Research Laboratory for Biotechnology and Biochemistry ( RLABB)

Wednesday, August 17, 2011

Calibration of Micropipettes

Objective:


1. To know different parts of Micropipettes.


2. To learn how to use Micropipettes.


3. To calibrate micropipettes used in Molecular Biology laboratory.


4. To learn how to take care of Micropipettes.



Introduction:



Micropipettes used to accurately measure small volumes of liquids (volumes typically vary from 1 to 1000 µL). The parts of typical micropipettes are shown below.







Accuracy and Precision


Pipettes and micropipettes can deliver accurate and precise volumes of solution. Our goal is to determine how accurate and how precise our pipette and micropipette are.


Accuracy is a measure of how close a measured value is to the accepted or “true” value. It is related to the percent error between the average volume of solution measured experimentally and the volume that was expected (the accepted value). Smaller percent error reflects higher accuracy. Percent error can be negative, indicating that the measured volume was smaller than the expected volume or positive, indicating that the measured volume was larger than the expected volume. For example, we are attempting to measure two different volumes of water with our micropipette and two with our graduated pipette. Perfect accuracy would have us measure the exact volume we desire each time. However, the volume of water that we actually measure will be close but probably different from these volumes. The farther away from the correct volume, the lower the accuracy of our pipettes and/or our technique will be. The formula for percent error is in the Statistical Functions portion of the Lab Manual Introduction.


Precision measures the closeness of a set of values obtained from identical measurements of the same quantity. It is the ability to repetitively measure the same volume of solution (whether it’s accurate or not). Precision is related to the standard deviation of a series of measurements of the same thing. For example, if the micropipette is set to the same volume (300 µL) and four measurements are taken at this volume, a standard deviation can be taken of these five measurements. The smaller the standard deviation, the more precise the micropipette is. We will use the standard deviation as a measure of the spread of potential errors in a given measurement. The formula for standard deviation is in the Statistical Functions portion of the Lab Manual Introduction. Standard deviation is usually reported with the average value like this:


In order to minimize the waste generated from experiments in this class, a number of the experiments involve a micropipette that can deliver between 100 and 1000 microliters (µL). The micropipettes are only to be used for this volume range. For larger volumes, a graduated pipette is in your locker (bulbs available from the stockroom or one of the side drawers).




Use of the micropipettes


When you push down gently on the plunger of the micropipette, you will feel a “stop” where the resistance increases. If you push a little harder, the plunger will move even further to a second stop. The first stop is used to suck up the correct volume. The second stop is used to completely expel the liquid you are measuring.


Liquid is never drawn into the barrel of the micropipette itself. An appropriate tip should always be placed firmly on the end. Since the principle by which the micropipette works is the creation of a vacuum in the tip, causing liquid to be drawn up, it is critical that the tip be on tight enough to make an air-tight seal. Having said this, do NOT jam the tips on so hard that they are hard to get off. The tips used for the 1000µL pipettes are usually blue.


The volume to be taken up is set by turning the plunger on the top of the micropipette and reading the numerical settings displayed. A setting of 100 µL is equal to 0.100 mL. A setting of 1000 µL is equal to 1.000 mL. Do not set the micropipette below 100 µL or above 1000 µL under any circumstances! Doing this is essentially the only way that the micropipettes can be broken.





When drawing liquid up into the micropipette, set the dial, and then pushes the plunger down to the first stop. While holding it there, put the end of the tip under the surface of the liquid to be measured and slowly, gently allow the plunger to return to its top position. If you go too fast, you will cause some liquid to spurt up into the micropipette barrel itself, which is bad for the micropipette, bad for your results and bad for your chemistry karma, whatever that is. This also lets air in, so the volume of fluid sucked up into the tip will be lower than the amount that you want. Never let the plunger snap up by it. Next, place the end of the tip where you want the liquid to go and push the down the plunger to the second stop to deliver the exact amount of fluid desired.


Although micropipettes are usually quite accurate when first purchased, they can eventually develop problems with use. We will spend some time checking the calibration of the micropipettes that we will be using throughout the semester to ensure that they are delivering a known volume of fluid. We will measure out different exact volumes of water with the micropipette, and using the density of water at the temperature of the water, we will determine the volume of fluid delivered based on the fluids mass.





There are many techniques and tips available that will optimize your pipetting performance and increase the reproducibility of your results. A brief description of each follows:



The Equipment



1) Tips - It is advocated that only high quality tips which optimize the pipette’s performance be used. A high quality tip is one that has a smooth uniform interior with straight even sides that prevents the retention of liquids and minimizes surface wetting. Also, the tip should have a clean, hydrophobic surface and a perfectly centered opening in order to ensure the complete dispensing of the sample. These tips should always securely interface with the nosecone, because if they do not fit correctly, the amount of liquid dispensed can be dramatically influenced.



