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CHAPTER 4

DOWNSTREAM PROCESSING OF PROTEASE

4.1 INTRODUCTION

After successful fermentation, when the fermented medium leaves

the controlled environment of the fermentor, it is exposed to drastic changes

in environmental conditions. It is therefore necessary that the product be

recovered as much as possible along with retention of its functional activity

for the desired application. This step is collectively termed as downstream

processing or bio-separation. These steps can account for up to 60 percent of

the total production costs, excluding the cost of the purchased raw materials

(Cliffe 1988). The downstream strategy depends on the desired end product

nature. The fermentation product may be extracellular (within the

fermentation broth) or intracellular (inside the cells). If the product of our

interest is of extracellular, cells are separated from the fermentation broth and

the products in dilute aqueous medium need to be recovered and purified. The

intracellular products can be released by rupturing the cells by various means

viz. mechanical or chemical and then they can be recovered and purified. The

downstream processing for intracellular enzyme will be similar to that of

extracellular products.

Partial purification/concentration of enzymes are generally carried

out by various techniques such as salting out (Towatana et al 1999), solvent

precipitation (Kumar et al 1999), spray drying (Namaldi et al 2006; Kamoun

et al 2008), flocculation (Collingwood et al 1988, 1989) and ultrafiltration

98

(Singh et al 2001). Apart from classical partial purification techniques,

alternative strategies such as aqueous two-phase extraction (ATPE) for

partitioning of protease have been reported recently (Chouyyok et al 2005). A

common impediment to the production of commercial enzymes is their

marginal stability in aqueous solution since water facilitates or mediates a

variety of chemical and/or physical degradation pathways operative during

protein purification, storage and delivery (Arakawa et al 1993). To improve

the shelf life of protease and make the preparation stable, dehydration

methods such as freeze and spray drying have been attempted.

In this chapter, studies have been carried out to develop and

compare important strategies on downstream processing such as salting out,

ultrafiltration, flocculation and spray drying of the alkaline protease from

Bacillus pumilus MTCC 7514. Since many industrial applications call for the

use of partially pure stable protease preparations, these methods are

developed keeping in view the storage stability, cost effectiveness, reduction

in number of downstream processing steps, higher recovery and end use of

protease.

4.2 MATERIALS AND METHODS

4.2.1 Medium for Protease Production

The optimized medium (Chapter II) used for protease production

was composed of (g/L): soya flour, 11; NaCl, 1.2; MgSO4.7H2O, 0.6;

KH2PO4, 0.6 and CaCl2.2H2O, 0.3. Inorganic salt solution was prepared,

autoclaved separately and added to the medium aseptically. Sodium

hydroxide (0.1 M) was used to adjust the initial medium pH to 7.0. The

enzyme developed as described in Chapter II was used for the study.

99

4.2.2 Enzyme Assay and Protein Estimation

Crude enzyme as well as the partially purified enzymes were

suitably diluted and activity was estimated by Kunitz method (1947) with

slight modification as mentioned in Chapter II and protein concentration of

the samples were determined according to Lowry et al (1951) using

crystalline bovine serum albumin (BSA) as the reference standard.

4.2.3 Ammonium Sulfate Precipitation

Proteins can be precipitated from crude enzyme by the addition of

concentrated salt solution like ammonium sulphate and this process is also

called “salting out”.

The fermented broth was centrifuged to remove the biomass and

cell free supernatant was used for the precipitation studies. 100 mL crude

enzyme was subjected to different levels of ammonium sulfate saturation

directly. It was stirred to dissolve the salt completely at 4 ºC for 30-40

minutes and then left overnight for saturation and complete precipitation of

enzyme. The resultant enzyme precipitate was collected by centrifuging

(Beckman centrifuge) (Figure A 4.1) at 10,000 rpm for 30 min at 4 ºC. The

enzyme precipitates from different fractions of ammonium sulfate

precipitation were dissolved in 0.1 M of Tris buffer, pH 9.0 and subsequently

quantified for protein and protease activity.

4.2.4 Ultrafiltration

Ultrafiltration (UF) is a membrane filtration in which hydrostatic

pressure forces a liquid against a semi permeable membrane. Solutes of

higher molecular weight than the pore size of membrane are retained, while

water and low molecular weight solutes pass through the membrane. This

100

separation process is used in industry and research labs, for purifying and

concentrating macromolecular solutions, especially protein solutions. The cell

free fermented broth was pre-filtered via microfiltration and then subjected to

ultrafiltration using a regenerated cellulose membrane with molecular weight

cut off (MWCO) of 10 KDa and the collected samples were subsequently

quantified for protein and protease activity. The membrane with a surface area

of 0.1 m2 (Millipore, India) was used to obtain about 5, 10 and 20 fold

concentrates. After the filtration, the cassettes are typically washed with 5 L

of 0.1 N NaOH followed by 5 L of 0.2% SDS and 2 L of 0.02% sodium azide

and then stored in 0.1 N NaOH solution at 4 ºC for further use.

4.2.5 Ultrafiltration and Ammonium Sulphate Precipitation

The cell free fermented broth (5 L) was subjected to ultrafiltration

using 10-kDa regenerated cellulose membrane with a surface area of 0.1 m2

(Millipore, India) at a pressure of 0.8-1.0 bar to obtain about five-fold

concentrate. To 100 mL aliquots of concentrate , ammonium sulphate was

added at 40, 60 and 80% saturation separately and equilibrated for 6-8 h at 4

C (cold room) and the precipitate was collected by centrifugation and

dissolved in 0.1 M Tris buffer, pH 9.0 and subsequently quantified for protein

and protease activity..

