Protein Metabolism

Just as dairy lactic acid bacteria are well suited to utilize lactose as a source of energy and carbon, they are also well adapted to use casein as a source of nitrogen. Lactic acid bacteria cannot assimilate inorganic nitrogen and, therefore, they must be able to degrade proteins and peptides to satisfy their amino acid requirements. The absolute requirement for a system to degrade milk casein was first demonstrated by McKay and Baldwin (1974), who showed that Lc. lactis C2, cured of a plasmid containing the proteinase gene, was unable to grow to high cell density in milk. However, if milk was supplemented with hydrolyzed milk protein, the plasmid-cured strain grew like the parental strain. We now know that dairy lactic acid bacteria have evolved highly efficient systems for reducing large casein subunits to smaller pieces and for supplying cells with all of the amino acids necessary for growth in milk. The proteolytic system consists of three main components. The first involves the proteolysis of casein to form a large collection of peptides. In the second step, peptides are transported into cells by one of several transport systems. Once inside the cell, peptides are further hydrolyzed by a diverse group of peptidases to form free amino acids which are ultimately either metabolized or assimilated into protein (Fig. 9).

A. Proteinase System

Although lactic acid bacteria vary considerably in their ability to degrade milk protein, most organisms possess similar systems, as typified by the extensively

Figure 9 Proteolytic system in lactococci. Milk casein is hydrolyzed by cell envelope-associated proteinase (PrtP) to form oligopeptides, which are transported across the membrane by the oligopeptide transport system (Opp). Intracellular oligopeptides are then hydrolyzed by a variety of peptidases (PepA, PepC, PepF, PepO, and PepX) to form amino acids. Dipeptides and tripeptides and free amino acids, also present in milk, are transported by dipeptide tripeptide transporters (DtpT, DtpP) and amino acid (AA) transporters. Dipeptides and tripeptides are further hydrolyzed to amino acids. (Adapted from Mierau et al., 1997 and Steele, 1998.)

Figure 9 Proteolytic system in lactococci. Milk casein is hydrolyzed by cell envelope-associated proteinase (PrtP) to form oligopeptides, which are transported across the membrane by the oligopeptide transport system (Opp). Intracellular oligopeptides are then hydrolyzed by a variety of peptidases (PepA, PepC, PepF, PepO, and PepX) to form amino acids. Dipeptides and tripeptides and free amino acids, also present in milk, are transported by dipeptide tripeptide transporters (DtpT, DtpP) and amino acid (AA) transporters. Dipeptides and tripeptides are further hydrolyzed to amino acids. (Adapted from Mierau et al., 1997 and Steele, 1998.)

studied proteolytic system of Lactococcus. For Lc. lactis and other dairy lactic acid bacteria, casein is the primary source of amino acid nitrogen, since the nonprotein nitrogen and free amino acids available in milk (<300 mg/L) are quickly depleted. Because Lc. lactis is a multiple amino acid auxotroph and requires as many as eight amino acids, casein hydrolysis is essential. Casein utilization by Lc. lactis begins with elaboration of a cell envelope-associated serine proteinase. This proteinase, PrtP, is expressed as a large (>200 kD), inactive preproprotein-ase. The leader sequence, which is responsible for directing the protein across the cytoplasmic membrane, is removed, leaving the remaining protein anchored to the cell envelope. However, the proproteinase is not active until it is further processed by the maturation protein, PrtM. The latter presumably acts by inducing an autocatalytic cleavage event that results in hydrolysis of the pro region of the enzyme, leaving a mature PrtP with a molecular mass of 180-190 kD.

Although the proteinases among different strains of Lc. lactis are all genetically related and show only minor differences with respect to their amino acid sequence, the specific casein substrates and hydrolysis products of PrtP enzymes from lactococci can vary considerably. For example, proteinases belong to group A (formerly Pin-type) hydrolyze asr, P-, and K-caseins, whereas group E proteinases (formerly PI-type) have a preference for P-casein and relatively little activity for aSi- and K-caseins. Still, the functional organization of the PrtP and PrtM system varies little among lactococci. Both are required for rapid growth in milk, and genes for both (prtP and prtM, respectively) are induced when cells are grown in low-peptide media (e.g., milk) and repressed in peptide-rich media.

Over 100 caseinolytic products result from action of PrtP on P-casein (Juil-lard et al., 1995) Most are large oligopeptides (4-30 amino acid residues) with a major fraction between 4 and 10 residues. Free amino acids, dipeptides, and tripeptides are not formed. The first and most abundant oligopeptides formed by PrtP are generated from the C-terminal end of P-casein (Kunji et al., 1998), and it now appears that initial hydrolysis events cause casein to unfold so that other cleavage sites are exposed.