2) Liquid Viscosity - Since the pipette was originally factory calibrated using water, any liquid that has a viscosity higher or lower than water will impact the volume dispensed. Viscosity differentials should be accounted for and taken into consideration in order to enhance the accuracy of the instrument.



3) Container - The material of construction for the extraction vessel is also important, since some materials tend to force water into a convex configuration while other materials force water into a concave configuration. Obviously, this can impact the amount of liquid drawn into the tip. A glass container is recommended since it tends to force water into a concave configuration which helps to reduce or eliminate variations due to this effect.



The Operator


1) Technique - Most end users have a tendency to believe that the volume delivery is completely


dependent on the setting of the micrometer dial. Obviously, this is not the case, since many factors associated with pipettes come into play.


• Position - Pipettes should be held vertical during the aspiration of liquids,however, some end users often hold pipettes at many different angles during a pipetting interval. Holding a pipette 30o off vertical can cause as much as 0.7% more liquid to be aspirated due to the impact of hydrostatic pressure. Always store pipettes in an upright position when not in use.



• Pre-Wetting/Pre-Rinsing Tips -Failing to pre-wet tips can cause inconsistency between samples


since liquid in the initial samples adhere to the inside surfaces of the pipette tip, but liquid from later samples does not. Also, if a new volume is dialed in on the pipette’s micrometer, you will receive better results at the new volume by taking the old tip off and placing a new one on the shaft before you commence pipetting.



• Release of Plunger - Releasing the plunger abruptly can cause liquid to be “bumped”inside the pipette during a liquid transfer application. This can cause liquid to accumulate inside the instrument which in turn can be transferred to other samples causing variability in sample volume and the potential for cross contamination. It is recommended that a smooth, consistent pipetting rhythm be employed since it helps to increase both accuracy and precision. After the liquid has been aspirated into the tip, the pipette should be placed against the wall of the receiving vessel and the plunger slowly depressed. This will help all of the liquid in the tip to be dispensed. After a pause of about 1 second, depress the plunger to the bottom or blowout position (if equipped) and remove the pipette from the sidewall by utilizing either a sliding action up the wall or a brief movement away from the wall (called “touching off”).



• Immersion Depth - The pipette tip should only be inserted into the vessel containing the liquid of be transferred about 1-3mm. If the tip is immersed beyond this, the results could be erroneously high. This is due to the fact that liquid could adhere to the tip and be transferred along with the aliquot in the tip. If the tip is not immersed far enough then air could be drawn into the tip which could yield results that are incorrect on the low end.



• Equilibration Time – Troemner recommends that the tip, the pipette, the liquid being transferred, and the transfer container itself all be allowed to equilibrate to the same temperature. This is done to lessen the effects of thermal expansion which can dramatically impact the delivered volume.



• Thermal conductance – Thermal energy can be transferred from the operator’s hand to the air within the pipette (dead air) or even to the internal components themselves. This can have a dramatic impact on the amount of liquid dispensed due to the effects of expansion and/or contraction. To lessen this effect, it is recommended that some type of thermally insulated gloves like latex or cloth be worn.



2) Pipette Micrometer Setting – It is important to avoid significantly overdialing or underdialing the recommended range of the pipette. Volume delivery performance may change radically and may become completely undefined.



The Environment


1) Temperature – The volume delivery performance specifications of pipettes have been referenced by most manufacturers at room temperature which is defined as 20-25ºC. Any deviation from this specification can affect the amount of liquid dispensed due to the expansion or contraction of the internal components. Temperature is probably the most important factor that influences pipette performance. In fact, the density of water in a gravimetric analysis is calculated as a function of temperature.



2) Barometric Pressure – Pressure is reduced by 1.06" Hg for every 1000' of elevation, however, barometric pressure has only a small effect on the density formula, so the error encountered in not correcting for elevation is often ignored.



