiotic

resistant Streptococcus. It exhibit excellent activity against clinical isolates of Strep.

pneumonia including penicillin resistant strains and other multi-drug resistant Gram-

positive pathogens. Nisin is used to treat bacteria mastitis, oral hygiene, treatment of

methicillin resistant Stap. aureus and enterococcal infection, enterocolitis, to inhibit skin

pathogens, prevention of periodontal disease, lung mucus clearing and in cosmetic,

deodorant, chewing gum and topical formulation (Blackburn et al, 1989, Blackburn and

Goldstein, 1995, Claypool et al, 1966, Cowel et al, 1971, Goldstein et al, 1998,

McConvile, 1995, Patel, 1995, Ryan et al, 2002, Severina et al, 1998; Valenta et al,

1996). Current research investigated that nisin may have application in treatment of head

and neck squamous cell carcinoma (Joo et al, 2012).

Nisin production

It is known that nisin production is influenced by many cultural factors such as producer

strain, compositions of the nutrient broth, pH, temperature, aeration (Parente and

Ricciardi, 1999) and even it is also affected by substrate inhibition, adsorption of nisin

onto the producer cells, and enzymatic degradation (de Vuyst and Vandemme, 1992;

Yang et al, 1992). Mattick and Hirsch (1947) first described a method of nisin production

using glucose and yeast extract with a nisin yield of only 80 IU/ml. Since then, many

scientific approaches of nisin production have been taken to improve the production rate

and productivity.

Producer strain

Nisin producing L. lactis strain were isolated from a variety of sources like raw or

fermented milk (Mitra et al, 2005; Mitra et al 2007), vegetables samples such as cabbage,

carrot, fermented vegetables like saukrout, fermented traditional foods like fermented

rice, from the river water etc. In 1994, de Vuyst and Vandamme screened 21 nisin-

producing and 6 non-producing strains of L. lactis for nisin production and immunity.

They found that the level of nisin production is different in different strains although they

have same number of nisin structural gene. According to Kim et al (1998), optimum

ceiling concentration of nisin production is different in different nisin producers and this

is due to the end product inhibition by the nisin.


27

Table 2.1. Comparative study of Natural nisin variants

Nisin Variants Nisin A Nisin Z Nisin Q Nisin F Nisin U

Gene nisA nisZ nisQ BacF nsuA

Producer organism L. lactis subsp.

lactis

L. lactis subsp.

lactis

L. lactis subsp.

Lactis L. lactis L. uberis

Formula C143 H246 N42 O45

S7

C141 H245 N41 O46

S7

C143 H249 N41 O46

S6

C140 H243 N41 O46

S7

C134 H226 N36 O40

S6

Absent amino acids DEFQRWY DEFQRWY DEFQRWY DEFQRWY DENQRVWY

Common amino acids CT CT CT CT T

Mass (Da) 3516.78 3493.74 3489.73 3479.71 3192.37

Net charge +5 +4 +4 +4 +4

Isoelectric point 8.52 8.51 8.51 8.51 8.51

Basic residues 5 4 4 4 4

Acidic residues 0 0 0 0 0

Hydrophobic residues 8 8 9 8 7

Polar residues 18 19 19 19 17

Aliphatic residues 6 6 8 6 5

Tiny residues 9 9 8 9 7

Boman Index -12.88 -14.86 -10.94 -15.74 -1.63

Hydropathy Index 0.41 0.41 0.52 0.4 45

Aliphatic Index 71.76 71.76 85.88 68.82 66.13

Instability Index 27.52 (stable) 17.08 (stable) 13.45 (stable) 13.45 (stable) 30.65 (stable)

Half Life

Mammalian : 20

hour

Yeast : 30 min

E. coli : >10

hour

Mammalian : 20

hour

Yeast : 30 min

E. coli : >10

hour

Mammalian : 20

hour

Yeast : 30 min

E. coli : >10

hour

Mammalian : 20

hour

Yeast : 30 min

E. coli : >10

hour

Mammalian : 20

hour

Yeast : 30 min

E. coli : >10

hour

Extinction Coefficient 250 M-1 cm-1 250 M-1 cm-1 0 M-1 cm-1 0 M-1 cm-1 0 M-1 cm-1

