Review ArticleThe Role of Microbial Aspartic Protease Enzyme in Food andBeverage Industries

Jermen Mamo and Fassil Assefa

Microbial, Cellular and Molecular Biology Department, College of Natural Science, Addis Ababa University, P.O. Box 1176,Addis Ababa, Ethiopia

Correspondence should be addressed to Jermen Mamo; jermenmamo@yahoo.com

Received 3 April 2018; Revised 16 May 2018; Accepted 29 May 2018; Published 3 July 2018

Academic Editor: Antimo Di Maro

Copyright © 2018 Jermen Mamo and Fassil Assefa. �is is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work isproperly cited.

Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total global enzymessale. According to the Nomenclature Committee of the International Union of Biochemistry andMolecular Biology, proteases areclassi�ed in enzymes of class 3, the hydrolases, and the subclass 3.4, the peptide hydrolases or peptidase. Proteases are generallygrouped into two main classes based on their site of action, that is, exopeptidases and endopeptidases. Protease has also beengrouped into four classes based on their catalytic action: aspartic, cysteine, metallo, and serine proteases. However, lately, threenew systems have been de�ned: the threonine-based proteasome system, the glutamate-glutamine system of eqolisin, and theserine-glutamate-aspartate system of sedolisin. Aspartic proteases (EC 3.4.23) are peptidases that display various activities andspeci�cities. It has two aspartic acid residues (Asp32 and Asp215) within their active site which are useful for their catalyticactivity. Most of the aspartic proteases display best enzyme activity at low pH (pH 3 to 4) and have isoelectric points in the pHrange of 3 to 4.5. �ey are inhibited by pepstatin. �e failure of the plant and animal proteases to meet the present global enzymedemand has directed to an increasing interest inmicrobial proteases. Microbial proteases are preferred over plant protease becausethey have most of the characteristics required for their biotechnological applications. Aspartic proteases are found in molds andyeasts but rarely in bacteria. Aspartic protease enzymes from microbial sources are mainly categorized into two groups: (i) thepepsin-like enzymes produced byAspergillus, Penicillium, Rhizopus, andNeurospora and (ii) the rennin-like enzymes produced byEndothia andMucor spp., such asMucor miehei,M. pusillus, and Endothia parasitica. Aspartic proteases of microbial origin havea wide range of application in food and beverage industries. �ese include as milk-clotting enzyme for cheese manufacturing,degradation of protein turbidity complex in fruit juices and alcoholic liquors, and modifying wheat gluten in bread by proteolysis.

1. Introduction

Enzymes are proteins produced by living organisms whichcatalyze the chemical reaction in greatly e�cient ways and areenvironment friendly. �ey have substantial advantages overchemical catalysts, in its speci�city, high catalytic activity, itscapability to work atmoderate temperatures, and the ability tobe produced in large amounts [1]. �e present high demandfor better use of renewable resources and the burden onindustry to work within an environment-friendly processencouraged the production of new enzyme-catalyst [1].

Proteases represent one of the three major groups of in-dustrial enzymes and occupy 60% of the total global enzyme

sale [1]. �ey have a wide range of application in various in-dustries to make a change in product taste, texture, and ap-pearance and in waste recovery. Besides this, they haveextensive applications in food industry, laundry detergents,leather treatment, bioremediation processes, and pharmaceu-tical industry.�eir depolymerization activity also plays amajorrole in nutrition. �e Novo industry of Denmark is among themajor protease producers of the world, which occupies 40% ofthe market share of proteases. It produces three types ofproteases, such as Aquaderm, NUE, and Pyrase, which are usedfor soaking, dehairing, and bating, respectively [1].

All cells, tissues, and organisms require proteolysis forgrowth and metabolism. Even a virus, the smallest nucleic

HindawiJournal of Food QualityVolume 2018, Article ID 7957269, 15 pageshttps://doi.org/10.1155/2018/7957269

acid-based self-replicating organism, typically requires ei-ther host cell proteolysis or enzymes coded by its own ge-netic material to provide processing of initial viral geneproducts. Microorganisms can be used as an excellent sourceof protease. *e incapability of plant and animal proteases tomeet the current global enzyme demand increased the interestfor the microbial protease. Microbial protease is preferredthan other sources because they possess almost all the char-acteristics desired for their biotechnological applications [1].

According to the Nomenclature Committee of the In-ternational Union of Biochemistry and Molecular Biology,proteases are classified in enzymes of class 3, the hydrolases, andthe subclass 3.4, the peptide hydrolases or peptidases. *e term“peptidase” is recommended by the Nomenclature Committeeof International Union of Biochemistry and Molecular Biologyto be used as synonymous with “peptide hydrolase” for any ofthe enzyme that hydrolyzes peptide bonds [1, 3].

However, proteases do not act in accordance with theuniversal enzyme nomenclature system due to their highstructural diversity and specificity. At present, proteases areclassified depending on three major criteria: (i) type of re-action catalyzed, (ii) chemical nature of the catalytic site, and(iii) evolutionary relationship with reference to structure [1].Currently, the term “peptidase” is also used equivalently with“protease” and “proteinase.” Peptidase was restricted to theenzymes included in subsubclasses EC 3.4.11–19, the exo-peptidases in the Enzyme Nomenclature (1984), while theterm “proteinases” was previously used for the enzymes in-cluded in subsubclasses EC 3.4.21–99 having the samemeaning as “endopeptidase.” However, the terms “protease”and “proteinase” are still preferred by many scientists [3].

1.1. Classification of Protease. Proteases are generally cate-gorized into two major groups based on their site of action,that is, exopeptidases and endopeptidases. Exopeptidases arethose proteases that cleave the peptide bond proximal to theamino or carboxy termini of the substrate (cleave N- orC-terminal peptide bonds of a polypeptide chain), whereasendopeptidases cleave peptide bonds distant from the ter-mini of the substrate (cleave internal peptide bonds) [3, 4].Proteases are also classified into acid, alkaline, and neutralproteases based on the pH at which they are active [1].

On the basis of their catalytic action, protease has beenalso grouped into four categories as aspartic, cysteine,metallo, and serine proteases. However, recently, three newsystems have been defined: the threonine-based proteasomesystem, the glutamate-glutamine system of eqolisin, and theserine-glutamate-aspartate system of sedolisin [2].

1.1.1. Serine Proteases. Serine proteases (EC 3.4.21) aredescribed by having a serine group in their active site. *eyare abundant and common among viruses, bacteria, andeukaryotes, indicating that they are useful for the organisms.Serine proteases are found in the exopeptidase, endopep-tidase, oligopeptidase, and omegapeptidase groups [4, 5].Most of the neutral and alkaline protease that are com-mercial serine proteases are produced from bacteria of thegenus Bacillus. Similar serine enzymes can also be produced

from other bacteria, such as *ermus caldophilus andDesulfurococcus mucosus, Streptomyces, Aeromonas, andEscherichia. Fungi species like Aspergillus oryzae similarlyproduce several serine proteases [1] (Table 1).

