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Production of trehalose by a dual enzyme system of immobilized maltose phosphorylase and trehalose phosphorylase
ELSEVIER
Production of trehalose by a dual enzyme system of immobilized maltose phosphorylase and trehalose phosphorylase Masahiro Yoshida,* Nobuyuki Nakamura,* *Research Institute of Nihon Shokuhin Chemistry,
Faculty
of Engineering,
and Koki Horikoshi’
Kako Co., Fuji, Shizuoka.
Toyo University,
Kawagoe,
Japan Saitama,
‘Department Japan
of Applied
Maltose phosphor&se (MP) and trehalose phosphotylase (TP) in the crude extractfrom a strain of Plesiomonas were adsorbed simultaneously on an anion-exchange resin to examine the continuous production of trehalosefrom maltose. The activity recoveries of both enzymes after immobilization were about 97% for MP, 33% for TP, and 39% for the trehalose-forming activity (TFA). The immobilized enzyme(s) expressed by TFA was stable at nearly neutral pH and showed about 94% of the original activity after a l-h incubation at 55°C and pH 7.0 after which the nonimmobilized enzymes completely lost their activities. About 10 mM or more of inorganic phosphate (Pi) was requiredfor TFA ofthe immobilized enzyme to achieve its fill activity, and apparent values of the Michaelis constant (KJ and maximum velocity (V_) for Pi were about 0.2 FLU and 94.2 kmol trehalose min-’ mg-’ protein at pH 6.2 and SO”C. respecu’vely. The maximum yield of trehalose from maltose after removing small amounts of glucose-l-phosphate (G-l-P) which was formed concomitantly during the reaction was about 60%. The optimum3ow rate achieving the maximum productivity of trehalose was about 1.0 hh ’ of the space velocity (SV) at 20% (w/v) maltose concentration at which the productivity was 252 mg trehalose hh ’ g - ‘. Half-lives of the immobilized enzyme in continuous operation at 0.2 h-t of SV andpH 6.2 in the presence of 10 ??ZMPi with 30% (w/k) corn syrup containing about 84% as dry basis of maltose as a substrate were calculated at about 164 days at 55°C. 28 &ys at 6O”C, ana’ 8 days at 65”C, respectively. Using the method reported here, the mother liquor for preparing crystalline trehalose from maltose can be produced conveniently on an industrial scale. 0 I998 Elsevier Science Inc.
Keywords: Trehalose; maltose phosphorylase; trehalose phosphorylase; EC 2.4.1.8: EC 2.4.1.64; Plesiomonas
Introduction Trehalose (cx-D-glucopyranosyl- 1, I-a-D-glucopyranoside) is a nonreducing disaccharide occurring widely in various microorganisms, insects, plants, and even in mammals as one of the reserved metabolites; it is also a protectant against environmental stresses such as desiccation, high osmolarity, frost, and heat.le4 Attempts to utilize this sweetening material are currently underway in several industrial fields as a stabilizer for proteins including enzymes against drying, freezing and heating during processes for producing foodstuffs, confectioneries, diagnostics and pharmaceuticals, and a moisturizer in cosmetics for human skin.5-9
We earlier isolated a bacterium capable of accumulating significant amounts of maltose phosphorylase (MP, maltose: orthophosphate glucosyltransferase, EC 2.4.1.8) and trehalose phosphorylase (TP, trehalose: orthophosphate glucosyltransferase, EC 2.4.1.64) simultaneously in the cells and demonstrated the industrial use of both enzymes to prepare trehalose from maltose.” In this work, some enzymatic characteristics of the immobilized enzyme composed of both phosphorylases from a strain of Plesiomonas are described to investigate the continuous production of trehalose on an industrial scale.
Materials and methods Address reprint requests to Dr. Nobuyuki Nakamura,
Research Institute of Nihon Shokuhin Kako Co., Tajima 30, Fuji, Shizuoka 417, Japan Received 2 February 1997; revised 17 June 1997; accepted 17 June 1997
Enzyme and Microbial Technology 22:71-75, 1998 Q 1998 Elsevier Science Inc. All rights reserved. 855 Avenue of the Americas, New York, NY 10010
Materials Maltose, trehalose, and a corn syrup (MC-90) composed of 1% (w/w, as dry basis) glucose, 84% maltose, 4% maltulose, 7%
0141-0229/98/$19.00 PII s01~41-0229(97)00132-4
Papers maltotriose, and 4% other larger saccharides were products of Nihon Shokuhin Kako Co (Tokyo, Japan). Glucoamylase (pure grade, 40 U rng- ‘, R&opus n&us) and egg white lysozyme (I U mgg’) were purchased from Seikagaku Kogyo Co (Tokyo, Japan) and QP Corporation (Tokyo, Japan), respectively. Soybean peptide (Hy-Soy J), yeast extract (AY-65), and caproic monoglyceride (Homotex PT) were obtained from Quest Corporation, Asahi Beer Shokuhin Co (Tokyo, Japan), and KAO Corporation (Tokyo, Japan), respectively. Hydrophilic and hydrophobic anion-exchange resins (Duolite A-561, A-568, and Amberlite IRA-94s) were from Rohm and Haas Co. Other anion-exchange resins (Diaion HPA 25, HPA 75, and Chitopearl BCW 3505) were from Mitsubishi Kagaku Co (Tokyo, Japan) and Fuji Bouseki Co (Tokyo, Japan), respectively. Other chemicals were reagent grade and available commercially.
Preparation of the crude enzyme A strain of Plesiomonas (SH-35, FERM P14465) was cultured aerobically in 200 1 of the medium (pH 7.3) composed of 1.5% (w/v) maltose, 0.05% trehalose, 1% Hy-Soy J, 1% AY-65, 1% NH,NO,, 0.3% urea, 0.3% K,HPO,, 0.02% MgSO, * 7H,O, and 0.003% MnCl, * 4H,O with a jar fermenter (300-l) under the following conditions: temperature, 35°C; aeration, 0.5 vvm; and agitation, 200 ‘pm. After 20 h, the culture broth (pH 6.6) was incubated at 35°C with 20 g of lysozyme in the presence of 1 mM EDTA and 0.5 kg of Homotex PT to extract both phosphorylases from the cells. After removal of the cell debris by filtration with diatomaceous earth clay as a filtration aid, the filtrate was concentrated to about i/o in volume with a UF membrane filter (OMEGA 30K, Filtron). The concentrated crude cell extract (29.1 U ml-’ of MP, 149.7 U ml-’ of TP, and 552 U ml-’ of TFA) was used as the enzyme source throughout this work. The specific activities of MP, TP, and TFA in the crude enzyme preparation were 5.0, 25.7, and 94.7 U mgg ’ protein, respectively.
