journal of life sciences and technologies vol. 4, no. 2 ... · pdf fileshahirah [2] from...

Purification of a Thermostable Lipase

Geobacillus thermodenitrificans nr68 (Lip.nr-68)

with Potential for Enzymatic Deinking

Nik Raikhan Nik Him

Bioprocess Engineering Department, Faculty of Chemical Engineering, Universiti Teknologi MARA

(UiTM), 40450 Shah Alam, Selangor, Malaysia

Email: [email protected]

Abstract—Lipase from Geobacillus thermodenitrificans nr68

(Lip.nr-68) has shown great enzymatic bio-deinking activity

towards a laser jet printed paper with deinkability

brightness test of 55%, a value that was slightly lower than

the value showed by a commercial lipase from Sigma (63%).

In regards of this quality, Lip.nr-68 was purified and

characterized to obtain the pure biological characteristic of

this catalyst. Airlift fermenter system was used with

optimum parameters of 65°C, pH of 6.8, and air flow rate of

1.00 L/min and inoculum size at 7.0% (v/v) in a cultivation

medium containing; glucose of 1.25% (w/v); yeast extract of

1.25% (w/v); NaCl 0.75% (w/v) and olive oil of 0.10% (v/v)

for 24 hours. The extracted extracellular crude lipase was

purified to homogeneity by using four-step procedures:

acetone precipitation, Sephadex G-100 filtration

chromatography and twice of DEAE Sefarose CL-6B anion

exchange chromatography by 22.1 times with a final yield of

25%. The molecular weight of the purified enzyme was

estimated to be 33.5 kDa after SDS-PAGE analysis.

Index Terms—lipase, enzymatic deinking, Geobacillus thermodenitrificans

I. INTRODUCTION

Lipase occupies a prominent place among biocatalysts

and has a wide spectrum of biotechnological applications,

very unique and appears to be a great catalyst for

hydrolyzing fats into fatty acids and glycerol at the water-

lipid interface as well as it can reverse the reaction in

non-aqueous media. Of so many of the lipase functions,

bio-deinking is currently being approached very well to

be practiced in the pulp ink-depolarization. For example,

commercial lipase has been extensively used in

enzymatic deinking of laser jet paper and in the recycling

of office waste paper such as inkjet and photocopy papers.

Unfortunately, these commercial lipase enzymes are

established lipase enzymes that has been accepted for

many other functions but very limited reports has been

published from newly isolated or lab-scale produced

microbial lipase for the same bio-deinking purposes.

Microbial lipases are produced mostly by submerged

culture or some solid state fermentation methods but the

second appears to be an interesting alternative for

Manuscript received April 10, 2016; revised July 18, 2016.

microbial enzyme production due to the possibility of

using residues and by-products of agro-industries as

nutrient sources and support for microorganism

development [1]. The use of by-products as substrates for

lipase production has added higher value to the process

and at the same time providing lower cost during the bio-

deinking process taking place. Shahirah [2] from

Universiti Malaysia Sarawak has been doing research on

bio-deinking of mixed office paper using the crude

enzymes produced by Bacillus amyloliquefaciens UMAS

1002 and Bacillus licheniformis P1 but the enzyme was

not purified. It is vital that very interesting enzymes

related to the bio-deinking isolated from the local sources

to be purified and to be characterized. This is in order to

overcome the issue of enzyme shortage as well as to

reduce the usage of the readymade commercial enzyme

that requires high cost.

The three major sources of raw material for such

recycling are newsprint, photocopied paper and inkjet

printed papers. Recycling of paper requires the removal

of the printing ink, also called deinking, from the used

paper to obtain brighter pulp. Printing on paper is

generally carried out by using impact or non-impact ink.

Impact ink, generally used for newsprint does not fuse

with the paper and therefore is easy to remove or disperse

during the deinking process [3]. This paper is focusing on a purification of lipase from

Geobacillus thermodenitrificans nr68 (Lip.nr-68), a

thermotolerant Malaysian isolated species [4], [5]. This

enzyme has been tested to have high capability on

enzymatic deinking and was significantly showed

comparable results to a commercial lipase (LS) from

Sigma with deinkability factor of 55%. This value is

slightly lower than the deinkability factoring of LS itself

(63%). Santosh & Anil [6] have reported the use of

alkaline active cellulose from Fusarium sp. and details

about the enzyme characteristics has been deeply

described for the purpose of enzymatic deinking. It is

important that more microbial lipases with the

deinkability characteristic to be isolated and being

purified in order to support the needs of more reliable

enzymes [7]. Therefore this paper is hoped to bring

important finding towards the success of microbial bio-

deinking to be shared among the related scientists.