1.1.2. Cysteine/*iol Proteases. Cysteine proteases (EC3.4.22) occur in both prokaryotes and eukaryotes [4, 5].*ere are about 20 families of cysteine proteases.*e activityof all cysteine proteases was determined on a catalytic dyadconsisting of cysteine and histidine.*e order of Cys andHis(Cys-His or His-Cys) residues varies among the families.Generally, cysteine proteases are active only in the presenceof reducing agents, such as HCN or cysteine. Cysteineproteases are broadly categorized into four groups based onthe specificity of their side chain: (i) papain like, (ii) trypsinlike with preference for cleavage at the arginine residue,(iii) specific to glutamic acid, and (iv) others. Papain cysteineproteases have optimum activity at neutral pH, while a fewof them like lysosomal proteases have maximum activity atacidic pH. *ey are sensitive to sulfhydryl agents such asPCMB but are not affected by DFP and metal-chelatingagents [4]. Cysteine proteases are not so broadly distributedas was seen with serine and aspartic proteinases [1] (Table 1).

1.1.3. Aspartic Proteases. Aspartic proteinases (EC 3.4.23) oraspartyl proteinases are endopeptidases having two asparticacid residues (Asp32 and Asp215, pepsin numbering) withintheir active site that are vital for their catalytic activity [7]. Itis commonly known as acidic proteases [4].

Acidic proteases have been categorized into threefamilies, that is, pepsin (A1), retropepsin (A2), and enzymesfrom pararetroviruses (A3). *ey have been placed in clanAA. Most of the aspartic proteases (Aps) show the bestactivity at low pH (pH 3 to 4) and have isoelectric points inthe pH range of 3 to 4.5 [4]. *ey are inhibited by a hex-apeptide from Streptomyces that contains two statin residuescalled pepstatin. Aspartic proteases are also sensitive todiazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) in the pres-ence of copper ions. Microbial acid proteases exhibit speci-ficity against aromatic or bulky amino acid residues on bothsides of the peptide bond, which is similar to pepsin, but theiraction is less stringent than that of pepsin [4, 7].

Acid proteases represent an essential group of enzymes,extensively used in food, beverage, and pharmaceuticalindustries. For most of these applications, the crude enzymesshould at least partially purified and free from substancesthat could alter the characteristics of the product [8].

Aspartic protease enzymes from microbial sources aremainly categorized into two groups: (i) the pepsin-likeenzymes produced by Aspergillus, Penicillium, Rhizopus,and Neurospora and (ii) the rennin-like enzymes producedby Endothia and Mucor spp., such as Mucor miehei,M. pusillus, and Endothia parasitica [4, 6] (Tables 1 and 2).

1.1.4. Metalloproteases. Metalloproteases (EC 3.4.24) arehighly diversified types of proteases. *ey contain enzymes

2 Journal of Food Quality

from different origins, such as collagenases from higher or-ganisms, hemorrhagic toxins from snake venoms, andthermolysin from bacteria. For their actions, they requiredivalent metal ion. A total of about 30 families of metallo-proteases have been documented, out of which 17 containonly endopeptidases, 12 contain only exopeptidases, and 1(M3) contains both endo- and exopeptidases [4, 5] (Table 1).

1.2. Mechanism of Action of Aspartic Protease. Asparticproteases (EC 3.4.23) are peptidases that exhibit variousactivities and specificities. *ey are found in animals, plants,fungi, and viruses. Aspartic proteases (Aps) have beenconnected to a wide range of physiological functions, in-cluding mammalian digestion of nutrients (e.g., chymosinand pepsin A), defense against pathogens, yeast virulence(e.g., candidapepsins), metastasis of breast cancer(e.g., cathepsin D), pollen-pistil interactions (e.g., cardosin A),control of blood pressure (e.g., renin), degradation of hemo-globin by parasites (e.g., plasmepsins), and maturation ofHIV proteins (retropepsin) [11].

Aspartic proteases (Aps) belong to the A1 pepsin familystructurally.*ey are synthesized as preproenzymes similar toother pepsin enzymes. *e proenzyme is secreted and au-tocatalytically activated after cleavage of the signal peptide.Commonly, the active enzymes contain a single peptide chainof about 320–360 amino acid residues with a molecular massof 32–36 kDa. Apsmostly have a β-strand secondary structurearranged in a bilobal conformation as confirmed by X-raycrystallographic analyses [11]. *e two lobes are homologous

to each other and have evolved by gene duplication. *ecatalytic center is located between the two lobes and containsa pair of aspartate residues, one in each lobe, that are essentialfor the catalytic activity [11]. *e retropepsin molecule hasonly one lobe, which consists of only one aspartic residue, andthe activity requires the formation of a noncovalent homo-dimer [4]. Generally, aspartic endopeptidases depend on itsaspartic acid residues for their catalytic activity [4].

In pepsin family enzymes, the catalytic Asp residues aremostly contained in an Asp-*r-X motif, where X is Ser or*r. *e role of these Asp residues is to activate a watermolecule that facilitates the nucleophilic attack on peptidebond of the substrate and also to find another water mol-ecule that is used in substrate binding by the formation ofhydrogen bond. *e catalytic center is enough to accom-modate as a minimum of seven residues of the polypeptidesubstrate. A flexible structure (flap) found at the entry of thecatalytic site controls the specificity of the enzyme [11]. APsare mostly active at acidic pH. *e optimum pH of asparticprotease is determined by the electrostatic potential at theactive site, which in turn is determined by the position andorientation of all residues near the active site [11] (Table 2).

2. Microbial Source of Aspartic Protease

*e failure of the plant and animal proteases to meet thepresent world demands of the enzyme has directed to anincreased interest in microbial proteases. *e presence ofdesired characteristics for biotechnological applications inmicrobial protease enzymes helps it to be preferred over

Table 1: *e characteristics of four types of protease [6].

Properties ECnumber

Molarmassrange(kDa)

pHoptimum

Temperatureoptimum

Metalrequirement

(s)

Active aminoacid(s) Major inhibitor(s) Major sources

Aspartyl orcarboxylprotease

3.4.23 30–45 3–5 40–55 Ca2+ Aspartate orcysteine Pepstatin

Aspergillus, Mucor,Endothia, Rhizopus,

Pencillium,Neurospora, animaltissue (stomach)

Cysteine orthiol protease 3.4.22 34–35 2–3 40–55 — Aspartate or

cysteineIodoacetamide,

p-CMB

Aspergillus, stem ofpineapple (Ananascomosus), latex offig tree (Ficus sp),papaya (Carica

papaya),Streptococcus,Clostridium

Metalloprotease 3.4.24 19–37 5–7 65–85 Zn2+, Ca2+ Phenylalanineor leucine

Chelating agentssuch as EDTA and

EGTA

Bacillus,Aspergillus,Pencillium,

Pseudomonas,Streptomyces

Serine protease 3.4.21 18–35 6–11 50–70 Ca2+Serine,

histidine, andaspartate

PMSF, DIFP,EDTA, soybeantrypsin inhibitor,phosphate buffers,indole, phenol, andtriamino acetic acid

Bacillus,Aspergillus, animal

tissue (gut),Tritirachium album(thermostable)

Journal of Food Quality 3

Tabl

e2:

Characteristicsof

variou

smicrobial

aspartic

proteases(EC3.4.23)[9,1

0].