4.2.6 Spray Drying

Spray drying was performed using a lab scale spray dryer

(Labultima, Mumbai, India) and pilot scale spray drier (Niro Atomizer,

Copenhagen, Denmark). Crude enzyme as well as the five-fold ultrafiltered

concentrate was subjected to spray drying by keeping the inlet and outlet air

temperatures at 110 ± 5 °C and 60 ± 5 °C, respectively. The inlet and outlet

air temperatures for pilot scale spray drying were maintained at 130 ± 5 °C

and 70 ± 5 °C respectively. During operation, solutions were fed at a constant

101

flow rate of 2 mL/min at the lab scale spray dryer and 200 mL/min at pilot

scale spray dryer by means of a peristaltic pump to a feeder nozzle, where

atomization of the solution was established using compressed air. Hot air

entered the drying chamber in the same direction as the descending spray

droplets (co-current operation). Additives such as maltodextrin and white

dextrin, salts [TATA common salt, (NH4)2SO4] and starch either individually

or in combination at different concentrations ranging from 5-15% w/v were

added to the enzyme solution prior to spray drying. Spray dried powder was

collected and stored at 4 °C and analyzed for protein and protease activity.

A – Feed reservoir

B - Spray Drying

C – Cyclone separator and product collector

Figure 4.1 Spray dryer (Labultima)

A

B

C

102

4.2.7 Flocculation Studies of Protease

4.2.7.1 Flocculation of crude enzyme

The cell free supernatant (500 mL) was distributed in five numbers

of beakers and required amount of flocculant (FeCl3) from stock solution

(20%, w/v) was added slowly to adjust the concentration to 0.2, 0.4, 0.6, 0.8

and 1.0% whilst stirring (magnetic stirrer). The flasks were then kept at 4 °C

for 20-30 min to facilitate flocculation and centrifuged at 10,000 rpm for 20

min to collect the pelleted flocs. The enzyme pellets were then dissolved in

required amount of Tris buffer and checked for protein content as well as

protease activity.

4.2.7.2 Ammonium sulphate saturation followed by flocculation

The cell free supernatant (crude enzyme) obtained after

centrifugation of the cell culture was collected. 100 mL of crude enzyme was

taken in each of different 250 mL beakers, subjected to 60 and 80% of

ammonium sulphate saturation separately and equilibrated for 6-8 h at 4 °C.

The required amount of each flocculant [FeCl3, AlCl3, Al2(SO4)3 and Ca(OH)2]

was taken from the designated stock solution and added slowly to the

saturated enzyme solution to bring the concentration to 0.05, 0.1 and 0.2%

(w/v) while stirring except Ca(OH)2 which was added to bring the

concentration of 0.5 and 1.0%. The beakers were then placed at 4 °C for 20-30

min. Following this, all the flocculated samples were centrifuged at 10,000

rpm for 10 min and the collected pellets were checked for protein content and

protease activity.

4.2.7.3 Effect of pH adjustment on flocculation and enzyme recovery

The crude enzyme was subjected to 80% ammonium sulphate

saturation in 250 mL beakers containing 100 mL of enzyme. Flocculant

103

(FeCl3) was taken from the stock solution and added to the saturated enzyme

solution to adjust the concentration to 0.05, 0.1 and 0.2% (w/v) while

maintaining the pH at 7.5-8.0 using sodium hydoxide (2 M). In another set of

same experimentation, pH (7.5-8.0) was adjusted by using calcium hydroxide

(2.7 M).

4.2.7.4 Experimental design and optimization by response surface

methodology

Response surface methodology (RSM) consists of a group of

empirical techniques devoted to the evaluation of relations existing between a

cluster of controlled experimental factors and the measured responses. A prior

knowledge and understanding of the process variables under investigation are

necessary for achieving a more realistic model (Dilipkumar 2011). Based on

the previous experiments and results obtained, ammonium sulphate saturation

level, concentration of flocculant (FeCl3) and saturation time were selected as

significant factors which affect protease enzyme recovery. The central

composite design with 5 coded values (- , -1, 0, +1 and + ) was used to

evaluate the effect of significant factors as well as their interactions on the

recovery of protease enzyme. The axial distance alpha ( ) was chosen to be

1.682 to make this design rotatable type. A set of 20 experiments (Table 4.11)

with six centre points was carried out. The analysis of experimental data was

performed using the Design Expert software (Version 7.0.0 Stat-Ease Inc,

Minneapolis, MN 55413, USA). All variables were taken at a central coded

value of zero. The minimum and maximum ranges of variables investigated

are listed in (Table 4.1). Upon the completion of experiments, the average

maximum recovery of protease enzyme was taken as the response (Y). The

experimental results of RSM were fitted via the response surface regression

procedure, using the following second order polynomial equation:

104

Y = ß0 + ßiXi + ßiiXi2

+ ßijXiXj

Where Y is the predicted response, XiXj are independent variables,

ß0 is the offset term, ßi is the ith linear coefficient, ßii is the ith quadratic

coefficient, and ßij is the ijth interaction coefficient. However, in this study,

the independent variables were coded as A, B and C. Thus, the second order

polynomial equation can be presented as follows:

Y = ß0 + ß1A + ß2B2 + ß3C + ß11A2

+ ß22B2+ ß33C

2

+ ß12AB + ß13AC + ß23BC

The statistical significance of the model equation and the model

terms were evaluated via the Fisher’s test. The quality of fit of the second-

order polynomial model equation was expressed via the coefficient of

determination (R2) and the adjusted R2. The fitted polynomial equation was

then expressed in the form of three-dimensional surface plots, in order to

illustrate the relationship between the responses and the experimental levels

of each of the variables utilized in this study.

