ZanfloA Novel Bacterial Heteropolysaccharide

16 Zanflo—A Novel Bacterial Heteropolysaccharide

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 27, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch016

K. S. KANG, G. T. VEEDER, and D. D. RICHEY Kelco, Division of Merck & Co., Inc., 8225 Aero Drive, San Diego, CA 92123

An exocellular bacterial polysaccharide called "xanthan gum" that was originally developed by the Peoria Lab of the USDA nearly two decades ago is at this time the only bacterial heteropolysaccharide that is being produced on a large commercial scale. The commercial success of xanthan gum is attributed to its many valuable and often unique properties for industrial applications and to an economic manufacturing process. Among our novel polysaccharides that are produced by many bacterial species that we have isolated in our screening program, a product which is now trade named ZANFLO is especially outstand ing in its fermentation efficiency and product properties. ZANFLO is produced by a bacterium that was isolated from a soil sample taken at Tahiti. The organism is a gram-negative, non -sporeforming rod with a size range of 0.75-1.0 by 1-2μ. The dimensions change during the fermentation. At the beginning of the fermentation they are large rods which quickly change to a coccobacillus 0.75-1.0μ in diameter. It is heavily encapsulated. Some of its biochemical characteristics are shown in Table I. This organism produces a positive lactose reaction within 24 hours. It possesses nitrate reductase, CMCase, urease and lysine decarboxylase. It can utilize citrate as a sole carbon source and will grow in the presence of 8% NaCl. Its optimum growth temperature is 30-33°C. and growth will occur at 45° C. In litmus milk this organism produces an acid curd with peptoniza tion and reduction of the litmus. At the present time we are evaluating these and other significant taxonomic tests to ascer tain the identity of this microorganism. This organism is quite specific about what carbon source it will use for optimum polysaccharide production. It produces an excessive amount of acid with glucose as a carbon source with only minimum polysaccharide synthesis. Even with pH control the conversion efficiency with glucose is still very low. Poly saccharide synthesis is better with sucrose or maltose or mixtures of these than with glucose, but the conversion efficiency is still poor. Improved polysaccharide synthesis is found with 211

Sandford and Laskin; Extracellular Microbial Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


212

EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

l a c t o s e and hydrolyzed s t a r c h . We t y p i c a l l y hydrolyze our s t a r c h s l u r r i e s with commercially a v a i l a b l e α-amylase p r e p a r a t i o n s . The p r e f e r r e d medium contains phosphate as a b u f f e r i n g agent, ammonium n i t r a t e and a soy p r o t e i n product as n i t r o g e n sources, and magnesium s u l f a t e i n a d d i t i o n t o the carbon source. The data i n F i g u r e 1 shows the r e s u l t s o f a t y p i c a l batch-type fermentation i n a p i l o t p l a n t fermentator. The inoculum s i z e i s t y p i c a l l y 5% with a 3% (as i s ) carbon source c o n c e n t r a t i o n . V i s c o s i t y development s t a r t e d a t approximately 7 hours and reached a maximum o f 4500 cps by 64 hours. Unless otherwise s p e c i f i e d , v i s c o s i t y measurements are made using a Model LVF B r o o k f i e l d viscometer with #4 s p i n d l e a t 60 rpm. The maximum c e l l p o p u l a t i o n o f about 1 χ 1 0 was reached i n 10 hours. By employing an automatic a g i t a t i o n and a e r a t i o n c o n t r o l l i n g system with an oxygen probe, the minimum d i s s o l v e d oxygen concen t r a t i o n was determined t o be 5-10% during the f i r s t 24 hours o f the fermentation. Recovery o f the product i s done by p r e c i p i t a t i o n with such organic s o l v e n t s as acetone, e t h y l a l c o h o l , i s o p r o p y l a l c o h o l , or v a r i o u s isomers o f butanol. A f t e r p r e c i p i t a t i o n , the polymer f i b e r s a r e d r i e d and m i l l e d t o a powder. The s t u d i e s on the chemical components o f t h i s p o l y saccharide were done on m a t e r i a l p u r i f i e d by f i l t r a t i o n o f ZANFLO s o l u t i o n s using diatomaceous earth as f i l t e r a i d followed by repeated r e p r e c i p i t a t i o n s w i t h IPA. The m a t e r i a l was found t o be 97% carbohydrate and 3% p r o t e i n . The p o l y s a c c h a r i d e was hydrolyzed using 2N H2SO4 and heated t o 100° C. f o r 5 hours. The components o f t h i s polymer were i d e n t i f i e d using paper chromatographic techniques. The molar r a t i o was determined using gas l i q u i d chromatography. In Table I I one can see the r e s u l t s o f t h i s chemical component a n a l y s i s . The carbohydrate p o r t i o n was found t o c o n t a i n glucose, g a l a c t o s e , g l u c u r o n i c a c i d , and fucose i n the molar r a t i o o f 3:2:1.5:1. Uronic a c i d accounts f o r approximately 20% o f the p o l y s a c c h a r i d e on a weight b a s i s . I t i s noteworthy t h a t fucose i s not commonly found as a s t r u c t u r a l c o n s t i t u e n t o f e x o c e l l u l a r b a c t e r i a l heteropolysaccharides. We are not y e t c e r t a i n as t o the r o l e o f p r o t e i n i n the p u r i f i e d p o l y s a c c h a r i d e . The completion o f our s t r u c t u r a l work w i l l provide the answers t o these questions. ZANFLO i s a h i g h - v i s c o s i t y polysaccharide, as shown i n the v i s c o s i t y c o n c e n t r a t i o n curve i n F i g u r e 2. I t i s c o n s i d e r a b l y higher than t h a t o f xanthan gum, a well-known b a c t e r i a l heterοp o l y s a c c h a r i d e widely used i n i n d u s t r y . T h i s d i f f e r e n c e becomes more outstanding a t higher c o n c e n t r a t i o n s . At a 1.5% gum con c e n t r a t i o n , the xanthan gum had a v i s c o s i t y of 2500 cps, while ZANFLO had a v i s c o s i t y o f 5000 cps. The r e s u l t s i n F i g u r e 3 show the e f f e c t o f heat on the ZANFLO p o l y s a c c h a r i d e . ZANFLO's v i s c o s i t y , l i k e t h a t o f many m i c r o b i a l p o l y s a c c h a r i d e s , i s d e f i n i t e l y a f f e c t e d by heat. As 1 0