3) Relative Humidity – This is the percentage of moisture in the air at a measured dry bulb temperature compared to the amount of moisture that the air can hold at that temperature if the air is 100% saturated. Relative humidity exerts a major influence on taking accurate measurements of volume delivery. Under dry conditions, which are defined as less than 30% RH, it is extremely difficult to ensure an accurate measurement due to the rapid evaporation rate. Conversely, excessive humidity which is defined as greater than 75% can cause a measurement to be erroneously high due to condensation. Therefore, generally accepted guidelines for pipette volume delivery specify that relative humidity be maintained within the range of 45%-75%. Relative humidity also has an effect on the delivery of air displacement pipettes specifically. This is due to the evaporation of liquid from the upper several factors to consider when calibrating a pipette or choosing a calibration service:



1) If you require the “as found” data, it is advisable to obtain this before any parts or components are replaced since this can drastically change your results.


2) Clean and inspect the instrument for any visible signs of wear and tear. Make sure that the instrument can be autoclaved before autoclaving, since this can seriously damage the pipette.


3) Replace the pipette’s seals and o-rings and any other part that shows signs of wear. Remember


to pay special attention to the piston and replace if it seems especially worn or bent.


4) Ensure that the o-rings and seals have seated properly by performing a leak test and a vacuum test.


5) Allow the pipette to stabilize in an environmentally controlled, vibration-free room for a 24 – hour period to eliminate the effects of thermal expansion.


6) Decide which calibration technique that you wish to employ (i.e. Addition, Addition-Tare, Subtraction, or Subtraction-Tare).


7) Prepare the balance by “exercising” it and modifying it to accept a liquid containing vessel. It is our recommendation to use a glass container, so that the liquid has a concave meniscus.


8) Since most manufacturers originally calibrate their pipettes between 20-25°C while using bidistilled, degassed water, it is our recommendation that these conditions are duplicated.


9) Wear some type of thermally insulated gloves to lessen the transfer of heat from your hand to pipette. Latex or cloth seems to work the best.


10) Begin the liquid transfer stage of the calibration procedure utilizing the appropriate technique that you have chosen to employ.


11) Record the weightings, so that they can be converted into volumetric readings at the end of the calibration procedure.


12) Make the conversion taking into account all pertinent environmental conditions. Usually these conditions are used to calculate a Z-factor which is in turn used to convert from a mass reading to a volumetric reading.



Calibrating Micropipettes:


The two most common techniques of calibrating pipettes are the gravimetric and colorimetric (a.k.a. photometric) methods. Of these, the gravimetric method is the most common and the most widespread in use today. This method requires a stringently controlled environment, a high precision balance, a highly skilled pipetting technician, and a rudimentary understanding of statistics. The principle of this method is simple in that, given a certain mass of water with a known specific gravity; its volume can then be predicted. The accuracy and precision of the pipette can then be assessed by using an appropriate statistical approach. This method can be performed one of four ways: Addition, Addition-Tare, Subtraction, or Subtraction-Tare.


1) Addition is perhaps the most common mode of pipette calibration and it is performed by using the cumulative weight of a liquid to determine the volume dispensed.


2) The Addition-Tare method is performed by taring the balance each time before dispensing.


3) The Subtraction method uses the total subtracted weight of a liquid to determine the volume aspirated by the pipetting device. In this technique, you tare the balance only once, at the beginning, then you aspirate volumes of liquid from the vessel, take cumulative (negative) weights, and then calculate the volume aspirated based on the difference between the current and previous total weights.


4) The Subtraction-Tare method entails taring the balance each time before removing liquid from the vessel.



Since this method is not fool-proof, all variables must be stringently controlled and accounted for in order to produce results that are statistically accurate. The second most common type of pipette calibration process is the colorimetric or photometric method. This method involves the analysis of volumes of diluted dye in a cell of known path length. According to the Beer–Lambert Relationship, if a beam of monochromatic light passes through homogeneous solutions of equal pathlength, the absorbance measured is proportional to the dye concentration. So, with this in mind, an unknown volume of dye can be pipetted into a known volume of diluent, the resulting dye concentration can be measured photometrically, and the volume can be calculated. This method is less prone to environmental influences, but it requires the use of standardized consumables. Obviously, this means that each lot of standardized dye must be very carefully manufactured and calibrated in order to produce results of high accuracy. However, once solutions are prepared, calibrated and shown to be stable, accurate results can be obtained even at volumes less than one microliter11.



Principle:


The volume delivered by the pipettes is determined by weighing the amount delivered and dividing this by the density of water (at RT and 4°C).