70

Journal of Life Sciences and Technologies Vol. 4, No. 2, December 2016

© 2016 Journal of Life Sciences and Technologiesdoi: 10.18178/jolst.4.2.70-74

II. MATERIAL AND METHODS

A. The Microbial Source

The microbe used was a thermotolerant Gram negative

Geobacillus thermodenitrificans nr68. This species was

isolated from a hot spring in Kelantan, Malaysia [4], [5].

The temperature of the pond and the soil was 48°C. This

species has been identified using APi-20E, series

biochemical tests, Gram staining test and 16S rRNA

analysis. Fig. 1 shows rod shape Gram negative

Geobacillus thermodenitrificans nr68 while Fig. 2 shows

the SEM micrograph of 24 hours Geobacillus

thermodenitrificans grown on nutrient agar slant [8].

Figure 1. Gram negative-stained Geobacillus thermodenitrificans nr68. Source: [4].

Figure 2. SEM micrograph of 24 hours Geobacillus

thermodenitrificans grown on nutrient agar slant. Source: [8].

B. Production and Media Development for Lip.nr-68

Circular airlift fermenter system was used with

optimum parameters of 65°C, pH of 6.8, air flow rate of

1.00 L/min and inoculum size at 7.0% (v/v). The

production medium contains 0.75% (w/v) glucose, 1.25%

(w/v) yeast extract, 0.45% (w/v) NaCl and 0.10% (v/v)

olive oil [4]. The specific growth, μ of the Geobacillus

thermodenitrificans nr68 was highest with 1.25% glucose

(w/v) and flow rate of 1.0 L/min (0.12 hour-1

). This

cultivation has the lowest doubling time, td with value of

5.77 hours, the rate of enzyme formation, dp/dt of 1.73

Uml-1

h-1

, a very high rate of specific enzyme production

(11700.0 U/g of mass), the rate of glucose used, ds/dt of

0.22 gL-1

h-1

and formation of enzyme per gram, 4110.0

U/g of glucose.

C. Enzyme and Assays

Lipase from Geobacillus thermodenitrificans nr68

(Lip.nr-68) was used. Enzyme activities were assayed by

olive oil emulsion method modified from Mustranta et al.

[7]. Substrate was prepared by homogenizing 30 mL of

olive oil with 70 mL of emulsification reagent. [5]. One

unit of lipase represented the release of 1µmol of fatty

acid per min respectively under the above assay condition

[9].

D. Protein Content Determination

Protein content in the cell-free supernatant was done

using bovine serum albumin as standard according to

method by Lowry [10].

E. Enzyme Purification

The collected extracellular crude Lip.nr-68 was

purified using four-step procedures: acetone precipitation

to concentrate the crude lipase, Sephadex G-100 filtration

chromatography and twice of DEAE Sefarose CL-6B

filtration chromatography.

F. Acetone Precipitation

80% (v/v) of cold acetone (4:1 analytical grade acetone

to dH2O at -20°C) was added into 200 ml of crude Lip.nr-

68, the mixture was vortex before placing at -20°C for

overnight [4]. It was then washed twice with new cold

acetone and was span at 13,000 x g for 10 minutes at 4°C

using Sorvall, RC-5C. The supernatant was carefully

poured off and pellet was dissolved in 5.0 ml of 0.2 M

(pH 6.8) phosphate buffer. The final washing supernatant

was poured off before samples were vortex. Crude

Lip.nr-68 showed an activity of 20.50 U/ml and after the

acetone precipitation it was 46.23 U/ml. The enzyme was

going through dialysis for 24h using a 10.0 mm wide

dialysis tube (Spectrum Medical Industries, 10,000 D)

prepared by Sugihara et al. [11]. Enzyme activity after

dialysis was recorded as 58.22 U/ml.

G. Gel-Filtration Chromatography

Sephadex G-100 (Sigma) was loaded into 80.0 cm ×

3.0 cm column equilibrated with 0.2 M phosphate buffer,

pH 6.8 and 1.0 ml concentrated Lip.nr-68 was then eluted

with the same buffer with a flow rate of 20 mL/hour.

Fractions of 5 mL were collected [12].

