The production of the enzyme dextransucrase by batch and continuous fermentation techniques

A review of the literature of work carried out on dextransucrase production, purification, immobilization and reactions has been carried out. A brief review has also been made of the literature concerning general enzyme biotechnology and fermentation technology. Fed-batch fermentation of the bacteria Leuconostoc mesenteroides NRRL B512 (F) to produce dextransucrase has formed the major part of this research. Aerobic and anaerobic fermentations have been studied using a 16 litre New Brunswick fermenter which has a 3-12 litre working volume. The initial volume of broth used in the studies was 6 litres. The results of the fed-batch fermentations showed for the first time that yields of dextransucrase are much higher under the anaerobic conditions than during the aerobic fermentations. Dextransucrase containing 300-350 DSU/cm3 of enzyme activity has been obtained during the aerobic fermentations, while in the anaerobic fermentations, enzyme yields containing 450-500 DSU/cm3 have been obtained routinely. The type of yeast extract used in the fermentation medium has been found to have significant effects on enzyme yield. Of the different types studied, the Gistex Standard was found to be the type that favoured the highest enzyme production. Studies have also been carried out on the effect of agitation rate and antifoam on the enzyme production during the anaerobic experiments. Agitation rates of up to 600 rpm were found not to affect the enzyme yield, however, the presence of antifoam in the medium led to a significant reduction in enzyme activity (less than 300 DSU/cm3). Scale-up of the anaerobic fermentations has been performed at up to the 1000 litre level with enzyme yields containing more than 400 DSU/cm3 of activity being produced. Some of the enzyme produced at this scale was used for the first time to produce dextran on an industrial scale via the enzyme route, with up to 99% conversion of sucrose to dextran being obtained. An attempt has been made at continuous dextransucrase production. Cell washout was observed to occur at dilution rates of greater than 0.4 h-1. Dextransucrase containing up to 25 DSU/cm3/h has been produced continuously.

Batch and continuous fermentation methods for the production of dextransucrase

The available literature concerning dextransucrase and dextran production and purification has been reviewed along with the reaction mechanisms of the enzyme. A discussion of basic fermentation theory is included, together with a brief description of bioreactor hydrodynamics and general biotechnology. The various fermenters used in this research work are described in detail, along with the various experimental techniques employed. The micro-organism Leuconostoc mesenteroides NRRL B512 (F) secretes dextransucrase in the presence of an inducer, sucrose, this being the only known inducer of the enzyme. Dextransucrase is a growth related product and a series of fed-batch fermentations have been carried out to extend the exponential growth phase of the organism. These experiments were carried out in a number of different sized vessels, ranging in size from 2.5 to 1,000 litres. Using a 16 litre vessel, dextransucrase activities in excess of 450 DSU/cm3 (21.67 U/cm3) have been obtained under non-aerated conditions. It has also been possible to achieve 442 DSU/cm3 (21.28 U/cm3) using the 1,000 litre vessel, although this has not been done consistently. A 1 litre and a 2.5 litre vessel were used for the continuous fermentations of dextransucrase. The 2.5 litre vessel was a very sophisticated MBR MiniBioreactor and was used for the majority of continuous fermentations carried out. An enzyme activity of approximately 108 DSU/cm3 (5.20 U/cm3) was achieved at a dilution rate of 0.50 h-1, which corresponds to the maximum growth rate of the cells under the process conditions. A number of continuous fermentations were operated for prolonged periods of time, with experimental run-times of up to 389 h being recorded without any incidence of contamination. The phenomenon of enzyme enhancement on hold-up of up to 100% was also noted during these fermentations, with dextransucrase of activity 89.7 DSU/cm3 (4.32 U/cm3) being boosted to 155.7 DSU/cm3 (7.50 U/cm3) following 24 hours of hold-up. These findings support the recommendation of a second reactor being placed in series with the existing vessel.

Biodeterioration of harvested sugar cane in Jamaica

The microbiological, physical and chemical changes which occur instored, harvested sugarcane were studied in Jamaica and the United Kingdom.The degree of deterioration was proportional to time of storage, and wasrevealed by a statistically significant reduction in sucrose content.Other symptoms included a fall in pH, and increases in reducing sugars,dextran, viscosity, and microbial count. Cut cane was universally infectedwith Leuconostoc mesenteroides, which reached a maximum count of 107 to 108organisms per ml. juice within. 3 to 4 days of harvest. Counts of othermicroorganisms were generally insignificant, except for occasional lactobacilli.A new dextran-forming species was named Lactobacillus confusus.Microorganisms isolated from deteriorated cane were screened for theirability to cause deterioration of a sterile, synthetic cane juice. L. mesenteroides strains were the most deteriogenic, but attempts toreproduce the symptoms of "sour" cane by inoculation of this organism intocut cane were only partially successful. L. mesenteroides was present in the soil and the epiphytic flora of the stalk. The principal vector of infection appeared to be the cutters' machete, especially in wet weather. Cane harvested by a chopper machine deteriorated more rapidly than hand-cut whole-stalks. Economic losses due to deterioration of harvested cane were estimated to be 9.2% of the initial recoverable sugar for the 1969 crop at Frome Estate, Jamaica. Dextran content was a useful indicator of cane biodeterioration. The dextran content of mill juices was correlated with rainfall, and significant correlations were obtained between dextran content and viscosity of mill syrups and the amount of sugar lost in final molasses; it also caused the formation of elongated crystals. Attempts to control sour cane by chemical and physical methods were unsuccessful, and it was concluded that the only solution is to mill cane within 24 hours of harvest. A novel method for removal of dextran from mill juices by enzymic treatment with dextranase was developed and patented.