16.

KANG

E T AL.


φ

Novel

Beer

Bacterial

Heteropolysaccharide

213

Viscosity

φ—Ο

ι

ι

ι

ι

1 0

20

Î0

40

Fermentation

Time

1 r

,0

(hours)

Figure 1. Fermentation parameters of the Zanflo fermentation


214

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Table I. NITRATE

+

REDUCTASE

CELLULASE

+

(Cx)

UREASE

+

AMYLASE

+

H S

+

2


Biochemical Characte

PRODUCTION

INDOLE

+

VOGES-PROSKAUER METHYL RED

+

LYSINE DECARBOXYLASE GELATIN

LIQUEFACTION

ACID AND GAS FROM CARBOHYDRATES GLUCOSE

+

LACTOSE

+

SUCROSE

+

MALTOSE

+

CELLOBIOSE

+

MANNITOL

+

INOSITOL

+

ADONITOL DULCITOL

Table II. Carbohydrate Composition of Zanflo URONIC

ACID

19%

GLUCOSE

39%

GALACTOSE

29%

FUCOSE

13%

100% ACETYL

4.5%



16.

KANG

E T AL.

—ι

Novel

Bacterial

J

Ο.25

215

Ι

1.00

2.00

POLYSACCHARIDE CONCENTRATION

20


60 TEMPERATURE (°C)

80

(%)

90

Figure 2.

Figure

3.