H. DEAE Sefarose CL-6B Anion Exchange

Chromatography (1)

Fractions of 10-20, 30-45 and 55-65 with the highest

activities of Lip.nr-68 after gel-filtration were used. They

were concentrated using ultrafiltration membrane

(minitans, mv cut-off 10,000) and were eluted into the

DEAE Sefarose CL-6B packed on 2.5 x 40 cm column at

16 ml/hour and fraction of 4.0 ml. The column was

before prepared according to [4]. Lip.nr-68 was eluted at

linearity of 0.50 L from 0 to 0.5 M NaCl with the same

buffer. The purity of Lip.nr-68 collected from fraction no

15-30 and 35-55 was studied.

I. DEAE Sefarose CL-6B Anion Exchange

Chromatography (2)

Lip.nr-68 collected from fraction no 15-30 and 35-55

were dialysis in 0.2 M phosphate buffer (pH 6.8) over-

night followed by steps in [6] but using 0 to 0.2 M NaCl.

Lip.nr-68 activity was recorded at fractions 30-50 and

one single band was seen on the SDS PAGE.

J. Determination of Lip.nr-68 GT Molecular Weight

Using SDS-PAGE

The molecular mass of the purified lipase was

determined by SDS-PAGE as described by Laemmli [13]

71


© 2016 Journal of Life Sciences and Technologies

using denatured protein and 12.5% of acrylamide gel.

Bromophenol stained; small molecular weight protein

markers were used (14.4-97.0 kDA, Pharmacia Biotech).

Rf was measured by length of Lip.nr-68 band movement

over length of marker. The gel was stained with silver

staining method as de-scribed by Bollag et al. [14]

(a)

(b)

(c)

Figure 3. (a) Profile of crude Lip.nr-68 using Sephadex G- 100, (b)

Sepharose CL-6B(1) and (c) Sepharose CL-6B(2).

III. RESULTS AND DISCUSSION

Geobacillus thermodenitrificans n68 is compatible to

the species reported by Anuradha et al., [1] and is a kind

of very special hyperthermotolerant that grow very well

at 45-70°C, at pH 6-7 but very greatly performing the

best quality of activities at pH 6.8 [4]. The growth too is

very much being affected by the content of NaCl and the

best range is between 0.25-0.30%. The typical cells of

Geobacillus thermodenitrificans nr68 is shown in the

above Fig. 1 and Fig. 2. The extracellular lipase from

Geobacillus thermodenitrificans nr68 was purified using

a four-steps procedures; ultrafiltration, Sephadex G-100

gel filtration chromatography and two (2) times of DEAE

Sefarose CL-6B anion exchange chromatography.

Acetone precipitation has yielded the amount of 89.9%

with Lip.nr-68 activity of 3768 U and has decreased to

86.4% or 3428.4 U after an overnight of dialysis. This

result is expected as dialysis leads to the net movement of

molecules from an area of higher concentration to a lower

concentration until equilibrium is reached, therefore

giving lower enzyme Unit activity. Fig. 3a shows profile

of crude Lip.nr-68 using Sephadex G-100 with 3 major

peaks from fractions 10-20, 30-45 and 55-65 with total

activity of 2550.6 U. Foreign non-lipase protein was

detected from fractions 5-10 and 30-60. Gel Sepharose

CL-6B(1) shows 2 peaks equivalent to Lip.nr-68 enzyme

protein peaks (Fig. 3b) and CL-6B(2) exhibited single

peak P1 by 22.1 times, with specific activity at 17.2 U/mg

proteins and yield of 25.0% at fraction 35-55 (Fig. 3c).

The purification of Lip.nr-68 is summarized in Table I.

Total protein decreased significantly during the process

but total activity was maintained and so the increment of

the specific activity of the Lip.nr-68. The same result has

been shown by Jesus et al. using lipase from Penicillium

restrictum [15]. From the result obtained, it is believed

that there was no hydrophobic interaction has involved

during elution of Lip.nr-68 in all three columns.

Figure 4. SDS-PAGE of thermostable Lip.nr-68. Lane 1: Denatured enzyme. Lane 2: Lip.nr-68 on G- 100. Lane 3: Lip.nr-68 on CL-6B(1). Lane 4: Lip.nr-68 on CL-6B(2), purified lipase. Lane 5:

Protein markers.

