Biotechnology Letters22: 1375–1378, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Purification and characterization of β-mannanase fromBacilluslicheniformis for industrial use

Jun Zhang, Zhimin He∗ & Kun HuLaboratory of Enzyme Technology, Chemical Engineering Research Center, Tianjin University, Tianjin 300072,P.R. China∗Author for correspondence (Fax:+86-22-27404757; E-mail: zhe@tju.edu.cn)

Received 5 May 2000; Revisions requested 2 June 2000; Revisions received 29 June 2000; Accepted 30 June 2000

Key words: Bacillus licheniformis, β-mannanase, purification

Abstract

An easily scaled-up technique has been designed to purifyβ-mannanase fromBacillus licheniformis. Using floc-culation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recoveryof 47% and a specific activity of 4341 U mg−1 protein. The enzyme had maximum activity at 60◦C and pH 7.0. Itwas stable at 50◦C and pH 6.0 for 6 h, but lost all of its activity when held at 70◦C and pH 6.0 for 1 h.

Introduction

β-D-Mannanase (1,4-β-D-mannan mannohydrolase,EC 3.2.1.78) hydrolyses 1,4-β-D-mannopyranosyllinkages of mannans, glucomannans, galactomannansand galactoglucomannans, yielding mannooligosac-charides which are growth factors forBifidobacteriumsp. andLactobacillussp. (Kobayashiet al. 1984).β-Mannanase is also useful for the enzymatic analysisof polysaccharide structures (McCleary & Matheson1986), food and feed processing industries, pulp andpaper industries and the stimulation of oil and gas well(Gomes & Steiner 1998).β-Mannanases occur widely in microorganisms

and possess different characteristics in terms of mole-cular weight, pI, optimum pH and temperature, etc.(Reese & Shibata 1965, Araujo & Ward 1990, Hos-sain et al. 1996, Gomes & Steiner 1998). Althoughmuch work has been done on the purification ofβ-mannanases (Shigenoriet al.1972, Akinoet al.1988),little information is available about the large-scalepreparation of these enzymes.

This paper proposes an industrial process for thepurification of β-mannanase fromBacillus licheni-formis and reports some properties of the purifiedenzyme.

Materials and methods

Materials

Locust bean gum (LBG) and bovine serum albumin(BSA) were obtained from Sigma. Konjac powdercame from Wufeng Industrial Co. (Dazhou Sichuan,China). Cross-flow ultrafiltration was performed inhollow fiber membrane (polysulfone membrane ofnominal MWCO 50 kDa and 10 kDa) obtained fromMotian Co. of Tianjin Institute of Textile Science andTechnology (Tianjin, China). DE-52 DEAE-cellulosecame from Whatman. All other reagents were ofanalytical grade.

Microorganism and cultivation

Bacillus licheniformiswas a gift from Prof WenboYang (Nankai University, Tianjin, China). The com-position of liquid medium was as follows (g l−1):konjac powder 20, meat peptone 30, corn steep liquor5, (NH4)2SO4 5, Na2HPO4 4, KH2PO4 0.3, MgCl20.6, CaCl2 3, FeSO4 0.01, Na2CO4 3, pH 7.0. Thebacterium was cultured in a 1000-ml flask contain-ing 200 ml of the above medium on a rotary shaker(180 rpm) at 30◦C for 24 h. The culture was thentransferred to 600 l of the same medium in a 1000-

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Table 1. Purification results ofβ-mannanase fromBacillus licheniformisa.

Procedure Total protein Total activity Specific activity Yield Purification

(mg l−1 of (105unit l−1 of (unit mg−1 protein) (%)b (fold)b

culture broth) culture broth)

Centrifugation 1280 1.68 131 100 1

Flocculation 660 1.72 260 102 2

Ultrafiltration (50 kDa) 251 1.44 574 86 (84) 4.4 (2.2)

Ultrafiltration (10 kDa) 101 1.22 1207 73 (85) 9.2 (2.1)

DEAE-cellulose (1st) 51.6 1.12 2171 67 (92) 16.5 (1.8)

