Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

BIOCATALYSIS AND BIOTRANSFORMATION

https://doi.org/10.1080/10242422.2019.1666829

RESEARCH ARTICLE

Aggregation properties of cold-active lipase produced by a psychrotolerant

strain of Pseudomonas palleroniana (GBPI_508)

a,b

a,c

a

Rahul Jain

, Neha Pandey and Anita Pandey

a

Centre for Environmental Assessment and Climate Change, G B Pant National Institute of Himalayan Environment and Sustainable

b

Development, Almora, India; Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, India;

c

Department of Biotechnology, Graphic Era Deemed to be University, Dehradun, India

ABSTRACT

ARTICLE HISTORY

The present study aims to exploit microbial potential from colder region to produce lipase

enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan envir-

onment, was investigated for the production of cold-active lipase emphasizing on its aggregation

properties. Plate based assays followed by quantitative production of enzyme was estimated

under different culture conditions. Further characterization of partially purified enzyme was done

for molecular weight determination and activity and stability under varying conditions of pH,

temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant

bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing.

Received 27 March 2019

Revised 23 July 2019

Accepted 5 September 2019

KEYWORDS

Pseudomonas; extremo-

philes; cold-active lipase;

aggregation;

Indian Himalaya

ꢀ

Maximum lipase production by GBPI_508 was recorded in 7 days at 25 C utilizing yeast extract

as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%)

in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by

bacterium showed aggregation which was confirmed by dynamic light scattering and native

PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active

bands of ꢁ50 kDa and ꢁ54 kDa. Optimum activity of partially purified enzymatic preparation was

ꢀ

recorded at 40 C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas,

ꢀ

maximum stability was recorded at pH values 7.0 and 11.0 at 25 C. Interestingly, lipase in the

partially purified fraction retained 60% enzyme activity at 10 C. Medium chain pNP ester (C10)

ꢀ

was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual

activity when incubated with different organic solvents (25% v/v) except toluene and dichloro-

methane which inhibited the activity below 50%. Partially purified enzyme was also stable in the

presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low

temperature conditions such as cold-active detergent formulations and cold bioremediation.

GRAPHICAL ABSTRACT

Introduction

Microbiota of cold environments and their products are

getting increased attention due to uniqueness in having

extremophilic characters. These environments, inhabited

by cold adapted microorganisms (psychrophiles and

psychrotolerants), are scattered all across the earth’s sur-

face. They contribute to various biogeochemical activities

in low temperature conditions. Polar environments, gla-

ciers, high altitude mountains and soils are the major

CONTACT Anita Pandey

anita@gbpihed.nic.in, anitapandey333@gmail.com

Centre for Environmental Assessment and Climate Change, G B Pant

National Institute of Himalayan Environment and Sustainable Development, Kosi-Katarmal, Almora, Uttarakhand 263 643, India

ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2

R. JAIN ET AL.

habitat for such microorganisms (Jain et al. 2017;

Maharana and Singh 2018). Himalayan mountains, well

recognized for its biodiversity, inhabit range of psychro-

tolerant or facultative psychrophilic microorganisms.

Microorganisms from these sites are reported to harbour

characteristics which are important from biotechno-

logical viewpoint (Jain and Pandey 2016; Pandey et al.

commercial detergents. Therefore, present study

reports production of cold-active lipase from a bacter-

ium Pseudomonas palleroniana isolated from

Himalayan soil. Optimization of production parame-

ters, partial purification and characterization of lipase

has been performed keeping aggregation properties

of enzyme under consideration.

2

016, 2018; Pandey and Yarz ꢀa bal 2019).

Isolation and screening of microorganisms for

production of cold applicable enzymes are becom-

ing an important area in research (Santiago et al.

Materials and methods

Bacterial isolation and culture maintenance

2

016). Lipases (EC 3.1.1.3) are ubiquitous biocatalysts

that hydrolyse triacylglycerol to free fatty acids and

glycerol in an oil-water interface. They also have the

property to reverse the reaction in aqueous and

non-aqueous solutions (Lee et al. 2015). Cold-active

lipases are important biocatalysts in biotechnology

sector due to their unique catalytic properties. The

enzymes from low temperature inhabiting microor-

ganisms show activity and stability at low tempera-

ture in comparison to their mesophilic counterparts.