Table 4.1 Experimental domain for the CCRD

Factors SymbolCoded levels

(- ) (-1) 0 (+1) (+ )

Ammonium sulphate (%

saturation)A 53.18 60 70 80 86.82

Saturation time (h) B 0.64 2 4 6 7.36

Ferric chloride (% w/v) C 0.01 0.04 0.08 0.12 0.15

105

4.2.7.5 Validation of the experimental model

The statistical model was validated with respect to all the three

variables within the design space. Experiments predicted by the numerical

optimization feature of the design expert software were conducted in

triplicates. One set of experiments predicted by the software for maximum

recovery of enzyme was conducted and experimental results obtained were

compared with predicted values.

4.2.8 Storage Stability of Spray Dried and Lyophilized Flocculated

Protease

The storage stability of spray dried (180 U/g) and dried flocculated

(3200 U/g) protease during storage of 360 days was carried out. The samples

were withdrawn at desired time interval and analyzed for the protease activity.

The 80% ammonium sulphate saturated crude enzyme was flocculated and the

enzyme pellet obtained was lyophilized (Figure A 4.2) to get dried form. The

protease activity in spray dried and lyophilized flocculated samples were

analyzed.

4.3 RESULTS AND DISCUSSION

Considering leather industry as a segment for the use of alkaline

protease, the crude enzyme was concentrated to make it suitable for storage as

well as to increase the shelf life of enzyme by preparing different

formulations of the enzyme by various partial purification techniques viz.,

spray drying, ultrafiltration, salting-out, flocculation and salting out followed

by flocculation.

106

4.3.1 Ammonium Sulfate Fractionation

Precipitation is the most commonly used method for the isolation

and recovery of proteins from crude biological mixtures (Kumar and Takagi

1999). The cell free supernatant obtained by the centrifugation of harvested

culture of Bacillus pumilus MTCC 7514 was subjected to different levels of

ammonium sulphate saturation directly and results obtained presented in

Table 4.2. The recovery of protease enzyme as well as specific activity

increased with the increase in level of ammonium sulphate saturation.

Maximum recovery of enzyme (52%) was achieved at 80% saturation with

1.95 fold increase in purification level and specific activity of 26.3 U/mg.

Table 4.2 Purification of crude protease enzyme with ammonium

sulphate precipitation

FractionVolume

(mL)

Activity

(U/mL)

Total

activity

(U)

Protein

(mg/mL)

Total

protein

(mg)

Specific

activity

(U/mg)

Yield

(%)

Purific

ation

fold

Crude

enzyme100 29.1 2910 2.16 216 13.47 100 1

(NH4)2SO4

20%10 1.7 17 0.18 1.8 9.4 0.58 0.7

(NH4)2SO4

40%10 11.7 117 0.84 8.4 13.93 4 1.03

(NH4)2SO4

60%25 52.3 1307.5 2.15 53.8 24.3 45 1.8

(NH4)2SO4

80%25 60.4 1510 2.3 57.5 26.3 52 1.95

Najafi et al (2005) have reported 72% protease enzyme recovery

with specific activity of 25 U/mg when the crude enzyme obtained from

Pseudomonas aeruginosa PD100 was precipitated with 70% ammonium

107

sulphate saturation. In another study, Shivshankar et al (2011) have reported

alkaline protease recovery of 69.5% at 40-70% ammonium sulphate saturation

for crude enzyme obtained by a new strain of Beauveria sp.

4.3.2 Ultrafiltration and Ammonium Sulphate Precipitation

The cell free supernatant (5L) containing protease enzyme of

activity 29.1 U/mL with specific activity of 13.47 was concentrated to 5-fold

via ultrafiltration. The activity of concentrated enzyme solution was 132.4

U/mL with specific activity of 30.6 U/mg (Table 4.3). When the concentrated

enzyme solution was subjected to different levels of ammonium sulphate

saturation (40, 60 and 80%), maximum recovery of enzyme (75.15%) was

observed at 80% saturation with specific activity of 25.2 U/mg. The enzyme

recovery increased with the increase in ammonium sulphate saturation level

though specific activity obtained with 60% saturation was less than the one

obtained with 40% and 80% saturation.

The specific activity of ammonium sulphate precipitated samples

after ultrafiltration was little less when compared to ultrafiltered enzyme

(Table 4.3), whereas it was observed that the enzyme recovery of the

ultrafiltered and ammonium sulphate precipitation was quite better compared

to direct precipitation of crude enzyme. Using ultrafiltration followed by

ammonium sulphate precipitation has 1) reduced the amount of ammonium

sulphate consumed 2) and increased the enzyme recovery with high specific

activity.