Num

ber

Enzyme

number

Acceptedname

Other

names

Source

Substrate

Optim

umpH

Optim

umtemp

Com

ment

1EC

3.4.23.8

Yeastproteinase

ASaccharopepsin

——

——

Now

EC3.4.23.25

2EC

3.4.23.9

Rhizopus

acid

proteinase

Rhizop

uspepsin

——

——

Now

EC3.4.23.21

3EC

3.4.23.10

Endothia

acid

proteinase

Endo

thiapepsin

——

——

Now

EC3.4.23.22

4EC

3.4.23.16

Retrop

epsin

HIV

aspartyl

protease,H

IVproteinase,

retrop

roteinase,HIV

-lp

rotease,HIV

-2protease

HIV

Viral

gagandgag-po

lproteinprecursor

5.5–

6.0

30(37°C)

5EC

3.4.23.18

Aspergillo

pepsin

I

Proteinase,

Aspergillu

sacid

protease,A

spergillu

sacid

proteinase,

Aspergillu

saspartic

proteinase,

Aspergillu

scarboxyl

proteinase,d

enapsin

,proctase

B,P,

proteinase

B,sumizym

eAP,

trypsin

ogen

kinase,

pepsin-typeaspartic

proteinase,carbo

xyl

proteinase

Foun

din

avarietyof

Aspergillu

sspecies

(imperfectfung

i)

Hydrolysis

ofproteins

with

broadspecificity

Differsfrom

substrate

tosubstratebu

tisin

therang

eof

1.6–

6.5

Differsbu

tinthe

rang

eof

30–5

0Fo

rmerly

includ

edin

EC3.4.23.6

6EC

3.4.23.19

Aspergillo

pepsin

II

ProteinaseA,proctase

A,A

spergillu

sniger

var.macrosporus

aspartic

proteinase,

nonp

epsin

-typeacid

proteinase

Isolated

from

Aspergillu

snigervar.

macrosporus

Preferentia

lcleavage

inBchainof

insulin

—30

° CFo

rmerly

includ

edin

EC3.4.23.6

7EC

3.4.23.20

Penicillo

pepsin

PeptidaseA,

Penicillium

janthinellu

maspartic

proteinase,a

cid

protease

A,

Penicillium

Xacid

proteinase

From

theim

perfect

fung

usPenicillium

janthinellu

m

Hydrolysis

ofproteins

with

broadspecificity

similarto

that

ofpepsin

A,p

referring

hydrop

hobicresid

ues

Differsbu

tinthe

rang

eof

2.5–

3.6

Differsbu

tinthe

rang

eof

50–7

5

Form

erly

EC3.4.23.7;

form

erly

includ

edin

EC3.4.23.6

4 Journal of Food Quality

Tabl

e2:

Con

tinued.

Num

ber

Enzyme

number

Acceptedname

Other

names

Source

Substrate

Optim

umpH

Optim

umtemp

Com

ment

8EC

3.4.23.21

Rhizop

uspepsin

Rhizopus

aspartic

proteinase,n

eurase,

proteinase,R

hizopus

acidprotease,R

hizopus

acid

proteinase

From

thezygomycete

fung

usRh

izopus

chinensis,R

.niveus

Hydrolysis

ofprotein

swith

broadspecificity,

prefershydrophobic

resid

ues,clo

tsmilk,and

activates

trypsinogen

3–4(trypsinogen);

3.5–

4.0(casein)

Differsfrom

substrate

tosubstratebu

tinthe

rang

eof

25and50

EC3.4.23.9

(formerly),EC

3.4.23.6

(formerly

includ

edin)

9EC

3.4.23.22

Endo

thiapepsin

Endothia

aspartic

proteinase,E

ndothia

acid

proteinase,

Endothia

parasitica

acid

proteinase,

Endothia

parasitica

aspartic

proteinase

From

theascomycete

Endothia

parasitica

Hydrolysis

ofproteins

with

specificity

similarto

that

ofpepsin

A,p

refers

hydrop

hobicresid

ues

—30

° C

EC3.4.23.10

(formerly),EC

3.4.23.6

(formerly

includ

edin)

10EC

3.4.23.23

Mucorpepsin

Proteinase,M

ucor

acid

proteinase,

Mucor

acid

protease,

Mucor

renn

in,M

ucor

aspartic

proteinase

Mucor

pusillus

empo

rase

from

ase

100,

Mucor

pusillus

renn

infrom

ase46TL

,Mucor

mieheirennin

Isolated

from

the

zygomycetefung

iMucor

pusillusand

M.

miehei

Hydrolysis

ofproteins,favou

ring

hydrop

hobicresid

ues,

clotsmilk

Differsbu

tinthe

rang

eof

3.5–

5.6

Differsbu

tinthe

rang

eof

40–6

3EC

3.4.23.6

(formerly

includ

edin)

11EC

3.4.23.24

Candidapepsin

Candidaalbicans

asparticproteinase,

Candidaalbicans

carboxyl

proteinase,

Candidaalbicans

secretoryacid

proteinase,C

andida

olea

acid

proteinase,

Candidaaspartic

proteinase,C

andida

olea

asparticp

roteinase

Imperfecty

east

Cand

idaalbicans

Hydrolyzedprotein

(preferentialc

leavage

atthecarboxyl

ofhydrop

hobicaa,

activ

ates

trypsin

ogen,

anddegrades

keratin

Variesbetween2.5

and5.5

42° C

(denatured

hemoglobin);4

5(at

pH3.0)

EC3.4.23.6

(formerly

includ

edin)

12EC

3.4.23.25

Saccharopepsin

Yeastendo

peptidase

A,S

accharom

yces

aspartic

proteinase,

aspartic

proteinase

yscA

,proteinaseA,

proteinase

yscA

,yeast

proteinase

A,

Saccha

romyces

cerevisia

easpartic

proteinase

A,y

east

proteinase

A,P

RA

Saccha

romyces

cerevisia

e

Hydrolysis

ofproteins

with

broadspecificity

forpeptidebo

nds

4–6.5

25° C

EC3.4.23.8

(formerly),EC

3.4.23.6

(formerly

includ

edin)

Journal of Food Quality 5

Tabl

e2:

Con

tinued.

Num

ber

Enzyme

number

Acceptedname

Other

names

Source

Substrate

Optim

umpH

Optim

umtemp

Com

ment

13EC

3.4.23.26

Rhod

otorulapepsin

Rhod

otorulaaspartic

proteinase,

Cladosporiu

macid

protease,

Paecilomyces

proteinase,

Rhodotorulaglutinis

aspartic

Proteina

se,

Rhodotorulaglutinis

aspartic,R

hodotorula

glutinisacid

proteinase

Rhodotorulaglutinis

andCladosporiu

msp.