Preparation of the immobilized enzyme The concentrated crude enzyme preparation( 1 1) was mixed at 4°C with 65 g as dry basis of Duolite A-561 which was equilibrated preliminarily with 20 mM potassium (K) phosphate buffer pH 7.0 by shaking at 160 rpm. After 7 h, the resin was collected by filtration and washed thoroughly with excess amounts of the same buffer. Activities of MP, TP, and TFA of the immobilized enzyme and the filtrate were determined by the standard assay methods. The content of protein absorbed on the resin which was determined by the method of Bradford” with bovine serum albumin as a standard was calculated from the deference of values in the filtrate before and after immobilization.
Enzyme assays The MP and TP activities were determined by the method of Kamogawa et al. ‘* with a slight modification. Because of different affinities of the two enzymes against Pi and inorganic arsenate, sodium arsenate buffer in the reaction mixture was replaced with K phosphate buffer pH 7.0 as follows. The reaction mixture containing about 160 mg as dry basis of the immobilized enzyme and 20 mM maltose or trehalose in 2.5 ml of 50 mM K phosphate buffer pH 7.0 was incubated at 50°C with shaking at 120 rpm for 10 min. After the incubation, an aliquot (0.5 ml) was withdrawn carefully without taking out the resin and then heated immediately in a boiling water bath for 5 min. Glucose in the aliquot was determined by the glucose oxidase-peroxidase method with mutarotase using a Glucose C&Test Wako (Wako Pure Chemicals, Japan). One unit (U) of MP or TP activity was defined as the amount of enzyme that formed 1 pmol of glucose min-’ from
72
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2000 ,
I
own 0
1
1
I
I
I
I
1
, 20
I
I
I
I
I
I
I()
2345676 Shaking time(h)
Figure 1 Adsorption capacity of Duolite A-561. The immobilization was performed under the conditions as described in the text. After certain periods, aliquots (0.5 ml) were withdrawn by filtration with a membrane filter (0.45 km), and the enzyme activities and protein content in the filtrate were determined. The enzyme activities and protein adsorbed on the resin were estimated from the differences in their original activities and protein content in the crude enzyme preparation. Symbols indicate MP (01, TP to), and protein (A)
maltose or trehalose under the conditions used. The TFA was determined under the following conditions. The reaction mixture composed of about 160 mg as dry basis of the immobilized enzyme and 1.5 ml of 20 mM K phosphate buffer pH 6.2 containing 450 mg of maltose was incubated at 50°C with shaking at 120 rpm for 30 min. The reaction digest (0.1 ml) was then withdrawn carefully without taking out the resin and heated immediately in a boiling water bath for 5 min. After hydrolysis of maltose in the digest with glucoamylase by the method reported”
followed by the removal of ionic materials with small amounts of an ion-exchange resin (Amberlite MB-3, Rohm and Haas Co.), the trehalose formed was determined by an HPLC method with a column (Ultron PS-80N, Shinwa Kako Co., Japan) under the following conditions: mobile phase, water; flow rate, 0.9 ml min- ‘; temperature, 50°C; and detector, RI. The retention times of trehalose, maltose, and glucose were 10.6, 11.0, and 13.1 min, respectively. As a reference, the TFA in the crude enzyme preparation was also determined under the same conditions by replacing the immobilized enzyme with 0.3 ml of the soluble enzyme. One U of the TFA was defined as the amount of enzyme that formed 1 p.mol of trehalose min- ’ from maltose under the conditions used.
Results Immobilization of the enzymes Among six anion-exchange resins tested, Duolite A-561 was selected as an adsorbent for immobilization of the two phosphorylases due to its better adsorption capacities and activity recoveries and its remarkable stabilization of both enzymes against heating (data not shown). Figure I shows the adsorption capacity of Duolite A-561, and Table 1 summarizes the immobilization of the two enzymes. Although about 17% protein (15.4 mg g-’ as dry basis of the resin) was in the crude enzyme preparation, the resin adsorbed about 378 and 1,454 U g-’ (as dry basis of the resin) of MP and TP. About 3 and 67% of their original activities were lost by immobilization, respectively. About 39% of the adsorbed TFA was recovered in activity. Even though, the activity recovery of TP was relatively low, and the activity ratio of MP/TP changed from about r/s to VI by
Trehalose production Table 1
Summary
by immobilized
enzyme: M. Yoshida et al.
of immobilization Activity (U g-‘)
MP TP TFA MP/TP
Applied
Adsorbed
Expressed
450 2,317 8,545 0.19
37% 1,454 3,633 0.26
366 485 1,425 0.75
Recovery (%) 96.8 33.4 39.2
O-6 00
0
immobilization. No effects on recovery of the activity of TP were observed by’the simultaneous presence of the saccharides and chemicals s&h as maltose, trehalose, sucrose, lactose, glucose, maltitol. sorbitol, EDTA, and dithiothreitol during immobilization.
Some enpmatic
characteristics
of the immobilized
enzyme To examine the stabilization of both phosphorylases by immobilization, the immobilized enzyme was incubated under various conditions of pH and temperature. As a reference, the thermal stability of TFA in the concentrated crude enzyme preparation was also determined under the same conditions. As shown in Figure ZA, the immobilized enzyme was most stable at nearly neutral pH and retained more than 90% of its original full activity in the range from pH 5.2-8.8 at 50°C 6.1-8.1 at WC, and 6.5-8.1 at 60°C under the conditions used, respectively. The enzyme(s) was stabilized significantly against heating by immobilization as shown in Figure 28. The TFA in the crude enzyme preparation lost most of its activity after l-h heating at 50°C and pH 6.2. On the other hand, the immobilized enzyme retained more than 90% of its original full activity even if the reaction was performed at 55°C and pH 6.2 in the
5
6
7 6 PH
9
5
-4
-2 0 2 11[Pfl(mW 20
4
6 25
Figure 3 Effect of the concentration of inorganic phosphate on t& trehalose-forming activity. About 160 mg (228 Ut as dry basis of the immobilized enzyme, after preliminary thorough washing with each concentration of K phosphate buffer pH 6.2, was incubated with 1.5 ml of the same buffer containing 450 mg of maltose at 50°C with shaking at 120 rpm for 30 min. After the incubations, the TFA was determined under the standard assay conditions. The value of K,,, and V,,, were also determined as described in the text (B)
absence of any saccharides as substrates and products. To examine the concentration of Pi required for trehalose production, the TFA of immobilized enzyme was determined in the presence of various amounts of Pi by the standard assay method. As shown in Figure 3A, at least 10 mM Pi was required to maximize the reaction velocity, and about 0.2 mM of Km and 94.2 U mg-’ protein of V,,,, were estimated by the method of Lineweaver and Burki3 as shown in Figure 3B. The relative TFA of immobilized or soluble enzyme which was calculated as 100% when the reaction was performed with 17% (w/v) maltose was determined with various amounts of this disaccharide as a substrate. As shown in Figure 4, the TFAs of both enzymes were significantly inhibited with increasing substrate concentration, and a relatively large inhibition was observed when the reaction was performed with the immobilized enzyme.