Viscosity vs. concentration

Effect of temperature Zanfio viscosity


on



216

you can see, the r e l a t i o n s h i p between v i s c o s i t y and temperature i s q u i t e l i n e a r . From t h i s graph one can c a l c u l a t e a decrease i n v i s c o s i t y of 25 cps/°C. as the temperature i s i n c r e a s e d . The ZANFLO c o n c e n t r a t i o n i s 1%. The v i s c o s i t y decrease i s temperature r e v e r s i b l e . The r e s u l t s i n F i g u r e 4 show the e f f e c t of pH on ZANFLO i n comparison to that of xanthan gum. The v i s c o s i t y of ZANFLO remains s t a b l e from a pH of 5 to 10 but decreases on e i t h e r side of t h i s range. One of the most s t r i k i n g p r o p e r t i e s of t h i s p o l y s a c c h a r i d e i s i t s c o m p a t i b i l i t y with c a t i o n i c dyes. Anionic gums, such as xanthan gum and many u r o n i c a c i d - c o n t a i n i n g p o l y s a c c h a r i d e s , r e a c t s t r o n g l y with c a t i o n i c dyes, such as methylene blue c h l o r i d e , to form a f i b r o u s p r e c i p i t a t e which l i m i t s t h e i r industrial applications. However, ZANFLO, even though i t cont a i n s a s u b s t a n t i a l amount of u r o n i c a c i d , does not p r e c i p i t a t e w i t h such c a t i o n i c dyes at any pH. I t was noted, nonetheless, that ZANFLO l o s t a l l or most of i t s v i s c o s i t y during the t e s t and f u r t h e r experimentation showed the r e a c t i o n with methylene blue to be i n f l u e n c e d by s a l t concent r a t i o n and pH. The r e s u l t s i n Table I I I demonstrate the e f f e c t of pH on the v i s c o s i t y of the ZANFLO-methylene blue complex using a c e t i c a c i d to a d j u s t the pH. The v i s c o s i t y from n e u t r a l i t y to at l e a s t 4.5 i s as low as water v i s c o s i t y . By the time a pH of 3.9 i s reached, the v i s c o s i t y s t a r t s to increase and reaches a maximum of 110 cps at a pH of 3.1. We a l s o n o t i c e d at t h i s time that the v i s c o s i t y would decrease to that of water again i f the pH were adjusted upward slowly with NaOH. I f one continues to add NaOH, the v i s c o s i t y w i l l begin to i n c r e a s e at a pH of 4.4-4.6 and i t can no longer be brought down to t h a t of water again. T h i s e f f e c t i s caused by the concentrat i o n of Na now present i n the s o l u t i o n . The r e s u l t s i n F i g u r e 5 show the e f f e c t of monovalent and d i v a l e n t c a t i o n s on a s i m i l a r system. Here, v a r i o u s concentrat i o n s of NaCl or MgCl2 were added to n e u t r a l s o l u t i o n s of ZANFLO c o n t a i n i n g MBC and having the v i s c o s i t y of water. The r e s u l t s show that a s t o i c h i o m e t r i c r e l a t i o n s h i p e x i s t s between Mg and Na i n r e s t o r i n g l o s t v i s c o s i t y to the s o l u t i o n . I t appears evident that an increase i n e l e c t r o l y t e c o n c e n t r a t i o n would increase competition with MB f o r the r e a c t i v e s i t e of polymer and t h e r e f o r e increase the v i s c o s i t y . The r o l e of pH i n t h i s phenomenon appears to be centered around the pKa of the uronic a c i d p o r t i o n of the polymer. Glucuronic a c i d has a pKa of 3.2. At pH values below t h i s , the number of r e a c t i v e s i t e s of the polymer from MB would decrease, and, hence, i n c r e a s e i n v i s c o s i t y . The mechanism involved i n c o m p a t i b i l i t y i s not understood at present. We speculate that perhaps the p r o t e i n or peptide moiety may p l a y an important r o l e i n masking the a n i o n i c groups of the gum or s t a b i l i z i n g the gum under these c o n d i t i o n s . +


KANG ET AL.

Novel

Bacterial


#


2,000 [-

\

/ 1,000 μ

ο.

-Ο-Ο

• s-io O

XANTHAN GUM

2

4

6

8

10

12

PH Figure 4.

Effect of pH on Zanflo viscosity

Table III. Effect of pH on the Viscosity of a Zanflo-Methylene Blue Chloride Complex in Solution PU 7.5

VISCOSITY: W0

CPS

(NO

MBC)

7.5

0 CPS (0.2%

5.8

0

CPS

4.2

0

CPS

3.9

25

CPS

3.1

110

CPS

MBC)



218


CATION CONCENTRATION (M) Figure 5.

Effect of monovalent and divalent cations on restoring viscosity to Zanflo-MBC solutions



16.

KANG ET AL.

Novel

Bacterial


219

Another p o s s i b i l i t y i s that the secondary o r t e r t i a r y s t r u c t u r e o f t h i s polysaccharide, which would give i t a d i s t i n c t i v e conformation i n s o l u t i o n , p r o t e c t s i t against the a c t i o n of c a t i o n i c agents such as methylene blue c h l o r i d e . In summary, we have i s o l a t e d a bacterium from the s o i l which produces l a r g e amounts o f a novel i n d u s t r i a l heteropolysaccharide. Because o f i t s unusual r h e o l o g i c a l p r o p e r t i e s and c o m p a t i b i l i t y with b a s i c dyes, ZANFLO has already e s t a b l i s h e d e x c e l l e n t a p p l i c a t i o n s i n p a i n t and shows e x c e l l e n t p o t e n t i a l i n other a p p l i c a t i o n areas.


ZanfloA Novel Bacterial Heteropolysaccharide

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