Cleaves

benzyloxycarbo

nyl-

Lys-+-A

la-A

la-A

laandactiv

ates

trypsin

ogen

2.0–

2.5(casein),

2.5–

2.7(casein,

hemoglobin),2

.5–3

.0(acid-denatured

hemoglobin)

Differsbetween55

and60

° CFo

rmerly

includ

edin

EC3.4.99.15

14EC

3.4.23.28

Acrocylindrop

epsin

Acrocylindricum

proteinase,

Acrocylindrium

acid

proteinase

Acrocylindrium

sp.

Preference

for

hydrop

hobicresid

ues

atP1

andP1

0and

actio

non

theBchain

ofinsulin

2.0(casein)

—

EC3.4.99.1

(formerly),EC

3.4.23.6

(formerly

includ

edin)

15EC

3.4.23.29

Polypo

ropepsin

Polypo

rusaspartic

proteinase,Irpex

lacteusaspartic

proteinase,Irpex

lacteuscarboxyl

proteinase

B

Basid

iomycete

Polyporustulip

iferae

(formerly

Irpex

lacteus)

Milk

-clotting

activ

ity,

broadspecificity,b

utfails

tocleave

Leu1

5-Ty

ror

Tyr16-Leuof

insulin

Bchain

2.8(hem

oglobin),4

.0(Phe-Leu-A

la-A

la)

——

16EC

3.4.23.30

syno

nyms

Pycnop

orop

epsin

Proteinase

ia,

Pycnoporus

coccineus

aspartic

proteinase,

tram

etes

acid

proteinase

Basid

iomycete

Pycnoporus

sanguineus,formerly

know

nas

P.coccineus

andTram

etes

sanguinea

Cleavingon

lythree

bond

sin

theBchain

ofinsulin

:Ala14

+Leu,

Tyr16+Leu,

andPh

e24+Ph

e

——

EC3.4.99.25

(formerly),EC

3.4.23.6

(formerly

includ

edin)

17EC

3.4.23.31

Scytalidop

epsin

Scytalidium

aspartic

proteinase

A,

Scytalidium

lignicolum

aspartic

proteinase,

Scytalidium

lignicolum

carboxyl

proteinase,

Scytalidium

lignicolum

acid

proteinase

Scytalidium

lignicolum

Hydrolysis

ofproteins

with

specificity

similarto

that

ofpepsin

Abu

talso

cleavesCys

(SO3H

)7Gly

andLeu1

7Val

intheBchainof

insulin

2–3.5(casein),3

.6(benzyloxycarbon

yl-

Phe-Glu-A

la-A

la)

50and55

—

6 Journal of Food Quality

Tabl

e2:

Con

tinued.

Num

ber

Enzyme

number

Acceptedname

Other

names

Source

Substrate

Optim

umpH

Optim

umtemp

Com

ment

18EC

3.4.23.32

Scytalidop

epsin

B

Scytalidium

aspartic

proteinase

B,Ganod

ermalucidu

mcarboxyl

proteinase,

Ganod

ermalucidu

maspartic

proteinase,

Scytalidium

lignicolum

aspartic

proteinase

B,SL

B

A2n

denzymefrom

Scytalidium

lignicolum,L

entin

usedodes

(sim

ilar

enzyme),G

anoderma

lucidu

m(sim

ilar

enzyme)

Hydrolysis

ofproteins

with

broadspecificity,

cleaving

Phe24Ph

e,bu

tnot

Leu1

5-Ty

randPh

e25-Ty

rin

the

Bchainof

insulin

2.0–

2.7(casein),

2.9–

3.2(hem

oglobin)

50,5

2,and65

—

19EC

3.4.23.33

Xanthom

onapepsin

Xan

thom

onas

aspartic

proteinase

proteinase,

Xan

thom

onas

aspartic

PCP,

pseudo

mon

ascarboxyl

proteinase

Pseudomon

assp.,

expressio

nin

E.coli,

Xan

thom

onas

sp—

2.7(casein,

acid-

denatured

hemoglobin),3

(acid-

denatured

hemoglobin,

casein)

50and55

Now

transferredto

EC3.4.21.101,

xantho

mon

alisin

20EC

3.4.23.35

Barrierpepsin

Barrierproteinase,

barproteinase

Saccha

romyces

cerevisia

e

Selectivecleavage

of-Leu

6 +Lys−

bond

inthemating

pherom

onea-factor

5–5.3

——

21EC

3.4.23.36

Sign

alpeptidaseII

Prem

urein-leader

peptidase,

prolipop

rotein

signal

peptidase,leader

peptidaseII,leader

peptidaseII

E.coli,

Enterobacter

aerogenes,

Staphylococcus

aureus

Hydrolyzes-Xaa-Yaa-

Zaa+

(S,

diacylglyceryl)C

ys−,

inwhich

Xaa

ishydrop

hobic

(preferablyLeu)

and

Yaa(A

laor

Ser)

and

Zaa(G

lyor

Ala)h

ave

Small,neutralside

chains

637

—

22EC

3.4.23.37

Pseudo

mon

apepsin

Pseudomon

assp.

pepstatin

-insensitive

carboxyl

proteinase,

pepstatin

-insensitive

carboxyl

proteinase

Pseudomon

assp.,

Xan

thom

onas

sp.

Hydrolysis

oftheB-

chainof

insulin

atGlu13-A

la-,Leu1

S-Ty

r-,P

he2S-Tyr-,and

angiotensin

Iat

Tyr4-

lle.A

good

synthetic

substrateisLys-Pro-

Ile-G

lu-Phe-(4-nitro)

Phe-Arg-Leu)

3(acid-denatured

hemoglobin,

casein)

50Now

EC3.4.21.100,

pseudo

mon

alisin

23EC

3.4.23.41

Yapsin

1Ye

astaspartic

protease

3,Ya

p3Saccha

romyces

cerevisia

e

Hydrolyzesvariou

sprecursorproteins

with

Arg

orLysinP1

,andcommon

lyArg

orLysalso

inP2

——

—

Journal of Food Quality 7

Tabl

e2:

Con

tinued.