108

Table 4.3 Purification of ultrafiltered protease enzyme with ammonium

sulphate precipitation

FractionVolume

(mL)

Activity

(U/mL)

Total

activity

(U)

Protein

(mg/mL)

Total

protein

(mg)

Specific

activity

(U/mg)

Yield

(%)

Purification

fold

UF

enzyme100 132.4 13240 4.33 433 30.6 100 1

(NH4)2SO4

40%25 62.6 1565 2.48 62 25.2 11.8 0.82

(NH4)2SO4

60%25 303.2 7580 14.2 355 21.4 57.2 0.7

(NH4)2SO4

80%25 398 9950 14.85 371.25 26.8 75.15 0.88

4.3.3 Ultrafiltration

The cell-free supernatant was concentrated via ultrafiltration to

different extents. It was observed that the percentage yield of enzyme was

92.33, 91.2, 88, 84.3 and 78 with 5, 10, 15, 20 and 60 fold concentrations

respectively (Table 4.4). The reduction of protease yield with increasing fold

concentration may be due to membrane fouling with time. Bezawada et al

(2011) have reported 83% of protease recovery with 5-fold concentration of

crude enzyme and suggested that reduction in 100% recovery cannot be

achieved due to loss of protease as deposit on the membrane and/or in the

tubes. The specific activity of enzyme increased with increase in fold

concentration and hence increases in purification fold. Although the yield of

enzyme was less (78%) at 60-fold concentration, specific activity was

comparatively more and the maximum among all. The optimum fold

concentration can be selected as 5 or 10, as the protease recovery is above

90%.

109

Table 4.4 Purification of protease enzyme by ultrafiltration

FractionVolume

(mL)

Activity

(U/mL)

Total

activity

(U)

Protein

(mg/mL)

Total

protein

(mg)

Specific

activity

(U/mg)

Yield

(%)

Purification

fold

Crude

enzyme30000 29.1 873000 2.16 64800 13.47 100 1

UF

(5 fold)6000 134.3 805800 4.09 24540 32.83 92.33 2.44

UF

(10 fold)3000 265.4 796200 6.47 19410 41 91.2 3.04

UF

(15 fold)2000 384 768000 7.74 15480 49.6 88 3.7

UF

(20 fold)1500 490.6 735900 8.89 13335 55.2 84.3 4.1

UF

(60 fold)500 1362 681000 20.2 10100 67.4 78 5.0

UF: Ultrafiltration

Similarly Saeki et al (1994) has reported 75.1% recovery of

protease by ultrafiltration of crude enzyme from Oerskovia xanthineolytica TK1.

4.3.4 Spray Drying

Spray drying of crude enzyme was carried out in the presence of

using single additive (MD, WD or starch) as well as by combination of

additives in different ratios (Table 4.5). Addition of additive is required in

order to increase the solid content of solution thereby minimizing material

loss. To reduce the degree of loss of enzyme activity during drying,

stabilizing agents, e.g., carbohydrates can be used. Two major mechanisms

have been put forward that can (at least in part) explain the stabilizing effect

of different carbohydrates: (a) carbohydrates replacing water with the protein

and (b) carbohydrates providing a glossy matrix. The water replacement

hypothesis states that in order to preserve the native structure of a protein, the

hydrogen bond formed between the protein and water molecules in aqueous

solution needs to be replaced by new hydrogen bonds in the dry states.

110

Carbohydrates have indeed been shown to form hydrogen bonds with proteins

in the solid state. The glassy state hypothesis states that in the rigid

amorphous matrix, it becomes difficult for proteins to change their shape and

hence they can maintain their activity (Namaldi et al 2006). Several reports

show that the addition of additives improved the stability as well as recovery

of enzymes (Belghith et al 2001; Namaldi et al 2006; Cui et al 2006).

In the present study it was observed that increasing the

concentration of MD from 10 to 15% in the crude filtrate did not interfere

much with the yield of enzyme (about 42%) whereas WD and starch at 10%

concentration led to yields of 38.6 and 40.6% respectively, which were little

less when compared to the recovery with MD. Many trials in combination

were carried out and results shown in Table 4.5. The maximum yield of

enzyme (about 70%) was achieved when WD and Tata salt were used in

combination at a final concentration of 15%. The same combination of

additives was used for the scale-up process. There was 67.2% recovery with

specific activity of 12.58 U/mg (Table 4.5) which clearly indicates that the

process can be used at industrial level.

Lopez-Diez et al (2000) have reported that MD forms an

amorphous phase with proteins during drying because of their relatively high

glass transition temperature (Tg) values. In the rigid amorphous matrix, it

became difficult for proteins to change their shape; hence, they could

maintain their activity (Fureby et al 1999). Namaldi et al (2006) have also

studied the effects of additives such as maltodextrin and glucose on spray

drying of serine alkaline protease from a recombinant Bacillus subtilis BGSC-

1A751 at concentrations of 0.5 to 2% (w/v) and reported a minimum loss in

activity in the presence of glucose at a concentration of 2% (w/v). Kamoun et

al (2008) reported spray drying of alkaline protease from Bacillus

licheniformis RP1 using PEG 0.5%, maltodextrin 1% and sucrose 1%.