B. Amino Acid and Peptide Transport Systems

Although it was once believed that extracellular peptidases must be present to degrade further these peptides before transport, it is now well established that extracellular hydrolysis of peptides formed by PrtP does not occur, at least not by peptidases. Instead, lactococci and other lactic acid bacteria possess an array of amino acid and peptide transport systems able to transport substrates of varying size, polarity, and structure. Some of these are highly specific, whereas others have rather broad substrate specificity. They also vary as to energy sources used to fuel active transport.

As described earlier, the concentration of free amino acids in milk is too low to support growth of lactic acid bacteria. Still, lactococci possess at least 10 amino acid transporters, most of which are specific for structurally similar substrates. If the medium contains an adequate concentration of free amino acids, these transport systems can deliver enough amino acids to the cytoplasm to support growth. However, it has been suggested that the primary function of these transporters may be simply to excrete or efflux excess amino acids from the cytoplasm to maintain appropriate intracellular pool ratios (Kunji et al., 1996). That is, if peptides are indeed the primary source of amino acids, then some amino acids, generated from intracellular peptidases (see later), may accumulate faster than they can be assimilated. These free amino acids could then diffuse out of cells down their concentration gradient via the amino acid transporter operating in the reverse or efflux direction. If efflux of an amino acid is accompanied by a coupling ion (e.g., proton extrusion), then a net increase in the PMF is obtained. It may even be possible for amino acid efflux to provide enough energy to drive uptake of peptides (Kunji et al., 1996).

In contrast to the amino acid transporters, peptide transport is clearly necessary for lactic acid bacteria to grow in milk. Three groups of peptide transport systems have been identified. Two of these, DtpT and DtpP, transport dipeptides and tripeptides. DtpT is a large (463 amino acid residues) monomeric, PMF-dependent transporter that has affinity for hydrophilic peptides. Mutants with a deletion in the dtpT gene have been obtained and are unable to express DtpT and transport some peptides. In a defined medium, dtpT mutants grew poorly; however, growth of these mutants in milk was unaffected, indicating that DtpT is not essential in milk. DtpP, the other transport system in lactic acid bacteria that transports dipeptides and tripeptides, is an ATP-dependent transporter that has high affinity for hydrophobic peptides (Foucaud et al., 1995). It also appears to be unnecessary for growth of lactococci in milk.

The third and most important peptide transport system in lactic acid bacteria is the oligopeptide transport system (Opp). Since dipeptides and tripeptides are not released from casein, neither DtpT nor DtpP is required for growth in milk; lactococci instead rely on oligopeptides and Opp to satisfy all amino acid requirements. Indeed, mutants unable to express genes coding for the Opp system are unable to transport oligopeptides and are unable to grow in milk (Kunji et al., 1995; Tynkkynen et al., 1993). Although it was initially not known which oligo-peptides were actually transported by Opp, many of the structural and genetic features of the Opp system in Lc. lactis are now well defined (Detmers et al., 1998). The Opp complex belongs to the ABC (ATP binding casette) family of transporters and consists of five subunits: two transmembrane proteins (OppB and OppC), two ATP binding proteins (OppD and OppF), and a membrane-linked substrate-binding protein (OppA). The five genes coding for Opp are organized as an operon in the order oppDFBCA. A gene coding for an oligopeptidase (pepO) is also located immediately downstream of oppA and is cotranscribed with the opp operon.

The Opp system transports a diverse population of oligopeptides. Although PrtP releases over 100 peptides from P-casein, only 10-14 peptides apparently serve as substrates for Opp. All of these oligopeptides contain more than 4 and fewer than 11 amino acid residues (Kunji et al., 1998). Detailed analysis revealed that they contain proportionally higher levels of valine, proline, and glutamate and moderate levels of alanine, leucine, isoleucine, lysine, and serine. Importantly, these oligopeptides provide all essential amino acids, with the exception of histidine, needed by lactococci for growth in milk.