Num

ber

Enzyme

number

Acceptedname

Other

names

Source

Substrate

Optim

umpH

Optim

umtemp

Com

ment

24EC

3.4.23.42

*ermop

sin—

*ermop

hilic

archeaon

Sulfolobu

sacidocaldariu

s

Similarin

specificity

topepsin

Apreferring

bulkyhydrop

hobic

aminoacidsinP1

and

P10

——

—

25EC

3.4.23.43

Prepilinpeptidase

—Manyspeciesof

bacteria

carrypili

Typically

cleaves

a–G

ly+Ph

e−—

——

26EC

3.4.23.44

Nod

avirus

endo

peptidase

BlackBe

etle

virus

endo

peptidase,flo

ckho

usevirus

endo

peptidase

From

several

nodavirusesthat

are

pathogensof

insects

Hydrolysis

ofan

asparaginy

lbon

d,typically

–Asn+Ala−

or–A

sn+Ph

e−

——

—

27EC

3.4.23.47

HIV

-2retrop

epsin

—HIV

-2—

——

—

28EC

3.4.23.48

Plasminogen

activ

ator

Pla

—Ye

rsinia

pestisthat

causes

plague

Con

vertsh

uman

Glu-

plasminogen

toplasmin

bycleaving

theArg560+Val

peptidebo

nd,a

lsocleavesarginy

lbon

dsin

otherproteins

——

—

29EC

3.4.23.49

Omptin

Protease

VII,p

rotease

A,o

mpT

protease,

proteina,protease

VII,O

mpT

Aprod

ucto

fthe

ompT

gene

ofEscherichiacoli

Has

avirtual

requ

irem

entforArg

intheP1

position

——

—

30EC

3.4.23.50

Hum

anendo

geno

usretrovirus

Kendo

peptidase

Hum

anendo

geno

usretrovirus

K10

endo

peptidase,

endo

geno

usretrovirus

HER

V-K

10pu

tativ

eprotease,

human

endo

geno

usretrovirus

Kretrop

epsin

HIV

-1Cleavageof

the

–SQNY+

PIVQ−

cleavage

site

——

—

31EC

3.4.23.51

HycIpeptidase

HycI,HycE

processin

gprotein

Escherichiacoli

Removes

a32-aaacid

peptidefrom

the

C-terminus

ofthe

precursorof

the

hydrogenase3

——

—

8 Journal of Food Quality

plant and animal proteases. Microorganisms represent anexcellent source of enzymes owing to their wide biochemicaldiversity and their susceptibility to genetic manipulation.About 40% of the total global enzyme sales are from mi-crobial sources [4]. Aspartic protease is found in molds andyeasts, but rarely in bacteria [6].

2.1. FungalAspartic Proteases. Fungi produce a wider range ofenzymes than do bacteria. For instance, Aspergillus oryzaeproduces all types of proteases such as acid, neutral, and alkalineproteases. *e pH ranges (pH 4 to 11) of fungi protease arewide, and this shows their broad substrate specificity. However,they have a lower reaction rate and low heat tolerance than dothe bacterial enzymes. Fungal enzymes can be convenientlyproduced in a solid-state fermentation process [4].

Due to the global scarcity of calf chymosin, fungal asparticproteases (Aps) have been used as milk-clotting enzymes inthe dairy industry for about 30 years. *e aspartic proteaseenzymes produced from Mucor miehei, Mucor pusillus, andCryphonectria (Endothia) parasitica and marketed under thetrade names Rennilase®, Fromase®, Novoren®, Marzyme®,Hannilase®, Marzyme®, and Suparen® are usually used for theproduction of various types of cheeses [11].*e production ofaspartic acid proteases that have a certain industrial appli-cation was also reported from Botrytis cinerea [12]. An al-ternative for milk clotting enzyme for cheese production wasalso reported from more than 100 fungal sources. Fungi thatproducemilk clotting enzyme are universal and easily isolatedfrom various environments [13].

Most of these extracellular fungal aspartic proteasesproduced from Aspergillus species. *ese include Aspergillusoryzae, Aspergillus fumigatus, Aspergillus saitoi, Aspergillus

awamori, and Aspergillus niger [5]. Aspartic proteases fromCandida albicans have been intensively studied due to itsrole in various forms of candidiasis. *e presence of secretedaspartic protease enzyme in C. albicans contributes to itsvirulence factor. *e major proteases secreted in vitro byC. albicans, C. parapsilosis, and C. tropicalis have beentermed as Sap2, Sapp1, and Sapt1, respectively [5].

A different study has revealed that C. albicans have atleast eight secreted aspartic protease genes (SAP genes). Outof these, SAP2 gene is the leading form expressed ina number of strains. SAP2 encodes 398-residue pre-proprotein that is processed to a 342-residue mature en-zyme, a typical aspartic proteinase having an optimum pHbetween 3 and 4 and inhibited by pepstatin A [14]. *eoptimum pH for fungal aspartic proteases is between 4 and4.5; however, they are stable at a pH range between 2.5 and6.0. Fungal aspartic protease enzymes are specifically usefulin the cheese-making industry due to their narrow pH andtemperature specificities [4] (Tables 2 and 3).

2.2. Bacteria. It has been widely assumed that bacteria donot produce clotting enzymes due to only a few researchworks conducted on bacteria [15]. But the study conductedon the genomes of two bacteria, Escherichia coli and Hae-mophilus influenzae, showed that the recombinant proteinsresulting from the expression of each of these DNA regionsare active aspartic proteinases [15].

A novel retropepsin-like enzyme (APRc) has been alsofound in two pathogenic species of Rickettsia such asR. conorii and R. rickettsia. *is APRc enzyme is particularlyinhibited by drugs clinically used to treat HIV infections,and therefore, this enzyme can be used as a target for

Table 3: Microbial sources of milk-clotting aspartic proteases [13].

Microorganisms PropertiesPleurotus sojur-caju (white rot fungi) Clotting activity under cheese-making conditions

Mucor bacilliformis High structural similarity to bovine chymosin lowerthermostablity than Rhizomucor miehei protease

*ermoascus aurantiacus Enzymatic hydrolysis of bovine casein differed largelyfrom proteolysis patterns generated by bovine chymosin

*ermomucor indicae-seudaticae N31 Crude enzymatic extract showed high milk-clottingand low proteolytic activity and low thermostability

Metschnikowia reukaufii Milk-clotting activity, successfully cloned intoEscherichia coli

Myxococcus xanthus

Molecular mass: 40 kDa, highest clotting activity atpH 6 and 37°C, acceptable yield and properties of thecurd in cheese-making experiments, successfully

cloned into Escherichia coli

Enterococcus faecalisSimilar electrophoretic patterns of hydrolyzed

k-casein as Rhizomucor miehei, effectively applied forcamembert cheese manufacture

Nocardiopsis sp. Milk-clotting ability of extracellular extracts, optimizationof enzyme yield by fermentation conditions

Bacillus subtilisRatio of milk clotting to proteolytic activity is

comparable with commercial fungal protease but hashigh thermostability

Bacillus licheniformis Shows typical milk-clotting kinetics

Journal of Food Quality 9

therapeutic intervention.*is implies that a retropepsin typeof aspartic protease enzymes is found in prokaryotes, sig-nifying that these enzymes may represent an ancestral formof these proteases [16]. Acid protease produced by Bacillussubtilis, which is GRAS (genetically regarded as safe), isgradually replacing chymosin in cheese making, and pro-tease produced from B. subtilis var. natto has showed milkclotting [4, 17]. Microbial aspartic protease produced fromBacillus amyloliquefaciens was also used for the productionof miniature cheddar-type cheeses [18] (Tables 2 and 3).

