BRIEF REPORT

Folia Microbiol. 38 (2), 159-160 (1993)

Chitinase and Changes A. HANZL~KOV,~ and A. JANDERA

Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4

of Microbial Community in Soil

Received November 23, 1992 Revised version Janualy 12, 1993

ABSTRACT. Enrichment of soil with chitin (0.6 %) significantly stimulated growth of chitinolytic microorganisms (the relative proportion was increased from 1.7 to 26.5 %) and the formation of chitinase in soil. In a soil enriched with chitin and glucose (0.6 %), the proportion of chitinolytic microorganisms remained similar to that in the nonenriched soil (1.4 %), the enzyme formation was negatively affected.

Studies of enzyme lysis of a fungal cell wall by microorganisms (Mitchell and Alexander 1963; Morrissey et al. 1973) have contributed to the understanding of the mechanism of decomposition of fungal walls in soil. The results obtained constitute a basis for application of lytic enzymes in the con- trol of some fungal soil pathogens (Mitchell 1963; Ordentlich et aL 1988). The lytic activity is char- acteristic for microorganisms producing fl-l,3-glucanase (EC 3.2.1.6) and chitinase (EC 3.3.1.14) (Skujins et al. 1965). In order to clarify these phenomena it is necesary to know also the conditions re- quired for the production of these enzymes as well as the occurrence of microorganisms producing these enzymes in soil.

In our previous studies (Jandera et al. 1987; Hanzlfkov~i et al. 1989) we reported that the air- dried soil exhibited a basal level of the chitinase activity (probably the activity of the enzyme complexed with soil clays and humus components) which was increased only in the presence of chitin but not of glucose or other substrates. This increase was not negatively influenced by glucose. In contrast, addi- tion of chitin with glucose resulted in a further increase of the chitinase activity. The maximal activity was always attained after 7-d cultivation with the substrate. In the present communication the effect of the above-mentioned nutritional factors on the structure of the soil microbial community with respect to the occurrence of chitinolytic microorganisms was investigated.

Samples of chernozem soil (soil isolated from the surface arable layer, contained 1.88 % C and 0.19 % N (C:N ratio 9.9), pH of the aqueous suspension (1:1) was 7.5) were used in the form of air- dried structural aggregates 0.2-2.0 mm in diameter. Soil cultivation (28 ~ 60 % water holding capacity) was described by Rygav~ and Macura (1972). The chitinase assay based on the concentration of N-acetylglucosamine was developed by Hanzl~ov/t et al. (1989). Cultivation of soil enriched with chitin (0.6 %) or with chitin and glucose (0.6 % each) took 7 d. Wetted soil was used as control. The total counts of microorganisms and the fraction of the chitinolytic ones in a soil sample were deter- mined by the dilution-plate technique on agar plates (mean of 4 plates) containing yeast and soil extracts, tryptone (Taylor 1951) and colloid chitin, if necessary (Hankin and Anagnostakis 1975). After 5 -8 d of incubation clear zones were seen in the chitin-containing opaque agar medium around the colonies able to degrade chitin.

Table I. Chitinase activity, total number of microorganisms, and number of chitinolytic microorganisms after 7-d cultivation of soil

Number of microorganisms x l 0 -7 per g soil

total chitinase producers

Soil enriched Chitinase activity with pkat/g soil

t i 20 13.2 a 0.43 0.007 2 Chitin, 0.6 % 56.9 3.4 0.9 Chitin and glucose

0.6 % each 85.8 1 030 14.0

aAlso chitinase basal level in dry soil.

Air-dried and wetted soil exhib- ited the basal chitinase activity (13.2 pkat/g) that remained constant during cultivation. About 1.7 % of the total number of microorganisms in a soil sample were able to decom- pose chitin (Table I).

When the soil was supplemented with chitin the total number of microorganisms increased 7.9 times, compared to nonenriched soil. The enrichment with chitin significantly stimulated multiplication of chitin- olytic microorganisms in the soil. Their number increased 125 times,

160 A. HANZL[KOV. A, and A. JANDERA Vol. 38

the relative proportion being as much as 26.5 %. At the same time, the formation of chitinase was also stimulated (43.7 pkat/g soil). The increase of the enzyme activity in soil was thought to be the result of its formation. The enrichment of the soil with chitin as a sole carbon and energy source reprcsents a selection pressure influencing the present microorganisms. After the enrichment, the activity of that part of the primary microbial community predominantly proliferating due to its ability to decompose chitin was observed.

In soil enriched with chitin and glucose, no change in the relative proportion of micro- organisms producing chitinase was detected (1.4 %). Glucose, a universal and readily available source of carbon and energy, made possible a nonspecific, 2400-fold increase of the amount of all present microorganisms, 303 times exceeding the amount observed in the presence of chitin. Under these conditions, the selection pressure of chitin could not manifest itself. In comparison with soil enriched only with chitin, the number of chitinolytic microorganisms was 15.6-fold greater whereas the chitinase formation (72.6 pkat/g soil) was enhanced no more than 1.7 times.

One can conclude that, if glucose and chitin were present simultaneously, the microorganisms producing chitinase synthesized only 11% of the enzyme as compared with the situation when only chitin was used for enrichment of the soil. The observed fact that the increase of the chitinase activity in the presence of chitin was not negatively affected by glucose could be attributed to the above- mentioned, glucose-stimulated, nonspecific multiplication of the soil microorganisms. It is, therefore, necessary to specify the conclusions of our previous study (Jandera et al. 1987; Hanzl~ovfi et al. 1989): the formation of chitinase, like that offl-l,3-glucanase (Jandera et al. 1987; Sotolovfi and Jandera 1985), is negatively affected by the presence of glucose.

The course of the enzyme formation is apparently related to nutritional conditions under which a heterogeneous microbial community grows (Macura and Kub~itovfi 1973) but a possible rela- tionship between the enzyme production and composition of the soil community cannot be excluded.

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