3040508070 Tempcwaum (“C)
Figure 2 Effects of pH and temperature on stability. About 160 mg (228 U) as dry basis of the immobilized enzyme was incubated for 60 min with 1.5 ml of 20 mM K phosphate buffer at 50, 55, and 60°C under various conditions of pH (A). As a reference, 0.3 ml (166 U) of the soluble enzyme was also incubated at pH 6.2 and various temperatures (B). After the incubation, the mixture was cooled immediately in an ice bath, and the remaining TFA was assayed by adding 450 mg of maltose to the mixture under the conditions described in the text and expressed as the relative activity when the value obtained by shaking at pH 7.0 and 0°C was 100%. Symbols in (A) indicate 50 (O), 55 (0). and 60°C (LI) and those in (B) were the immobilized (0) and soluble (0) enzymes
Figure 4 Effect of maltose concentration on the trehaloseforming activity. About 160 mg (228 U) as dry basis of the immobilized enzyme or 0.3 ml (166 U) of the soluble enzyme was incubated at 50°C for 30 min with 1.5 ml of 20 mM K phosphate buffer pH 6.2 containing various amounts of maltose. After the incubation, the TFA was determined under the standard assay conditions and expressed as the relative activity when the value obtained with 17% (w/v) maltose as a substrate was 100%. Symbols show the immobilized (0) and soluble (0) enzymes
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5
10 15 20 25 lncubstlon time (hr)
30
35
Formation of trehalose by the action of immobilized enzyme The formation of trehalose by the batchwise reaction was examined with the immobilized enzyme to determine the maximum yield of this saccharide from maltose. As shown in Figure 5, about 60% as dry basis of trehalose and small amounts of glucose were formed from maltose in the final stage of the reaction after removing G-l-P. Figure 6 shows the relationship between the feed rates and the amounts of trehalose produced with changing maltose concentration. The maximum yield of trehalose from maltose in continuous operation was almost the same as that obtained by the batchwise reaction even if the substrate concentration was varied from lo-30% (w/v), and SV values to obtain the maximum yield of this saccharide at 55°C were 0.5 hh’ with lo%, 0.25 hh’ with 20%, and 0.05 hh’ with 30% maltose solutions, respectively. The optimum SV value achieving the maximum productivity was about 1.0 hh’
0
t
I
I
I
1
I
0.2
0.4
0.6
0.6
1
1.2
spaceveloclty(h-1)
Figure 6 Effect of flow rate on the formation of trehalose in continuous column operation. Maltose solutions (1030%, w/v) containing 10 mM K phosphate buffer pH 6.2 were passed through the column of immobilized enzyme (10 ml, 3.56 g as dry basis) at 55°C under various conditions of flow rate. The trehalose content in the effluent was determined by the HPLC method described in the text. Symbols show 10 W, 20 (0). and 30% (a) maltose solutions
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15
20
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Operstlon time (days)
Figure 5 Formation of trehalose from maltose by the batchwise reaction. About 160 mg (228 U) of the immobilized enzyme was incubated with 2 ml of 10 mM K phosphate buffer pH 6.2 containing 600 mg as dry basis of maltose at 50°C. Aliquots (0.1 ml) were withdrawn and heated immediately in a boiling water bath for 5 min. Saccharides in the reaction digest before and after hydrolysis with glucoamylase were determined by the HPLC method described in the text. Symbols show trehalose (01, maltose (O), and glucose (A)
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1998, vol. 22, January
Figure 7 Stability in continuous operation. About 30% (w/v) corn syrup (MC-90) containing 10 mM K phosphate buffer pH 6.2 was passed through the column of immobilized enzyme (IO ml, 3.56 g as dry basis) at 55 (0). 60 (O), and 65°C (A) at 0.2 hh’ of SV. The trehalose content in the effluent was determined by the HPLC method described in the text
using 20% maltose solution; the productivity was 252 mg trehalose hh ’ g- ’ of the dry resin. The stability of the immobilized enzyme in continuous operation was evaluated at 55,60, and 65°C with a corn syrup containing about 84% maltose as a substrate. As shown in Figure 7, half-lives of the immobilized enzyme at the above-mentioned temperatures were calculated at about 164, 28, and 8 days, respectively.