Materials:


1. Balance (digital, upto 0.0001gm),


2. Micropipettes (1-10, 10-100, 100-1000µL),


3. Distilled water,


4. Aluminium foil



Procedure:


1. Turn on the balance


2. Place aluminium foil (prepared to cup shaped) on the pan of balance carefully


3. Zero the balance by tareing from its keyboard


4. Pipette out DW onto the foil and observe the weight of water on the data display


5. Zero the balance by tareing from its keyboard, again


6. Pipette out DW with the same pipette onto the foil and observe again the weight of water


7. Repeat steps 5 and 6 for more than 30 attempts



Observation:


Example: Pipetting of 10µL


Table: Weight (d=M/V) and corresponding volume (l) of water (d=1) delivered by micropipette




























S. no.


1


2


3


4


5


6


7


…………………………………..


30


Experimental volume (µL)


11


12


09


10


13


09


08



11



Calculation/ Statistics:


Table 2: Determination of standard deviation (Sd) of volume of DW delivered by the micropipette






















































Experimental volume of DW (µL)-X


True Volume (µL) (expected volume)- X bar


(X-X bar)


(X-X bar)2


11





12





09





10





13





09





08





Calculate for all readings


11





S(X-X bar)2



Sd= Ö S(X-X bar)2/Ö n-1



Interpretation:


………………………………………………………………………………………………


Note: Ensure confidently for no handling error.



Reference:


1. Experiment 1: Volumetric Measurement; Using Micropipettes and Graduated Pipettes Adapted from the CSUS Biochemistry (Chem 162) Lab Manual, Fall 2002.


2. The Science Learning Center at the University of Michigan-Dearborn TROEMNER Pipette Sttandards handbook Raising the standard


3. Tiwari K.B and Ghimire P. (2010) A Practical Handbook for Microbial Genetics and Molecular Biology. First Edition, Kantipur College of Medical Sciences, Sitapaila, Kathmandu.


Saturday, August 8, 2009

Biochemical Characterization of Yeast Isolates from Murcha

Kiran Babu Tiwari1,2*, Manindra Lal Shrestha1, and Vishwanath Prasad Agrawal1,2

1Universal Science College, Pokhara University, Maitidevi, Kathmandu, Nepal
2Research Laboratory for Agricultural Biotechnology and Biochemistry, Maitidevi,
Kathmandu, Nepal

*Corresponding author: Kiran Babu Tiwari, Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal. Email: kiranbabu.babukiran@gmail.com


Abstract

Eight Murcha samples were collected from different localities of Nepal. Altogether 31 yeasts (unicellular fungi) were isolated in Potato-Dextrose Agar (PDA). Compared to Saccharomyces cerevisiae (white/round/raised, Gluose/Fructose/Galactose/Sucrose/Maltose - positive, Mannose/Arabinose/Lactose/Citrate/Urea - negative), the yeasts were characterized morphologically and biochemically. Of the total isolates, eight (25.8%) were similar to S cerevisiae for their colonial properties. All isolates could assimilate Glucose, Maltose and Starch; and none of them could utilize citrate as carbon source. Higher proportions of the strains were able to assimilate Lactose (7, 22.6%), Mannose (6, 19.4%) and Arabinose (5, 16.2%) compared to S. cerevisiae. Further, nine (29.0%), five (16.2%) and one (3.2%) were unable to assimilate Galactose, Fructose and Sucrose respectively. Out of seven (22.6%) urea positive strains, two were able to hydrolyze urea strongly as nitrogen source.

Saturday, July 25, 2009

A Novel Class of Protease from Choerospondias axillaris (Lapsi) Leaves

Sudeep Karki1, Rupendra Shakya1 and Vishwanath P.Agrawal1,2

1Universal Science College and 2Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal.

Abstract

A novel protease from leaves of Choreospndias axillaris has been reported. C. axillaris , locally called Lapsi is dioceous, deciduous fruit – bearing large tree, having multiple daily uses. In an attempt to find method for determining sex of Lapsi at seedling stage , we stumbled upon a unique protease that has thwarted our effort to find sex - related protein. Thus protease is highly thermo – stable and acid resistant. Its preparation can be autoclaved without significant loss in activity. Its activity can be repeatedly precipitated by trichloroacetic acid. It possesses a Km value of 29 µM and Vmax 52.63 pmoles/min for bovine serum albumin as the substrate. It is catalytically so powerful that the level of soluble protein in leaf is below 20 µg per g dry weight. Lapsi leaf protease is an specific endopeptidase attacking peptide bonds that have phenylalanine, tyrosine, alanine and threonine / aspartic acid residues.

Introduction

Choerospondias axillaries:(Locally called “lapsi”), is a large, deciduous fruit-bearing tree of the family Anacardiaceae.A native of the Nepal hills (850–1900m) Lapsi wood is used as light construction timber and fuel wood; seed stones are used as fuel in brick kilns and the bark has medicinal value. Nepal is unique in processing and utilizing lapsi fruits. The fruits are rich in vitamin C content.