These enzymes are active not only in cold condi-

tions but also show stability in presence of organic

solvents, metal ions, etc. (Wiese et al. 2010; Javed

et al. 2018). The application of these biocatalysts

has been recognized in various industrial as well as

environmental sectors such as synthesis of fine

chemicals, food additives, detergent formulations,

pharmaceuticals, production of biodiesel and biopol-

ymers and in the bioremediation of contaminated

soils and water (Maiangwa et al. 2015; Hassan

et al. 2018).

The bacterium was originally isolated from the rhizo-

sphere of wheat growing in Uttarakhand Himalaya at

an altitude of 1900 m asl in Kalimati village, Dist.

Chamoli, Uttarakhand. Ten-fold serial dilution of soil

was performed for isolation of bacterium as pure cul-

ture. The bacterium was further maintained in tryp-

ꢀ

tone yeast extract (TY) agar at 25 C for routine

ꢀ

experiments and in glycerol stocks at ꢂ20 C for pres-

ervation purpose.

Bacterial characterization and identification

Morphology and microscopic features of bacterium

were recorded after growth on TY agar following an

incubation of 24 h. Nikon Eclipse 50i microscope was

used to study the cell size and Gram nature of the

bacterium. Routine biochemical tests including oxi-

dase, catalase, urease, nitrate reduction, indole pro-

duction and carbohydrate utilization were done using

standard assays. Plate based assays were performed

for screening the production of hydrolytic enzymes

including protease, amylase, and lipase/esterase. The

physiological limits of the bacterium to withstand dif-

Bacteria or fungi have been mostly reported as pro-

ducer of cold-active lipases (Sahay and Chouhan

2018). Although, lipases of bacterial origin are consid-

ered to be more robust for their applicability in harsh

industrial conditions and this has accelerated search

for new bacterial lipases. Pseudomonas is known to be

one of the dominant inhabitants in Himalayan soils

and also known for its low temperature tolerance. As

various commercial lipases have been produced from

Pseudomonas spp. (Rahman et al. 2006); bacteria

belonging to this genus still offer opportunities for

cold-active lipases screening. With the increasing food

waste and waste-water rich in lipids and oils in cold

environments, there is an increasing concern over

their remediation in a sustainable way and use of cold

adapted bacterial strains and lipases is a possible

answer to this environmental problem (Wang et al.

ꢀ

ferent temperatures (5–45 C) and pH conditions (pH

1

.0–14.0) were assessed as described in Jain and

Pandey (2016).

For identification of bacterium using 16S rRNA

gene sequencing, genomic DNA was isolated and PCR

amplification of 16S rRNA gene was performed using

universal eubacterial primers. The amplified product

was purified and sequencing of the gene was per-

formed (courtesy: NCCS Pune, India). The resulting

sequence was used for the identification of bacterium

using EzTaxon database (Kim et al. 2012). Further, a

phylogenetic tree of bacterium was also reconstructed

using MEGA v6 (Tamura et al. 2013). The bacterial cul-

ture and its 16S rRNA gene sequence are deposited in

National Centre for Microbial Resource, NCCS, Pune,

India and NCBI, respectively.

2012; Hassan et al. 2018). Also, cold washing can be

effectively achieved using detergents containing such

cold-active lipases which is a necessary component in

BIOCATALYSIS AND BIOTRANSFORMATION

3

Scanning electron microscopy (SEM)

(Bradford 1976) using bovine serum albumin as stand-

ard. Specific activity in culture supernatant and active

fraction was also calculated.

The bacterial cells were also studied using SEM after

its growth in TY broth as well as lipase production

medium (Jain et al. 2017). The bacterium was incu-

bated for 24 h in respective media and cells were

The partially purified lipase fraction was subjected

to gel filtration chromatography using Sephadex G100

for determining the aggregation behaviour of the

enzyme. Attempts for disaggregation of the enzyme

were made by treating the sample with various

reagents including acetonitrile (5%), sucrose (5 M),

Tween 20 (0.5%), NP-40 (0.5%), Brij-35 (0.05%), isopro-

panol (5%), taurocholic acid (1%) and analysing with

dynamic light scattering using Zetasizer Nano

extracted by centrifugation at 10000 rpm for 10 min at

ꢀ

4

C. Pellet was then fixed with 2.5% glutaraldehyde

ꢀ

prepared in 0.05 M phosphate buffer for 4 h at 4 C.