Discussion The conventional methods for the preparation of trehalose, i.e., the extraction from yeast cells and the isolation from culture filtrates of a few microbial strains are not practical in a case larger than the pilot plant-scale because of their low productivities, complicated production procedures, and technical difficulties in utilizing large amounts of the by-products as other valuable materials.‘4,‘” Several enzymatic attempts have been made using a few disaccharide phosphorylases and trehalase to improve the production procedure, but they also could not overcome technical disadvantages of low productivity and enzyme instability, thus making them unsuitable for industrial use.16.” Recently, characterization of novel microbial trehalose-forming enzymes and their application on trehalose production have been reported by some investigators.“-” The simplified production procedures described in these recent reports showed better yield of trehalose than those reported previously, but it was still technically difficult to utilize large amounts of the by-products formed after removing a major part of trehalose in the mother liquor by crystallization. We previously demonstrated the industrial use of intracellular thermostable MP and TP from a strain of Plesiomonas to prepare trehalose from maltose.‘c The production procedure was simplified by changing the substrate from maltose to starch. About 30% by weight of trehalose was obtained as crystals.‘* We also showed that the remaining by-product in which about 25-30% by weight of trehalose still remained could be utilized repeatedly as a mother liquor for crystallization by reacting with both phosphorylases again. In accordance with this finding, we thought that
Trehalose production the continuous column reaction with the immobilized enzyme was a better way for regeneration of the above by-product to the starting mother liquor than that by the batchwise reaction with the soluble enzyme. In this work, we screened anion-exchange resins which were available commercially and without legal restrictions for use by the food processing industry as adsorbents to prepare the immobilized MP and TP from a strain (SH-35) of Plesiomonas. Although the activity recovery of TP was relatively low as compared with that of MP, Duolite A-561 was selected as a suitable adsorbent for preparing the immobilized enzyme because of its better adsorption capacities and activity recoveries of the enzymes, and its remarkable effect for stabilization against heating which exceeded those of other resins (data not shown). Relatively large TFA was observed at nearly 1.0 of the activity ratio of MP/TP (data not shown). Seventy-five percent of the ratio observed with the immobilized enzyme prepared in this work was larger than those with other resins such as Diaion HPA 75 and Chitopearl BCW 3505 (data not shown). Generally, various sweeteners are enzymatically produced from starch at 5070°C and pH 4-7 to avoid microbial infection and nonenzymatic isomerization during saccharification. The immobilized enzyme prepared in this work can also be used under the conditions described above. The concentration of Pi required for TFA of the immobilized enzyme to maximize its reaction velocity was almost the same as that for the soluble enzyme (data not shown). Furthermore, although a lower TFA was observed with a higher substrate concentration, the maximum yield of trehalose from maltose by the immobilized enzyme was also almost the same as that obtained by the soluble enzyme (data not shown). This was true even if the reaction was performed with the high concentration of maltose solution actually used in the starch processing industry. The long-term stability of immobilized enzyme was sufficient for practical use in the continuous production of trehalose in column operation on an industrial scale. The breeding of a microbial strain and the improvement of fermentation to enhance enzyme productivity are in progress to obtain an immobilized enzyme(s) showing a higher specific activity.
M., Chalmers, K., and Reed, R. H. The role of trehalose in dehydration resistance of Succharomyces cerevisiae. FEMS
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Koster, K. L. and Leopald, A. C. Sugars and desiccation tolerance in seeds. Phnt Physiof. 1988, 88, 829-832 Wiemken, A. Trehalose in yeast. stress protectant rather than reserve carbohydrate. Antonie van Leeuwenhoek 1990,58,209 -2 I7 Crowe. J. H.. Carpenter. J. F.. Crowe. L. M., and Anchordoguy, T. J.
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Kato. M.. Miura. Y.. Kettoku, M., Shindo. K., lwamatsu, A., and Kobayashi. K. Purification and characterization of thermostable trehalose synthase from the thermoacidophilic archaebacterium Biosci.
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Maruta, K.. Nakada, T.. Kubota. M.. Chaen H.. Sugimoto. T.. Kurimoto, M.. and Txujisaka. Y. Formation of trehalose from maltooligosaccharides by a novel enzymatic system. Biosci. Biorechnol.
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enzyme: M. Yoshida et al.
Are freezing and dehydration similar stress vectors’? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 1990, 27, 219-23 1 Colaco, C., Sen, S., Thangaveln, M., Pinder, S., and Roser, B. Extraordinary stability of enzymes dried in trehalose: Simplified molecular biology. Biotechnologv 1992, 10, 1007-1011 Kitahara. A. K., Suzuki. Y., Zhan, C. W., Wada, H., and Nishimura, Y. Preservation of skin free-flap using trehalose. .I. Surg. Res. 1996, 62, 130-134 Jsowa. N., Hitomi. S., and Wada, H. Trehalose-containing solutions enhance preservation of cultured endothelial cells. Ann. Thoruc. Surg. 1996, 61,542-545 Hirata. T., Fukuse, T., Lin, C. J., Muro, K.. Yokomise, H.. Yagi, K.. Inui, K., Hitomi, S., and Wada, H. Effect of trehalose in canine lung preservation. Surgery 1994, 11% 1). 102-107 Roser, B. Trehalose, a new approach to premium dried foods. Trends Food Sci. Technol. 1991. July, 166-169 Yoshida. M.. Nakamura. N., and Horikoshi, K. Production and application of maltose phosphorylase and trehalose phosphorylase by a strain of Plesiomonas. Jap. J. Appl. Glyvsci. 1995, 42, 19 -25 Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 1976, 72, 248-254 Kamogawa, A., Yokobayashi. K.. and Fukui. 7. Purification and properties of maltose phosphorylase from LactobaciUu.~ brevis. Agric. Biol. Chem. 1973, 37( 12). 28 13-28 19 Webb, J. L. General principles of inhibition. In: En,-virte atzd Metabolic Inhibitors Vol. 1. Academic Press, New York. I963 Kinefuchi. M., Yamazaki, A., and Yamamoto. K. Extraction of trehalose from Saccharomyws cerel+siae by high-pressure treatment. 0~0 Toshitsu Kugaku Kaishi 1995. 42(3). 237-242 Kizawa. H.. Miyazaki, J., Yokota. A., Kanagae, Y.. Miyagawa. K.. and Sugiyama. Y. Trehalose production by a strain of Micrococcus varians. Biosci. Biotechnol. Biochem. 1995, 59(8). 1522- 1527 Murao. S., Nagano. H.. Ogura. S., and Nishino. T. Enzymatic synthesis of trehalose from maltose. A,qric. Biol. Chem. 1985. 49(7). 2113-2118 Nakano, H.. Moriwaki. M.. Washino. T.. and Kitahata. S. Formation of trehalose and its 2-deoxy analogs through condensation by a trehalase from Loboshaenr sp. Riosci. Riotechnrrl. Biochrm. 1994, 58(8). 1435-1438 Fukuda, S., Sugimoto, T., Kurimoto, M.. and Tsujisaka. Y. Existence of a novel enzyme converting maltose into trehalose. Bimci. Biotechnol. Biochem. 1995, 59( 1 1J, 2 189-2 190 Nakada, T.. Ikegami. S.. Chaen. H.. Kubota. M., Fukuda. S.. Sugimoto, T.. Kurimoto. M.. and Tsujisaka. Y. Purification and characterization of thermostable trehalose synthase from the thermoacidophilic archaebacterium Sulfr~lobus rtcidoca/dariu,s. Biosci.
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References I. Gadd. G. 2.
5.