111

Table 4.5 Purification of protease enzyme by spray drying

Additives

Wt. of

powder

(g)

Activity

(U/g)

Total

activity

(U)

Protein

(mg/mL)

Total

protein

(mg)

Specific

activity

(U/mg)

Yield

(%)

Crude

enzyme

(100 mL)

-- 27 2700 2.03 203 13.3 100

MD 10% 5.61 202 1133 24.15 135.5 8.36 42

MD 15 % 11.56 94.45 1091.8 12.86 148.7 7.3 40.44

WD 10% 5.4 193.1 1042.7 23.6 127.4 8.19 38.6

Starch 10% 7.28 151.1 1099 18.85 137.2 8.01 40.70

MD 10%,

Starch 5%11.93 114.31 1363.7 12.92 154.1 8.85 50.51

WD 5%;

Tata Salt

10%

11.96 154.9 1852.6 13 155.5 11.9 68.6

WD 3%;

Tata Salt

12%

11.36 166.14 1887.4 13.3 151.1 12.5 69.9

WD 5%;

Starch 5%;

Tata Salt 5%

9.9 136.36 1350 14.5 143.6 9.4 50

WD 5%;

Starch 5%;

AS 5%

11.8 119 1404.2 12.1 142.8 9.83 52

WD 5%;

Starch 2.5%;

Tata Salt

7.5%

8.95 176.8 1582.4 17.16 153.6 10.3 58.6

Crude

enzyme (10

L)

-- 27 270000 2.03 20300 13.3 100

WD 3%;

Tata Salt

12%

955 190 181450 15.1 14420.5 12.58 67.2

For crude enzyme – activity expressed as U/mL.

112

The best combination of additives obtained from spray drying of

crude enzyme was selected and employed for ultrafiltered enzyme. The result

of spray drying is shown in Table 4.6. The maximum recovery of about 67%

with specific activity of 20 U/mg was observed when additives were used at a

concentration of 15% (WD, 3% and Tata salt 12%) while 63.6% of protease

recovery was obtained at a concentration of 10% (WD, 3% and Tata salt 7%).

Neubech and Hatboro (1980) have also described a similar downstream process

for enzyme, which comprises of concentrating liquid enzyme solution by

ultrafiltration and then subjecting to spray drying using water insoluble salts such

as tribasic calcium phosphate and suspenders/thickeners such as starch in

combination. The concentration of salt used by them ranged from 10-20% (w/v).

During the process of scale-up with 15% additive (WD, 3% and

Tata salt 12%) a protease recovery of 60% was observed which was 6% less

compared to recovery obtained at small scale level of spray drying. The

reason for loss of activity may be due to high inlet and outlet temperatures (70

and 130 ºC, respectively) used at pilot level. The loss of enzyme activity in spray

drying at a high inlet temperature was also reported by Namaldi et al (2006).

Table 4.6 Spray drying of five-fold ultrafiltered enzyme concentrate

Additives Wt. of

powder

(g)

Activity

(U/g)

Total

activity

(U)

Protein

(mg/mL)

Total

protein

(mg)

Specific

activity

(U/mg)

Yield

(%)

UF enzyme

(100 mL)

-- 132.4 13240 4.33 433 30.6 100

WD 3%; Tata

Salt 12%

11.8 750 8850 37.5 442.5 20 66.8

WD 3%; Tata

Salt 7%

7.5 1123 8422.5 48.6 364.5 23.1 63.6

UF enzyme

(2.8 L)

-- 132.4 370720 4.33 12124 30.6 100

WD 3%; Tata

Salt 12%

328 681 223368 41 13448 16.6 60.3

For UF enzyme – activity expressed as U/mL.

113

4.3.5 Flocculation of Crude Enzyme

Table 4.7 represents the effect of different concentrations of ferric

chloride on the recovery of protease enzyme from crude filtrate. Although,

recovery of protease from crude filtrate by flocculation increased with the

increase in ferric chloride concentration from 0.2 to 0.8%, it reduced with

further increase in concentration up to 1%. The maximum recovery of

enzyme (13%) was at 0.8% (w/v) concentration of ferric chloride followed by

1%. The loss in enzyme activity may be due to the enzyme denaturation as

addition of ferric chloride to crude filtrate reduces the pH to 3.0-4.0, and in

turn the stability of enzyme. The fold purification was drastically reduced at

all concentrations of flocculant indicating the denaturation of enzyme. While

in contrast, Colligwood et al (1988, 1989) have reported approximately 80%

recovery of enzymes by flocculation with Fe (III) and Al (III).

Table 4.7 Purification of protease enzyme with flocculant FeCl3

FlocculantConcentration

(%, w/v)

Yield

(%)

Purification

fold

FeCl3

0.2 0.8 0.17

0.4 7 0.25

0.6 11.1 0.42

0.8 13 0.47

1.0 11.6 0.36

4.3.6 Ammonium Sulphate Precipitation Followed by Flocculation

A study on the effect of flocculants at different concentrations on

the recovery of protease enzyme was carried out. Crude enzyme filtrate with

60 and 80% ammonium sulphate saturation was used. Table 4.8 shows the

effect of different flocculants at various concentrations on enzyme recovery

114

from 80% ammonium sulphate saturated enzyme. Ferric chloride gave

maximum enzyme recovery of 93.8% with 2.6-fold purification when used at

concentration of 0.05%. Further increase in concentration leads to reduced

enzyme recovery as well as fold purification.

Aluminium chloride also gave maximum recovery of enzyme

(95.6%) at 0.05% whereas further increase in concentration reduced the

recovery of enzyme. In contrast to ferric and aluminium chloride, alum

showed the lowest enzyme recovery (87.9%) at 0.05% concentration while

the enzyme recovery increased with the concentration of alum attaining a

maximum (98.9%) at 0.2% concentration (Table 4.8). Calcium hydroxide did

not make any difference at concentrations of 0.05 to 0.2% and the precipitates

were dispersed in the liquid phase and hence, the concentration was increased

to 0.5%, which gave an enzyme recovery of 76.2%. Further increase in

concentration to 1%, did not significantly increase the recovery while the

purification fold was slightly reduced to 2.36 from 2.52.

