Thermostable Saccharidases - ACS Symposium Series (ACS

Chapter 4

Thermostable Saccharidases New Sources, Uses, and Biodesigns Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch004

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J. Gregory Zeikus , Chanyong Lee , Yong-Eok Lee , and Badal C. Saha 1

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Michigan Biotechnology Institute, Lansing, M I 48910 Department of Biochemistry and Department of Microbiology and Public Health, Michigan State University, East Lansing, M I 48824 2

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Thermostable saccharidases are required for industrial processing of starch and lignocellulosic fibers. Thermophilic microbes have not been used as sources for industrial enzymes and the molecular biochemistry of enzyme thermophilicity is not understood. We have purified and characterized thermostable (70°C) saccharidases from thermoanaerobic bacteria including: ß-amylase, amylopullulanase, α -glucosidase, glucose isomerase (GI), and endoxylanase. Thermostable saccharidase synthesis was regulated in Thermoanaerobacter to produce fructose directly from starch. The GI gene from Thermo anaerobacter was cloned and sequenced and site-directed mutagenesis was employed to explain enzyme catalysis and to design an enzyme active at 70°C and pH 5.5. Endoxylanase of Thermoanaerobacter was cloned and characterized in relation to a xylanosomal cellular organization and a source for fiber modification. Future studies on these novel saccharidases will focus on the molecular basis of thermo philicity, coordination with other hydrolases and industrial uses. Saccharidases have a broad base for biotechnology applications (see Table I). Future industrial applications of saccharidases requires solving several problems including: finding new product uses, enhancing activity and stability, and lowering production costs in food- or environmentally-safe hosts. Our approach has been to develop thermophilic enzymes that are temperature and pH compatible with process conditions, to screen for new microbes, to clone and produce thermophilic enzymes in industrial hosts, and to enhance enzyme activity and stability by genetic design. The major current industrial use for saccharidases is in the manufacture of high fructose corn syrup from starch. The current process requires thermostable enzymes and three processing steps because the enzymes used are not compatible at the same pH and temperature (see Figure 1). Industry is looking to improve the process by developing: 1) an improved a-amylase that works at low pH that has a low Ca**

0097-6156/91/0460-0036$06.00/0 © 1991 American Chemical Society

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Thermostable Saccharidases

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Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch004

Table I. Saccharidase Biotechnology

Applications:

Starch processing Baking and food processing Brewing and fermentation Forest products Detergents Specialty chemicals Waste treatment

Current Problems:

Limited uses Unstable biocatalysts Low efficiency and product yield High cost and safety

Approaches:

Increase thermostability and environmental compatibility Increase specificity and activity Develop thermophilic enzymes •screen for new microbes •clone and overexpress in industrial hosts •site-direct mutagenesis

requirement; and, 2) an improved glucose isomerase (GI) that works at acidic pH and at higher temperature (i.e., 70°C) so as to improve the final chemical equilibrium concentration of fructose. Thermophilic enzymes are active and stable at high temperature (> 60°C) but they are generally inactive and extremely stable at low temperature (< 25°C). The molecular basis has not been elaborated to explain such thermophilicity. In general, thermophilic enzymes do not denature at high temperature and their activity is higher due to the Q rule where a 10°C increase results in a doubling of chemical activity. We have chosen to explore enzymes in thermophilic anaerobic bacteria, because these kinds of microorganisms were believed to have been the first forms of life on earth and have evolved under energy limited conditions that place stress on selection of enzymes with high catalytic efficiency (2). Thermoanaerobes contain a diverse array of enzymes with unique properties and their enzyme outfits now serve as models for understanding the biodegradation of polymers such as cellulose (5). Our early studies dealt with characterization of cellulasefromClostridium thermocellum (4, J), the first described thermoanaerobe. More recently, we have characterized the saccharidases in three new non-cellulolytic thermoanaerobic species (6-72). Table II compares the general properties of thermophilic saccharidases identified in C. thermosulfurogenes strain 4B (6), C. thermohydrosulfuricum strain 39E (7), and Thermoanaerobacter strain B6A (13). It is worth noting here that 10


