Preparation of glucosamine by hydrolysis of chitosan with commercial α-amylase and glucoamylase

Preparation of glucosamine by hydrolysis of chitosan with commercial α-amylase and glucoamylase 1 2 first-author 1 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jinkimnh@yahoo.com †E-mail:jinkimnh@yahoo.com jinkimnh@yahoo.com Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang 222005, China Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang 222005, China School of Marine Science and Technology, Huaihai Institute of Technology, Lianyungang 222005, China School of Marine Science and Technology, Huaihai Institute of Technology, Lianyungang 222005, China Food Safety and Inspection Service, Office of Field Operations, US Department of Agriculture, Albany, NY 12203, USA Food Safety and Inspection Service, Office of Field Operations, US Department of Agriculture, Albany, NY 12203, USA Objective: 2 In order to overcome the defects of chemical hydrolysis approach to prepare glucosamine, an enzymatic hydrolysis method was developed. Methods: 2 Glucosamine was prepared by hydrolyzing chitosan, employing α-amylase initially, and subsequently, glucoamylase. Results: 2 The optimal hydrolyzing conditions were as follows: reaction time, 4 h; pH, 5.0; temperature, 50 °C; and, α-amylase, 80 U/g for the initial reaction. Subsequently, glucoamylase was added in the presence of α-amylase. The optimal reaction conditions were found to be: reaction time, 8 h; pH, 4.5; temperature, 55 °C; and, glucoamylase, 4 000 U/g. The hydrolysates were subject to filtrating, concentrating to about 20% (w/w), precipitating with five volumes of ethanol, and drying at 60 °C for 2 h. The content and the yield of glucosamine in the dried precipitate were 91.3% (w/w) and 86.2% (w/w), respectively. Conclusions: 2 The method developed in this study is a promising option in the preparation of glucosamine. Glucosamine α-Amylase Glucoamylase Hydrolysis Introduction 1 Glucosamine, an amino monosaccharide, has been used for the treatment of arthritis (Reginster et al., 2001 2001 ; Nakamura, 2011 2011 ), inflammation (Nagaoka et al., 2011 2011 ), cancer (Oh et al., 2007 2007 ), and orthopedic diseases (Minami et al., 2011 2011 ). Glucosamine can usually be obtained by chemical hydrolysis of chitosan (Zhang et al., 2007 2007 ; Xing et al., 2009 2009 ). The chemical hydrolysis often involves acid and has some defects: harsh hydrolysis conditions, chemical modifications of glucose ring, and others (Wang et al., 2008 2008 ). Enzymatic methods have advantages over chemical approach, such as mild reaction conditions and high specificity, as well as high yield, in the products of glucosamine, without modifications of glucose ring. Many enzymes with different specificities have been reported for their abilities to hydrolyze chitosan at a comparable level, such as cellulase, pectinase, pepsin, papain, neutral protease, lipase, and α-amylase (Xia et al., 2008 2008 ; Wu, 2011 2011 ). Based on our previous studies, glucoamylase, which belongs to exo-amylase, also has the ability to hydrolyze chitosan (Pan and Wu, 2011 2011 ). In this paper, we report the preparation of high glucosamine-containing product by hydrolyzing chitosan with α-amylase and glucoamylase. The optimal reaction conditions were investigated, and the composition of the product produced by hydrolysis was studied. Materials and methods 1 Materials 2 Chitosan was obtained from Nantong Biochemical Co. (Jiangsu, China). Its degree of de-acetylation and molecular weight ( M w ) were 91.7% and 4.1×10 5 , respectively. α-Amylase, with activity at 4 000 U/mg, and glucoamylase, with activity at 50 000 U/mg were obtained from Fuchen Chemical reagents Co. (Tianjin, China). Hydrolysis of chitosan using α-amylase followed by glucoamylase 