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Texas A&M Department of Chemical Engineering

The Production of Lactase

by

Phillip Thane, Chris Spletter, Teddy Beitler,

Brad Bevolo, and Kaitlyn Coker

CHEN 282: Section 501

Lactase, the common name for beta-D-galactoside galactohydrolase, is an enzyme critical

to the human digestion of lactose which is present in dairy products. Lactase hydrolyzes lactose

into glucose and galactose, which are sugars more easily used by the body. (See Appendix B for

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an illustration of this reaction). As people age, the lactase enzymes they are born with can

become denatured, resulting in lactose intolerance. Some people, especially individuals that are

not of European descent, are born without an adequate level of lactase and need to supplement

their diet with lactase supplements or consume lactose free products in order to avoid

gastrointestinal distress. (See Appendix C for a population density map illustrating lactose

intolerance across the world). With around thirty to fifty million Americans currently

experiencing lactose intolerance and a blossoming market for lactase in developing countries,

lactase production will become increasing impactful to society [5]. Thus, the study of lactase

production and attempts at improving its production are imperative.

Current Methods:

Production of lactase is sensitive to the species used to create it. Certain bacteria and

fungi function more efficiently and are more likely to survive under certain environmental

conditions such as different temperature and free proton concentration. Not all enzymatic lactase

produced is the same and some can function in different conditions based on production

methods. For example, the human stomach is far too acidic for some enzymes to survive and

function so they become denatured and, therefore, useless. So the enzyme must be delivered in a

way that will allow it to survive metabolism.

Microorganisms and Reactor Design:

The two most common species to produce lactase are the yeast Kluyveromyces lactis and

the fungi Aspergillus Niger. Current commercial production of lactase focuses on the use of non-

pathogenic fungi which has achieved yields of roughly one kilogram per liter. This uses a

fermentation procedure carried out in a submerged culture on solid or semi-solid medium which

are arranged in large trays. After growth, an extensive post processing procedure is followed

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during which the enzyme is filtered, purified, and concentrated. The entire process must be

closely monitored to prevent contamination as regulated by the Enzyme Technical Foundation, to

ensure the product is safe to consume. The majority of enzyme supplements in United States

drug stores and groceries are produced with fungus.

Yeast based production of lactase proceeds slightly differently with fed batch processes

running at various agitation rates to stimulate growth. The yeast adapts to the medium, which

includes lactose that is used as a source of energy to produce the enzyme lactase. In this way the

yeast can utilize lactose as a carbon and energy source. Unfortunately purification proves to be

difficult due to filter clogging from various protein and sugars in the solution. Yeast application

is currently limited as there are only two known strains that can produce lactase and production

from these generally uses newer methods.

Production methods continue to be improved upon and tested in a quest to find the most

efficient and highest yield procedures. According to the China technology exchange, production

of fungi can be improved by about six time’s current standards through the use of gene

engineering to improvements in thermal stability and ruggedness of the enzyme [3]. Yeast, on the

other hand, has been the focus of recent studies as difficulty with filtering and purification is

solved and new fermentation methods are optimized. A recent study using an agitated batch

reactor running at various speeds and constant aeration to ensure oxygen saturation has produced

notable improvements in yield at a pilot plant in Chile [1]. As enzyme producers try to gain a

competitive edge it will continue to cause diversification of production methodology using yeast,

fungus, or possibly both sources.

Extraction

Lactase is grown inside cells and thus must be extracted from cells by the use of

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solutions, enzymes, or sonication. It is important to note that extraction lysis, or kills, the cells.

Extraction by solution follows the same procedure regardless of solvent. The solvent and

a pH buffer is added to the cells and incubated for a set amount of time followed by

centrifugation to remove the cells. The centrifugation step is rather quick, within five minutes at

four thousand rpm in the cited study, the time constraint is the incubation. Depending on solvent

the process can take anywhere from one to twenty one hours. Chlorophyll for instance reaches a

peak amount of released lactase after one hour, but does not show the greatest enzyme activity.

Whereas methanol and butanol had a twenty one and five hour incubation time respectively.

This time constraint greatly limits the applicability of extraction by solution.

Enzyme extraction, although efficient, almost sixty-one percent recoveries in some cases,

is very expensive and as a result sees little if any use industrially. Furthermore over time the

enzymes used in extraction will also cause a decrease in activity of the lactase enzyme. The

method however is very streamlined as the enzymes used for extraction are simply grown

together with the lactase, shortening process steps.