Table 4.8 Purification of protease enzyme with combination of

ammonium sulphate precipitation (80%) and flocculation

(FeCl3)

FlocculantsConcentration

(%, w/v)

Yield

(%)

Purification

fold

Ferric chloride (FeCl3)

0.05 93.8 2.60

0.1 90.8 2.31

0.2 88 2.09

Aluminium Chloride (AlCl3)

0.05 95.6 3.1

0.1 88 2.25

0.2 90 2.33

Alum [Al2(SO4)3]

0.05 87.9 3.39

0.1 91.3 3.1

0.2 98.9 2.5

Calcium hydroxide [Ca(OH)2]

0.5 76.2 2.52

1.0 77.38 2.36

115

Table 4.9 shows the effect of flocculation on the recovery of

enzyme from crude enzyme filtrate treated with 60% (saturation) ammonium

sulphate employing different flocculants and their concentrations. Yields of

protease enzyme with the first three flocculants (ferric chloride, aluminium

chloride and alum) were comparatively less (72 to 86%) when compared to

that obtained with flocculation of 80% (saturation) ammonium sulphate

saturated crude filtrate. The enzyme recovery by flocculation with calcium

hydroxide was found to be similar for both 60% and 80% (saturation)

ammonium sulphate precipitated samples. However the fold purification was

higher at 3.95 in the former case.

Table 4.9 Purification of protease enzyme with combination of

ammonium sulphate precipitation (60%) and flocculation

(FeCl3)

FlocculantsConcentration

(%, w/v)

Yield

(%)

Purification

fold

Ferric chloride (FeCl3)

0.05 72 3.3

0.1 86 2.82

0.2 80 2.5

`Aluminium Chloride (AlCl3)

0.05 85 5

0.1 84 4.68

0.2 76 4.24

Alum [Al2(SO4)3]

0.05 82 5.7

0.1 84 5

0.2 82 4.06

Calcium hydroxide [Ca(OH)2]0.5 76 3.95

1.0 75 3.86

116

4.3.7 Effect of pH Adjustment on Flocculation and Enzyme Recovery

Flocculation of 80% (saturation) ammonium sulphate treated crude

filtrate was carried out under controlled pH. The pH was adjusted to the initial

pH of crude enzyme (7.5-8.0). The yield of enzyme reduced when pH was

adjusted either by sodium or calcium hydroxide during flocculation at 0.05%

concentration of ferric chloride but gradually increased with increase in ferric

chloride concentration. The maximum enzyme yield (96%) was observed at

0.2% ferric chloride concentration when pH was maintained with calcium

hydroxide. This indicates that the pH maintenance of ammonium sulphate

saturated enzyme solution during flocculation did not significantly contribute

to enzyme recovery as 94% recovery was observed at low concentration of

flocculant without pH adjustment.

Table 4.10 Effect of pH adjustment on protease enzyme recovery

Flocculation

condition

FeCl3 (0.05%, w/v) FeCl3 (0.1%, w/v) FeCl3 (0.2%, w/v)

Yield

(%)

Purification

fold

Yield

(%)

Purification

fold

Yield

(%)

Purification

fold

No pH control 94 2.7 92 2.35 90 2.24

pH controlled

with sodium

hydroxide

56 1.94 78 1.95 93 2.34

pH controlled

with calcium

hydroxide

65 1.68 84 1.94 96 2.1

4.3.8 Optimization of Significant Variables by RSM

Present study was aimed to determine the optimum level of

significant variables on the recovery of enzyme from saturated ammonium

sulphate treated crude filtrate. This method offers a number of important

117

advantages such as the significant variable effects, obtaining the optimum

values and also developing a system model with considerably less

experimental requirements (Wu et al 2007). The level of the saturation time

was selected based on the literature reported by Farinas et al (2011) whereas

levels of other two factors were chosen based on previous experiments. The

experiments comprising the factorial design were carried out as per the design

matrix developed by the design expert software. An estimate of the main

effect was obtained by a change from low (-1) to high (+1) level of the

corresponding factor. The experimental conditions along with predicted

enzyme recovery obtained from the regression equation for the 20

combinations are shown in Table 4.11. The process performance was

measured by enzyme recovery response. The results demonstrated that highest

recovery of enzyme (97.42%) from saturated crude filtrate was observed in

the 7th

run and the lowest enzyme recovery (84.87%) was observed in run 5.

A second order regression equation showed the dependence of

enzyme recovery on the selected factors. The parameters of the equation were

obtained by multiple regression analysis of the experimental data. The coded

model used to generate response surfaces for the analysis of variable effects

on enzyme recovery was expressed in terms of second order polynomial

equation:

Enzyme Recovery (Y) = 93.12 + 3.87(A) + 1.64 (B) + 0.93 (C) - 0.18 (A) (B)

- 0.5 (A) (C) - 1.48 (B) (C) - 2.22 (A2) - 0.39 (B

2) - 0.13 (C

2)

Where enzyme recovery is the response (Y) and A, B and C are the

level of ammonium sulphate saturation, saturation time and FeCl3

concentration, respectively.