2 Chitosan was dissolved in acetic acid (HAc) solution (1%, v/v) to make a solution of a concentration of 1% (w/w). The pH was adjusted to 5.0 using 1 mol/L NaOH. α-Amylase (80 U/g) was added into a reactor containing 500 ml of chitosan solution and the reactor was kept in a thermostatic water bath at 50 °C for up to 6 h. To the above hydrolysates, 4 000 U/g glucoamylase was added and the pH was adjusted to 4.5 using 1 mol/L HAc. The hydrolysis was continued at 55 °C for additional 12 h. Aliquots were periodically taken from the reaction mixture, filtrated, and heated to 95 °C for 15 min to terminate the reaction. Precipitating with ethanol 2 The reaction mixture was filtrated through Whatman GF/A filter paper, concentrated to about 20% (w/w) in a vacuum rotary evaporator (Jiangyin Dry Equipment Manufacture Co., Ltd., Jiangyin, Jiangsu, China) at 60 °C, and then precipitated with five volumes of ethanol. The precipitates were dried at 60 °C for 2 h (Wu et al., 2009 2009 ). Analytical methods 2 The pH of the solution was recorded using a digital pH meter (Model PHS-3C; CD Instruments, China). The reducing sugars were estimated by the method of Somogyi and expressed by dextrose equivalent (DE) value (Nelson, 1944 1944 ). DE value was used as an index of enzyme activity in the reaction mixture. The composition of the sugar in the resulting product was analyzed employing Waters 600 high-performance liquid chromatography (HPLC) equipped with a double column system. The first column (Sugarpark 1, 6.5 mm i.d.×300 mm) used pure water as mobile phase at a flow rate of 0.5 ml/min and the column temperature was maintained at 85 °C. The second column (SpherisorbNH 2 , 4.6 mm i.d.×250 mm) used acetonitrile/water (70/30, v/v) as mobile phase at a flow rate of 1.0 ml/min and the column temperature was 30 °C. The detector used was a high sensitive refractive index detector (Model ERC-7515 A, ERC Inc., Japan). The detector sensitivity was 4 μRIU and the inject volume was 10 μl (Wu et al., 2009 2009 ). Results and discussion 1 Hydrolysis of chitosan by α-amylase and glucoamylase 2 α-Amylase, an endo-acting hydrolase, was expected to hydrolyze β-1,4 linkages in chitosan in a random fashion and to yield water-soluble chitooligosaccharides and glucosamine. Subsequently added enzyme, glucoamylase, an exo-acting hydrolase, was expected to attack the substrates from the non-reducing end, producing glucosamine. Mechanism of hydrolysis of chitosan by α-amylase and glucoamylase to produce glucosamine can be illustrated as shown in Fig. 1 1 . First, chitosan is hydrolyzed by α-amylase to produce chitooligosaccharides. Second, chitooligosaccharides are hydrolyzed by glucoamylase to produce the final glucosamine. Effect of time on hydrolyzing chitosan 2 As shown in Fig. 2a 2a , DE values increased sharply within 3 h, slightly from 3 h to 4 h, and did not increase further after 4 h. Therefore, the optimal reaction time with α-amylase was judged to be 4 h. When glucoamylase was added to this reaction mixture, the maximum DE value was achieved at 8 h reaction time, and there was no subsequent increase in DE value between 8 and 12 h. Therefore, the optimal reaction time with glucoamylase was considered to be 8 h (Fig. 2b 2b ). (a) Initial reaction conditions with α-amylase: pH 5.0, 50 °C, 80 U/g of α-amylase; (b) Subsequent reaction conditions with glucoamylase: pH 4.5, 55 °C, 4 000 U/g of glucoamylase. Data are shown as mean±SD ( n =9) Effects of pH, temperature, and enzyme amount on hydrolyzing chitosan 2 The pH and temperature of a reaction mixture can influence α-amylase and glucoamylase activities, and subsequently chitosan hydrolysis. Enzyme concentration may also be important for efficient hydrolysis. In the initial hydrolysis by α-amylase, the reaction was