Absorbance 280nm 7.58 7.58 7.68 7.58 8.33


28

Nutrients

Lactococci are nutritionally fastidious microorganisms and they required complex

organic medium for growth (de Vuyst and Vandamme, 1994a) and nisin production is

related to growth and biomass production as it is produced as a primary metabolites

(Buchman et al, 1988; de Vuyst and Vandamme, 1994; Parente and Ricciardi, 1999).

Therefore, nisin production is highly affected by type and level of carbon, nitrogen and

phosphate.

Carbon source

Both type and concentration of carbon sources play an important role in the growth and

nisin production in L. lactis strains. Many carbon sources support growth and nisin

production but glucose was the most preferred carbon source for the nisin production in

L. lactis strains. Certain carbohydrates stimulate nisin production maximally and sucrose,

xylose, lactose and maltose were reported as the most efficient carbon sources in strain

ATCC 11454 (Chandrapati and O’Sullivan, 1998) and LM 0230 (Yu et al, 2002), strain

JCM 7638 (Chinachoti et al, 1997), strain A 164 (Cheigh et al, 2005) and strain W8 and

CM1 (Mitra et al, 2005; Mitra et al, 2007) respectively. Sucrose stimulates nisin

production maximally in a number of nisin producing strains. Hengstenberg (1977)

suggested that sucrose was rapidly utilized by the nisin producer strain because of its

highly efficient phosphoenolpyruvate-dependent phosphotransferase system (PTS) for

sucrose uptake, transport, and metabolism. In the presence of sucrose sucrose-specific

proteins that are induced are sucrose-specific uptake protein Enzyme II, a sucrose 6-

phosphate hydrolase and a fructokinase (Thompson and Chassy, 1981; Thompson et al,

1991).

Nitrogen source

Growth and nisin production are dependent on the organic nitrogen sources. In the L.

lactis NIZO 22186, maximum nisin production obtained with cotton-seed meal (2,500

IU/ml), and more than 2,000 IU/ml with yeast extract and fish meal (de Vuyst and

Vandamme, 1993). Guerra et al, (2001) found that a combination of whey with yeast

extract and casitone increased nisin production. Kim et al, (1997) reported that nisin

production increases with increasing organic acid content in the medium.


29

Mineral source

Nisin production is differentially affected by both anions (phosphate) and cations (Mg2+

and Ca2+) in different strains. Nisin production is strongly affected by KH2PO4 in L. lactis

NIZO 22186 (de Vuyst and Vandamme, 1993) and L. lactis ATCC 11454 (Li et al,

2002). Nisin production was improved in L. lactis ATCC 11454 in the presence of

MgSO4, 7H2O (Meghrous et al, 1992). In strain IO-1 calcium increased nisin production

but magnesium or phosphate did not (Matsusaki et al, 1996).

pH and temperature

The optimal pH for nisin production is usually at 5.5-6.0 (Meghrous et al, 1992; and

Matsusaki et al, 1996), which is lower than optimal pH for growth. The optimum pH of

nisin production varies in different L. lactis strains. The optimal pH for nisin Z

production by strain IO-1 is pH 6.0 in xylose medium (Chinachoti et al, 1997) and pH 5.5

in glucose medium (Matsusaki et al, 1996). Cabo et al, (2001) demonstrated that a pH-

drop gradient enhanced nisin production approximately four-fold when pH of

fermentation broth was adjusted back to 7.0 every 6 h in L. lactis strain IIM Lb.1.13. In

another experiment carried out by Mitra et al, (2007) it was demonstrated that an initial

medium pH of 11.0 results in approximately three-fold higher nisin production by the

strain L. lactis W8.

The optimal temperature for growth of L. lactis was reported to be at 30-37°C in L. lactis

strains (Meghrous et al, 1992, Matsusake et al, 1996, Cheigh et al, 2002, Mitra et al,

2005; Mitra et al, 2007). The optimal temperature for nisin production was reported at

30°C.