Agrawal and Kesari (1992) were first to observe strong proteotytic activity in Lapsi leaves. Lapsi Protease was found to be active even under autoclave condition. Dekhang and Sharma (2006) reported optimium pH of 7 for the protease. The protease is not inhibited at all by phenylmethanesulfonylfluoride ( PMSF ) and 20-30% inhibited by sodium iodoacetic acid, thus revealing that protease is not a serine protease. No smaller proteolytic products of BSA could be seen in SDS-PAGE using silver staining indicating that the protease is not exopeptidase, Protease activity can be repeatedly precipitated by 0.2 M tricloroacetic acid TCA (Singh and Giri 2007). In order to find an insight into the mechanism of protease action, the present research was carried out

______________________________

The research was done for the fulfilment of degree requirement for B. Sc. Biochemistry (SK and RS)


Materials and Methods

Preparation of Lapsi Leaf Powder : Dried leaves of lapsi were blended in a glass blender to get fine powder.

Partial purification and concentration of protease : It involved following steps.

1. Washing of the lapsi powder (5gm) with 50 ml acetone
2. Extraction of acetone washed powder with 10 ml phosphate buffer (0.1M of pH 7).
3. Heat treatment of the extract at 70. ºC for 30min
` 4. TCA precipitation of protein in the extract by adding TCA (2.45M) to heat treated solution to a final concentration of 0.2M followed by keeping in freeze for 10min, centrifuging at 10000rpm/10min and dissolving the pellet dissolve in 200μl of phosphate buffer.

Determination of proteolytic activity :

A typical reaction mixture cntaining 28.75μg of protein and 50 μg BSA in the total volume of 200μl of 0.1M pH 7 phosphate buffer was incubated for 30 min . The proteolytic activity is measured by 3 following methods.

1. Direct Method : The reaction was stopped by adding Bradford reagent for determination of BSA ( Bradford 1976, Saleemudin 1980 ).

2. Indirect Method : The reaction was stopped by adding 2.45M TCA to a final concentration is 0.2M, centrifuged at 10,000rpm/10min and pellet dissolved in 200μl of phosphate buffer (0.1M of pH-7) and protein determined by Bradford method.

3. Determination of amino acid produced in reaction .by Ninhydrin method :
To the reaction mixture (100μl), 0.9ml distilled water and 4ml Ninhydrin (0.5% in ethanol) reagent were added and heated at 80ºC for 10 min, cooled and absorbance measured at 570nm. Alanine was used as standard.

Detection of amino acids by paper chromatography :
Ascending paper chromatography of the reaction mixture (60μl 0.1% isopropanol) was done on Whatman paper 1 using Butanol: Glacial acetic acid: water (40:10:50 V/V) as the solvent system. Chromatogram was dried in air for 6 hrs.Amino acid spots were made visible by spraying the chromatogram with 0.2% Ninhydrin reagent. Then the purple coloured zones were marked and Rf value calculated.


Determined RF value of Standard Amino acid used.

Amino-Acids Rf-value Amino-Acids Rf-value
Alanine 0.35 Threonine 0.27
Tryptophan 0.63 Valine 0.8
Leucine 0.89 Aspartic acid 0.25
Iso-leucine 0.86 Serine 0.15
Phenylalanine 0.85 Cystine 0.05
Glycine 0.1 Arginine 0.07
Histidine 0.08 Glutamic acid 0.2
Tryrosine 0.45 Lysine 0.06

Results and Discussion

In order to measure the effect of enzyme concentration on protease activity, the double TCA precipitated enzyme (50μl) preparation (5.75ug/10μl) was used. It was noticed that enzyme activity was linear up to 28.75μg of protein. In order to determine the effect of time of incubation on protease activity, 50μl of double TCA precipitation enzyme (28.75μg) of protein and 50μg of the substrate (BSA) were used and incubated for various periods. Result obtained showed that the proteolytic activity was linear only up to 30min. Therefore, further experiments were carried out using 28.75μgm of protein and 30mins of incubation.In order to determine the Km and Vmax by Lineweaver-bulk plot, the effect of BSA concentration on enzyme activity was measured.. It was found that protease has Km of 29.1μM for BSA and Vmax of 52.63pmoles/min.A low value of Km indicates that the substrate is tightly bound to enzyme.

Since BSA has 607 amino acids , 607nmoles of amino acids should be produced per nmoles of BSA degraded. In order to check this equivalency, the amino aid contents of reaction mixture were analysed ( Table 1 ).