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ENZYMES IN BIOMASS CONVERSION


Stage

Liquefaction

Enzyme

Bacterial Atpha-Amytase

80%) from starch, a true debranching pullulanase that does not hydrolyze a-1,4 bonds is required to work with the 6-amylase. Table IV shows the biochemical features of thermostable cc-glucosidase purified 140-fold from C. thermohydrosulfuricum (Saha and Zeikus, unpublished work). The enzyme has a 162,000 molecular weight and displayed an optimum temperature for activity of 75°C. Notably, the protein preparation hydrolyzed both a-1,6 and a-1,4 linkages. This enzyme appears to play an important role in starch degradation by C. thermohydrosulfuricum because it can hydrolyze the degradation intermediates formed by the organism's unique amylopullulanase. Xylose-Glucose Isomerase Characterization The regulation of glucose isomerase was studied in Thermoanaerobacter strain B6A (20). This species produces thermostable amylase and 6-galactosidase constituitively; whereas, xylose/glucose isomerase is inducible and catabolite repressed by glucose (20). Figure 3 shows the temperature, activity, and thermostability response for these three enzymes. These thermophilic enzymes are not active at < 30°C and display optimal activity at > 60°C. They are active and stable at £ 65°C and can function together at pH 6.5 with > 70% activity for each enzyme. Because all these enzymes were produced in xylose grown cells and they displayed activities compatible at 65°C and pH 6.5, these conditions were used to develop single-step processes for production of fructose sweetener from soluble starch and maltodextrin or milk lactose (see Figure 3). Consequendy, fructose sweetener production from soluble starch or milk lactose can be achieved with a single-step enzymatic process. The xylose-glucose isomerase was purified to homogeneity from C. thermosulfurogenes (Lee, C.-Y and Zeikus, J. G., Biochem. J., in press); and, its general



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Reaction time(h) Figure 2. Time course of maltose production from maltodextrin (DE 10) by B-amylase and pullulanase: 1, B-amylase at 75°C; 2, B-amylase at 75°C for 24 h and then B-amylase and pullulanase at 60°C; 3, pullulanase at 60°C for 24 h and then B-amylases at 75°C; 4, B-amylase and pullulanase at 60°C for 24 h and then temperature raised to 75°C. Arrow indicates time of addition of second enzyme and/or changing of temperature. Enzyme used (units/g substrate); B-amylase, 200; pullulanase, 50. Reprinted with permission from ref. 18. Copyright 1989 John Wiley & Sons.


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100

Glucose isomerase

80 60

^ ^

40 20

>-

0

>

100

H O

< LU > < -J

LU CC

80 60 40 20 0 100 80 60 40 20 0

TEMPERATURE (°C) Figure 3(A). Comparison of temperature optima for activities of glucose isomerase, amylase, and /?-galactosidase. Enzymes were assayed with cell extract from xylose-grown cells. A 100% activity value corresponds to 0.60, 0.58, and 0.46 U/mg for glucose isomerase, amylase, and /?-galactosidase, respectively. Cell extracts in 50 mM sodium phosphate buffer (pH 7.0), 100 mM sodium acetate buffer (pH 5.5), and 100 mM sodium phosphate buffer (pH 6.0) for glucose isomerase, amylase, and ^-galactosidase, respectively, were preincubated at the indicated temperatures, prior to the assay for residual enzyme activities. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.


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TIME (MIN) Figure 3(B). Comparison of temperature optima for stabilities of glucose isomerase, amylase, and 0-galactosidase. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.


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Table IV. Biochemical Characteristics of Thermostable a-Glucosidase from Clostridium thermohydrosulfuricum 39E Molecular weight Optimum pH Optimum temperature (°C) pH stability Km (mM) maltose isomaltose panose maltotriose pNPG Metal ion requirement activity stability Half-life at 60°C (h) at 70°C (min) at 80°C (min) Specificity Inhibitor