This was followed by sample washing with distilled

water, thrice. A thin film of bacterial sample was lay-

ered on to a microscopic slide and dehydrated using

light source. Sample was coated with gold by using

JFC 1600 gold coater and viewed under JEOL JSM-

(Malvern Instruments). Protein samples diluted (1:4) in

Tris–Cl buffer (50 mM, pH 8.0) was used for DLS meas-

6610LV scanning electron microscope.

ꢀ

urements at 25 C. The treated samples were also sub-

jected to native PAGE analysis followed by zymogram

analysis (Prim et al. 2003) using 4-methyl umbelliferyl

butyrate (Sigma) as substrate to observe the effect of

various treatments on aggregation.

Qualitative and quantitative lipase production

Lipase production by the bacterial isolate was first

screened on tributyrin agar and confirmed using

rhodamine B-olive oil agar. Quantitative estimation of

lipase production at different temperature and pH was

done in lipase production medium enriched with 1%

olive oil. Effect of oil source (1%), organic nitrogen

SDS-PAGE and zymogram

SDS-PAGE (12.5%) was performed in presence and

absence of reducing agent (b-mercaptoethanol) using

gel system of Laemmli (1970). After electrophoresis,

gel was washed in a solution of 2.5% Triton X-100

(0.5%) and inorganic nitrogen (0.3%) source, and non-

ionic detergents (1%) on the lipase production by bac-

terium was also investigated. In all cases, original

medium with 1% olive oil served as control. The pro-

duction of lipase enzyme was estimated till 7 days of

incubation. The lipase activity was assayed each day in

the clear, cell free supernatant using 4-nitrophenyl

dodecanoate/laurate (pNPL) (Sigma, USA) as substrate

by the method described in our previous study (Jain

et al. 2017). Enzyme activity was expressed in terms of

enzyme unit which is defined as the enzyme required

to release 1 lM of pNP per min under standard

assay conditions.

(only in case of non-reducing SDS-PAGE) in Tris–Cl

(50 mM, pH 8.0) buffer to remove SDS from gel and

zymogram was developed as described in the previous

section. Same gel after activity was also stained with

coomassie brilliant blue R-250 (CBB) while reducing

gel was directly stained with CBB after electrophoresis.

Molecular weight was determined by comparing active

bands with standard pre-stained molecular weight

markers (Puregene, Genetix Biotech) and protein

bands in CBB stained gels.

Partial purification of lipase and

aggregation studies

Enzyme characterization

Partially purified preparation of the bacterial active lip-

ase obtained after ammonium sulphate fractionation

was further used for the characterization studies.

The crude enzyme in supernatant was precipitated

using solid ammonium sulphate. Two fractions i.e.

0

–50% (fraction A) and 50–80% (fraction B) were col-

lected after centrifugation for 20 min at 15000 rpm.

Both the fractions were then solubilized in Tris–Cl

Effect of temperature and pH

(

50 mM, pH 8.0) buffer and then dialysed overnight in

The lipase assay in Tris-Cl buffer (pH 8.0) was per-

formed by incubating the reaction mixture at different

temperatures (10–70 C) to determine optimum tem-

the same buffer with multiple change of buffer ensur-

ing complete removal of salt. All these experiments

were carried out at 4 C to prevent loss of proteins.

Enzyme activity was measured in both fractions and

only active fraction was considered for further experi-

ments. Protein was estimated by Bradford method

ꢀ

perature for lipase activity. For stability of lipase in dif-

ferent temperature conditions, enzyme was incubated

ꢀ

at different temperatures (10–70 C) for 1 h. Lipase

assay was then performed to calculate the residual

4

R. JAIN ET AL.

activity. Similarly, optimum pH for lipase activity was

determined after performing the lipase assay in differ-

ent pH buffers (pH 4.0–12.0). For stability experiment,

the enzyme was incubated with different pH buffers

for 2 h at 25 C and residual activity was calculated

using standard assay.

Table 1. Morphological, biochemical and physiological charac-

teristics of GBPI_508.