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1829-l
834
Yoshida, M.. Nakamura, N.. and Horikoshi. K. Production of trehalose from starch by maltose phosphorylase and trehalose phosphorylase from a strain of P/e.\inmonus. SranWSfiirke 1997. 49, 2 l-26
Enzyme Microb.
Technol.,
1998, vol. 22, January
75
Production of trehalose by a dual enzyme system of immobilized maltose phosphorylase and trehalose phosphorylase Masahiro Yoshida,* Nobuyuki Nakamura,* *Research Institute of Nihon Shokuhin Chemistry,
Faculty
of Engineering,
and Koki Horikoshi’
Kako Co., Fuji, Shizuoka.
Toyo University,
Kawagoe,
Japan Saitama,
‘Department Japan
of Applied
Maltose phosphor&se (MP) and trehalose phosphotylase (TP) in the crude extractfrom a strain of Plesiomonas were adsorbed simultaneously on an anion-exchange resin to examine the continuous production of trehalosefrom maltose. The activity recoveries of both enzymes after immobilization were about 97% for MP, 33% for TP, and 39% for the trehalose-forming activity (TFA). The immobilized enzyme(s) expressed by TFA was stable at nearly neutral pH and showed about 94% of the original activity after a l-h incubation at 55°C and pH 7.0 after which the nonimmobilized enzymes completely lost their activities. About 10 mM or more of inorganic phosphate (Pi) was requiredfor TFA ofthe immobilized enzyme to achieve its fill activity, and apparent values of the Michaelis constant (KJ and maximum velocity (V_) for Pi were about 0.2 FLU and 94.2 kmol trehalose min-’ mg-’ protein at pH 6.2 and SO”C. respecu’vely. The maximum yield of trehalose from maltose after removing small amounts of glucose-l-phosphate (G-l-P) which was formed concomitantly during the reaction was about 60%. The optimum3ow rate achieving the maximum productivity of trehalose was about 1.0 hh ’ of the space velocity (SV) at 20% (w/v) maltose concentration at which the productivity was 252 mg trehalose hh ’ g - ‘. Half-lives of the immobilized enzyme in continuous operation at 0.2 h-t of SV andpH 6.2 in the presence of 10 ??ZMPi with 30% (w/k) corn syrup containing about 84% as dry basis of maltose as a substrate were calculated at about 164 days at 55°C. 28 &ys at 6O”C, ana’ 8 days at 65”C, respectively. Using the method reported here, the mother liquor for preparing crystalline trehalose from maltose can be produced conveniently on an industrial scale. 0 I998 Elsevier Science Inc.
Keywords: Trehalose; maltose phosphorylase; trehalose phosphorylase; EC 2.4.1.8: EC 2.4.1.64; Plesiomonas
Introduction Trehalose (cx-D-glucopyranosyl- 1, I-a-D-glucopyranoside) is a nonreducing disaccharide occurring widely in various microorganisms, insects, plants, and even in mammals as one of the reserved metabolites; it is also a protectant against environmental stresses such as desiccation, high osmolarity, frost, and heat.le4 Attempts to utilize this sweetening material are currently underway in several industrial fields as a stabilizer for proteins including enzymes against drying, freezing and heating during processes for producing foodstuffs, confectioneries, diagnostics and pharmaceuticals, and a moisturizer in cosmetics for human skin.5-9
We earlier isolated a bacterium capable of accumulating significant amounts of maltose phosphorylase (MP, maltose: orthophosphate glucosyltransferase, EC 2.4.1.8) and trehalose phosphorylase (TP, trehalose: orthophosphate glucosyltransferase, EC 2.4.1.64) simultaneously in the cells and demonstrated the industrial use of both enzymes to prepare trehalose from maltose.” In this work, some enzymatic characteristics of the immobilized enzyme composed of both phosphorylases from a strain of Plesiomonas are described to investigate the continuous production of trehalose on an industrial scale.
Materials and methods Address reprint requests to Dr. Nobuyuki Nakamura,
Research Institute of Nihon Shokuhin Kako Co., Tajima 30, Fuji, Shizuoka 417, Japan Received 2 February 1997; revised 17 June 1997; accepted 17 June 1997
Enzyme and Microbial Technology 22:71-75, 1998 Q 1998 Elsevier Science Inc. All rights reserved. 855 Avenue of the Americas, New York, NY 10010
Materials Maltose, trehalose, and a corn syrup (MC-90) composed of 1% (w/w, as dry basis) glucose, 84% maltose, 4% maltulose, 7%
0141-0229/98/$19.00 PII s01~41-0229(97)00132-4
Papers maltotriose, and 4% other larger saccharides were products of Nihon Shokuhin Kako Co (Tokyo, Japan). Glucoamylase (pure grade, 40 U rng- ‘, R&opus n&us) and egg white lysozyme (I U mgg’) were purchased from Seikagaku Kogyo Co (Tokyo, Japan) and QP Corporation (Tokyo, Japan), respectively. Soybean peptide (Hy-Soy J), yeast extract (AY-65), and caproic monoglyceride (Homotex PT) were obtained from Quest Corporation, Asahi Beer Shokuhin Co (Tokyo, Japan), and KAO Corporation (Tokyo, Japan), respectively. Hydrophilic and hydrophobic anion-exchange resins (Duolite A-561, A-568, and Amberlite IRA-94s) were from Rohm and Haas Co. Other anion-exchange resins (Diaion HPA 25, HPA 75, and Chitopearl BCW 3505) were from Mitsubishi Kagaku Co (Tokyo, Japan) and Fuji Bouseki Co (Tokyo, Japan), respectively. Other chemicals were reagent grade and available commercially.
Preparation of the crude enzyme A strain of Plesiomonas (SH-35, FERM P14465) was cultured aerobically in 200 1 of the medium (pH 7.3) composed of 1.5% (w/v) maltose, 0.05% trehalose, 1% Hy-Soy J, 1% AY-65, 1% NH,NO,, 0.3% urea, 0.3% K,HPO,, 0.02% MgSO, * 7H,O, and 0.003% MnCl, * 4H,O with a jar fermenter (300-l) under the following conditions: temperature, 35°C; aeration, 0.5 vvm; and agitation, 200 ‘pm. After 20 h, the culture broth (pH 6.6) was incubated at 35°C with 20 g of lysozyme in the presence of 1 mM EDTA and 0.5 kg of Homotex PT to extract both phosphorylases from the cells. After removal of the cell debris by filtration with diatomaceous earth clay as a filtration aid, the filtrate was concentrated to about i/o in volume with a UF membrane filter (OMEGA 30K, Filtron). The concentrated crude cell extract (29.1 U ml-’ of MP, 149.7 U ml-’ of TP, and 552 U ml-’ of TFA) was used as the enzyme source throughout this work. The specific activities of MP, TP, and TFA in the crude enzyme preparation were 5.0, 25.7, and 94.7 U mgg ’ protein, respectively.