162,000 5.0 - 5.5 75 5.0-6.0 1.85 2.95 1.722 0.58 0.31 none none 46 110 35 hydroxyzes both a-1,4 and a-1,6 linkages acarbose

features are shown in Table V. The protein was a tetramer with a subunit molecular weight of 50,000. The enzyme was very thermostable and displayed an optimal activity at 80°C. C. thermosulfurogenes xylose isomerase was cloned, expressed, and over-produced in both E. coli and food-safe B. subtilis (27). Heat treatment of recombinant B. subtilis extracts at 85°C for 10 min enabled 90% protein purification of the thermophilic glucose isomerase. The cloned enzyme was active and stable and used to produce fructose at pH 7.0, 70°C and with industrial saccharide concentra tions (see Figure 4). The glucose/xylose isomerase gene of C. thermosulfurogenes was sequenced and analyzed in relation to gene structure, function and similarity to other xylose isomerase sequences (22). Figure 5 shows that C. thermosulfurogenes xylose isomerase displayed higher homology to thermolabile xylose isomerase of B. subtilis (70%) than thermostable xylose isomerase of Streptomyces (24%). The putative catalytic domain of the thermoanaerobe xylose isomerase was deduced from gene sequence homologies of both Bacillus and Streptomyces xylose isomerase and indicated that His was in the thermophilic enzyme active site. Table VI indicates that His in the native enzyme was inhibited by diethylpyrocarbonate, but not in site-directed mutant enzymes His -» Phe^ or in His -» Gln . Notably, the function of histidine 101 was ascertained to stabilize the transition state by hydrogen bonding and not in base-mediated ring opening for endiol formation. This biochemical evidence supports chemical/x-ray structural evidence of Collyer and Blow (25) on the 101

101

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Time ( hours ) Figure 4. Time course of glucose conversion into fructose using heat-treated glucose isomerase obtained from recombinant B. subtilis determined at 70°C and pH 7.0 using 35% (w/w) glucose solution and 10.8 units of enzyme/g of dry substance. Reprinted with permission from ref. 21. Copyright 1990 American Society for Microbiology.


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Thermolabile

t enzymes

E. coli

50%

B. subtilis

70%

I Thermostable t enzymes

50%

C.

thermosulfurogenes

1 23% 24%

_L

65% S. violaceoniger

22%

Arthrobacter

66% — 68% •

Ampullariella

Figure 5. Summary of amino acid sequence homology between different xylose isomerases. The percent of homology was calculated by using the University of Wisconsin Genetics Computer Group, version 5, program (Devereux, L, Haeberli, P., and Smithies, O. Nucleic Acids Res. 12, 387-395, 1984). Reprinted with permission from ref. 22. Copyright 1990 American Society for Biochemistry and Molecular Biology.


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Table V. Biochemical Characteristics of C. thermosulfurogenes Strain 4B Xylose/Glucose Isomerase


Property Molecular weight Optimum pH Optimum temperature (°C) Temperature stability (°C, 1 hr) Kcat (1/min) Isoelectric point pH Subunit composition Km (mM) xylose glucose V (U/mg) xylose glucose

200,000 7.0 80 85 1040 4.9 tetramer 20 140

m a x

15.7 5.2

Table VI. Effect of Diethylpyrocarbonate (DEPC) on Thermoanaerobe Glucose Isomerase Activity of the Wild-Type and Mutant Enzymes* Residual Activity (%) Enzyme

0 mM DEPC

1 mM DEPC

10 mM DEPC

Wild-type (His )

100

8

1

His-^Phe^

100

10

3

His -> Gln

100

100

100

101

101

•Purified enzymes (80 |ig for wild-type and His-Phe^ mutant enzyme and 800 |Xg for His-Gln mutant enzyme) in 50 mM sodium phosphate buffer (pH 7.0) containing 5 mM MgS0 and 0.5 mM CoCl were incubated with indicated concentrations of DEPC at room temperature for 30 rnin. The residual glucose isomerase activity was assayed as described in Materials and Methods and expressed as percentage of specific activity found in the control without DEPC. Reprinted with permission from ref. 22. Copyright 1990 American Society for Biochemistry and Molecular Biology. 101

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revised catalysis mechanism involving a hydride shift that is now proposed for glucose/xylose isomerase. Figure 6 shows the increased pH stability of the sitedirected Gln mutant over the wild-type His enzyme. To date, these studies on glucose/xylose isomerase of thermoanaerobes have enabled improvement in two 101