Characteristics

Morphology

Observations

Off white, round and convex colony

ꢀ

(

2–3 mm) at 25 C

ꢀ

Microscopy

Biochemical

Gram negative, scattered, tiny rods, size

1.85 ± 0.28 mm

Oxidase, catalase, urease (weak) positive;

nitrate reduction, indole production

negative; facultative anaerobic

Dextrose, inositol, sucrose, lactose, inulin,

and mannitol negative

¼

Effect of cations and inhibitors

Carbohydrate utilization

Hydrolytic enzymes

þ

Effect of mono and multivalent cations including (K ,

Protease and lipase positive;

þ

2þ

þ

2þ

3þ

amylase negative

Na , Cu , Cd , Mg , Ca , Sr , Zn , Co , Al

,

ꢀ

Physiological

characterization

Temperature tolerance from 5 to 45 C

3

ꢀ

and Fe ) on lipase activity were studied by perform-

ing the lipase assay in the presence of these ions

(Optimum 25 C), pH tolerance from 2.0

to 14.0 (optimum pH 6.0–7.0), salt

tolerance up to 15%

(1 mM and 5 mM). Similarly, inhibitors and detergents

Accession numbers

Culture: MCC2692; Nucleotide: KR259219

including EDTA (1 mM), DTT (1 mM), CTAB (1%), Triton

X-100 (1%), SDS (1%), and Tween 60 (1%) were also

examined for their effect on enzyme activity. Lipase

activity in absence of any treatment served as control.

The bacterium was moderately halophilic with salt tol-

erance up to 15%. The characteristics of GBPI_508 are

presented in Table 1. Overall, GBPI_508 was observed

as a psychrotrophic halotolerant bacterium along with

wide pH tolerance ability.

Effect of organic solvents and substrate specificity

1

6S rRNA gene sequencing followed by database

Organic solvents including methanol, ethanol, n-pro-

panol, n-butanol, acetone, dichloromethane, aceto-

nitrile, toluene, and hexane were examined for

determining their effect on lipase stability. Each solv-

ent (25% v/v) was incubated with enzyme for 30 min

and residual activity (%) in absence of any solvent was

calculated. For substrate specificity, p-nitrophenyl

linked esters of varying chain length including pNP

octanoate (C8), pNP decanoate (C10), pNP dodeca-

noate (C12), pNP myristate (C14), and pNP palmitate

search using EzBioCloud (https://www.ezbiocloud.net/

resources/16s_download) identified bacterium as

Pseudomonas palleroniana (99.30%). Phylogenetic ana-

lysis of GBPI_508 revealed its close similarity with sev-

eral psychrophilic pseudomonads (Figure 1).

Scanning electron microscopy

Bacterial cells under the SEM showed different charac-

teristics when gown in TY broth and lipase production

broth. Actively growing and lipase secreting cells, har-

vested from lipase production medium, had increased

cell dimensions (length ꢃ dia ¼ 2.17 ꢃ 1.26 mm) in

comparison to those harvested from TY broth

(C16) were used for lipase assay.

Statistical analysis

One-way ANOVA followed by Duncan’s test was per-

formed to measure statistical difference (p < 0.05) in

enzyme activity among different treatments. Mean

and standard error (SE) were calculated using

Microsoft excel 2013.

(

1.85 ꢃ 0.72 mm) (Figure 2). Dense cell population in

lipase production medium was observed as the media

facilitated maximal bacterial growth while hydrolysing

olive oil.

Lipase production

Results

Maximum lipase production by GBPI_508 was

recorded at 25 C (28.81 U/ml, 7th day) which

Bacterial characterization and identification

ꢀ

The bacterial isolate GBPI_508 produced round, off-

white colonies on TY agar at 25 C after 24 h of incu-

increased gradually till 7 days of incubation and

remained consistent thereafter (28.50 ± 0.25 U/ml, 8th

day). At 35 C, lipase production increased initially

ꢀ

bation. The bacterium showed dark coloured diffusible

pigmentation when incubated beyond 24 h. The Gram

negative bacterium measured an average cell length

of 1.85 ± 0.28 mm. Temperature and pH tolerance of

which decreased on later incubation. Slow but con-

tinuous secretion of lipase by GBPI_508 was recorded

ꢀ

at 4 C and 14 C even beyond 7 days of incubation

time (Figure 3(a)). Initial pH of medium significantly

(p < 0.05) affected lipase production. Maximum lipase

ꢀ

bacterium ranged from 5 C to 45 C (optimum 25 C)

and pH 2.0–14.0 (optimum pH 6.0–7.0), respectively.

BIOCATALYSIS AND BIOTRANSFORMATION

5

Figure 1. Phylogenetic tree of GBPI_508 reconstructed using Neighbour Joining Method (Bootstrap ¼ 1000). Image in the top

left shows GBPI_508 colonies on TY agar.