Table 1 : Comparison of experimental and the theoretical values of amino acid content of reaction mixture

BSA (μg)
a BSA degraded(μg)
b Theoretical value of amino acids produced
( nmoles)
c Experimental value of amino acids produced ( nmoles)
D c/d Calculated no. of free amino aacids
10 5.5 48.207 13.6 0282 171
20 12 105.180 28 0.266 161
50 26.8 234.904 65 0.277 168
100 60.4 529.410 145 0.273 166

*For BSA degradation, reaction mixture containing 28.75μg of enzyme and equired amount of BSA in total volume of 200μl of 0.1 M pH 7 phosphate buffer .After 30 min of incubation 100 μl of the mixture was used for protein determination using direct method.
*molecular weight of BSA 69323.4 Da, 1 mole of BSA contains 607 moles of amino acids
( Hilger et al. ) ; 50μg of BSA contains 0 .722 nmoles of BSA.
*For amino acid determination, to the reaction mixture (100 μl ) 0. 8.ml water was added and ninhydrin method used.

It was observed that upon proteolysis one molecule of BSA yielded only 161 -171 molecules of amino acids ( Table 1 ) suggesting that in addition to free amino acids 70 – 75 % BSA is degraded to smaller peptides which cannot be observed in SDS-PAGE as well as not amenable to the Ninhydrin method.

In order to determine the identity of released amino acids during proteolysis of BSA ,we embarked upon the paper chromatography of the reaction mixture . In paper chromatogram, only 4 spots could be seen. Comparing with the Rf-values of standard amino acids ( Fig. 1 ) we concluded that amino acids produced by degradation of BSA are phenylalanine, tyrosine, alanine and threonine / aspartic acid.

Table 2. Rf valuue of amino acids

Sample(enzyme) Rf-value Amino acid
Spot A 0.26 Thr , Asp
Spot B 0.35 Ala
Spot C 0.44 Tyr
Spot D 0.85 Phe

So, we conclude that lapsi protease is very special type of protease. It’s not a exopeptidase, it is an endopeptidase with very specific activity attacking peptide bonds containing phenylalanine, tyrosine, alanine, threonine / aspartic acid residues. A serious literature search shows that such type of protease has not been reported in the literature.

References

Agrawal VP, Keshari and Singh D, (1992) Study of Lapsi (Choreospondias axillaris) Protease Unpublished result.

Bradford MM (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72: 248-254.

Dekhang RN and Sharma G (2006) Study of Protease from the leaves of Choreospondias axillaries (Lapsi). Submitted to Universal Science College, Biochemistry Department for the fulfilment of degree requirement of . B. Sc. Biochemistry.

Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Hentges F ( 2001 ) Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. J. Clin. Exp. Immupol. 123 (3), 387-394. http://www.ncbi.nlm.nih.gov/protein/3336842?report=genpep

Saleemudin M, Ahmad H and Hussain A. (1980) A Simple, Rpapid and Sensitive Procedure for the Assay of endoprotease using Coomassie Brilliant Blue G-250. Analytical Biochemistry 105: 202-205.

Singh R and Giri S (2007) Characterization and purification of protease from leaves Choerospondias Axillaris (Lapsi).Submitted to Universal Science College, Biochemistry Department for the fulfilment of degree requirement of B.Sc. Biochemistry.

Friday, July 17, 2009

Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

Shrestha KO1, Adhikari S1, Tiwari KB1,2 and Agrawal VP1,2

1Universal Science College, Maitidevi, Kathmandu, Nepal

2Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal


Abstract

Lactose utilizing yeasts have a potential application in the lactose pollution management in brewery and dairy industries as the organisms can grow at environmental conditions well. Eleven strains of lactose positive yeast were isolated from murcha samples collected from local markets in Kathmandu. Because of highest lactose positive activity among the isolated strains, L4 was selected for production, partially purification and characterization of β-galactosidase. Mass culture of the strain was treated with 2% (v/v) Chloroform to disrupt the cells. The enzyme was purified with Acetone precipitation method to 7.7 fold activity with 77.6% yield; specific activity was found to be 0.054 nmol/min/mg of protein. Optimum pH and temperature were determined to be 6.6 and 37°C respectively with the reaction time period 180 min. Michaelis-Menten constant (Km) value of the enzyme was found to be 8.798 mM of O-nitrophenyl-β-D-galactopyranoside.