118

Table 4.11 Central composite design matrix for the experimental design

and predicted response for enzyme recovery

RunLevel of AS

saturation (%)

Saturation

time (h)

FeCl3

(%, w/v)

Actual

value, Y

Predicted

value, Y

1 80 2 0.12 94.3 93.81

2 60 6 0.12 88.75 90.01

3 70 4 0.01 92.14 89.86

4 70 7.36 0.08 96 94.18

5 60 2 0.04 82.47 84.87

6 70 4 0.08 99.51 91.14

7 80 6 0.12 94.68 97.42

8 60 6 0.04 89.5 88.48

9 70 4 0.15 92.4 92.43

10 70 4 0.08 93.11 91.14

11 86.82 4 0.08 95.56 97.38

12 70 4 0.08 93.28 91.14

13 80 6 0.04 93.72 95.89

14 53.18 4 0.08 77.11 84.91

15 80 2 0.04 91.15 92.28

16 70 4 0.08 89.32 91.14

17 70 4 0.08 91.6 91.14

18 70 0.64 0.08 87.05 88.11

19 70 4 0.08 92.1 91.14

20 60 2 0.12 91.35 86.40

Adjusted R2= 0.5389 Predicted R

2 = -0.0734

4.3.8.1 Model fitting

Analysis of variance (ANOVA) was used to examine the adequacy

of the fitted quadratic model which was tested using Fisher’s statistical

analysis and the results are shown in Table 4.12. The model F-value of 3.47

119

and p-value of <0.0329 imply that the model is significant. Model terms with

Prob >F (less than 0.05) are considered significant while those greater than

0.1 are insignificant (Li et al 2008). The lack of fit F value of 0.88 implies

that lack of fit is not significant relative to the pure error suggesting that the

obtained experimental responses sufficiently fit with the model.

The R2 value provides a measure of how much of variability could

be examined by the experimental factors and their interactions. A good model

explains for most of the variations in the response. The closer the R2 value to

1.0, the stronger the model and better the response prediction (Reddy et al

2008). The R2 value of 0.76 showed that almost 75% of the variation could be

accounted by the model; hence, the model was good.

Table 4.12 ANOVA for Response Surface Quadratic Model

SourceSum of

squares

Degrees of

freedom

Mean

squareF-value

Prob

(P)>F

Model 344.42 9 38.27 3.47 0.0329a

A-AS 204.19 1 204.19 18.50 0.0016

B-ST 36.84 1 36.84 3.34 0.0977

C-FeCl3 11.77 1 11.77 1.07 0.3261

AB 0.27 1 0.27 0.025 0.8781

AC 2.02 1 2.02 0.18 0.6777

BC 17.47 1 17.47 1.58 0.2370

A2

71.27 1 71.27 6.46 0.0293

B2

2.18 1 2.18 0.20 0.6663

C2

0.23 1 0.23 0.021 0.8889

Residual 110.38 10 11.04

Lack of Fit 51.74 5 10.35 0.88 0.5530b

Pure Error 58.64 5 11.73

Total 454.80 19

R2 = 0.7573

a – significant

b – not significant

120

Adequate precision measures signal to noise ratios and a ratio

greater than 4 is desirable. The adequate precision value of 6.99 for enzyme

recovery indicates that the model can be used to navigate the design space.

Also, the cofficient of variance (CV) indicates the degree of precision with

which the treatments are compared, and the low value of CV showed the

reliability of experiments. In this study, a relatively lower value of cofficient

of variation (CV = 3.64) suggested a good precision and reliability of the

experiment.

4.3.8.2 Three dimensional graphs

The three dimensional (3D) response surface plots is the graphical

representation of the regression equation used to investigate the interaction

among variables and to determine the optimum level of each factor for the

maximum enzyme recovery. The 3D plot shown in Figure 4.2 to 4.4 was

based on the function of level of two variables keeping other variables at their

optimum level. Figure 4.2 represents the effect and interaction of ammonium

sulphate and saturation time on the recovery of enzyme when the other factor

FeCl3 was kept constant at 0.08% concentration. A linear increase in enzyme

recovery was observed when saturation time and ammonium sulphate

concentration were increased. There was no significant increase in enzyme

recovery beyond 75% of ammonium sulphate concentration. Therefore, for

maximum enzyme recovery the ammonium sulphate concentration could be

maintained at moderate level i.e. 70-75%.

Figure 4.3 depicted the 3D response surface plot showing the effect

and interaction of FeCl3 and ammonium sulphate concentration when

saturation time was kept constant at its mid value of 4 h. Similar to Figure 4.2,

a linear increase in enzyme recovery was observed when both ammonium

sulphate and ferric chloride concentrations were increased whereas

121

ammonium sulphate shows more effect on enzyme recovery compared to the

ferric chloride.

The response surface plot showing the effect and interaction of

FeCl3 and saturation time by keeping the other factor (AS) constant at its mid

value of 70% (Figure 4.4) shows that enzyme recovery was linearly increased

with the increase in FeCl3 concentration and saturation time, whereas lower

and higher level of both ammonium sulphate and saturation time did not result

in higher enzyme recovery. The shape of the response surface curved showed

a moderate interaction between these two variables.

Design-Expert® Software

Enzyme recovery99.51

77.11

X1 = A: ASX2 = B: Saturation time

Actual FactorC: FeCl3 = 0.08

60.00

65.00

70.00

75.00

80.00

2.00

3.00

4.00

5.00

6.00

77

82.75

88.5

94.25

100

E

nzym

e r

eco

ve

ry

A: AS B: Saturation time

Figure 4.2 Response surface plot showing the effect of ammonium

sulphate (AS) and saturation time on enzyme recovery

122

Farinas et al (2011) have reported higher recovery of

endoglucanase by 80% ammonium sulphate saturation at 10 ºC after 3 h of

incubation and shown that saturation time had a positive effect on the enzyme

recovery. The yield of precipitation with ammonium sulphate increased to an

optimum value of 65% after 12 h of saturation time. The positive effect of

saturation time on enzyme recovery can be explained by the fact that a longer

aging time increases the probability of nuclei formation due to collision

among the molecules. Once the nuclei reach a critical size, they continue to

grow (Farinas et al 2011).