found to be optimal at pH 5.0 (Fig. 3a 3a ), 50°C (Fig. 4a 4a ), and 80 U/g α-amylase of reaction mixture (Fig. 5a 5a ). In contrast, other reports have described optimal conditions for chitosan hydrolysis using α-amylase at pH 5.4 (Zhou et al., 2003 2003 ) and 7.0 (Yu and Wang, 2008 2008 ), and temperatures of 45 °C (Yu and Wang, 2008 2008 ) and 50 °C (Zhou et al., 2003 2003 ). These varying reported optimal pH and temperatures may be due to differences in substrates, α-amylase sources, and reaction time. In the second stage of hydrolysis by the addition of glucoamylase, the reaction was found to be optimal at pH 4.5 (Fig. 3b 3b ), 55 °C (Fig. 4b 4b ), and 4 000 U/g glucoamylase of reaction mixture (Fig. 5b 5b ). (a) Initial reaction conditions with α-amylase: 4 h, 50 °C, 80 U/g of α-amylase; (b) Subsequent reaction conditions with glucoamylase: 8 h, 55 °C, 4 000 U/g of glucoamylase. Data are shown as mean±SD ( n =9) (a) Initial reaction conditions with α-amylase: 4 h, pH 5.0, 80 U/g of α-amylase; (b) Subsequent reaction conditions with glucoamylase: 8 h, pH 4.5, 4000 U/g of glucoamylase. Data are shown as mean±SD ( n =9) (a) Initial reaction conditions with α-amylase: 4 h, pH 5.0, 50 °C; (b) Subsequent reaction conditions with glucoamylase: 8 h, pH 4.5, 55 °C. Data are shown as mean±SD ( n =9) Characterization of the product in the resulting hydrolysate 2 The sugars in the resulting hydrolysate were glucosamine (91.3%, w/w), N -acetyl-glucosamine (7.2%, w/w), and chitooligomers (1.5%, w/w) (Fig. 6 6 ), indicating that the amount of glucosamine in the product was very high. The yield of glucosamine was 86.2% (w/w). Although the glucosamine content was comparable to that obtained by Zhang et al. ( 2007 2007 ) (89.14%–97.78%, w/w), the yield achieved in this study was higher than that achieved by those researchers (64.86%, w/w). Conclusions 1 Glucosamine can be prepared by hydrolyzing chitosan, using α-amylase followed by glucoamylase. The optimal conditions with α-amylase were as follows: reaction time, 4 h; pH, 5.0; temperature, 50 °C; and α-amylase, 80 U/g. In the subsequent hydrolysis by glucoamylase in the presence of α-amylase, the optimum conditions were found to be: reaction time, 8 h; pH, 4.5; temperature, 55 °C; and glucoamylase, 4 000 U/g. The hydrolysates were subject to filtrating, concentrating to 20% (w/w), and precipitating with five volumes of ethanol. The precipitates obtained were dried at 60 °C for 2 h. The content of glucosamine in the product and the yield of glucosamine were 91.3% (w/w) and 86.2% (w/w), respectively. References C Minami , M Hata , Y Tamai , M Hashida , T Takayama , S Yamamoto , M Okada , T Funatsu , T Tsuka , T Imagawa , 等 . Clinical application of D -glucosamine and scale collagen peptide on canine and feline orthopedic diseases and spondylitis deformans . Carbohydr Polym , 2011 . 84 ( 2 ): 831 - 834 . DOI: 10.1016/j.carbpol.2010.06.021 http://doi.org/10.1016/j.carbpol.2010.06.021 . Clinical application of D-glucosamine and scale collagen peptide on canine and feline orthopedic diseases and spondylitis deformans I Nagaoka , M Igarashi , J Hua , Y Ju , S Yomogida , K Sakamoto . Recent aspects of the anti-inflammatory actions of glucosamine . Carbohydr Polym , 2011 . 84 ( 2 ): 825 - 830 . DOI: 10.1016/j.carbpol.2010.04.007 http://doi.org/10.1016/j.carbpol.2010.04.007 . Recent aspects of the anti-inflammatory actions of glucosamine H Nakamura . Application of glucosamine on human disease—osteoarthritis . Carbohydr Polym , 2011 . 84 ( 2 ): 835 - 839 . DOI: 10.1016/j.carbpol.2010.08.078 http://doi.org/10.1016/j.carbpol.2010.08.078 . Application of glucosamine on human disease—osteoarthritis N Nelson . A photometric adaptation of the Somogyi method for the determination of glucose . J Biol Chem , 1944 . 