Amylase Bacillus macerans

Considerable effort has been spent on working out methods for the production of B. macerans amylase, and although high yields have been obtained in some cases, there is need for improvement in this re.spect. 

The usual laboratory method for obtaining B. macerans amylase is to culture the organism on an autoclaved potato or oatmeal medium in the presence of calcium carbonate at 37-45° for 2-4 weeks. The culture fluid (filtrate or centrifugate) contains enzyme which can be stored as such, 

The ability of culture filtrates to produce Schardinger dextrins (identified as their crystalline iodine complexes) has been used as a means for the identification of B. macerans. So far no other source, microbial or otherwise, has been foimd for this unique enzyme. 

Of the schemes which have been proposed for the assay of the unique amylase activity of B. macerans, the simplest and most direct is the Tilden and Hudson microscopic test.  

Although the Tilden-Hudson test is very useful it has certain drawbacks in precise work, (a) The exact time at which needles appear is difficult to determine, (b) Enzyme activity is slowed but not stopped by the addition of iodine solution in fact, it is possible to add starch to a mixture of enzyme and iodine solution and observe the gradual formation of the crystalline dextrin-iodine complexes, (c) The apparent activity depends very much on the presence of such foreign materials as salts, n-glucose, maltose, etc., which may be present in the enzyme solution. In some enzyme preparations which seem to have fair activity, the needles are never observed. 

---

Thus, Freudenberg concluded that the cyclodextrins are not preformed in starch, but that their formation is made possible by the helicity of the starch chain. Freudenberg s hypotheses concerning the starch structure (that is, the amylose fraction) and the Bacillus macerans amylase mechanism have been confirmed by X-ray crystallography and chromatographic techniques. ... 

In the authors experiments, the cycloamyloses by very prolonged action of the enzyme did vanish with the production of maltose. It is not clear if this is an action of the Bacillus macerans amylase or of another enzyme present in the bacillus. After a very long time the yield of fermentable sugar was about 60% of the starch. 

The action of /8-amylase on starch partially degraded by the Bacillus macerans amylase was determined (Table XXVII). The amount of maltose decreases with decreasing degree of polymerization. When the... 

Several methods can be used for separation. A current favorite is to use a series of compounds called cyclodextrins, which have been found to associate preferentially with one form of optical isomer more than the other. Figure 31-9 shows a diagram of alpha cyclodextrin. They are obtained by the action of Bacillus macerans amylase on starch to form homogeneous cyclic alpha- 1,4-D-glucopyranose units. Alpha is the hexa ring, beta is the... 

CDs are produced from starch by the action of Bacillus macerans amylase or the eitzyme cyclodextrin transglycosylate (CTG) [16-19]. The latter enzyme can be used to produce CDs of specific sizes by controlling the reaction conditions. In the past few years, enantiomers have been resolved using peralkylated a-, and y-CDs dissolved in polysiloxanes and coated within glass or fused silica capillary tubing [20, 21]. Subsequently, the CDs were linked to the solid supports. In 1979, Harada etal. [22] polymerized and crosslinked a CD with a gel support, and the CSP developed was tested for the chiral resolution of mandelic acid and its derivatives. Various workers have subsequently bonded all three CDs with different solid supports [23-33]. Of course, these CDs bonded to gel support have been used for the chiral resolution of different racemates, but they suffer from certain drawbacks because of their poor mechanical strength and efficiency in both GC and HPLC. An improvement to these... 

Cyclodextrins are obtained when starch is digested with bacillus macerans amylase. They appear not to have any biological significance. They cannot be used as carriers as the complexes are too unstable - on the way from one side of the path to the other the complex would decompose. 

---