C. Peptidases

The third and final step of protein catabolism involves peptidolytic cleavage of Opp accumulated peptides. Over 20 different peptidases have been identified and characterized, either biochemically and/or genetically, in lactococci and lactoba-cilli (Table 2). Both endopeptidases (those that cleave internal peptide bonds) and exopeptidases (those that cleave at terminal peptide bonds) are widely distributed. Of the latter, only aminopeptidases have been reported; carboxypeptidases apparently are not produced. In general, concerted efforts of endopeptidases, aminopeptidases, dipeptidases, and tripeptidases are required fully to utilize peptides accumulated by the Opp system. Although there was once considerable

Table 2 Peptidases from Lactic Acid Bacteria

Peptidase

Abbreviation

Substrate or specificity3

Aminopeptidase A

PepA

Glu/Asp — (X)n

Aminopeptidase C

PepC

X — (X)n

Aminopeptidase L

PepL

Leu eE x Leu eE x — X

Aminopeptidase N

PepN

X — (X)n

Aminopeptidase P

PepP

X — Pro — (X)n

Aminopeptidase X Pyrrolidone carboxylyl peptidase

PepX Pcp

X — Pro — (X)n Glu — (X)n

Dipeptidase V

PepV

X — X

Dipeptidase D

PepD

X — X

Tripeptidase T

PepT

X — X — X

Proiminopeptidase

PepI

Pro — X — (X)n

Prolidase

PepQ

X — Pro

Prolinase

PepR

Pro — X

Endopeptidase F

PepF

(X)n — X — X — X — (X)n

Endopeptidase O

PepO

(X)n — X — X — (X)n

Endopeptidase E

PepE

(X)n — X eE x — (X)n

Endopeptidase G

PepG

(X)n — X eE x — (X)n

aThe positions of the hydrolyzed peptide bonds are shown by arrows.

aThe positions of the hydrolyzed peptide bonds are shown by arrows.

debate on the location of these peptidases, it is now well accepted, based on genetic as well as physical evidence (e.g., lack of signal peptides and anchor sequences, cell fractionation, and immunogold labeling experiments), that they are intracellular enzymes. Substrate size and specificity and other properties of peptidases from lactic acid bacteria have been of considerable interest, not only because of their physiological importance but also because of the significant role peptidases play in cheese manufacture and ripening.

1. Endopeptidases

Several endopeptidases have been described, including PepF and PepO in Lc. lactis (Monnet et al., 1994) and PepE, PepG, and PepO in Lb. helveticus (Christensen et al, 1999). Most of these endopeptidases are metalloenzymes that contain sequences typical of zinc-binding domains and hydrolyze oligopeptides of varying lengths as substrates. It is interesting to note that some endopeptidases (e.g., PepF) have pH optima in an alkaline range (7.5-9.0) and that peptidase activity at pH levels typical of ripened cheese (e.g., < pH 6) are very low. Thus, the contribution of some of these enzymes either to cell physiology or to cheese ripening may be minor. In addition, growth of endopeptidase mutants in milk is not affected (Mierau et al., 1993; Monnet et al., 1994).

2. Dipeptidases and Tripeptidases

Dipeptides and tripeptides that accumulate from the medium or that are formed from intracellular peptidolytic cleavage of oligopeptides are subsequently hy-drolyzed by dipeptidases and tripeptidases. Several of these have been purified and genes have been cloned (see Table 2) (Christensen et al., 1999). Although these enzymes vary with respect to their biochemical and physical properties, it appears, based on their substrate specificites, that some dipeptidases and tripepti-dases serve important functions. Several dipeptidases are also prolinases or proli-dases and hydrolyze peptides having N- or C-terminal proline residues. For example, PepQ from Lc. lactis and PepR from Lb. helveticus hydrolyze the dipeptides X-Pro and Pro-X, respectively (Boothe et al., 1990; Varmanen et al., 1996). Another peptidase that hydrolyzes proline-containing dipeptides and tripeptides has also been described (Baankries and Exterkate, 1991). The PepT tripeptidase from lactobacilli has preference for hydrophobic tripeptides (Savijoki and Palva, 2000). The role of these peptidases in cheese ripening will be discussed later.

3. Aminopeptidases

Aminopeptidases, enzymes that hydrolyze N-terminal peptide bonds and release N-terminal amino acids, are the most widespread peptidases found in lactic acid bacteria. Mierau et al. (1997) classified aminopeptidases based on their specific ity. The "general" or broad-specificity aminopeptidases, PepN and PepC, hy-drolyze peptides ranging in size from 2 to 12 amino acids, and, in general, have little activity on proline-containing dipeptides. They are well conserved among lactococci and lactobacilli.

Because P-casein is proline rich, many of the PrtP-generated oligopeptides contain proline. As noted above, proline-containing peptides are often poor substrates for general peptidases. ''Specific-task'' aminopeptidases (e.g., PepA, PepX, PepP, PepR, and PepI), in contrast, can hydrolyze these proline-containing peptides. Like other peptidases, these aminopeptidases vary as to substrate size and specificities. The substrates of PepP from Lc. lactis, for example, are oligopeptides containing from 4 to 10 amino acids and having the sequence X-Pro-Pro-(X)n (Mars and Monnet, 1995). In contrast, PepX from Lc. lactis hydrolyzes similar oligopeptides but in addition can also act on tripeptides, as well as some non-proline-containing peptides (Mayo et al., 1991). Both the general and specific aminopeptidases are especially important during cheese manufacture, since many oligopeptides contribute to bitter-flavored cheese if not degraded. The implications of proline-containing and other bitter peptides in cheese and their effect on flavor is discussed later.