2.3. Viruses. *e concern to viral proteases has been startedas a result of its involvement in the cause of certain fataldiseases, such as AIDS and cancer. Several types of viruseshave serine, aspartic, and cysteine peptidases. All of thevirus-encoded peptidases are endopeptidases, but there areno metallopeptidases in viruses [4]. *e crystal structures ofaspartic proteases from retroviruses such as HIV and Roussarcoma have been widely studied and determined since1989 [5]. *e aspartyl proteases of retroviruses have a greatrole in viral assembly and replication. *e aspartyl proteasesof retroviruses are homodimers and expressed as a part ofthe polyprotein precursor. *e mature protease is releasedby autolysis of the precursor [4].

Microbes serve as an ideal source of protease enzymes,even though they are widespread in nature. *e rapidgrowth, the lesser space required for cultivation, and the easefor genetic manipulation to generate new enzymes withimproved properties make microbes desirable for proteaseenzyme production [4] (Table 2).

3. Application of Microbial Aspartic Protease

*e proteases have been considered as one of the mostessential groups of enzymes in enzyme industry and havevarious applications in different industries, such as de-tergents, foods, pharmaceuticals, and leather [19].

Microbial acid proteases have an application mostly inthree industries: food, beverage, and pharmaceuticals [5].However, there is limited evidence available on the appli-cation of aspartic proteinases other than cheese industry thathas been the preferred area of application to date [7].

3.1. Application in Dairy Industry. *e major application ofacid proteases is for cheese production in dairy industry.*emicrobial milk-coagulating proteases belong to a class ofacid aspartate proteases and have molecular weights between30,000 and 40,000 [4]. *e major role of acid proteases incheese production is to hydrolyze the specific peptide bond(the Phe105-Met106 bond) to generate para-K-casein andmacropeptides. Chymosin is preferred due to its highspecificity for casein, which accounts for its exceptionalperformance in cheese production. *e aspartic proteasesproduced by microbes such asMucor miehei, B. subtilis, andEndothia parasitica, which is GRAS (genetically regarded assafe), are gradually replacing chymosin in cheese making [4].

*e enzymatic coagulation of milk is a two-phase pro-cess; any variation in the chemical environment can affect

the two phases of reaction separately [7]. In the first phase,calf rennet (CAR) and most microbial proteases clot milkwith the cleavage of K-casein at the Phenylalanine105-Methionine 106 bond, which liberates hydrophilic glyco-peptide (residues 106–169) that enters into the whey andpara-K-casein (hydrophobic) [7, 20]. Proteinase from Cry-phonectria parasitica cleaves the S104-F105 bond. Rennincan also hydrolyze other milk proteins (αs1-, αs2-, andβ-caseins and α-lactalbumin) at a lower rate. Fungal pro-teases cause extensive nonspecific hydrolysis of bothK-casein and para-K-casein as compared to rennin in whichits activity is limited to the hydrolysis of K-casein with theformation of only macropeptide and para-K-casein [20].

*e second phase is nonenzymatic in which para-K-casein and other caseins aggregate under the influence ofCa2+, which eventually leads to gel formation. *ese twosteps of milk-clotting activity overlap with each other wherethe aggregation of micelles starts before the end of theenzymatic process [20].

*e production of UF (ultrafiltrated) white soft cheeseusing fungal rennin (1 ml fungal rennin/100ml milk) fromRhizomucor miehei NRRL 2034 in the laboratory showedvery close properties to calf rennet cheese used as a control.*e cheese produced with fungal rennet revealed bettervalues of soluble nitrogen (SN), total volatile fatty acids(TVFAs), tyrosine, and tryptophan than control cheese.Moreover, the sensorial examination conducted on cheeseproduced with fungal rennet showed that the experimentalcheese had a soft body, smooth texture, and desirable tasteduring cold storage for 2 months [21, 22].

*e production of miniature cheddar-type cheeses usingmicrobial rennet from Bacillus amyloliquefaciens (clottingenzyme (MCE)) and calf rennet (CAR) did not have sig-nificant differences in gross composition with the exceptionof pH. *e level of αs1-casein and β-casein hydrolysis de-termined by urea-PAGE was equivalent for both cheesesamples. *e concentration of peptides in 2 cheese sampleswas increased during the course of ripening. However, theratio of hydrophobic to hydrophilic peptides was higher inCAR-C than in MCE-C.*eMCE-C was softer than CAR-Cas a result of higher protein hydrolysis. Microbial rennetfrom B. amyloliquefaciens contributed to higher photolyticrates that reduced ripening time [18].

In the study that compares the milk-clotting activity ofproteinase produced by B. subtilis var. natto, Rhizopus oli-gosporus, and commercial rennet, the curd formed by thecommercial rennet had the highest viscosity and curdtension and the shortest clotting time followed by the curdproduced by proteinase from Rhizopus among the threeenzymes. *e highest proteolytic activity was recorded bythe enzymes from B. subtilis, while the highest milk-clottingenzymes were noticed in commercial rennet. Observationsof microstructures by scanning electron microscope (SEM)showed that the three-dimensional network of curd formedby commercial rennet was denser, firmer, and smootherthan others [17]. Fresh cheese produced using anochratoxin-free extracellular acid protease from Aspergillusniger FFB1 and reconstituted cowmilk as a substrate showedsimilar basic characteristics (pH 4.5, acid taste, and white

10 Journal of Food Quality

color) as cheeses produced with calf rennet [23]. *e con-centration of free amino acid (FAA) and physiochemicalcharacteristics were similar in the Turkish, white, brinedcheese produced using calf rennet and microbial rennetfrom Rhizomucor miehei [24].

*e experimental study conducted on cheese producedby calf rennet and microbial rennet from Rhizomucor mieheiafter 90 days of ripening period revealed that the physico-chemical characteristics and the contents of the total bittertasting amino acids (Phe, Leu-Ile, Val, and Pro) were similarfor both types of cheeses. Phe, Leu-Ile, Gln, Val, Pro, and Alawere the principal free amino acids (FAAs) in the whitecheeses at all stages of ripening [25].

*e study during 2 months under pickling conditions ofdomiati cheese produced using the M. mucedo KP736529enzyme (E-cheese) and commercial calf rennet (C-cheese) ascontrol showed that the yield and chemical properties ofE-cheese were better than those of C-cheese. *e fungalenzyme (MCE) showed higher proteolytic activity than calfrennet with the absence of bitter taste defects. *e organo-leptic scores of E-cheese were also higher than C-cheese [26].

In the study conducted at Hyderabad, the milk-clottingenzyme produced from Rhizomucor miehei was subjected toammonium sulfate and acetone precipitation and then used forcheese production. *e result indicates that higher quantity ofcheese (280 g/500ml milk) was obtained by ammonium sulfateprecipitation than with acetone precipitation (220 g/500mlmilk). But the milk curdling time was found to be rapid withthe enzyme obtained by acetone precipitation than that ob-tained by saturated ammonium sulfate precipitation [27].