Figure 2. Scanning electron micrograph of GBPI_508 cells harvested from (a) TY broth and (b) lipase production medium.

Figure 3. Lipase production by isolate GBPI_508 at different temperatures and pH conditions.

6

R. JAIN ET AL.

production, after 7 days of incubation, was observed

at pH 9.0 (35.95 U/ml) followed by pH 7.0, while no

significant difference (p < 0.05) was recorded in lipase

production at pH 5.0 and pH 11.0 (Figure 3(b)).

Table 2. Effect of nutrient components on lipase production

by the isolate GBPI_508.

Lipase production at

ꢀ

Nutrient source

25 C (U/ml ± SE) in 7 days

ꢄ

ab,†

Control

Oil source (1% v/v)

28.81 ± 0.40

Bacterial isolate utilized olive oil (in control) as the

preferred carbon source as well as inducer of lipase

but soybean oil was utilized almost equally with no

significant difference (p < 0.05). In comparison to con-

trol, corn oil could not enhance the enzyme produc-

tion, while tributyrin was the least preferred inducer

for lipase production. Similarly, sodium nitrate as inor-

ganic and yeast extract as organic (both in control)

were the most preferred nitrogen sources for

GBPI_508 to produce maximum lipase. Other nitrogen

sources inhibited the production of lipase. Organic

nitrogen played a crucial role in lipase secretion as the

production almost ceased in its absence. No significant

increase in lipase production was observed as com-

pared to control when Triton X-100 was added to pro-

duction medium while all other detergents inhibited

the lipase secretion by GBPI_508. Effect of various

nutrients on lipase production is summarised in

Table 2.

cd

Corn oil

Soybean oil

Tributyrin

24.67 ± 2.98

bc

27.07 ± 1.35

g

7.83 ± 0.83

Inorganic N-source (0.3% w/v)

Ammonium nitrate

Ammonium chloride

Ammonium sulphate

No Inorganic-N source

Organic N-source (0.5% w/v)

Peptone

Malt extract

Casein

Urea

No organic-N source

Non-ionic detergent (1% v/v)

Triton X-100

Tween 20

Tween 40

de

21.46 ± 0.28

de

21.73 ± 0.98

ef

19.81 ± 0.39

de

22.76 ± 2.34

f

17.29 ± 0.42

hi

3.21 ± 0.35

de

22.35 ± 0.57

de

21.34 ± 0.45

i

2.2 ± 0.25

a

30.59 ± 0.17

de

22.56 ± 0.47

gh

6.08 ± 0.06

g

Tween 80

7.14 ± 0.28

ꢄ

Control indicates original LPM with 1% olive oil (pH 7.0).

Similar alphabets indicate no significant difference in enzyme activity,

calculated using DMRT (p < 0.05).

†

Table 3. Effect of different treatments on aggregation of pro-

teins produced by GBPI_508 analysed using DLS.

Treatment

Z Average (d.nm)

Control

Acetonitrile (5%)

Sucrose (5 M)

Tween 20 (0.5%)

NP-40 (0.5%)

114.60

102.50

171.80

172.20

55.38

Partial purification and aggregation studies

The lipase activity was recorded only in fraction A,

therefore, used for further enzymatic characterizations.

Specific activity in fraction A was measured as

Brij-35 (0.05%)

Isopropanol (5%)

Taurocholic acid (1%)

114.60

121.40

109.50

4

17.97 U/mg in comparison to 190.58 U/mg in crude

Value in the bold indicates decrease in protein aggregation in presence

of NP-40.

supernatant. Initially, attempts for purification of lipase

were made using gel filtration, ion exchange and

hydrophobic interaction chromatography with several

modifications. Aggregation of protein in the concen-

trated fraction, hampered all such attempts. Further,

various reagents used to disaggregate the proteins

were successful only to a small extent. NP-40 at a con-

centration of 0.05% was able to reduce the size of

protein aggregates to 55.38 d.nm in comparison to

also obtained when SDS-PAGE was performed under

reducing conditions (Figure 4).

Enzyme characterization

The lipase enzyme produced by the bacterium

ꢀ

showed optimal activity at 40 C with more than 60%

ꢀ

1

14.60 d.nm for control as analysed by DLS (Table 3).