DEAE-cellulose (2nd) 18.2 0.79 4341 47 (70) 33.1 (2)

aTen l sample was withdrawn from the culture broth and centrifuged as comparison with subsequent purifiedβ-mannanase. The residual culture broth was flocculated at pH 6.0 by 80 mg chitosan l−1 with agitation (80–100 rpm)for 5 min. The supernatant was diafiltrated against water with membrane of MWCO 50 kDa. The permeate wasconcentrated and then diafiltrated against phosphate buffer (pH 6.0, 25 mM) by membrane of MWCO 10 kDa. Theconcentrate was loaded on a DEAE-cellulose column (5 cm× 50 cm) previously equilibrated with the above buffer,at a flow rate of 90 cm h−1, until A280 of the eluent reached 50% of the initial enzyme solution.β-Mannanase wasrecovered in the flow-through, adjusted to pH 7.0, then applied to another DEAE-cellulose column (5 cm× 50 cm)pre-equilibrated with phosphate buffer (pH 7.0, 25 mM), step-eluted with 0.15 M NaCl at a flow rate of 30 cm h−1.b Values in parentheses are relative to the previous steps.

Fig. 1. Effects of pH (A) and temperature (B) onβ-mannanase ac-tivity. (A) The enzyme reaction was carried out in a pH range of4–10 (50 mM citrate, pH 4–6; 50 mM phosphate, pH 6–8; 50 mMglycine-NaOH, pH 9–10) at 60◦C for 10 min. (B) The enzyme reac-tion was performed at various temperatures from 40◦C to 70◦C for10 min under pH 7.0. 100% value represents 4302 U mg−1 protein.All data are the average of triplicate samples and are reproduciblewithin ± 10%.

l fermenter and cultivated at 30◦C for 42 h withagitation (200 rpm) and aeration (600 l min−1).

Enzyme assay

A reaction mixture containing 0.9 ml 0.5% (w/v)lcust bean gum (LBG) in phosphate buffer (pH 7.0,50 mM) and 0.1 ml of suitably diluted enzyme solu-tion was incubated at 60◦C for 10 min. The amountof reducing sugar liberated was determined by the 3,5-dinitrosalicylic acid method. One unit ofβ-mannanaseactivity was defined as the amount of enzyme whichreleased 1µmol of reducing sugar equivalent toD-mannose per minute under the above conditions.

Fig. 2. Effects of pH (A) and temperature (B) onβ-mannanase sta-bility. (A) The enzyme solution was maintained at 50◦C for 1 hat various pHs. (B) The enzyme solution was incubated at 50◦C,60 ◦C and 70◦C for different time periods at pH 6.0. In both casesthe remaining activity was measured by the standard assay method.100% value represents 4086 U mg−1 protein. All data are the meanvalue of three replicates and are reproducible within± 10%.

Protein assay

Protein content was measured by the method of Brad-ford with BSA as standard.

Purification ofβ-mannanase

The complete process of purification is illustrated inScheme 1.

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Scheme 1.Purification procedure ofβ-mannanase fromBacillus licheniformis.Unit operations are in rectangles and intermediate fractions arein italics.

Results and discussion

Purification ofβ-mannanase

The purification ofβ-mannanase from the extracellu-lar culture broth ofBacillus licheniformisis summa-rized in Table 1. Chitosan, a natural polyelectrolyte,was chosen for its effectiveness and safe status (Rams-denet al.1998). This cationic flocculant could interactby charge neutralization and polymer bridging mech-anisms with components in the fermentation broth ofBacillus licheniformis(the final pH value was about6.2), resulting in large, fluffy and non-sticky flocswhich could easily be removed by centrifugation. Thepurification comprises six steps, each of which candeal with large volume of sample and can easily bescaled up. The purified enzyme can be used in food orpharmaceutical industries.

Activity and stability ofβ-mannanase

β-Mannanase was most active at pH 7.0 (Figure 1A)and maintained more than 95% of original activityfrom pH 5.5 to pH 7.5 at 50◦C for 1 h (Fig-ure 2A). The reason whyβ-mannanase activity wasslightly higher at pH 9.0 than pH 8.0 is under inves-tigation. The enzyme activity was optimal at 60◦Cunder the experimental conditions (Figure 1B) andappeared thermostable at 60◦C with half-life of3 h (Figure 2B). However, it is better to choose50 ◦C in mannooligosaccharides production usingβ-mannanase because the enzyme was rather stable at50 ◦C. No enzyme activity was observed after in-cubation at 70◦C for 1 h. These properties werein accordance with mostβ-mannanases produced bybacterium.

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Acknowledgement

Prof Zhimin He would like to thank the financialsupport from the Ministry of Human Resource ofChina.

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