FOOD AND INDUSTRIAL MICROBIOLOGY

Module 9. Microbial process strategies

Lesson 29
INDUSTRIAL ENZYMES AND VITAMIN B12

29.1 Introduction

Major industrial enzymes from bacteria, molds and yeasts are listed below:

Table 29.1 Major industrial enzymes from bacteria, molds and yeasts

29.2 Chymosin

Chymosin is also known as rennet or chymase and is used in the manufacture of cheese. Over 90% of the chymosin used today is produced by E. coli, and the fungi, Kluyveromyces lactis and Aspergillus niger. Genetically engineered chymosin is preferred by manufacturers because while it behaves in exactly the same way as calf chymosin, it is purer than calf chymosin and is more predictable. Furthermore, it is preferred by vegetarians and some religious organizations.

29.2.1 General principle of chymosin production using rDNA technology

In higher eukaryotes, most chromosomal genes have intron sequences which interrupt the coding sequences for translation to proteins. When these sequences are transcribed in eukaryotic cells, the introns are spliced out of the mRNA transcript and subsequent translation gives proteins with correct amino acid sequences. Because bacterial cells have no such splicing mechanism, genes obtained from eukaryotic chromosomes (genomic genes) cannot be correctly expressed in bacteria. Therefore, the general procedure for cloning any eukaryotic gene in a bacterial host is to synthesize complementary DNA (cDNA) by reverse transcription of mRNA from which introns have been spliced out.

Highly specialized cells within tissues (for example, the mucosal layer of abomasum), frequently contain large amounts of mRNA that codes for pre prochymosin. This provides the basis for isolation of the specific mRNA to make cloning easier. The total RNA from the mucosal layer of abomasum is isolated and the mRNA is fractionated by oligo(dT) cellulose affinity chromatography. The mRNA is reverse transcribed to cDNA and subsequently cloned in a suitable vector and expressed in a suitable bacterial or yeast host.

The gene for chymosin has been successfully cloned and expressed using a number of plasmid or modified plasmid vectors and host organisms such as E. coli, Bacillus subtilis, Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus spp, baby hamster kidney cells, and so forth. Several forms of chymosins have been expressed, including preprochymosin, met-prochymosin, and met-chymosin as heterologous proteins made in E. coli. Expression as fusion protein is often more efficient than direct expression. Chymosin is especially well suited for expression as a fusion protein which is autocatalytically reactivated to chymosin during product recovery, ensuring that the final product has the proper amino terminus.

The method for Chymosin A produced from Escherichia coli K-12 containing calf prochymosin A gene is explained below

29.2.1.1 Construction of production strain

E. coli K12 JA198 strain was subjected to several genetic manipulations to construct the recipient strain for the expression plasmid carrying the prochymosin A gene. The expression plasmid was derived from the widely used cloning vector pBR322. cDNA coding for bovine chymosin A was previously cloned and characterized. The prochymosin gene was divided into three sections, each terminated by a unique restriction endonuclease recognition site. Each section was assembled from several synthetic oligonucleotides synthesized in an automated DNA synthesizer. Each assembled section was subcloned into a pBR322 vector, transformed into E. coli, and amplified. All three subcloned sections were assembled together in the correct order to reconstruct the prochymosin gene, which was inserted into pBR322 vector. The gene was attached to the vector DNA through the ribosomal binding site and the E. coli tryptophan (trp) promoter. The created expression vector was transformed into the recipient strain GE81. The plasmid carries the ampicillin resistance gene as a selective marker for bacterial transformants carrying the prochymosin gene.

29.2.1.2 Fermentation

The production strain is grown in an aqueous solution containing carbohydrates, nitrogen, mineral salts and miscellaneous inorganic and organic compounds.

29.2.1.3. Recovery

The solid prochymosin is liberated from the producing organism by cell disruption and harvesting of "inclusion bodies" by centrifugation or membrane concentration. The harvested inclusion bodies are washed with phosphate buffer solution containing 1-4 M urea. The residual E. coli are inactivated by holding at pH less than 2.0 for at least one hour. The inclusion bodies are then dissolved by addition of urea to a concentration of 7-9 M and pH adjustment to 10.0 - 11.0. Subsequently, the solution containing prochymosin is diluted with a buffer, and the pH is reduced to 8.5- 9.5, followed by a 2-hour period to allow renaturing of the prochymosin, which is subsequently activated to chymosin by adjusting the pH to 1.8 - 2.2 and holding for one hour. Following readjustment of the pH to 5.5 - 6.0, the chymosin is purified via absorption on a suitable anion-exchange resin followed by elution with a buffer containing 1 M sodium chloride. Increasing effort is being devoted to improve the expression of chymosin in bacteria and yeasts; better yeast expression systems are particularly needed. Protein engineering of chymosin is still another area of interest. Protein engineering can potentially enhance activity of chymosin and beneficially modify the pH optima.

