Chapter 7
Novel Thermostable Saccharidases from Thermoanaerobes Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch007
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Badal C. Saha , Saroj P. Mathupala , and J. Gregory Zeikus 1
Michigan Biotechnology Institute, Lansing, MI 48910 Department of Biochemistry and Department of Microbiology and Public Health, Michigan State University, East Lansing, MI 48823
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We have purified and characterized several new saccharidase activities including β-amylase, amylopullulanase, α-glucosidase, and cyclodextrinase from thermoanaerobes. The β-amylase was stable and active at 75°C. The amylopullulanase displayed higher affinity towards pullulan than starch; and it contained a putative single active site for cleavage of both α-1,4 and α-1,6 linkages. It produced maltotriose from pullulan, and DP2, DP3 and DP4 from starch. The α-glucosidase had an apparent MW of 162,000 and cleaved both maltosyl and isomaltosyl polymers with a decreasing affinity for longer chains. The cyclodextrinase hydrolyzed various cyclodextrins with decreasing activity rates displayed on α-CD > β-CD > γ-CD. The unique biochemical properties and process features of these thermophilic enzymes that have potential usefulness for development of new saccharide biotechnologies are described.
Amylolytic enzymes are an important group of industrial enzymes. Three types of enzymes are involved in the production of sugars from starch: (1) endo-amylase (aamylase), (2) exo-amylase (β-amylase, glucoamylase) and (3) debranching enzymes (pullulanase, isoamylase). The starch bioprocessing usually involves two steps liquefaction and saccharification. First, an aqueous slurry of starch (30-40%, DS) is gelatinized (105°C, 5-7 min) and partially hydrolyzed (95°C, 2 h) by highly thermostable α-amylase to DE (dextrose equivalent) 5-10. The optimum pH for the reaction is 6.0-6.5 and calcium is also required. Then in the saccharification step, glucoamylase and pullulanase are used (60°C, pH 4.0-4.5,48 h) to produce more than 95-96% glucose; β-amylase and pullulanase can also be used (55°C, pH 5.0-5.5, 4872 h) to produce around 80-85% maltose. Thus, there is a need for thermostable saccharolytic enzymes to run the saccharification reaction at a higher temperature. Thermophiles often possess thermostable enzymes and interest in thermoanaerobic
0097-6156/91/0458-0086$06.00/0 © 1991 American Chemical Society
Friedman; Biotechnology of Amylodextrin Oligosaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
7. SAHA ET AL.
Novel Thermostable Saccharidases from Thermoanaerobes
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch007
bacteria has increased because of their unexamined potential as a source of thermostable and thermoactive enzymes including saccharidases. Thermoanaerobic bacteria may then serve as gene sources for cloning thermostable enzymes into aerobic industrial hosts (1). Our group initiated a screening program for thermostable saccharidases from diverse thermoanaerobic species. As a result, some new organisms have been isolated from hot spring areas and some novel highly thermophilic saccharidases have been discovered. Table I summarizes our efforts on obtaining some unique saccharidases from thermoanaerobes. Clostridium thermo-
Table I. Thermophilic Saccharidases from Thermoanaerobes Source (Enzyme)
Optimum Temp.
Optimum pH
Thermal stability (up to °C)
pH Stability
Clostridium thermosulfurogenes strain 4B B-amylase a-glucosidase Glucose isomerase
75
5.5-6.0
80
-
-
85
7.0
86
3.5-6.5
-
5.5-8.0
Clostridium thermohydrosulfimcum strain 39E Amylopullulanase a-glucosidase Cyclodextrinase Glucose isomerase
90 75 65 85
5.5-6.0 4.0-6.0 6.0 8.0
90 75 60 85
4.5-5.5 5.0-6.0 5.5 6.0-8.0
Thermoanaerobacter strain B6A Amylopullulanase Glucogenic amylase β-galactosidase Xylanase Cyclodextrinase Glucose isomerase
75 70
4.5-5.5 5.0-5.5
70 70
5.0-6.0 4.5-6.0
65 75 60 80
6.0-6.5 5.5 6.0 7.0
60 65 60 85
5.0-7.0 5.0-7.0 6.0 5.5-8.0
sulfurogenes strain 4B produces an extracellular β-amylase and intracellular glucose isomerase. Clostridium thermohydrosulfimcum strain 39E produces amylopullu lanase, α-glucosidase, glucose isomerase and cyclodextrinase activities. Thermo anaerobacter strain B6A produces amylopullulanase, glucogenic amylase, Bgalactosidase, cyclodextrinase, glucose isomerase and xylanase. In this chapter, we will focus our research efforts on the amylo-saccharidases from these thermo anaerobes.