Preparation of the immobilized enzyme The concentrated crude enzyme preparation( 1 1) was mixed at 4°C with 65 g as dry basis of Duolite A-561 which was equilibrated preliminarily with 20 mM potassium (K) phosphate buffer pH 7.0 by shaking at 160 rpm. After 7 h, the resin was collected by filtration and washed thoroughly with excess amounts of the same buffer. Activities of MP, TP, and TFA of the immobilized enzyme and the filtrate were determined by the standard assay methods. The content of protein absorbed on the resin which was determined by the method of Bradford” with bovine serum albumin as a standard was calculated from the deference of values in the filtrate before and after immobilization.
Enzyme assays The MP and TP activities were determined by the method of Kamogawa et al. ‘* with a slight modification. Because of different affinities of the two enzymes against Pi and inorganic arsenate, sodium arsenate buffer in the reaction mixture was replaced with K phosphate buffer pH 7.0 as follows. The reaction mixture containing about 160 mg as dry basis of the immobilized enzyme and 20 mM maltose or trehalose in 2.5 ml of 50 mM K phosphate buffer pH 7.0 was incubated at 50°C with shaking at 120 rpm for 10 min. After the incubation, an aliquot (0.5 ml) was withdrawn carefully without taking out the resin and then heated immediately in a boiling water bath for 5 min. Glucose in the aliquot was determined by the glucose oxidase-peroxidase method with mutarotase using a Glucose C&Test Wako (Wako Pure Chemicals, Japan). One unit (U) of MP or TP activity was defined as the amount of enzyme that formed 1 pmol of glucose min-’ from
72
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2000 ,
I
own 0
1
1
I
I
I
I
1
, 20
I
I
I
I
I
I
I()
2345676 Shaking time(h)
Figure 1 Adsorption capacity of Duolite A-561. The immobilization was performed under the conditions as described in the text. After certain periods, aliquots (0.5 ml) were withdrawn by filtration with a membrane filter (0.45 km), and the enzyme activities and protein content in the filtrate were determined. The enzyme activities and protein adsorbed on the resin were estimated from the differences in their original activities and protein content in the crude enzyme preparation. Symbols indicate MP (01, TP to), and protein (A)
maltose or trehalose under the conditions used. The TFA was determined under the following conditions. The reaction mixture composed of about 160 mg as dry basis of the immobilized enzyme and 1.5 ml of 20 mM K phosphate buffer pH 6.2 containing 450 mg of maltose was incubated at 50°C with shaking at 120 rpm for 30 min. The reaction digest (0.1 ml) was then withdrawn carefully without taking out the resin and heated immediately in a boiling water bath for 5 min. After hydrolysis of maltose in the digest with glucoamylase by the method reported”
followed by the removal of ionic materials with small amounts of an ion-exchange resin (Amberlite MB-3, Rohm and Haas Co.), the trehalose formed was determined by an HPLC method with a column (Ultron PS-80N, Shinwa Kako Co., Japan) under the following conditions: mobile phase, water; flow rate, 0.9 ml min- ‘; temperature, 50°C; and detector, RI. The retention times of trehalose, maltose, and glucose were 10.6, 11.0, and 13.1 min, respectively. As a reference, the TFA in the crude enzyme preparation was also determined under the same conditions by replacing the immobilized enzyme with 0.3 ml of the soluble enzyme. One U of the TFA was defined as the amount of enzyme that formed 1 p.mol of trehalose min- ’ from maltose under the conditions used.
Results Immobilization of the enzymes Among six anion-exchange resins tested, Duolite A-561 was selected as an adsorbent for immobilization of the two phosphorylases due to its better adsorption capacities and activity recoveries and its remarkable stabilization of both enzymes against heating (data not shown). Figure I shows the adsorption capacity of Duolite A-561, and Table 1 summarizes the immobilization of the two enzymes. Although about 17% protein (15.4 mg g-’ as dry basis of the resin) was in the crude enzyme preparation, the resin adsorbed about 378 and 1,454 U g-’ (as dry basis of the resin) of MP and TP. About 3 and 67% of their original activities were lost by immobilization, respectively. About 39% of the adsorbed TFA was recovered in activity. Even though, the activity recovery of TP was relatively low, and the activity ratio of MP/TP changed from about r/s to VI by
Trehalose production Table 1
Summary
by immobilized
enzyme: M. Yoshida et al.
of immobilization Activity (U g-‘)
MP TP TFA MP/TP
Applied
Adsorbed
Expressed
450 2,317 8,545 0.19
37% 1,454 3,633 0.26
366 485 1,425 0.75
Recovery (%) 96.8 33.4 39.2
O-6 00
0
immobilization. No effects on recovery of the activity of TP were observed by’the simultaneous presence of the saccharides and chemicals s&h as maltose, trehalose, sucrose, lactose, glucose, maltitol. sorbitol, EDTA, and dithiothreitol during immobilization.