Introduction

Murcha is a mixed starter inoculum, used in production of local alcoholic beverages in India, Tibet, Nepal, Bhutan etc. (Tsuyoshi et al., 2005). Murcha is a round cake, which is mildly acidic and has a pH around 5.2 containing 13% w/w moisture and 0.7% w/w ash (dry weight basis). The Murcha cakes contain mixed microbial population viz. molds, yeasts and bacteria (Tamang and Sarkar, 1995).

Yeasts are the world's premier industrial microorganisms, which have wide exploitation in the production of foods, beverages and pharmaceuticals. Alcoholic beverages are one of the major products in the world’s market. Yeasts can contaminate different dairy products because they have relatively low water activity (aw, 0.88) (Frazier and Westhoff, 1995), can easily grow at room temperature and can utilize (assimilate and/or ferment) a variety of carbohydrates (Nahvi and Moeini, 2004), eg. pentoses, hexoses, disaccharides and, rarely polysaccharides (Barnett et al., 1990). The capacities of the organisms, thus, can be exploited to manage the biodegradeable wastes of the food, dairy and beverage industries. Most of the yeasts, except Saccharomyces spp., can grow on cellulosic materials, however; only few genera are able to degrade starch. Among disaccharides, lactose is one of the most refractory carbon substrate to most of the yeasts. Among hundreds of genera, only few are lactose positive, viz. many Cryptococcus spp. and Trichosporon spp.; some Debaryomyces spp., Kluyveromyces spp. and Myxozyma spp.; and occasionally Bullera spp., Candida spp., Rhodotorula spp. and Tremella spp. (Barnett et al., 1990). Lactose is one of the major components in whey in cheese industry that is non-friendly in the environment (Nahvi and Moeini, 2004). As they can grow at 25-300C, yeasts can be exploited to manage lactose pollution (Sarova and Nikolova, 2002).

Yeast has been considered the predominant microbial enzyme source for food applications. Lactose utilizing yeast are good source of β-galactosidase. With rigorous search, Tiwari et al (2008) elucidated the presence of abundant lactose positive yeasts in murcha samples collected from various parts of Nepal. Hence, the study was designed to collect various yeasts capable to utilize lactose efficiently, and production, purification and characterization of β-galactosidase.

Materials and Methods

Isolation: Murcha samples were collected from local markets in Kathmandu, Nepal. The pure culture of yeast strains were isolated by serial dilution methods in Potato Dextrose Agar (PDA) (20% potato extract, 2% dextrose, and 1.5% agar, pH 4.5) incubating for 48 hr at 270C. The purified isolates were stored on PDA slant at 40C. The pure culture of the isolates was Gram stained for microscopic morphology.

Sugar assimilation test: The sugars used were glucose, fructose, sucrose, maltose, mannitol, galactose, lactose, and arabinose using a basal medium (4.5gm yeast extract, 7.5gm peptone and 20gm sugar in 1-lit distilled water) with phenol red (1mg/ml) as indicator. The pH was adjusted to 7.0-8.0. The medium was dispensed into tubes and sterilized by autoclaving. The pure culture of the isolates was incubated at 270C till 72 hr. in the tubes and the result was indicated by change of color from red to yellow (Shrestha and Sharma, 1995). Control tubes were used in each set to monitor contamination.

Yeast Propagation: Sterile basal media containing ammonium sulphate, yeast extract and potassium dihydrogen phosphate was prepared with pH 4.5. Filter sterilized lactose (0.05M) was added to the 100 ml broth and yeast was inoculated and was incubated in waterbath shaker at 30°C for 48 hour.

Enzyme Extraction: The broth was centrifuged at 7000 rpm for 10 min. and the pellet was resuspended in 80ml phosphate buffer of pH 6.8. Chloroform (2%, v/v) was added and incubated overnight at 28°C. Incubated cells were centrifuged at 10000 rpm for 20 min. Supernatant was collected and one volume of acetone was added. The mixture was shaken in vertex shaker and centrifuged at 10000 rpm for 20 min. Pellets were collected and dissolved in 16ml of phosphate buffer with pH 6.8.

Enzyme Activity: β-galactosidase assay was done with O-nitrophenol β-D-galactopiranoside (ONPG) as substrate. Two test tubes were filled with 3ml of 0.01M of ONPG prepared in supplemented phosphate buffer of pH 6.8. Enzyme extract (1ml) was added to the test solution and reaction progress was determined spectrophotometrically against the blank tube at 420nm for every 30 min. for 3 hours. The reaction was quenched by adding 1ml of 0.5M sodium carbonate.