Agitation and aeration

As L. lactis are the aerotolerant organism, they can grow well in the presence and

absence of atmospheric O2. Therefore, nisin production typically does not require

aeration and agitation but a slow agitation is needed to achieve a homogeneous

suspension (de Vuyst and Vandamme, 1994) and it also increases nisin production.

Sugar transport in Lactococcus lactis Lactococcus lactis, having great industrial importance, are generally used as starter

culture in fermentation industry and as a model organism for the production of

biotechnologically relevance proteins. They are homofermentative, Gram-positive


30

bacteria with simple energy and sugar metabolism and completely dependent upon the

sugar fermentation for their energy. The L. lactis converts sugars via the glycolytic

pathway to pyruvate for the generation of energy through substrate level phosphorylation.

The metabolism of carbohydrates begins with the transport of sugars through the semi

permeable cytoplasmic membrane and are by (i) the phosphoenolpyruvate:

(carbohydrate)-phosphotransferase system (PTS), which is involved in both transport and

phosphorylation of a sugar at the expense of phosphoenol pyruvate (PEP), driven by

group translocation (Postma et al, 1993); (ii) ion-linked sugar transport: secondary

transport systems, driven by an ion gradient (Poolman, 1993); and (iii) carbohydrate

transport ATPases: primary transport systems, driven by ABC transport systems (Fath

and Kolter, 1993) (Fig. 2.13). Among the above three transport machinery, PTS system is

the most efficient and well studied sugar transport system. The PTS system consists of

enzyme I (EI), the HPr protein and several sugar-specific enzymes II (EII). According to

the literature, the uptake of glucose in L. lactis strains is mediated by mannose-PTS

system (PTS man) (Thompson, 1987)) and subsequently phosphorylated to glucose 6-

phosphate by EIIA. The other transport systems of glucose in some strains are by

glucose-PTS system or via permease systems (Thompson and Saier Jr., 1981; Thompson

et al, 1985). After translocation glucose is phosphorylated and enters in to the glycolysis

pathway. Whereas the transport of lactose can occurs via a lactose-PTS or by a permease

to yield lactose 6-phosphate (de Vos et al, 1990; de Vos and Vaughan, 1994). The lactose

6-phosphate is then hydrolyzed by β-galactosidase to glucose and galactose. Glucose is

then entering to the glycolysis pathway. The translocation of galactose is mediated by

galactose-PTS or by galactose specific permease system. The metabolism of galactose

occurs by tagatose 6-phosphate pathway or by Leloir pathway (galactose 1-phosphate

pathway) when translocated by PTS-gal or permease system, respectively (Grossiord et

al, 1998; Thomas et al, 1980; Thompson, 1980). The transport of sucrose occurs via

sucrose-PTS system and phosphorylated to sucrose 6-phosphate. After translocation,

sucrose 6-phosphte is hydrolyzed by sucrose 6-phosphate hydrolase to glucose 6-

phosphate and fructose (Thompson and Chassy, 1981). The glucose 6-phosphate entering

into the glycolysis pathway and fructose is phosphorylated to fructose 6-phosphate by an

ATP-dependent fructokinase (Thompson et al, 1991) and then enters to the glycolysis

pathway. The genes responsible for the transport and metabolism of sugars are present in


31

the genome. Therefore, the metabolism of sugars is under the control of specific sets of

genes which are expressed in the presence of that specific sugar. The phenomenon by

which bacteria utilized the sugars is called carbon catabolite repression. The PTS system

plays an important role in the carbon catabolite repression in bacteria. The catabolism of

sugars in Gram-positive lactic acid bacteria are regulated by CcpA (catabolite control

protein) which is a pleiotropic regulator that mediates the global transcriptional response

to rapidly catabolizable carbohydrates.

Fig. 2.13. Transport and metabolism of carbohydrates in Lactococcus lactis (Neves et al, 2005).