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American Chemical Society Library 115516th St., N.W. Leatham and Himmel; Enzymes in Biomass Conversion Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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PH Figure 6. Plot of relative log of apparent V versus pH for Gln mutant (filled circles) and wild-type (open circles) xylose isomerases. Apparent V values at different pH were determined from a Lineweaver-Burk plot The scale of relative log Vmax indicates the fraction of each experimental value at different pH relative to the maximal value. Both enzymes were stable under the assay conditions used. Reprinted with permission from ref. 22. Copyright 1990 American Society for Biochemistry and Molecular Biology. m a x

101

m a x

app


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industrial enzyme parameters: higher thermostability for increased fructose concentration by improved chemical equilibrium with highly thermostable glucose isomerase and higher acid stability for lower process costs and less by-product color formation with genetically engineered glucose isomerase.


Xylanase Characterization The work to be summarized here is preliminary and will be published elsewhere (manuscripts in preparation). Thermoanaerobacter strain B6A produces a xylanase complex that is inducible by xylose and catabolite repressed by glucose. Noticeably, when mixed with insoluble xylan, glucose-grown cells do not bind xylan; whereas, xylose-grown cells bind very tightly. Cationized ferritin was used to identify xylanosome structures in xylose grown cells which are similar to cellulosomes observed in C. thermocellwn. Thermoanaerobacter strain B6A produces thermostable cell-bound endoxylanase, which is also excreted. A genomic library with cosmid pHC79 was constructed with digested DNA from Thermoanaerobacter strain B6A. One xylanase clone, pXAl-H4 was obtained by screening for clear zones on Remazol brilliant blue xylan plates. This clone contained thermophilic endoxylanase activity. Heat treatment of E. coli HB101 containing expressed plasmid pXAI-H4 at 65°C for 30 min resulted in >50% purification of the thermophilic endoxylanase. The biochemical properties of the partially purified (90%), cloned, thermoanaerobe endoxylanase differed from the native enzyme in displaying lower thermophilicity (by 10°C) and it was not glycosylated (see Table VII). The main end products of xylan hydrolysis by the enzyme were xylose and xylobiose. The endoxylanase gene from Thermoanaerobacter strain B6A was sequenced and structural interpretations were deduced from computer analysis of the amino acid sequence (see Table VIII). The endoglucanase is hydrophilic and has compact secondary structure with 30 B-turns and five potential glycosylation sites. Potential industrial uses for thermostable xylanase include diverse food and feed manufacturing processes and pulp, paper and fiber manufacturing processes (see Table IX). Table VII. Biochemical Properties of Thermoanaerobacter Strain B6A Endoxylanase Cloned in E. coli

Molecular weight pH optimum pH stability Temperature optimum (°C) Thermal stability (°C) Metal requirements (for activity or stability Main end products from xylan hydrolysis

55,000 5.5 5.0 - 6.0 60 70 (< 30 min) none xylose and xylobiose


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Table VIII. Structural Interpretations Deduced From Amino Acid Sequence Computer Analysis of the Thermoanaerobacter Strain B6A Endoxylanase Gene

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The protein is hydrophilic based on overall amino acid composition.

2.

The protein contains five potential glycosylation sites.

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The protein is a very compact molecule with a secondary structure having > 20 6-turns.

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Protein thermostability may be related to glycosylation and compactness but not cysteine disulfide bonds.

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The enzyme is distinct from other sequenced xylanases and shows < 15% homology to others characterized including C. thermocellum.

Table IX. Potential Industrial Application Targets for Thermostable Xylanases

I.

Food and feed manufacture A. Improved baking with high fiber materials B. Clarification of fruit juices and wine C. Improved nutritional and product properties of cereal fibers D. Production of food thickeners

II.