Design-Expert® Software

Enzyme recovery99.51

77.11

X1 = A: ASX2 = C: FeCl3

Actual FactorB: Saturation time = 4.00

60.00

65.00

70.00

75.00

80.00

0.04

0.06

0.08

0.10

0.12

77

82.75

88.5

94.25

100

E

nzym

e r

eco

ve

ry

A: AS C: FeCl3

Figure 4.3 Response surface plot showing the effect of ammonium

sulphate (AS) and ferric chloride (FeCl3) on enzyme

recovery

123

Design-Expert® Software

Enzyme recovery99.51

77.11

X1 = B: Saturation timeX2 = C: FeCl3

Actual FactorA: AS = 70.00

2.00

3.00

4.00

5.00

6.00

0.04

0.06

0.08

0.10

0.12

87

90.25

93.5

96.75

100

E

nzym

e r

eco

ve

ry

B: Saturation time

C: FeCl3

Figure 4.4 Response surface plot showing the effect of saturation time

and ferric chloride (FeCl3) on enzyme recovery

4.3.8.3 Validation of the experimental model

An additional experiment in triplicate was carried out with the

combination of factors (79.4% AS, 6 h saturation time and 0.04% ferric

chloride) predicted by the software for maximum recovery of enzyme to

validate the accuracy of model. The average enzyme recovery of 99% was

observed which was in accordance with the predicted value of 96.8% and

hence the model is reasonably good.

124

4.2.9 Storage Stability of Spray Dried and Lyophilized Flocculated

Protease

The protease preparations obtained by flocculation of ammonium

sulphate precipitated enzyme followed by lyophilization and spray dying were

stored at 4 ºC and at room temperature (30±5 ºC). The enzyme stability results

are shown in Table 4.13. Lyophilized flocculated protease exhibited better

shelf life of more than a year at 4 ºC and exhibited a shelf life of 210 days at

room temperature. The shelf life of spray dried enzyme was 270 days at 4 ºC

but reduced to 150 days at room temperature.

Table 4.13 Stability studies of protease preparations

No. of days

SDP (Spray dried

powder)

LFP (Lyophilized flocculated

poweder)

4 ºC R.T. 4 ºC R.T.

0 180 180 3200 3200

7 180 180 3200 3184

15 180 178 3200 3190

30 178 178 3180 3188

60 178 175 3172 3152

90 176 171 3168 3108

120 176 167 3162 3064

150 175 162 3154 3012

180 174 158 3140 2963

210 170 152 3112 2880

240 166 147 3088 2815

270 162 139 3062 2746

300 155 130 3030 2650

330 148 121 2998 2568

360 140 108 2965 2400

125

Namaldi et al (2006) have also evaluated the storage stability of

spray dried protease at 4 ºC for a period of 6 months and reported that some

of the spray dried protease powder retained more that 90% of their activity

even after 6 months however the protease stability at room temperature was

not studied by them. The protease under study has shown better shelf life at 4

ºC as well as room temperature which makes it suitable from the industrial

point of view.

4.4 CONCLUSIONS

This chapter deals with the partial purification and improved

recovery of enzyme from crude filtrate obtained from the submerged

fermentation by Bacillus pumilus MTCC 7514. The partial purification

techniques employed were i) ammonium sulphate precipitation, ii) spray

drying, iii) ultrafiltration, iv) ultrafiltration followed by precipitation/spray

drying and v) flocculation, ammonium sulphate precipitation followed by

flocculation. The silent findings of this chapter are as shown:

Ammonium sulphate precipitation (80% saturation) of crude

enzyme and concentrated crude enzyme via ultrafiltration

resulted in the maximum enzyme recovery of 52 and 75.15%,

respectively.

Maximum recovery of 92.33% with specific activity of 32.83

was obtained with five-fold concentration of crude enzyme

whereas 78% of recovery with specific activity of 67.4 was

observed at 60-fold concentration.

Spray drying of crude enzyme yielded a maximum recovery of

69.9% with 3% WD and 12% Tata salt as additives at small

level whereas scaling up of the process yielded a similar

enzyme recovery of 67.2%.

126

Spray drying of UF enzyme yielded maximum recovery of

66.8% at small level whereas scaling up of the process yielded

an enzyme recovery of 60.3%.

An optimized protocol employing ammonium sulphate

precipitation and flocculation indicated that complete enzyme

(~100%) could be recovered from the crude filtrate.

The protease preparation obtained by spray drying exhibited

better stability and shelf life of 9 months (at 4 ºC) and 5 months

(at room temperature) respectively while protease preparation

obtained by lyophilization of precipitated enzyme exhibited

shelf life of 1 year (at 4 ºC) and 7 months (at room temperature)

respectively.

Among the above downstream processes, the combination of

ammonium sulphate precipitation (80% saturation) followed by flocculation

with 0.04% FeCl3 gives the maximum yield (99%) with very minimal loss of

enzyme, which could be lyophilized to obtain dry powder for further low-end

applications such as dehairing of hide and skin and also for high-end

application like pharmaceuticals.