Sonication is the most effective and widely used method of extraction. The cells are

ruptured gently by bombardment of sonic waves introduced into the solution. This method not

only sees the quickest extraction but highest activity because there are no added reagents or

enzymes to disrupt the enzyme. An important effect of sonication from a process perspective is

the subsequent decrease in viscosity of the fluid as a result of the cells releasing all their proteins.

So centrifugation following sonication will be easier, even if by only a slight margin [2].

Purification Techniques:

Due to the medium in which lactase must be grown, the solution in which it is suspended

is littered with polysaccharides and oligosaccharides. Poly-and oligosaccharides are very

4


problematic when it comes to filtration. They are long repeat chain carbohydrate molecules that

can either be linear molecules or branched molecules. Their long, branched nature makes them

difficult to filter. In fact, polysaccharides are the molecules that make up many things in nature

such as cellulose (cotton and paper fibers), chitin (which makes up the exoskeletons of many

insects), and pectin (which make up most cell walls of the non-woody parts of plants) [cite]. As

one can imagine, these compounds would be quite effective at clogging filter cakes and a

clogged filter prevents efficient isolation of the desired product, lactase. For this reason,

chromatography is enlisted to remove the lactase from the suspended solution. By using the

appropriate chromatographic methods, research has shown that nearly all the poly- and

oligosaccharides can be removed from solution. Based on research from US Patent 7,955,831 a

desirable concentration of poly-and oligosaccharides left in solution after chromatography has

been performed is 10 g poly-oligosaccharides/kg solution [cite]. This solution is then filtered to

increase purity once the bulk of the filter blocking poly- and oligosaccharides have been

removed.

The filtration of lactase is typically involved when lactase is incorporated into a dairy

product, such as milk, in an in-line production scheme. The lactase-saccharide solution is

typically fed into a filter, where the solution is cleaned of impurities. It is then fed to the dairy

product where the lactase breaks down the lactose. However, as stated before, without

chromatography the filter cake becomes clogged easily. Thus, implementing chromatography

before the filtration step will keep the process running smoothly and help to prevent “down-

time,” in which product is not being made.

According to investigation from US Patent 7,955,831 the most effective chromatographic

method for removing the poly- and oligosaccharides is with the use of anion and cation exchange

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resins [9]. Lactase binds to the resin and travels up the chromatography column, therefore

leaving the solution and most of the poly- and oligosaccharides behind. Denaturing (recovery)

of the lactase from the chromatography column also proves to be simple when anion and cation

exchange resins are used. To denature the lactase from the resin, the ionic strength of the resin is

decreased, releasing the lactase.

In an example experiment run by Rudolf Franciscus Wilhelmus Cornelis Van Beckhoven

and Petrus Andreas Van Paridon, the inventors of US Patent 7,955,831, chromatographically

purified lactase was tested against a common commercial product, Maxilact, to determine which

had a higher glucose concentration. The purified lactase had less than 2 grams of poly- and

oligosaccharides/kg solution, while the Maxilact had 56.62 grams poly- and oligosaccharides/kg

solution. Thus, the chromatographically purified lactase is much more pure than the

commercially available one [9]. In a second experiment, chromatographically purified lactase

was pushed through a sterile hand filter, similar to a syringe. The chromatographically purified

lactase was easily moved through the filter. However, when the syringe was filled with Maxilact,

the lactase-solution would not proceed through the filter. In a third experiment, the viscosity of

the chromatographically filtered lactase was compared to that of the Maxilact. The viscosity of

the Maxilact was recorded to be 170 mPa while the viscosity of the purified lactase was 40 mPa

[9].

The ability of lactase to be easily filtered is essential in many processes in which lactase

is a major product. Based upon the above experiments, purified lactase obviously presents itself

as the more advantageous choice for running processes, highlighting why chromatography plays

such a vital role in the current production and purification of lactase.

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Improvements

Purification:

Improvements in the purification focus on more efficiently providing a purer product than

current methods. The most efficient procedure for purification is to begin by removing the

biggest and most abundant impurities and gradually increase the sensitivity of the purification

equipment until the most sensitive equipment and separation is used. An acceptable sequence is

starting with a continuous disc-stack centrifuge, through a more sensitive centrifuge, then using

chromatography based on two different driving forces to provide maximum product purity.