153 ( 3 ): 375 - 380 . . A photometric adaptation of the Somogyi method for the determination of glucose HJ Oh , JS Lee , DK Song , DH Shin , BC Jang , SI Suh , JW Park , MH Suh , WK Baek . D -Glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K . Biochem Biophys Res Commun , 2007 . 360 ( 4 ): 840 - 845 . 17624310 DOI: 10.1016/j.bbrc.2007.06.137 http://doi.org/10.1016/j.bbrc.2007.06.137 . D-Glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K SK Pan , SJ Wu . Preparation of water soluble chitosan by hydrolysis with commercial glucoamylase containing chitosanase activity . Eur Food Res Technol , 2011 . 233 ( 2 ): 325 - 329 . DOI: 10.1007/s00217-011-1524-7 http://doi.org/10.1007/s00217-011-1524-7 . Preparation of water soluble chitosan by hydrolysis with commercial glucoamylase containing chitosanase activity JY Reginster , R Deroisy , LC Rovati , RL Lee , E Lejeane , O Bruyere , G Giacovelli , Y Henrotin , JE Daore , C Gossett . Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomized, placebo-controlled clinical trial . Lancet , 2001 . 357 ( 9252 ): 251 - 256 . 11214126 DOI: 10.1016/S0140-6736(00)03610-2 http://doi.org/10.1016/S0140-6736(00)03610-2 . Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomized, placebo-controlled clinical trial WP Wang , YM Dua , YL Qiu , XY Wang , YJ Hu , JH Yang , J Cai , JF Kennedy . A new green technology for direct production of low molecular weight chitosan . Carbohydr Polym , 2008 . 74 ( 1 ): 127 - 132 . DOI: 10.1016/j.carbpol.2008.01.025 http://doi.org/10.1016/j.carbpol.2008.01.025 . A new green technology for direct production of low molecular weight chitosan SJ Wu . Preparation of water soluble chitosan by hydrolysis with commercial α-amylase containing chitosanase activity . Food Chem , 2011 . 128 ( 3 ): 769 - 772 . DOI: 10.1016/j.foodchem.2011.03.111 http://doi.org/10.1016/j.foodchem.2011.03.111 . Preparation of water soluble chitosan by hydrolysis with commercial α-amylase containing chitosanase activity SJ Wu , HQ Chen , QY Tong , XM Xu , ZY Jin . Preparation of maltotriose by hydrolyzing of pullulan with pullulanase . Eur Food Res Technol , 2009 . 229 ( 5 ): 821 - 824 . DOI: 10.1007/s00217-009-1118-9 http://doi.org/10.1007/s00217-009-1118-9 . Preparation of maltotriose by hydrolyzing of pullulan with pullulanase WS Xia , P Liu , J Liu . Advance in chitosan hydrolysis by non-specific cellulases . Bioresour Technol , 2008 . 99 ( 15 ): 6751 - 6762 . 18328693 DOI: 10.1016/j.biortech.2008.01.011 http://doi.org/10.1016/j.biortech.2008.01.011 . Advance in chitosan hydrolysis by non-specific cellulases RE Xing , S Liu , L Wang , SB Cai , HH Yu , JH Feng , PC Li . The preparation and antioxidant activity of glucosamine sulfate . Chin J Oceanol Limnol , 2009 . 27 ( 2 ): 283 - 287 . DOI: 10.1007/s00343-009-9135-x http://doi.org/10.1007/s00343-009-9135-x . The preparation and antioxidant activity of glucosamine sulfate HY Yu , S Wang . Study on enzymatic production of low-molecular weight chitosan . Food Sci , 2008 . 29 464 - 466 . (in Chinese) . Study on enzymatic production of low-molecular weight chitosan LJ Zhang , DY Hou , G Xin , H Gu , SY Zhao . Study on preparation and antioxidation effects of D -glucosamine hydrochloride . Food Sci , 2007 . 28 103 - 105 . (in Chinese) . Study on preparation and antioxidation effects of D-glucosamine hydrochloride G Zhou , ZP He , GH Dun , ZY Huang , XC Tan . Enzyme kinetics of amylase and cellulose on hydrolyzing chitosan . Mar Sci , 2003 . 27 ( 11 ): 59 - 62 . (in Chinese) . Enzyme kinetics of amylase and cellulose on hydrolyzing chitosan 10.1631/jzus.B1100065 Project (No. 2009HS07) supported by the Open Fund of Jiangsu Key Laboratory of Marine Biotechnology, China 2011-03-03 2011-10-17 2011-07-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 12