Some enpmatic
characteristics
of the immobilized
enzyme To examine the stabilization of both phosphorylases by immobilization, the immobilized enzyme was incubated under various conditions of pH and temperature. As a reference, the thermal stability of TFA in the concentrated crude enzyme preparation was also determined under the same conditions. As shown in Figure ZA, the immobilized enzyme was most stable at nearly neutral pH and retained more than 90% of its original full activity in the range from pH 5.2-8.8 at 50°C 6.1-8.1 at WC, and 6.5-8.1 at 60°C under the conditions used, respectively. The enzyme(s) was stabilized significantly against heating by immobilization as shown in Figure 28. The TFA in the crude enzyme preparation lost most of its activity after l-h heating at 50°C and pH 6.2. On the other hand, the immobilized enzyme retained more than 90% of its original full activity even if the reaction was performed at 55°C and pH 6.2 in the
5
6
7 6 PH
9
5
-4
-2 0 2 11[Pfl(mW 20
4
6 25
Figure 3 Effect of the concentration of inorganic phosphate on t& trehalose-forming activity. About 160 mg (228 Ut as dry basis of the immobilized enzyme, after preliminary thorough washing with each concentration of K phosphate buffer pH 6.2, was incubated with 1.5 ml of the same buffer containing 450 mg of maltose at 50°C with shaking at 120 rpm for 30 min. After the incubations, the TFA was determined under the standard assay conditions. The value of K,,, and V,,, were also determined as described in the text (B)
absence of any saccharides as substrates and products. To examine the concentration of Pi required for trehalose production, the TFA of immobilized enzyme was determined in the presence of various amounts of Pi by the standard assay method. As shown in Figure 3A, at least 10 mM Pi was required to maximize the reaction velocity, and about 0.2 mM of Km and 94.2 U mg-’ protein of V,,,, were estimated by the method of Lineweaver and Burki3 as shown in Figure 3B. The relative TFA of immobilized or soluble enzyme which was calculated as 100% when the reaction was performed with 17% (w/v) maltose was determined with various amounts of this disaccharide as a substrate. As shown in Figure 4, the TFAs of both enzymes were significantly inhibited with increasing substrate concentration, and a relatively large inhibition was observed when the reaction was performed with the immobilized enzyme.
3040508070 Tempcwaum (“C)
Figure 2 Effects of pH and temperature on stability. About 160 mg (228 U) as dry basis of the immobilized enzyme was incubated for 60 min with 1.5 ml of 20 mM K phosphate buffer at 50, 55, and 60°C under various conditions of pH (A). As a reference, 0.3 ml (166 U) of the soluble enzyme was also incubated at pH 6.2 and various temperatures (B). After the incubation, the mixture was cooled immediately in an ice bath, and the remaining TFA was assayed by adding 450 mg of maltose to the mixture under the conditions described in the text and expressed as the relative activity when the value obtained by shaking at pH 7.0 and 0°C was 100%. Symbols in (A) indicate 50 (O), 55 (0). and 60°C (LI) and those in (B) were the immobilized (0) and soluble (0) enzymes
Figure 4 Effect of maltose concentration on the trehaloseforming activity. About 160 mg (228 U) as dry basis of the immobilized enzyme or 0.3 ml (166 U) of the soluble enzyme was incubated at 50°C for 30 min with 1.5 ml of 20 mM K phosphate buffer pH 6.2 containing various amounts of maltose. After the incubation, the TFA was determined under the standard assay conditions and expressed as the relative activity when the value obtained with 17% (w/v) maltose as a substrate was 100%. Symbols show the immobilized (0) and soluble (0) enzymes
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10 15 20 25 lncubstlon time (hr)
30
35
Formation of trehalose by the action of immobilized enzyme The formation of trehalose by the batchwise reaction was examined with the immobilized enzyme to determine the maximum yield of this saccharide from maltose. As shown in Figure 5, about 60% as dry basis of trehalose and small amounts of glucose were formed from maltose in the final stage of the reaction after removing G-l-P. Figure 6 shows the relationship between the feed rates and the amounts of trehalose produced with changing maltose concentration. The maximum yield of trehalose from maltose in continuous operation was almost the same as that obtained by the batchwise reaction even if the substrate concentration was varied from lo-30% (w/v), and SV values to obtain the maximum yield of this saccharide at 55°C were 0.5 hh’ with lo%, 0.25 hh’ with 20%, and 0.05 hh’ with 30% maltose solutions, respectively. The optimum SV value achieving the maximum productivity was about 1.0 hh’
0
t
I
I
I
1
I
0.2
0.4
0.6
0.6
1
1.2
spaceveloclty(h-1)
Figure 6 Effect of flow rate on the formation of trehalose in continuous column operation. Maltose solutions (1030%, w/v) containing 10 mM K phosphate buffer pH 6.2 were passed through the column of immobilized enzyme (10 ml, 3.56 g as dry basis) at 55°C under various conditions of flow rate. The trehalose content in the effluent was determined by the HPLC method described in the text. Symbols show 10 W, 20 (0). and 30% (a) maltose solutions
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Operstlon time (days)
Figure 5 Formation of trehalose from maltose by the batchwise reaction. About 160 mg (228 U) of the immobilized enzyme was incubated with 2 ml of 10 mM K phosphate buffer pH 6.2 containing 600 mg as dry basis of maltose at 50°C. Aliquots (0.1 ml) were withdrawn and heated immediately in a boiling water bath for 5 min. Saccharides in the reaction digest before and after hydrolysis with glucoamylase were determined by the HPLC method described in the text. Symbols show trehalose (01, maltose (O), and glucose (A)
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1998, vol. 22, January
Figure 7 Stability in continuous operation. About 30% (w/v) corn syrup (MC-90) containing 10 mM K phosphate buffer pH 6.2 was passed through the column of immobilized enzyme (IO ml, 3.56 g as dry basis) at 55 (0). 60 (O), and 65°C (A) at 0.2 hh’ of SV. The trehalose content in the effluent was determined by the HPLC method described in the text
using 20% maltose solution; the productivity was 252 mg trehalose hh ’ g- ’ of the dry resin. The stability of the immobilized enzyme in continuous operation was evaluated at 55,60, and 65°C with a corn syrup containing about 84% maltose as a substrate. As shown in Figure 7, half-lives of the immobilized enzyme at the above-mentioned temperatures were calculated at about 164, 28, and 8 days, respectively.