Determination of optimum time duration for reaction: Seven test tubes were filled with 3ml of 0.01M ONPG prepared in supplemented phosphate buffer of pH 6.8. Enzyme extract (1ml) was added in each tube and incubated at 37°C. By adding 1ml of 0.5M sodium carbonate, the reaction was quenched at every 30 min interval for respective tube and reaction progress was measured at 420nm against the blank.

Determination of optimum pH: Seven different test tubes were taken and 3ml of 0.01M ONPG prepared in buffer of pH 5.5, 6.0, 6.2, 6.4, 6.6, 6.8 and 7.0 was added to each respective test tube. Enzyme extract (1ml) was added to each test tube and incubated at 37°C for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Determination of optimum temperature: Seven test tubes were taken and 3ml of 0.01M ONPG prepared in phosphate buffer of pH 6.6 was added to each test tube. Enzyme extract 1ml was added to each test tube and incubated separately at 0°C, 15°C, 28°C, 37°C, 40°C, 45°C and 50°C respectively for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Determination of KM value: ONPG solution (0.01M) was prepared in phosphate buffer of pH 6.6, and 0.6ml, 1.2ml, 1.8ml, 2.4ml, 3.0ml of the substrate was added in a series of five test tubes respectively. The final volume was set to 3ml by adding phosphate buffer of pH 6.6. Enzyme extract 1ml was added to each test tube and incubated at 37°C for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.


Results:

L4 strain those posses highest lactose positive activity was selected for production, partially purification and characterization of β-galactosidase. The enzyme was purified with Acetone precipitation method to 7.7 fold activity with 77.6% yield; specific activity was found to be 0.054 nmol/min/mg of protein (Table 1). Optimum pH and temperature were determined to be 6.6 (Fig. 1) and 37°C (Fig. 2) respectively with the reaction time period 180 min (Fig. 3). Michaelis-Menten constant (Km) value of the enzyme was found to be 8.798 mM of O-nitrophenyl-β-D-galactopyranoside (Fig. 4).

Discussion:

Yeasts are one of the important organisms having a wide industrial application. Most of the genera are unable to assimilate lactose as carbon source (Barnett et al., 1990). A higher abundance of the yeasts capable to produce β-galactosidase and thus assimilate lactose explored the importance of Nepalese yeast cakes (Murcha). Nahvi and Moeini (2004) reported three (3/30, 10.0%) β-galactosidase positive strains in Iran. In this study out of 148 isolates, only 11 were lactose positive.

The β-galactosidase from the yeast isolated from marcha on this study was characterized by their pH optimum at neutral or on the weekly acidic side. In general, β-galactosidase from yeast and bacteria have pH optimum near the neutral region, whereas the enzyme from molds act well at more acidic level as reported by Wierzbicki and Kosikowski (1973). The β-galactosidase of yeast was relatively heat labile, but they hydrolyze substrate well at lower temperature. The optimal hydrolysis was attended at 37°C and nearly 60% of hydrolyzing ability was maintained at 14°C. An enzyme workable at low temperature as possible is preferred for treatment of foods like milk. The Km value with ONPG as a substrate is slightly higher than reported in K. lactis by Kim and Lim (1981).

β-galactosidase commercially can be extracted from yeast. Its primary commercial use is to break down lactose in milk to make it suitable for people with lactose intolerance. β-galactosidase is also used in the manufacture of ice cream. Because glucose and galactose are sweeter than lactose, β-galactosidase produces a more pleasant taste. Lactose positive yeast strains can be used for removal of whey pollutants, Single cell protein (SCP) and ethanol production and treatment of lactose in dairy industry (Nahvi and Moeini, 2004).

Whey is the aqueous fraction of milk generated a by-product of cheese manufacturing which is produced in large amounts. The main solute in cheese whey is lactose present at a concentration of about 4.5-5% (Rohm et al., 1992; Zadow, 1992). Because of its high organic content, dumping directly to the environment causes serious contamination problems. As a solution, bioconversion of whey into SCP or ethanol has been performed in several countries (Gonzales, 1996; Irvine and Hill, 1985; Mawson, 1994). SCP could be produced from whey with employing of yeasts from different species including Kluyveromyces spp., Candida spp. and Trichosporon spp. as they are naturally able to metabolize lactose (Castillo, 1990). Although species of yeasts may differ considerably in their physiology, those of industrial importance have enough physiological characteristics in common to permit generalizations, provided that it is kept in mind that there will be exceptions to every statement made (Frazier and Westhoff, 1995). The findings explored that these yeasts may be new strains and lead to a search of yeast strains having high β-galactosidase activity (Nahvi and Moeini, 2004) as more efficient bioactive agents.

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