The first step is centrifugation, which will remove the yeasts and the large filter-clogging

materials. A disc-stack centrifuge should remove a bulk of the bigger, denser particles. This uses

the large size of the yeast as an advantage rather than a hindrance. Using chromatography first

does not take advantage of the different densities of the yeasts, enzymes, and sugars. Although

using chromatography first is an acceptable method, it is expensive to do and again, does not

take advantage of the intensive properties of the particles. Chromatography is best used as a final

purification rather than an intermediary step.

Since the enzyme will be used for human consumption, maximum sterility and

purification is critical. Filtration is not a sterile process because of the open air nature and should

be excluded from the purification sequence. As an alternative, the product can be fed through a

faster, more sensitive centrifuge after the primary purification process. Not only will this keep

the product sterile, but it will allow the avoidance of clogging in filtration. Avoiding filtration

can also reduce cost given that the centrifuge is well designed by eliminating spending on filter

cake or downtime due to clogging.

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After the two centrifugation processes, chromatography can be performed last to remove

the remaining impurities, since it is the most selective and therefore can remove the impurities

that have similar properties to lactase. First, the chromatography method based on the size

exclusion principle is to be used. This is followed by the affinity chromatography method to

ensure maximum purity is obtained.

Chromatography Scheme:

Using “swing-style” preparative chromatography columns, more product can be

processed at a time. Since chromatography columns must be prepared prior to use and single

columns cannot accept steady streams of the semi-pure product, a large number of

chromatography columns can be utilized to achieve a more continuous and efficient separation.

If a single column was run at steady-state, the particle bands would eventually overlap and the

separation would be pointless.

Based on the residence time of the enzyme solution in the column and the target amount

of purified product per unit time, the number and size of columns can be calculated. These

columns will stand in a circle around the feed ready to accept a portion of the stream from a

revolving feed mechanism. Around this active circle of columns an inactive ring of columns is

being rinsed and packed in preparation to accept the fresh stream. This second ring has exactly

the same number of columns as the first, and a column in the second ring is connected to the

column in the first by a bar that rotates one hundred and eighty degrees. Once the inactive

purifier is ready, it is swung into the active circle, trading places with its complementary partner

just as the revolving feed mechanism pours solution into it. See appendix A for a visual

representation of this separation scheme.

This swing style mechanism can be used for the size exclusion as well as the chemical

8


affinity chromatography steps, but the number of columns in each scheme may be different due

to differences in retention times for each method.

Business Model

In the following section we will outline a business model for production and sale of

commercially produced Lactase.

Inputs:

The substrate feeding the bacteria in the batch reactor will be a variable mixture known

as YM medium which is a culture media that contains malt extract, peptone, yeast extract

distilled water, and lactose as Dickson, Dickson, and Markin used in their lactase production [2].

Reactor:

We will be growing the enzyme on Kluyveromyces Marxianus in a batch reactor. Lactase

is a primary metabolite and will be produced during the growth phase of the reactor, therefore

our target is to minimize the lag phase and increase the duration of the growth phase to increase

enzyme production. To accomplish this; two reactors will be run with staggered run times so that

yeast already in the growth phase can be taken from a running reactor and added to the startup of

the offline reactor. As soon as deceleration starts the slurry will be removed and separated.

Substrate will be put in the reactor at an initial amount that provides optimum product yields in

the minimum time. This amount can only be determined from experimental data. This would be

done during several smaller scale trial runs as well as adjustment during initial startup with

actual conditions.

Purification:

Sonification will be used to remove the lactase from inside the yeast cells. From the

sonicator, the slurry will be sent through two subsequent centrifugation processes to separate the

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products and then through two different chromatographic processes. The swing style

chromatography columns will be used in order to allow the chromatography to keep up with the

rest of the flow processes. After chromatography, the lactose will go through further processing,

(drying, testing, and packaging) before being put to market.

Final Manufacturing:

After purifying the produced lactase, the enzyme needs to be collected and allocated into

pills in portions that can be ingested directly. The purified enzyme will be run through a

hydroxylapatite column and an ion exchange chromatography column to concentrate the product

[2]. Commercially available lactase supplements range in amounts from 1500 to 9000 FCC units.