3.2. Application in the Bakery Industry. Wheat flour isa major component of baking processes. It comprises aninsoluble protein called “gluten” that regulates the doughproperty. Endo and exoproteinases from Aspergillus oryzaehave been applied to improve the wheat gluten by limitedproteolysis [4]. Moreover, fungal aspartic proteases have alsobeen broadly used in the production of food seasonings andthe improvement of protein-rich foods such as bread andrelated foodstuffs [5].

*e fermentation of liquid dough made fromwheat flourwith the combination of enterococci and Rhizopus oryzaeproteases (dough B) for 48 h revealed that it had threefoldhigher concentration of water-soluble peptides than thechemically acidified dough (CAD) used as the control. *econcentration of free amino acids was also being higher indough B. *e SDS-PAGE analysis showed that gliadins werealmost fully degraded in dough B, while albumin and glu-tenin fractions were incompletely hydrolyzed [28]. In an-other study, gluten treated with pepsin had shown a bandless than 10 kDa, while gluten treated with pronase, chy-motrypsin, and papain showed two bands corresponding to40 and 10 kDa by HPLC analysis. *ese results suggest thatless protease-resistant peptides exist in gluten treated withpepsin as compared to gluten treated with pronase, chy-motrypsin, and papain [29].

*e experimental gluten-free pasta (E-GFp) producedfrom gluten-free sourdough after fermentation by lactic acid

bacteria and fungal proteases showed higher chemicalscores, essential amino acid profile, biological value, andnutritional index than those of durum wheat pasta (C-DWp). *is might be due to proteolysis during sour-dough fermentation. *e in vitro protein digestibility ofE-GFp has also resulted in the highest value. *e sensorycharacteristic of E-GFp was acceptable as shown by sensoryanalysis [30].

*e hydrolysis of wheat gluten by the acid protease fromAspergillus usamii under optimized conditions has greatlyincreased the solubility of wheat gluten. Enzymatic hydrolysisof wheat gluten resulted in a radical increase in emulsifyingactivity index (EAI), water, and oil-holding capacity. *emolecular weight determination study also showed that mostof the peptides above 10 kDa have been hydrolyzed intosmaller peptides. Furthermore, the functional properties ofwheat gluten improved after hydrolysis [31].

*e treatment of nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments by prolyl endopeptidasefrom Aspergillus niger (AN-PEP) successfully degraded all thenine epitopes in the stomach pH range at considerable lesserdosage than the digestive enzyme supplements. *e digestiveenzyme supplements showed comparable proteolytic activi-ties with near neutral pH optima and modest gluten de-toxification properties as determined by ELISA [32].

3.3. Application in the Beer Industry. *e visual aspects ofbeer such as clarity, color, and foam are vital for consumers.Foam affects the consumers’ views, flavor, and mouth feelabout the beer. Brewers desire the presence of sufficient,stable, white, and finely textured foam to satisfy consumers’concern [33].

Haze formation is a major problem in beer production,as it affects the qualities of the end product [34]. During theproduction of beer, proteins and polyphenols extracted fromthe plant tissue may interact and form haze [35]. Beer hazecomprises numerous components: the most common or-ganic parts are proteins (40–75%), polyphenols (in com-bination with proteins), and, to a smaller percentage,carbohydrate (2–15%) [34]. *e group of proteins that con-tributes to haze formation is called “cystine-rich proteoses”(albumins and barley hordeins) [36].

*ere are two types of haze: the cold break (chill haze)and the age-related haze. *e cold break haze is formed at0°C and disappears at higher temperatures. If cold break hazedoes not dissolve, age-related haze, which is irreversible,develops. Chill haze is produced after polypeptides andpolyphenols bound noncovalently. Permanent haze is pro-duced in a similar way primarily, but covalent bonds aresoon formed and insoluble complexes are created that willnot dissolve when heated [34]. Both chill haze and age-related haze formation are not desired by the consumers, asthey show the oldness and staleness of beer and alter thephysical stability of beer [36, 37].

*e production of undesirable chill haze in the final beerproduct can be removed by several ways. *ese includehydrolyzing the undesirable proteins in finishing the op-eration, adsorbing proteins using silica adsorbents or silica

Journal of Food Quality 11

hydrogels, and/or using polyvinylpolypyrrolidone to removepolyphenols that contribute to protein condensation re-actions [36]. *e application of proline-specific endopro-teinases (i.e., Brewers Clarex®, DSM, France) that target onthe degradation of haze-active proteins (i.e., hordeins) re-duces the formation of storage haze in final beer product.*ese hydrolyzed proteins are unable to condense withpolyphenols and hence do not form haze [37]. *e additionof a brewer’s yeast that secretes a proteinase enzyme that candegrade haze-forming proteins into brewer’s wort duringwort fermentation is also a possible alternative to removechill-proofing beer [36].

*e addition of acid proteinase from Saccharomycopsisfibuligera 1570 and Torulopsis magnoliae 1536 along withbrewer’s yeast into brewer’s wort in bench-scale fermenta-tions conducted at 200°C showed that the final bottled beerwas resistant to haze formation with a slight reduction in thefinal ethanol concentration [36]. *e addition of proteinaseA (0.5mU/ml concentration) to the sweet wort and in-cubating for 144 h at 250°C also showed a significant de-crease in hydrophobic character of wort. Moreover, theactivity of proteinase A contributed for about 47% reductionin hydrophobic nature of high gravity sweet wort.*is resultimplies that proteinase A alters the hydrophobic characterrather than the molecular size of the wort polypeptide [34].Similarly, the use of commercial protease enzyme fromBacillus subtilis correspondingly increased the level of totalsoluble nitrogen, levels of α-amino nitrogen, wort color, andextract recovery levels in wort when mashing with 100% rawbarley malt. However, as the level of protease increased, theefficiency of the protease decreased [38].

*e incubation of beer wort with acidic proline-specificprotease from Aspergillus niger in small-scale brewing ex-periment extensively hydrolyzed the proline-rich proteins andproduced a peptide fraction unable to form a haze. Sub-sequent pilot plant trials also verified that the addition of thisacidic enzyme even at low levels during wort fermentationefficiently inhibits chill haze formation in bottled beer. Resultsfrom the determination of beer foam stability indicated thatthe enzyme treatment did not affect the beer foam [35].

*e addition of protease enzyme during beer fermentationalso has a significant benefit beyond haze removal. *e ex-pression of aspartyl protease in recombinant yeast used for fuelethanol production improves the capability of the yeast tometabolize soluble proteins and leads to significant increase inethanol production. Furthermore, the recombinant yeaststrains exhibited advanced growth rate, viability, and loweryields of by-products such as glycerol and pyruvic acid [39].Likewise, the use of the multicomponent protease enzyme(Flavourzyme) with brewer’s yeast strainWeihenstephan 34/70for beer fermentation showed a significant increase in nitrogenavailability during the course of beer fermentation [40].

3.4. Application in Wine Industry. *e production of clearwine, particularly for white wines, is one of the very im-portant parameters from the consumer’s perspective.*erefore, maintaining the stability of wine before bottling isa challenging and crucial step in the winemaking process.