Although it appears that no single peptidase is essential for cell growth, inactivation of multiple peptidases clearly is detrimental to growth in milk. Apparently, absence of a particular peptidase that degrades a particular peptide is not a very serious problem, since alternative peptides and peptidases are readily available. However, if several peptidases are missing, the rate of peptide hydrolysis would be expected to decrease. Indeed, when cells containing multiple mutations in pepO, pepN, pepC, pepT, and pepX were grown in milk, growth rates were reduced more than 10-fold (Mierau et al., 1996). That mutants reached final cell densities comparable to that of parent strains suggests that enough essential amino acids are eventually released by other peptidases.

D. Role of Protein Metabolism in Cheese Manufacture and Cheese Ripening

Although the PrtP system and components of the peptide transport and hydrolysis steps are essential for starter culture growth and activity, they also have important implications during cheese manufacture. Recent identification and characterization of many of the genes involved in protein metabolism have made it possible to construct mutants defective in a single enzymatic or transport activity. Comparing such mutant strains with the isogenic parent has provided a clearer picture of the role of various proteinase components on cheese properties.

Several studies have established that cheese made with strains deficient in proteinase activity lack flavor, have poor texture, and otherwise age poorly (Law et al., 1993). Thus, products of starter culture proteinases, combined with prod ucts of residual coagulant and milk proteases, impart desirable cheese flavor, either directly or by serving as substrates for additional reactions (Fox and Law, 1991; Urbach, 1995). However, despite the necessary role of PrtP in developing desirable aged cheese flavor, casein hydrolysis by PrtP also releases several peptides which are bitter. In general, bitter peptides are hydrophobic and their hydrolysis requires specific peptidases. Starter culture strains that possess the appropriate peptidases necessary to degrade these peptides are often considered as being ''nonbitter'' strains, as opposed to ''bitter'' strains that lack those enzymes and produce bitter cheese. Several peptidases have been proposed to have de-bittering activity (Baankreis et al., 1995; Tan et al., 1993). Experiments using peptidase mutants have provided in vivo evidence for the debittering role of peptidases. Cheese made with PepN or PepX mutants was bitter and had lower organoleptic quality (Mierau et al., 1997).

Although it is clear that bitterness, or lack of bitterness, is an important determinant of cheese flavor and quality, other aspects of protein metabolism undoubtedly influence the properties of aged cheese. Free amino acids and small peptides are thought to contribute to ''nutty'' and ''sweet'' flavor notes typical of Swiss, Parmesan, and other cheeses, whereas products of amino acid catabo-lism are primarily responsible for Cheddar cheese flavor (Fox and Wallace, 1997). Among degradation products formed from amino acids, methanethiol and other sulfur-containing compounds are considered to be essential in many cheese varieties, especially those that are surface ripened (Urbach, 1995; Weimer et al., 1999). Most of these sulfur compounds evolve from methionine and, for Cheddar, are produced by starter as well as nonstarter bacteria. Catabolism of aromatic and other amino acids by lactic acid bacteria certainly results in a large number of volatile compounds, some of which may be desirable, but others may be considered as flavor defects. However, the specific means by which metabolism of amino acids occurs and how products of nitrogen metabolism contribute to cheese flavor and quality await further study.

E. Lactic Acid Bacteria as Flavor Adjuncts

Once it was realized that peptidases from lactic acid bacteria could reduce bitterness and improve cheese flavor, several investigators began to identify suitable strains and to use them as culture adjuncts in cheese making. Species used as adjuncts include starter culture strains of Lc. lactis as well as nonstarter strains of Lb. casei, Lb. helveticus, and Lb. delbrueckii subsp. bulgaricus. In general, these strains have high peptidase activity. Since addition of such strains to cheese could also increase acid production, adjunct cultures are often prepared or used so that actual growth is minimized or prevented, while retaining their enzymatic activities. For example, using lactose-nonfermenting variants ensures that adjunct cells will not produce significant acid. Another way to deliver culture adjuncts is to heat- or freeze-shock the cells, treatments that cause cells to lose acid-forming ability, before addition to milk or curd or to lyse early in the ripening process. Cell extracts can also be added directly, and commercial products containing peptidase-rich extracts have been developed and are used for accelerated cheese-ripening programs.

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