Pulp, paper, and fiber manufacture A. Biobleaching of kraft pulps B. Biopulping of wood C. Purification of fibers for rayon manufacture

Direction for Future Study We are continuing protein engineering studies on the xylose isomerase of C. thermosulfurogenes with the aim to: 1) redesign an enzyme via site-directed mutagenesis to be a "true" glucose isomerase (i.e., have a higher catalytic efficiency towards glucose than xylose); 2) redesign this thermophilic enzyme into a mesophilic enzyme (i.e., high activity at low temperature) via site-directed mutagenesis so as to explain the molecular basis for thermophilicity (high activity at high temperatures).


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The glucose isomerase is very hydrophobic and high in alanine which may contribute to its thermophilic character. We are continuing molecular analysis of Thermoanaerobacter strain B6A xylanase structure and function in relation to: 1) establishing the component enzyme basis for a xylanosomal organization; 2) to establish the putative synergistic interactions between different but potentially juxtapositioned xylanase activities; and, 3) establishing a role for glycoconjugate structure and protein secondary structure in thermophilicity. Finally, we are continuing studies on establishing process uses for thermophilic xylanases in the manufacturing of foods, feeds, paper and fibers. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Zeikus, J. G. In Biotechnology: Science, Education and Commercialization; Vasil, I. K., Ed.; Elsevier Science Publishing Co., Inc.: New York, New York, 1990; pp 23-39. Zeikus, J. G. Enzyme Microb.Technol.1979, 1, 243-252. Lamed, R.; Bayer, E.; Sana, B. C.; Zeikus, J. G. Proc. 8th Intl. Biotech. Symp. Paris, 1988, pp 371-383. Ng, T. K.; Zeikus, J. G. Appl. Environ. Microbiol. 1981, 42, 231-240. Ng, T. K.; Zeikus, J. G. Biochem. J. 1981, 199, 341-350. Schink, B.; Zeikus, J. G. J. Gen. Microbiol. 1983, 129, 1149-1158. Zeikus, J. G.; Ben-Bassat, A.; Hegge, P. W. J. Bacteriol. 1980, 143, 432-440. Hyun, H. H.; Zeikus, J. G. Appl. Environ. Microbiol. 1985, 49, 1162-1167. Hyun, H. H.; Zeikus, J. G. Appl. Environ. Microbiol. 1985, 49, 1168-1173. Hyun, H. H.; Zeikus, J. G. J. Bacteriol. 1985, 164, 1146-1152. Hyun, H. H.; Shen, G. J.; Zeikus, J. G. J. Bacteriol. 1985, 164, 1153-1161. Hyun, H. H.; Zeikus, J. G. J. Bacteriol. 1985, 164, 1162-1170. Weimer, P. J.; Wagner, C.; Knowlton, S.; Ng, T. K. Arch. Microbiol. 1984, 138, 31-36. Shen, G.-J.; Saha, B. C.; Bhatnagar, L.; Lee, Y.-E.; Zeikus, J. G. Biochem. J. 1988, 254, 835-840. Saha, B. C.; Shen, G.-J.; Zeikus, J. G. Enzyme Microbiol. Technol. 1987, 9, 598-601. Saha, B. C.; LeCureux, L. W.; Zeikus, J. G. Anal. Biochem. 1988, 175, 569572. Saha, B. C.; Zeikus, J. G. Process Biochem. 1987, 22, 78-82. Saha, B. C.; Zeikus, J. G. Biotech. Bioeng. 1989, 34, 299-303. Saha, B. C.; Zeikus, J. G. Enzyme Microb. Technol. 1990, 12, 229-231. Lee, C.-Y.; Saha, B. C.; Zeikus, J. G. Appl. Environ. Microbiol. 1990, 56, 2895-2901. Lee, C.-Y.; Bhatnagar, L.; Saha, B. C.; Lee, Y.-E.; Takagi, M.; Imanaka, T.; Bagdasarian, M.; Zeikus, J. G. Appl. Environ.Microbiol.1990, 56, 2638-2643. Lee, C.-Y.; Bagdasarian, M.; Meng, M.; Zeikus, J. G. J. Biol. Chem. 1990, 265, Collyer, C. A.; Blow, D. M . Proc. Natl. Acad. Sci. 1990, 87, 1362-1366.

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Thermostable Saccharidases - ACS Symposium Series (ACS

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