Aspergillus niger 변이주를 이용한 xylanase 생산

박양순, 강성우, 김승욱*

고려대학교 생명공학원, 고려대학교 화공생명공학과*

Xylanase production by Aspergillus niger mutant

Yang Soon Park, Seong Woo Kang, Seung Wook Kim*

Graduate School of Biotechnology, Korea University

Department of Chemical and Biological Engineering, Korea University*

Introduction

Xylanases catalyze the hydrolysis of xylan, the major constituent of hemicellulose. Applications of xylanases can be found in the food, feed, pharmaceutical, pulp and paper industry. Several different strategies to enhance enzyme production in various microorganisms have been successfully applied.

Filamentous fungi are particularly interesting xylanases producers from an industrial point of view, due to the fact that they excrete xylan-degrading enzymes into the medium, thus eliminating the need for cell disruption. Furthermore, xylanase levels from fungal cultures are typically much higher than those from yeasts or bacteria. In addition to xylanases, fungi typically produce several auxiliary enzymes which are necessary for debranching of the substituted xylans.

The aim of this study is to optimize a culture medium for the production of xylanase by Aspergillus niger mutant in submerged culture. For optimization of culture medium, statistical experimental designs were employed.

Materials and Methods

Aspergillus niger KK2 mutant was selected from A. niger KKS through mutagenesis (Kang et al., 1999). Seed culture grown on 2% (w/v) malt extract was incubated in a rotary shaking incubator at 28℃ for 2 days. The basal medium consisted of 2% ground rice straw, 1% wheat bran, 1% CSL(Corn Steep Liquor), 0.05% IYE(Industrial Yeast Extract), 0.5% KH2PO4, 0.05% CuSO4·5H2O, 0.01% CoSO4·7H2O. The pH before sterilization was adjusted to 7.0. Seed culture (5%, v/v) was used as inoculum in shake-flask cultures. Shake-flask cultures were grown in 250 ml Erlenmeyer flasks containing 50 ml of medium in a rotary shaking incubator at 28℃ and 200 rev/min for 7 days.

A statistical analysis method was used to optimize production medium. In order to find the most effective factor on the production of xylanase, ANOVA(analysis of variance) was used. And we obtained an optimal concentration of the factor with RSM(Response Surface Methodology).

Xylanase activity was assayed in 1.5 ml of a reaction mixture containing 0.5 ml of diluted enzyme solution and 1.0 ml of supernatant of 2.0% (w/v) xylan (birchwood, sigma Co) in 0.05M citrate buffer (pH 4.8) . The mixture was incubated at 50。C for 20 min. After the incubation period, the reducing sugars were determined by the dinitrosalicylic acid method (Miller 1959) with xylose as standard.

Result and discussion

When xylanase was produced in the basal medium, a maximum activity of xylanase (385 IU/ml) was obtained at 5 days. The optimal medium obtained from the common 'one-factor-at-a-time' method for the production of xylanase was determined to be 3% ground rice straw, 1% wheat bran, 5% CSL, 0.05% IYE, 0.5% KH2PO4, 0.05% CuSO4·5H2O and 0.01% CoSO4·7H2O. When the strain was grown in the optimized medium, xylanase production (439 IU/ml) was increased about 14% compared to basal medium. Fig. 1 shows the time course of xylanase production in basal and optimal media by A. niger mutant.

From preliminary experiments made in order to examine the effects of different medium components on xylanase production, five factors which play the most important role in the xylanase synthesis were chosen. These were the concentration of rice straw, wheat bran, CSL, IYE, and KH2PO4. After experiments were done according to a 25-1 fractional factorial design, results were analyzed using ANOVA. It showed that rice straw, IYE and CSL were key factors. The concentration of rice straw, wheat bran and KH2PO4 were fixed at 3%, 1%, 0.05%, respectively. The optimal concentration of media components was determined using response surface methodology with the two variable nitrogen sources(Table 1). Fig. 2 shows a three-dimensional diagram and contour plot of the calculated response surface. From the result, the maximum point of the model can be obtained, which was 6.42% of CSL, 0.15% of IYE. The model predicted a maximum response of 669.5 IU/ml for this point. To conform the results, experimental rechecking was done by shake flask experiments using a medium representing this maximum point and a value of 660 IU/ml was obtained. The good correlation between these two results verifies the validity of the response model and the existence of an optimal point. Xyanase production (660 IU/ml) could be increased by 48% when the strain was grown on the medium that was developed by means of statistical design compared with the optimal medium composition obtained from the common 'one-factor-at-a-time' method. The optimum composition of the nutrient medium was found  to be 6.42% CSL and 0.15% IYE.

Fig. 1. Time course of xylanase production by A. niger mutant

in the shake flask.

(○ : basal medium, ● : optimized medium)

Fig. 2. Dependence of xylanase activity on the concentration

of CSL and IYE.

Table 1. Range of values and result of the 2□full factorial design

Component	Variable	coded values
		- 1.414	-1	0	+1	1.414
C S L	X1	3.2%	4%	6%	8%	8.8%
I Y E	X2	0.06%	0.08%	0.14%	0.2%	0.22%

Run	Variable		Xylanase activity (IU/ml)
	X1	X2	Assay
1	-	-	574
2	+	-	613
3	-	+	601
4	+	+	636
5	-1.414	0	585
6	1.414	0	606
7	0	-1.414	550
8	0	1.414	594
9	0	0	640
10	0	0	694

Rice straw: 3g/100□, Wheat bran:  1 g/100□, KH2PO4: 0.5 g/100□,

CoSO4·7H2O: 0.01g/100□, CuSO4·5H2O: 0.05g/100□ fixing

Acknowledgement

This study was supproted by research grants from the KOSEF through the Applied Rheology Center at Korea University, Korea.

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