1970_bruijn_decomposition products of enzymatic

8/3/2019 1970_Bruijn_Decomposition Products of Enzymatic

1/4

Proceedings of The South ~f r i c a n ugar Technologists' Association -June 1970

DECOMPOSITION PRODUCTS OF ENZYMATICSTARCH- HYDROLYSIS

by J. BRUIJNSugar Milling R esearch Institute

IntroductionIn the Rabe process, starch is mechanically re-moved by a flotation process without leaving anyresidue in the juice.The sugar industry now applies an enzymatic pro-cess to reduce the quantity of starch in raw sugar.This process is operated in such a way that theunaffinated raw sugar contains approximately 100ppm of starch. The hydrolysis products, however,remain in the syrup and may be partly included in

the crystal.Various authors have published data on the hydro-lysis of starch by B. subtilis a-am ylase ', ', 3.Robyt and French used various glucans, amongothers amylose and amylopectin. They added to thesesubstrates (conc. 0.1-1 %) 0.1 amylase unit * per mgof glucan.The oligosaccharide distribution which theyobtained in the digest is shown in Table 1.TABLE IHydrolysis with B subtilis a-Amylase

Product of Hydrolysis Amylose Amylopectin60 min. 180 min 60 min 180 mino / 0 / o / 0 1

0 1G( 3 3G4G5GG,Larger than G,

* One amylase unit as defined by French is that amount ofenzyme which reduces the blue value of a 1% solublestarch solution at 40C in one minute. At present oneunit of enzyme activity (U) is that amount that willcatalyse the transforma tion of lpm ol. of substrate (orlp equivalent of the group attacked) per minute underoptimum conditions of pH and temperature4.Unfortunately it is rather difficult to analyseaccura tely for all ,the hydrolysis products of starchin syrup, juice or any other sugar-house product.

ExperimentalEnzyme DigestNo effort was made to treat amylose and amy-lopectin separately as the factory only treats the com-plete starch mixture.The factory conditions were simulated by usinga potato starch solution which was boiled fo r twentyminutes an d a utoclaved for five minutes a t .3 bar. .

The concentration was adjusted to 400 mg/l and the %p H to 6.5. Ten m g/l each of sodium chloride andcalcium chloride were added . Bactamyl D.200 wasadded in the range 5-15 mg/l to the starch solution,which had been heated to 65C. The hydrolysis wascarried out in such a way that approximately90-95 % of the starch was decomposed.Under these circumstances 5 mg/l of enzymepowder gave an increase in reducing power of 0.25mg glucose equivalent per minute.Little increase in hydrolysis was obtained between -30 and 120 minutes. Alcohol was added to the mix-ture (1.6 times the volume) to stop the reaction andthe solution was evaporated on a rotary filmevaporator. Final drying was carried o ut in adesiccator.

Quantitative determination of OligosuccharidesThe oligosaccharides were separated by paperchromatography at room temperature using butanol:ethanol: water (2:l:l) as eluant. The quantitativedetermination was a modification of the method ofWhistler & Hicksons.Guide strips were developed in silver nitratereagent.7 After the centre of the paper had beencut in between the fractions, the individual oligo-saccharides were extracted in water by digesting thepapers for one hour. As it was found that filterpaper fibres interfered with the determination, theextract was filtered through a . 4 5 ~ illipore filter,washed and ma de up to a suitable volume. T o analiquot of the filtrate (normally 10 ml) 2 ml hydro-chloric acid (10 N) was added and the mixture washeated for twenty-five minutes in a boiling waterbath.After careful neutralisation using Brom o ThymolBlue (0.005%) as an indicator, the oligosaccharides

were subsequently determined as glucose, accordingto the method of Hagedorn and Jensen'.A chromatogram of the oligosaccharides is shownin Fig. 1-Determination of polysacchar ides.Oligosaccharides larger than G, (maltooctaose) areno t ~ easy to separate by paper chromatography,although it is possible. Thin layer chromatographywill separate to G,,-G,,.Higher carbohydrate polymers are best separatedby gel chromatography. This does not separa te eachindividual sugar but separates groups of com-ponents according to their molecular weight.The gel used was Sephadex G SO*, which separates* Pharma cia Fine Chemicals Uppsala Sweden.


2/4

Proceedings of The Sourh African Sugar Technologists' Association - une 1970

polysaccharides in the range Mw 500-10,000, ex-cluding the higher Mw. T he column used was 1.7x 43 cm and 1 ml fractions were collected. Th eeluants used were either 0.15 M sodium chloridesolution or distilled water. In these experiments nodifference was observed between the two. On e ml ofeluant containing 1 mg of carbohydrate was placedin the column and washed into the gel with a fewml of eluant. Subsequently the reservoir with eluantwas connected and the run started. Th e calibrationof the column was carried out with Dextran T40*(Mw=40,000) Dextran T,, (Mw=10,000) andglucose (Mw =180). Th e analysis of the fractionswas carried out by a phenol-sulphuric acid method5.

FIGURE 1

Results an d DiscussionT he distribution of oligosaccharides formed dur -ing enzymatic hydrolysis is given in Table I1 below:TABLE II~ l i ~ o s a c c h a r i d eomppsition of a starch hydrolysate after treat-ment with B. subtilis a-amylase Potato Starch conc: 400 mg/I5 PPm 10ppm 25 ppmenzyme enzyme enzyme

% % %1.8 4.0 8.04.8 7.8 8.78.8 10.2 10.0

~ o t a iecoveredcarbohydrate in. % 99 97 101Thi s distribution of oligosaccharides compares wellwith those published by Robyt and French. Themain oligosaccharides formed are G,, Go, G,. Withhigher enzyme concentrations the amount of G,(glucose) increases and G , decreases.