TABLE I. SUMMARY OF PROTEIN PURIFICATION OF LIP.NR-68

Purification

Steps

Protein

Total

(mg)

Total

Lip.nr-

68

Activity

(U)

Lip.nr-

68

Specific

Activity

(U/mg)

Yield

(%)

Purity

in fold

Crude lipase 5744.20 3868.0 0.67 100.00 1.00

Acetone

precipitation

1880.2 3868.0 2.00 89.9 2.31

Lip.nr-68 after 24 h

dialysis

1020.2 3768.0 3.36 86.4 3.51

Sephadex

G-100

745.0 3428.4 4.17 78.4 4.38

Sefarose

CL6B-1

156.9 3112.8 8.49 33.5 10.88

Sefarose

CL6B-2

56.89 1332.5 17.2 25.0 22.1

To determine the molecular weight of an unknown

protein (the Lip.nr-68), the protein sample was separated

on the same gel with a set of molecular weight standards.

In this paper, we were using lower molecular weight

standard with set of markers varies in size from

phosphorilase b (97 kDa), albumin (66 kDa), ovalbumin

(45 kDa), carbonic anhydrase (30 kDa), tripsin inhibitor

72



(20.1 kDa) and α-lactalbumin (14.4 kDa) [4], [15]. After

running the standards and the unknown size of Lip.nr-68

protein samples, the gel was processed with the

Coomassie Brilliant Blue R-250 and then de-stained for

about 12 to 14 hours to visualize the protein bands. The

single Lip.nr-68 protein band is shown in Fig. 4.

The Lip.nr-68 is calculated to have a molecular weight

of 33.5 kDa by using Rf method (Fig. 5). When using a

uniform density, the relative migration distance of a

protein known as Rf, (where f is a subscript) is negatively

proportional to the log of its mass, so we could estimate

the unknown protein molecular weight using the plotted

graph. After running the gel, determination of the relative

migration distance (Rf) of the protein standards and the

unknown size Lip.nr-68 protein was calculated using the

following equation:

Migration distance of the dye front

Plotting the Rf will generate a linear plot for most

proteins when the protein samples are fully denatured and

the gel percentage is appropriate for the molecular weight

range [15]. Alternatively, appropriate software may also

be used to determine the Rf values of the resulting bands

to compare with the calculated value in Fig. 5. As

expected, the Lip.nr-68 has a moderate protein size. This

result is significant because according to the researchers

who are working on microbial lipases, most reported

lipase protein sizes are between 20-60 kDa [10]. Lipase

from Bacillus are reported to have low molecular weight

of ~20 kDa [2] and whereas thermophilic lipase BTID-B

from Bacillus thermoleovorans ID-1 purified by Lee et al.

[7] was about 43 kDa. The Lip.nr-68 protein size is with

great advantages because they are more stable to wide

temperature range. This characteristic is believed to be

supported by the smaller changes (unfolding) in the

protein’s tertiary structure [8]. Interpolating the value

from Fig. 5 has been giving the size of the Lip.nr-68 but

it is important to verify the accuracy of the calculated

value as this method may somehow contributed to about

3% to 4% of inaccuracy [4]. This is due to the presence of

polypeptides such as glycol- and lipoproteins that usually

leads to erroneous results since they are not fully coated

with SDS and thus, would not behave as expected [16].

Figure 5. Rf for markers and Lip.nr-68 with the molecular weights.

Lip.nr-68 has been calculated to have a moderate size molecular

weight of 33.5 kDa.

IV. CONCLUSION

Lipase enzyme produced by locally isolated

hyperthermotolerant Geobacillus thermodenitrificans

nr68 (Lip.nr-68) with high potential to be further used to

deink laser-printed paper has been purified with 22.1 fold.

The molecular weight of Lip.nr-68 was confirmed as 33.5

kDa. It was a moderate protein size but a very significant

size to other microbial lipases as reported by other

researchers. To further study the bio-deinking capability,

we are looking forward to start a new trial, using fully

immobilized cells for newspaper bio-deinking.

ACKNOWLEDGMENT

The authors gratefully acknowledge the Ministry of

Higher Education (MOHE) for the Research

Acculturation Grant Scheme (RAGS), Universiti

Teknologi MARA (UiTM) and UiTM Research

Management Centre (RMC) for the fund and all the

facilities used during the research.

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73



Rf = Migration distance of the protein

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Dr. Raikhan is with 12 years of research experiences in biochemistry,

kinetics, microbiology, biotechnology, enzymatic studies, esterification,

fermentation, anti-cancer, anti-fungal and lipase base work. Holding PhD in Industrial Biotechnology and majoring in Enzyme and Chemical

Engineering. Currently working with biosensors, bio-deinking and

bioprocess team.

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