Tài liệu Báo cáo khóa học: Non-specific depolymerization of chitosan by pronase and characterization

Non-specific depolymerization of chitosan by pronase

and characterization of the resultant products

Acharya B. Vishu Kumar1

, Lalitha R. Gowda2 and Rudrapatnam N. Tharanathan1

1

Department of Biochemistry and Nutrition, 2

Department of Protein Chemistry and Technology, Central Food Technological

Research Institute, Mysore, India

Pronase (type XXV serine protease from Streptomyces

griseus) efficiently depolymerizes chitosan, a linear bfi1,4-

linked polysaccharide of 2-amino-deoxyglucose and

2-amino-2-N-acetylamino-D-glucose, to low-molecular

weight chitosans (LMWC), chito-oligomers (degree of poly￾merization, 2–6) and monomer. The maximum depolymeri￾zation occurred at pH 3.5 and 37 C, and the reaction obeyed

Michaelis–Menten kinetics with a Km of 5.21 mgÆmL)1 and

Vmax of 138.55 nmolesÆmin)1

Æmg)1

. The molecu lar mass of

the major product, LMWC, varied between 9.0 ± 0.5 kDa

depending on the reaction time. Scanning electron

microscopy of LMWC showed an approximately eightfold

decrease in particle size and characterization by infrared

spectroscopy, circular dichroism, X-ray diffractometry and

13C-NMR revealed them to possess a lower degree of acety￾lation, hydration and crystallinity compared to chitosan.

Chitosanolysis by pronase is an alternative and inexpensive

methodtoproduceavarietyofchitosandegradationproducts

that have wide and varied biofunctionalities.

Keywords: chitosan; chito-oligomers; low-molecular weight

chitosan; pronase; structure.

Chitosan is the de-N-acetylated derivative of chitin, a linear

polysaccharide of b1fi4-linked 2-deoxy-2-acetamido-D-glu￾cose units [1], that constitutes the exoskeleton of inverte￾brates and is one of the components of the cell walls of

fungi. It has wide and varied applications in medicine,

agriculture, pharmaceuticals and the food industry [1,2],

which is attributed to the biofunctionality of the amine

moiety that confers both cationic (polyelectrolyte) and

chelating properties. Despite being biocompatible, nontoxic

and multifunctional, the use of chitosan in vivo is hampered

by its high-molecular mass and high viscosity even at low

concentrations [2]. Therefore, a prerequisite for efficient

utilization of chitosan is its depolymerization to low￾molecular weight chitosans (LMWC), chito-oligomers and

monomer. The depolymerized products find additional

applications as hypo-cholesterolemic, antitumorigenic,

antimicrobial, immuno-enhancing agents, and also in the

treatment of osteoarthritis, gastritis, etc. [3–5]. A 9 kDa

LMWC suppressed Escherichia coli activity whereas that of

LMWC 5 kDa showed antihyperlipemic and hypocholes￾terolemic effects [2]. Chito-oligomers with a degree of

polymerization (DP) > 6 showed antitumor activity

towards Sarcoma-180 and Meth-A tumors, and caused

activation of defence responses in plants. Chitotriose

exhibited maximum inhibitory effect towards angiotensin

converting enzyme (ACE) [4].

Chitosan can be depolymerized by acid or enzymatic

hydrolysis. The former is harsh, time consuming, modifies

the products and forms a large quantity of monomers [6].

In contrast, enzymatic hydrolysis produces specific

products as the reaction can be precisely controlled.

Chitosanase, the enzyme of choice due to its specificity,

degrades chitosan to chito-oligomers, but is, however,

very expensive and unavailable in bulk for commercial

exploitation [7].

The earlier concept of a ratio of one enzyme to one

substrate/group of related substrates is no more a reality in

most of hydrolases as evidenced in recent literature. b1fi4

Glucanase, although specific for b1fi4-linked glucans, can

hydrolyze mannans and cellobiose [8]. Chitosanase from

Myxobacter A-1, Streptomyces griseus HUT 6037, Bacillus

sp.7-M and Bacillus megaterium degrades carboxymethyl￾cellulose. A b1fi3/1fi4 glucanase from Bacillus circulans

WL-12 depolymerizes chitosan to low-molecular mass

products [9–12]. Susceptibility of chitosan to various

nonspecific enzymes like wheat germ lipase, lysozyme,

papain, cellulase, hemicellulase, b-glucosidase, etc., has

been reported earlier, although enzyme purity was in doubt

and could be contaminated with chitosanase [13,14].

Recently, we have shown that a homogeneous isozyme of

Aspergillus niger pectinase could depolymerize chitosan

quantitatively to yield LMWC and chito-oligomers [15].

The objective of the present study was to demonstrate yet

another example of enzyme nonspecificity in catalyzing the

cleavage of completely unrelated substrates. An electropho￾retically pure pronase preparation was used to depolymerize

chitosan to LMWC, chito-oligomers and monomer, and

structural characterization of the depolymerized products is

presented herein.

Correspondence to R. N. Tharanathan, Department of Biochemistry

and Nutrition, Central Food Technological Research Institute,

Mysore ) 570 013, India.

Fax: + 91 821 2517233, Tel.: + 91 821 2514876,

E-mail: [email protected]

Abbreviations: DP, degree of polymerization; LMWC, low-molecular

weight chitosan.

Enzyme: Pronase (type XXV serine protease from Streptomyces

griseus; EC 3.4.24.31)

(Received 30 October 2003, revised 12 December 2003,

accepted 22 December 2003)

Eur. J. Biochem. 271, 713–723 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2003.03975.x