Biochemical Characteristics β-Amylase, β-Amylase (EC 3.2.1.2, a-l,4-D-glucan maltohydrolase, saccharogenic amylase) is an exo-acting saccharidase which cleaves alternative a-l,4-ghicosidic
Friedman; Biotechnology of Amylodextrin Oligosaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch007
88
BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES
linkages in starch from the non-reducing end and produce β-maltose. β-Amylase occurs widely in many higher plants and is also produced by microorganisms. C. thermosulfitrogenes 4B is the only anaerobe reported so far that produces extracellu lar β-amylase. The enzyme is stable up to 80°C and optimally active at 75°C (2). β-Amylase synthesis in this organism is inducible and subject to catabolic repression. A hyperproductive mutant was developed which produced 8-fold more β-amylase in starch medium than the wild type (3). The effect of culture conditions and metabolite levels on the production of thermostable β-amylase with the overproducing mutant of C. therrnosulfurogenes 4B was investigated in continuous culture (4). The β-amylase activity level reached 90 units/ml at the dilution rate of 0.07/h in 3% starch medium. Growth inhibition by acetate and low enzyme productivity at low growth rates limited the further increase in enzyme production level. Nipkow et al. (5) then developed a microfîltration cell-recycle pilot system for continuous production of β-amylase by the thermoanaerobe. The concentration of β-amylase rose to 220 units/ml in the reactor, which was 5.5-fold more than under comparable conditions in a chemostat The β-amylase from C. thermosulfitrogenes 4B was purified 811-fold to homogeneity from the culture broth by ultrafiltration, ethanol treatment, DEAËSepharose CL-6B column chromatography and gelfiltrationon Sephacryl S-200 (6). The purified enzyme had a specific activity of 4215 units/mg protein. It was a tetramer (MW 210,000) having an isoelectric point at pH 5.1. The enzyme displayed K and K values for boiled soluble starch of 1.68 mg/ml and 400,000/min, respectively. It was antigenically distinctfromsweet potato and barley β-amylases. The β-amylasefromC. therrnosulfurogenes 4B readily and strongly adsorbed onto raw starch (7). pCMB treated β-amylase lost its activity towards raw or gelatinized starch but preserved the ability to adsorb onto raw starch. The adsorbed β-amylase was gradually releasedfromstarch in liquid phase during hydrolysis at 75°C. The degradation of raw starch by β-amylase was greatly enhanced by the addition of pullulanase. The optimum pH for raw starch hydrolysis by β-amylase was 4.5-5.5, whereas, that of soluble starch hydrolysis was 5.5-6.0. Raw starch adsorbed βamylase and soluble β-amylase showed similar rates of hydrolysis in reaction mixtures. It was found that the adsorbed β-amylase can be easily desorbedfromraw starch by using soluble starch or maltodextrin as elutant The soluble starch treated β-amylase could not adsorb onto raw starch which suggests that the soluble and insoluble substrate binding sites of the β-amylase may be the same. The β-amylase was easily purified to homogeniety by simple raw starch adsorption-desorption techniques and octyl-Sepharose chromatography (5). A comparison of certain physicochemical characteristics of this β-amylase with some other microbial βamylases is given in Table Π. A gene coding for the β-amylase of C. thermosulfitrogenes 4B was cloned into Bacillus subtilis and its nucleotide sequence was determined (72). The β-amylase was translatedfromthe monocistronic mRNA as a secretory precursor with a signal peptide of 32 amino acidresidues.The deduced amino acid sequence of the mature β-amylase contained 519 residues with a MW of 57,167. The amino acid sequence showed 57, 32 and 32% homology with those of B. polymyxa, soybean and barley β-amylases. The hydrophobicity of severalregionsin the amino acid sequence of C. thermosulfitrogenes 4B β-amylase was found to beremarkablyhigh as compared with that of the correspondingregionsof the B. polymyxa β-amylase. m
cat
Friedman; Biotechnology of Amylodextrin Oligosaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch007
7. SAHA ET AL.
Novel Thermostable SaccharidasesfromThermoanaerobes
Amylopullulanase. Pullulanase is a debranching enzyme which specifically attacks the α-1,6 glucosidic linkages of pullulan and starch. Pullulan is a linear polymer of about 250 maltotriosyl units linked together by a-1,6 linkages. Pullulan degrading enzymes can now be classified intofivegroups (Table IS). Amylopullulanase, a new class of enzyme, hydrolyzes a-1,6 linkages of pullulan like normal pullulanase but unlike pullulanase which cleaves only a-1,6 linkages in starch, this enzyme cleaves a-1,4 linkages of starch (79). We have suggested the name amylopullulanase. Recently, thermostable pullulanase activity has been reported in a number of microorganisms such as C. thermohydrosulfiuicum (27-25), C. thermosaccharofyti (24), Clostridium sp. (25), Thermus sp. (26), Thermus aquaticus (27), The anaerobium Tok6-Bl (28), T. brockii (29), Thermoanaerobacter strain B6A (3 finmi (23), Thermobacteroides ethanolicus (24), T. acetoethylicus (24), Th my ces thalpophilus (31), and thermophilic Bacillus sp. (32). The pullulanase from C thermohydrosulfiuicum (21,22), Thermoanaerobium Tok6-Bl (28), T. brock Thermus sp.(26), Thermoanaerobacter sp. B6A (30) and thermophilic Bacillus sp (32) have already been demonstrated to be of the amylopullulanase type. The synthesis of amylase in C. thermohydrosulfiuicum 39E (ATCC 33223) was inducible and subject to catabolic repression (27). Catabolic repression resistant mutants were isolated which displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production and starch consumption (33). In chemostat cultures, both wild type and mutant strains produced amylopullulanase at
Table IL Comparison of Properties of β-Amylase from Different Microbial Sources Property
B. cereus B. polymyxaB. megatariumC. thermosulfurogenes var. 1,11(10) (ID 4B mycoides (6) (9) 35,000
44,000
58,000
210,000
Optimum pH
7.0
7.5
6.5
6.0
Optimum temp (°Q
50
45
40-55
75
6.0-9.0
4.0-9.0
5.0-7.5
3.5-5.0