The results of the gel chromatography of thestarch hydrolysate are shown in Fig. 2. The calibra-tion of the column is shown in Fig. 3.Th e fractions 56-90 were collected in a separateanalysis and combined. After evapo ration of theeluant the residue was separated by paper-chro-matography and proved to contain the oligo-saccharides G,-G, in the same proportions as found 'before. T he first peak, which was 30% of the totalamount of carbohydrate present in the enzymatichydrolysate has its maximum at fraction 32. Th eDextran T, , shows a maximum at fraction 28. Theaverage Mw of the polysaccharides in the enzymatichydrolysate is for this reason < 10,000.ConclusionThe hydrolysis of starch B. subtilis a-amylase re-sults in two groups of carb ohydrates. On e containsa mixture of oligosaccharides (70%) in which G,,G, and G, are predominant. With higher quantitiesof enzyme the amount of G, decreases. T he secondgroup are polysaccharides (30%) with an averageMw


3/4

Proceedings of Th e South African Sugar Technologists' Association - une 1970- E 4 9 0 n~

2-

I -

-120 40 60 80 100FRACTION NUMBER

FIGURE 2: Gel chromatography of a starch hydrolysate uslng Sephadex G50.

- E490 n~2-

Mw 40,000

I I I I I I I

20 40 60 ' 80 100FRACTION NUMBERFIGURE 3: Calibration of a Sephadex G50 column wit h Dextr an T4O and glucose.


4/4

Proceedings of The South African Sugar Tzchnologists' Association - une 1970 97Acknowledgementhanks are due to Mrs. M. Roberts for the ex-perimental work.

References1. F. J. Bates PoIarimetry, Saccharimetry and the Sugarsp. 198 U.S.A. Gov. Print. Office Washington (1942).2. J. Robyt and D. FrenchArch Biochem & Biophys 100 451 (1963).3. W. Banks e.a. Carbohydrate Research 12 79 (1970).4. Co mmission on Enzymes, Internat ional Unio n of Bio-chemistry 1961.Enzyme units p. 7 Pergamon Press, Oxford (1961).5. M. Dubois e.a.Analyt. Chem. 28 350 (1956).6. J. Osugi Rev. Phys. Chem. Jpan 31 957 (1958).7. W. E. Trevelyan e.a. Nature 166 52 (1950).8. R. L. Whistler & J. C. Hickson.Analy. Chem. 27 1514 (1955).

DiscussionMr. Alexander (in the chair): Do any of theoligosaccharides in t he hydrolysate co-crystallise with

sucrose?'Mr. Bruijn: I have not done any work on this. Buta subject under investigation at the moment is to tryan'd fin,d out why som e factories p ~ od u ce longatedcrystals, which, accord'ing to an Australian publica-tion, are due to dextran.We have analysed massecuites, which gaveelongated crystals, for their total polysaccharide con-tent but found no difference in the total quantitycompared with other massecuites.At present I am isolating the total quantity ofpolysaccharides an'd putting them through columnsto try and find any difference in molecular weight.Mr. Comrie: Mr. Bruijn mentions that the oligo-saccharides G,, to G Z j were separated by thinlayer chromatograph y, using silica gel. We have hadno success with this method.Also, did he ulse streaking techniques for his paperchromatography, or only the spolts as shown on theslides?I must congratulate him on Table 11. where histotal carlbohydrate recovery was near 100%.Mr. Bruijn: I did carry out thin layer chromato-graphy, but not very successfully, hence I ulsed paperchromatography.Dr. Murray: I would like to know more about thekinetics of star ch hydrolysils. The kinetics shown byM r. Smith appear to be zero order under his experi-mental conditions.

However, the kinetics shown by Mr. Bruijn's ex-periments under factory con'ditions are not of zeroor(der. Now in a norm al chemical reaction, if theorder changes then the products of the reaction maychange and, of course, if lthe products change thismeans that the subsequent chemical and physicalbehaviou r of the system will change. If then oneproduces dcifferent products by changing hydrolysisconditions then miglht this not have a n effect on thecrystal~lisation of sucrose an,d impurities in tercalatedat a later stage?Does the mechanism of starch hydrolysis vary withreaction conditions?Mr. Bruijn: A lot has been published about themechanism of the amylase attack on starch but Iwould have to study this to answer your question.Mr. Cox: Starch hydrolylsis has the characteristicsof a first orde r reaction. On e of the characteristicsof this type of re action is that it ha s a fixed anddefinite half life; therefore the percentage of starchhydrolysed in mixtures will remain constant, sinceuntil the point is reached at which all the actual siteson the enzyme are saturated, the speed of reactionwill increase in proportion to the concentration ofstarch present. When all the active sites are saturatedthe kinetics change to zero orde r. This does notentail however that the molecularity of the reactionhas changed, only that the availability of active sites.which under constant enzyme concentration will re-main constant, has become the limiting factor. Underthese conditions the m echanism of the reaction shouldremain exactly the same as when there still remainedsome active sites avail~able,hat is, as when the kineticswere still of rhe first mder.Dr. Matic: Over ninety percent of the originalmaterial was hydrolysed as determined by the starch-iodine colour determination. However, thirty percentof the hydrolysate according to gel-chromatogaphyhad an average molecular weight of 10.000 and there-fore it would be expected to give blue colour. Thesetwo results seem to be conltradictory.Mr. Bruijn: It has been pulblished that maltooligosaccharides with a molecular weight greater than10 give a coloured iodine complex.The re seems to be a discrepancy in the results. Itis however likely that the malto oligosaccharide hasto be unbranched to give this colour. In thehydrolysate obtained most of the larger oligo-saccharides obtained will be branched.

1970_bruijn_decomposition products of enzymatic

Documents