29.3 Vitamin B₁₂

Vitamin B₁₂ is needed to help maintain healthy red blood cells and nerve cells, as well as aid in the production of DNA. As the body is capable of storing large amounts of B12 in the body, deficiencies occur infrequently, but they do happen. A person with a B12 deficiency is susceptible to several different types of anemia, low blood pressure, dementia and muscle weakness. Other disorders that have been tied to low vitamin B12 levels are Alzheimer's disease, breast cancer, fatigue and heart disease.

It seems probable that the only primary source of vitamin B12 in nature is the metabolic activity of the microorganisms. It is synthesized by a wide range of bacteria and Streptomycetes, though not to any extent by yeasts and fungi. While over 100 fermentation processes have been described for the production of vitamin Bl2 only half a dozen have apparently been used on a commercial scale.

Fermentation processes using Bacillus megatherium, Streptomyces olivaceus and other species, Propionibacterium freundreichii, and P. shermanii. The processes using the Propionibacterium species are the most productive and are now widely used commercially. Both batch and continuous processes have been described.
It is important to select microbial species which make the 5, 6-dimethyl benzimidazolylcobamide exclusively. Several manufacturers have been led astray by organisms that gave high yields of the related cobamides including pseudo-vitamin B₁₂Streptomyces cells. The vitamin B12 activity is released from the cells by acid, heating, cyanide or other treatments. Addition of cyanide solutions decomposes the coenzyme form of the vitamin in and results in the formation of the cyanocobalamin. (adeninylcobamide). The natural form of the vitamin is Barker's Coenzyme where a deoxyadenosyl residue replaces the cyano group found in the commercial vitamin. Practically all of the cobamides formed in the fermentation are retained in the cells, and the first step is the separation of the cells from the fermentation medium. Large high speed centrifuges are used to concentrate the bacteria to a cream, while filters are used to remove

The cyanocobalamin is adsorbed on ion exchange resin IRC-50 or charcoal, and is eluted. It is then purified further by partition between Phenolic solvents and water. The vitamin is finally crystallized from aqueous-acetone solutions. The crystalline product often contains some water of crystallization.

The most commercial sound procedure produces B12 produced industrially by microbial fermentation, using almost exclusively Pseudomonas denitrificans and Propionibacterium spp. Contrary to Pseudomonas, Propionibacteria are food-grade. Processes using Propionibacterium species thus have the advantage that they allow to formulate natural vitamin B12 together with the biomass in which it is produced. Such processes avoid the conversion of natural vitamin B12 into the cyanocobalamin form by chemical processes including cyanidisation followed by extraction and purification steps using organic solvents. The chemical conversion step and any subsequent purification steps cause this production process to be expensive, unsafe to the operators and environmentally unfriendly.

Several Propionibacterium species are capable to produce vitamin B₁₂ in large scale fermentation processes. The process is described as a two-stage fermentation with a 72-88 hours anaerobic fermentation followed by a 72-88 hours aerobic phase. The vitamin B₁₂ concentration in the cells rapidly increases in the aerobic phase, with typical values of 25-40 mg vitamin B₁₂/ l . Anaerobic growth followed by an aerobic phase with limited growth is important for economic production of vitamin B₁₂ using Propionibacterium species. This requirement, however, limits the amount of biomass to 25-35 g/l as described above. Several attempts have been made to overcome the barrier of propionic acid toxicity in order to increase biomass and thereby the yield of vitamin B₁₂.

Alternated anaerobic-aerobic phases are e.g. suggested to reduce the amount of acids. In the aerobic phase the propionic acid is converted to less toxic acetic acid, with simultaneous formation of vitamin B₁₂. The relative yield of vitamin B12 has been increased, but the final titre is rather low. This is probably due to inhibition early in the synthesis of vitamin B₁₂ and/or other oxygen related products limiting the synthesis of vitamin B₁₂. The final vitamin B₁₂ produced with this method is 9 mg/l compared to 4.5 mg/l with the fully separated anaerobic and aerobic phases. Both values are rather low for vitamin B₁₂ production with Propionibacterum.