Discussion The conventional methods for the preparation of trehalose, i.e., the extraction from yeast cells and the isolation from culture filtrates of a few microbial strains are not practical in a case larger than the pilot plant-scale because of their low productivities, complicated production procedures, and technical difficulties in utilizing large amounts of the by-products as other valuable materials.‘4,‘” Several enzymatic attempts have been made using a few disaccharide phosphorylases and trehalase to improve the production procedure, but they also could not overcome technical disadvantages of low productivity and enzyme instability, thus making them unsuitable for industrial use.16.” Recently, characterization of novel microbial trehalose-forming enzymes and their application on trehalose production have been reported by some investigators.“-” The simplified production procedures described in these recent reports showed better yield of trehalose than those reported previously, but it was still technically difficult to utilize large amounts of the by-products formed after removing a major part of trehalose in the mother liquor by crystallization. We previously demonstrated the industrial use of intracellular thermostable MP and TP from a strain of Plesiomonas to prepare trehalose from maltose.‘c The production procedure was simplified by changing the substrate from maltose to starch. About 30% by weight of trehalose was obtained as crystals.‘* We also showed that the remaining by-product in which about 25-30% by weight of trehalose still remained could be utilized repeatedly as a mother liquor for crystallization by reacting with both phosphorylases again. In accordance with this finding, we thought that
Trehalose production the continuous column reaction with the immobilized enzyme was a better way for regeneration of the above by-product to the starting mother liquor than that by the batchwise reaction with the soluble enzyme. In this work, we screened anion-exchange resins which were available commercially and without legal restrictions for use by the food processing industry as adsorbents to prepare the immobilized MP and TP from a strain (SH-35) of Plesiomonas. Although the activity recovery of TP was relatively low as compared with that of MP, Duolite A-561 was selected as a suitable adsorbent for preparing the immobilized enzyme because of its better adsorption capacities and activity recoveries of the enzymes, and its remarkable effect for stabilization against heating which exceeded those of other resins (data not shown). Relatively large TFA was observed at nearly 1.0 of the activity ratio of MP/TP (data not shown). Seventy-five percent of the ratio observed with the immobilized enzyme prepared in this work was larger than those with other resins such as Diaion HPA 75 and Chitopearl BCW 3505 (data not shown). Generally, various sweeteners are enzymatically produced from starch at 5070°C and pH 4-7 to avoid microbial infection and nonenzymatic isomerization during saccharification. The immobilized enzyme prepared in this work can also be used under the conditions described above. The concentration of Pi required for TFA of the immobilized enzyme to maximize its reaction velocity was almost the same as that for the soluble enzyme (data not shown). Furthermore, although a lower TFA was observed with a higher substrate concentration, the maximum yield of trehalose from maltose by the immobilized enzyme was also almost the same as that obtained by the soluble enzyme (data not shown). This was true even if the reaction was performed with the high concentration of maltose solution actually used in the starch processing industry. The long-term stability of immobilized enzyme was sufficient for practical use in the continuous production of trehalose in column operation on an industrial scale. The breeding of a microbial strain and the improvement of fermentation to enhance enzyme productivity are in progress to obtain an immobilized enzyme(s) showing a higher specific activity.
M., Chalmers, K., and Reed, R. H. The role of trehalose in dehydration resistance of Succharomyces cerevisiae. FEMS
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Koster, K. L. and Leopald, A. C. Sugars and desiccation tolerance in seeds. Phnt Physiof. 1988, 88, 829-832 Wiemken, A. Trehalose in yeast. stress protectant rather than reserve carbohydrate. Antonie van Leeuwenhoek 1990,58,209 -2 I7 Crowe. J. H.. Carpenter. J. F.. Crowe. L. M., and Anchordoguy, T. J.
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Kato. M.. Miura. Y.. Kettoku, M., Shindo. K., lwamatsu, A., and Kobayashi. K. Purification and characterization of thermostable trehalose synthase from the thermoacidophilic archaebacterium Biosci.
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enzyme: M. Yoshida et al.
Are freezing and dehydration similar stress vectors’? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 1990, 27, 219-23 1 Colaco, C., Sen, S., Thangaveln, M., Pinder, S., and Roser, B. Extraordinary stability of enzymes dried in trehalose: Simplified molecular biology. Biotechnologv 1992, 10, 1007-1011 Kitahara. A. K., Suzuki. Y., Zhan, C. W., Wada, H., and Nishimura, Y. Preservation of skin free-flap using trehalose. .I. Surg. Res. 1996, 62, 130-134 Jsowa. N., Hitomi. S., and Wada, H. Trehalose-containing solutions enhance preservation of cultured endothelial cells. Ann. Thoruc. Surg. 1996, 61,542-545 Hirata. T., Fukuse, T., Lin, C. J., Muro, K.. Yokomise, H.. Yagi, K.. Inui, K., Hitomi, S., and Wada, H. Effect of trehalose in canine lung preservation. Surgery 1994, 11% 1). 102-107 Roser, B. Trehalose, a new approach to premium dried foods. Trends Food Sci. Technol. 1991. July, 166-169 Yoshida. M.. Nakamura. N., and Horikoshi, K. Production and application of maltose phosphorylase and trehalose phosphorylase by a strain of Plesiomonas. Jap. J. Appl. Glyvsci. 1995, 42, 19 -25 Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 1976, 72, 248-254 Kamogawa, A., Yokobayashi. K.. and Fukui. 7. Purification and properties of maltose phosphorylase from LactobaciUu.~ brevis. Agric. Biol. Chem. 1973, 37( 12). 28 13-28 19 Webb, J. L. General principles of inhibition. In: En,-virte atzd Metabolic Inhibitors Vol. 1. Academic Press, New York. I963 Kinefuchi. M., Yamazaki, A., and Yamamoto. K. Extraction of trehalose from Saccharomyws cerel+siae by high-pressure treatment. 0~0 Toshitsu Kugaku Kaishi 1995. 42(3). 237-242 Kizawa. H.. Miyazaki, J., Yokota. A., Kanagae, Y.. Miyagawa. K.. and Sugiyama. Y. Trehalose production by a strain of Micrococcus varians. Biosci. Biotechnol. Biochem. 1995, 59(8). 1522- 1527 Murao. S., Nagano. H.. Ogura. S., and Nishino. T. Enzymatic synthesis of trehalose from maltose. A,qric. Biol. Chem. 1985. 49(7). 2113-2118 Nakano, H.. Moriwaki. M.. Washino. T.. and Kitahata. S. Formation of trehalose and its 2-deoxy analogs through condensation by a trehalase from Loboshaenr sp. Riosci. Riotechnrrl. Biochrm. 1994, 58(8). 1435-1438 Fukuda, S., Sugimoto, T., Kurimoto, M.. and Tsujisaka. Y. Existence of a novel enzyme converting maltose into trehalose. Bimci. Biotechnol. Biochem. 1995, 59( 1 1J, 2 189-2 190 Nakada, T.. Ikegami. S.. Chaen. H.. Kubota. M., Fukuda. S.. Sugimoto, T.. Kurimoto. M.. and Tsujisaka. Y. Purification and characterization of thermostable trehalose synthase from the thermoacidophilic archaebacterium Sulfr~lobus rtcidoca/dariu,s. Biosci.
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5.
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834
Yoshida, M.. Nakamura, N.. and Horikoshi. K. Production of trehalose from starch by maltose phosphorylase and trehalose phosphorylase from a strain of P/e.\inmonus. SranWSfiirke 1997. 49, 2 l-26
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