A stable white wine is a clear wine that is free from anyprecipitates at the time of bottling till to consumption. Hazywine with precipitate is formed as a result of microbialinstability, tartrate instability, and protein heat instability[41]. Microbial stability is attained prior to bottling by theaddition of sulfur dioxide and filtration, whereas tartratestability is accomplished by three techniques, such as coldstabilization, ion exchange resins, and/or electrodialysis [41].

Heat unstable grape protein could remain and causehazy appearance in final wine products. Grape proteins inwines that exist as unstable under certain conditions canaggregate into light-dispersing particles and cause wines toappear turbid. Specifically, grape pathogenesis-related (PR)proteins, such as thaumatin-like proteins (TLPs) and chi-tinases, contribute for wine haze formation [42]. Otherproteins like β-glucanases have also been associated withhaze formation, while they are much less abundant thanchitinases and TLPs in wine. But the role of β-glucanases inwine haze formation is not widely studied [41].

*e mechanism of haze formation is started by theunfolding and aggregation of grape-derived wine proteins.*e experimental study proved that wine protein unfoldingand aggregation are the two discrete events occurred in wineprocessing. Heat experiment has shown that proteins canunfold as soon as the wine is heated, while haze is formedafter wine cooling [41].

In commercial winemaking, the stability of proteins isaccomplished by the addition of bentonite. Bentonite isa clay cation exchanger that binds with proteins andeliminates them from wine through precipitation, and it hasbeen commonly used in oenology as a fining agent since the1930s [41, 42]. Proteins that bound with bentonite settle tothe bottom of wine tanks as lees, and it covers 3−10% of thetotal wine volume. Wine is recovered from bentonite leesusing rotary drum vacuum filtration, specialized lees fil-tration equipment, or centrifugation processes [41]. Ben-tonite fining has some negative characteristics such asdilution of the wine by the bentonite slurry, elimination ofwine flavor, high labor expenses, trouble in bentonite spentdisposal, and loss of wine quality [42].

For the abovementioned reasons, alternative methodsfor stabilizing white wine have been extensively studied[41, 42]. Several alternatives have been suggested, like the useof other adsorbents, ultrafiltration, and flash pasteurization,but none of them has proven suitably effective to replacebentonite. One ideal solution to this issue would be the useproteolytic enzymes capable to degrade the heat unstableproteins [42].

Acid protease enzyme is suitable for the degradation ofthe turbidity complex produced from proteins in fruit juicesand alcoholic liquors [6]. Some fungal aspartic proteaseshave been used to hydrolyze proteins that cause turbidity injuices and wine. *ese are the protease BcAP8 from Botrytiscinerea and the aspergillopepsin I from Aspergillus saitoi(commercially marketed as Molsin F by Kikkoman Corp.,Japan), both used in the winery as they successfully removehaze-forming proteins and hence reducing bentonite re-quirements [19]. *e addition of AGP (a combination ofAspergillopepsin I (EC 3.4.23.18) and Aspergillopepsin II

12 Journal of Food Quality

(EC 3.4.23.19)) into two clarified grape juices (Chardonnayand Sauvignon blanc juices) with heat treatment (at 750°C,1min) and without heat treatments prior to fermentationexhibited about 20% total protein reduction as compared tothe control wine. However, it showed the best activity whenthe enzymes were combined with juice heating (≈90% totalprotein reduction). But the more heat-stable grape proteins(i.e., those do not contribute to wine hazing) were not af-fected by the treatments and hence account for theremaining 10% of proteins still found in the solution afterthe treatments. *e major physicochemical parameters andsensorial characteristics of wines produced with AGP werecomparable with the control [42].

*e application of Aspergilloglutamic peptidase (AGP)(commercially known as Proctase and formerly known asAspergillopepsin II) to clarified grape juice prior to flashpasteurization and fermentation resulted in heat-stable winesthat were completely free from haze-forming proteins. *eresults of chemical and sensory analysis also indicated thatthere were no significant changes in physicochemical pa-rameters of wine preference. *is combined use of proteasewith flash pasteurization has been shown to be effective atindustrial scale, and consequently, the use of AGP in wine hasbeen recently accepted in Australian winemaking. *e overallcost of AGP application is comparable to bentonite treatment,and this makes AGP a potentially cost-effective and com-mercially viable alternative for bentonite [41].

*e clarification of blackcurrant juice with acid protease(Enzeco and Novozyme 89L) after precentrifugation andcold storage showed a significant reduction in haze devel-opment. *e addition of Enzeco protease (conc. 0.025 g/L)and gallic acid (conc. 0.050 g/L) into blackcurrant juice andallowing it to react in the juice for 90min at 50°C showed thelowest levels of haze formation after 28 days of storage at20°C [43]. In other studies, the treatment of cherry juice withprotease (Enzeco, enzyme preparations derived from As-pergillus spp.) resulted in a considerable reduction in im-mediate turbidity but had low clarification impact during thesubsequent cold storage [44]. *e combined treatment ofwines with heat (90°C for 1 minute) and enzymes (Trenolinblank, a mixed pectolytic and proteolytic enzyme solutionand porcine pepsin) also reduced 40%–80% of the proteinlevel in wines [45].

*e treatment of banana juice extracted from the bananapulp by two selected commercial protease enzymes (zumi-zyme and papaine) during banana winemaking indicated thatthe wine turbidity was significantly reduced as compared tothe control. *ese proteases had also shown a substantialeffect on the protein haze removal after periods of one weekand four weeks. Moreover, a longer incubation period leads toa higher decrease in turbidity. Based on the sensorial analysis,the overall organoleptic quality of the wine did not showsignificant variation than the control (p> 0.05) [46].

*e application of BcAP8 (Botrytis aspartic protease)from the grape fungal pathogen Botrytis cinerea, intoAustralian Semillon and Sauvignon blanc juices, noticeablydegraded chitinase, a major class of haze-forming proteinswithout heat denaturation. *erefore, BcAP8 could poten-tially benefit winemakers by removing haze-forming

proteins under normal winemaking conditions [12]. *eextracellular acid protease secreted by Saccharomyces cer-evisiae PlR1 during alcoholic fermentation was also found tobe active against grape proteins (molecular mass≈ 25 kDa) at38°C and pH 3.5 [47].

In another study, the wines treated with protease enzymehave been shown to have a higher amino acid contents thanthe nonenzyme-treated wines except for arginine and his-tidine. *ese results indicated that protease treatment couldenhance the concentration of assimilable nitrogen, whichwas one of the important nutrients for yeast in wine fer-mentation [48].

4. Conclusions

Aspartic protease enzyme has a wide range of application infood and beverage industries, such as cheese industry,bakery industry, and beer and wine industry. *e microbialaspartic protease enzyme produced from bacteria and fungiwere used for the production of curd in cheese manufacturing,for haze removal in brewery and winery, and for modificationof bread in the bakery.

Conflicts of Interest

*e authors declare that they have no conflicts of interest.

References

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