(15 FCC roughly equal to 1 mg) It would be economical to produce three tiers of product with

concentrations of about 1500, 4500 and 9000 FCC units. This would provide consumers with

light, moderate or severe lactose intolerances with options for treatment without having to alter

our production process. Once lactase is concentrated, it can be portioned out by weight by an

automatic scale into the different quantities for sale.

Retail:

Lactose intolerance affects different populations with varying severity. People of

European origin have the lowest levels of intolerance, while up to ninety five percent of people

of Asian descent will have developed a shortage of lactase by adulthood [8]. With around thirty

to fifty million Americans currently experiencing lactose intolerance and a blossoming market

for lactase in Asian countries, lactase production will become increasingly beneficial to society

[8]. Lactose intolerance also heavily impacts the southern half of South America and South

Africa, with upwards of eighty percent of the population affected [5]. This can also be seen

visually in the distribution density map in Appendix C. Making use of this information, it would

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be beneficial to focus production facilities in Asia and South America to facilitate the local

markets and then have exports to America and some other countries where there is an available

but more limited market.

This business model was constructed under the notion that cost is not an issue.

Eliminating cost allows for the business model to be idealized to realize maximum recovery and

activity of the enzyme. Despite this idealization, the presented model gives a notion of what

real world production would look like.

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Works Cited

1. Acevedo, F., J.C. Gentina, N. Bojorge, I. Reyes, and A. Torres. Development of a Pilot Plant Fermentation Process for the Production of Yeast Lactase. School of Biochemical Engineering University of Catalica De Valparaiso. Web. 26 Apr. 2012. <http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1996.tb33256.x/pdf>.2. Beccera, Manuel. "Extraction of Intracellular Proteins from Kluveromyces Lactis." Foot Technical 39 (199): 135-39. Department of Molecular and Cellular Biology at Zapatiera University, Spain. Web. 28 Apr. 2012. <http://www.ftb.com.hr/39/39-135.pdf>.3. CTEX. "Latase Production Technology." China Technology Exchange (2010). China Technology Exchange. Web. 26 Apr. 2012. <http://en.ctex.cn/article/HightsProjects/201002/20100200000166.shtml>.4. Dickson, R. C., L. R. Dickson, and J. S. Martine. "Purification and Properties of an Inducible Beta Galactoside Isolated from the Yeast Kluveromyces Lactis." Journal of Bacteriology 137 (1979): 51-61. ASM.org. Web. 26 Apr. 2012. <http://jb.asm.org/content/137/1/51.full.pdf+html>.5. "Lactose Intolerance." DSM. Web. 26 Apr. 2012. <http://www.dsm.com/le/en_US/maxilact/html/lactose_intolerance.htm>.6. Seyis, Isil, and Nilufer Aksoz. "Production of Lactase by Trichoderma Sp." Food Technology 42.2 (2004): 121-24. Print.7. Tarantino, Laura M., and Office of Food Additive Safety. "Agency Response Letter GRAS Notice No. GRN 000132." Letter to Ms. Caddow. 12 Dec. 2003. FDA.gov. Food and Drug Administration, 22 Mar. 2012. Web. 26 Apr. 2012. <http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm153949.htm>.8. Wexner Medical Center. "Lactose Intolerance." Ohio State University Wexner Medical Center. Web. 26 Apr. 2012. <http://medicalcenter.osu.edu/patientcare/healthcare_services/digestive_disorders/lactose_intolerance/Pages/index.aspx>.

9. Wilhelmus Cornelis Van Beckhoven, Rudolf Franciscus, and Petrus A. Van Paridon. Purified Lactase. DSM IP Assets B.V., assignee. Patent 7955831. 7 June 2011. Print.

Appendix A

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Appendix B

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http://www.lsbu.ac.uk/biology/enztech/images/lactase.gif

The above reaction illustrates the role of lactase in the deconstruction of lactose. The bond between the exo position oxygen and the adjacent ring is broken and both rings are oxidized with an alcohol creating the two sugars. The reaction, although simple, takes place very slowly without the lactase enzyme to catalyze the reaction.

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Appendix C

http://www.oliverbenjamin.net/dudespaper/wp-content/uploads/2009/06/lactose-intolerance-map.jpg

The above map indicates lactose intolerance by a percentage of population by country. It is to be noted that China in particular has a high incidence rate.

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