Carbon source mediated nisin production

Nisin, as a safe natural food preservative has been used in more than 80 countries in the

world and approved by WHO, FDA and other regulatory agencies across the world. It has

a GRAS (Generally Recognized As Safe) status and has an E.C. number E.C. 22. Nisin

has a global market as a food preservative. A major limitation of its application in food is

the high cost of the commercial nisin. For commercialization of nisin, further research is

required to ensure low-cost production of nisin using cheap substrate. Lactococcus lactis

is the fastidious organism and requires a number of specific nutrients and physiological

condition for the growth and nisin production (Kozak and Dobranski, 1977). In L. lactis

subspecies lactis the amino acids serine, threonine and cysteine stimulate nisin


32

biosynthesis (de Vuyst, 1995). Many investigator have utilized complex media for the

production of nisin, which contains costly chemicals like yeast extract, peptone, tryptone

etc. The optimal media for growth and nisin production contained (w/v), 2.68% sucrose,

0.5% tryptone, 1% yeast extract, 0.3% tween-80, 0.02% MgSO4, 0.8% NaCl, 1.91%

K2HPO4 and 0.05% ascorbic acid (Zhou et al, 2008). However this medium is too

expensive for the commercial nisin production (de Man et al, 1960, Guerra and Pastrana,

2002; Steele and McKay, 1986). Other highly productive media, such as MRS

(developed by de Man, Rogosa, and Sharp) are also too expensive (Daba et al, 1993),

primarily due to high costs for yeast extract and purified minerals. The commercial nisin

A is produced on milk based medium (de Vuyst and Vandamme, 1994) and is used as a

preservative in some foods (Delves-Broughton, 1990). The addition of nisin in many food

products or for use in bulk fermentation processes remains too expensive (Ogden et al,

1988). Therefore, many scientists have used various by products like sugar molasses

(Egorov et al, 1980), mussel-processing waste (Guerra, 2002), hydrolyzed fish viscera

(Vasquez et al, 2008), skimmed milk (Jozala et al, 2007), cheese whey (Guerra et al,

2001), whey permeate and 3% fat milk (Mitra et al, 2009). Limitations of these by

products are high downstream purification, inconsistent supply, quality and/or price along

with resulting variability in nisin yields. In addition to nutrient availability, control of pH

also affects the growth rate and nisin production (Yang and Ray, 1994).

Recently, nisin is being used as therapeutics in treating multi-drug resistant

human pathogens because of the gradual emergence of antibiotic resistant human

pathogens. Nisin may replace the use of antibiotics as it is safe for human consumption,

non toxic and there is no report of development of nisin resistant Gram-positive bacteria

as it has dual mode of action. Therefore, nisin is an interesting candidate for future use.

Recently, nisin controlled gene expression system and nisin biosynthetic genes

are exploited for overproduction of nisin. This is achieved either by genetic engineering

of nisin genes or by induction of nisin genes with a specific carbon source. In 2005,

Cheigh et al, demonstrated improved nisin Z production by increasing the copy number

of nisRK or nisFEG involved in nisin Z biosynthesis. They also showed increased nisin Z

production by induction of nisin Z genes by lactose. Another study conducted by Lv et al,

2005, proposed the possibility of producing more nisin by controlling the sucrose

concentration at appropriate levels. Mitra et al, (2007) demonstrated a distinct behavior


33

of L. lactis W8 to grow and produce nisin in maltose medium with an initial ph of 11.0.

Nisin production is thus thought to be stimulated by certain carbohydrates and it is strain

specific. These observations raise the question of whether lactose, sucrose and maltose

can induce nisin biosynthesis through a separate regulatory mechanism. In an

investigation by Chandrapati et al, (2002), nisin A of L. lactis ATCC 11454 was found to

be induced by lactose and the nisA promoter was induced with galactose maximally in a

nisin-independent regulatory mechanism. They also demonstrated that nisin-independent

induction of nisA promoter utilizes two TCT direct repeat in the upstream of nisA

promoter. In the present investigation the role of sucrose on induction of nisin genes in L.

lactis KL has been investigated.

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