Same was also confirmed using native PAGE analysis

data not shown).

activity retained at low temperature (10 C). Activity

ꢀ

decreased drastically beyond 40 C. In contrast, max-

ꢀ

(

imum stability of enzyme was recorded at 20 C.

ꢀ

Enzyme lost its activity when incubated at 50 C and

beyond (Figure 5(a)). Surprisingly, enzyme was almost

equally active in pH ranging from 7.0 to 12.0 while

below pH 7.0 activity of enzyme was reduced. Crude

enzymatic preparation showed maximum stability at

two pH i.e. pH 7.0 and pH 11.0. The stability was

observed to be more than 80% at pH 4.0. The higher

activity and stability in alkaline pH suggest the

GBPI_508 lipase to be an alkaline lipase (Figure 5(b)).

SDS-PAGE and zymogram

SDS-PAGE performed in non-reducing condition fol-

lowed by development of zymogram showed two

active lipase bands. Two bands corresponding to the

zymogram were obtained after staining same gel with

CBB having molecular weight of approximately 50 kDa

and 54 kDa (isoenzymes I and II). Similar results were

BIOCATALYSIS AND BIOTRANSFORMATION

7

Effect of metal ions and inhibitors indicated that

GBPI_508 lipase is resistant to these factors at low

concentration. While examining the effect of metal

ions, more than 75% activity was recorded in most

cases when reaction was performed in presence of

1

mM concentration of metal ions. When concentra-

tion was increased to 5 mM, >75% enzyme activity

þ

2þ

was retained only in presence of K , Na , and Cu

,

while in rest cases the activity reduced to <70%.

Surprisingly, inhibitors including EDTA (1 mM), DTT

(1 mM), CTAB (1 mM), and SDS (1%) increased the lip-

ase activity in comparison to control. Triton X-100 and

Tween 60 caused inhibition of the enzyme activity to

8

6.40% and 71.47%, respectively. The results of the

effects of metal ions and inhibitors on GBPI_508 lipase

activity are shown in Table 4.

GBPI_508 lipase stability decreased when incubated

with organic solvents of decreasing polarity. More

than 75% residual activity was recorded only in case

of methanol, ethanol and acetone while other solvents

decreased the solubility below 75%. Minimum activity

Figure 4. Determination of molecular weight of lipase pro-

duced by isolate GBPI_508 using SDS-PAGE followed by zymo-

gram analysis. Lane 1 – SDS-PAGE under non-reducing

conditions; 2 – SDS-PAGE under reducing conditions; 3 –

Zymogram showing two active lipase bands; M – Standard

protein marker.

(23.58%) was recorded in presence of toluene (Figure

6(a)). Lipase of GBPI_508 showed maximum activity

towards mid chain pNP acyl esters (C10). 5-fold

increased activity of lipase of GBPI_508 was recorded

towards pNP decanoate in comparison to pNP dodeca-

noate as well as pNP octanoate whereas higher chain

acyl (C14 and above) esters were the least hydrolysed

substrates (Figure 6(b)).

Discussion

Bioprospection of extremophiles for enzyme produc-

tion is getting continuous attention as these enzymes

possess higher stability and activity at unusual condi-

tions unlike mesosphilic enzymes. Cold tolerance and

activity of enzymes at low temperature is desirable in

various industrial applications. Such requirements can

be fulfilled either by molecular engineering of

enzymes to increase their catalytic properties (Javed

et al. 2018) or by bioprospecting microbes of low tem-

perature origin for cold-active enzymes. Himalayan

environment provides a unique habitat for isolation of

similar bacterium surviving extreme conditions

(Pandey et al. 2006; Joseph and Ramteke 2013; Jain

and Pandey 2016). Providing the fact that Himalaya

are the youngest mountains, there remains a lot in

exploring its microbial diversity. Therefore, a newly

isolated cold tolerant bacterium, Pseudomonas paller-

oniana (GPPI_508) producing cold-active lipases, has

been characterized in the present study. The bacter-

Figure 5. Effect of (a) temperature and (b) pH on activity and

stability of partially purified GBPI_508 lipase.

ꢀ

ium ability to survive in low temperature of 5 C as

8

R. JAIN ET AL.

Table 4. Effect of metal ions and inhibitors on activity of lipase produced by GBPI_508.

Relative activity (%±SE)

Metal ions

1mM

5mM

Inhibitors

Control

EDTA (1 mM)

DTT (1 mM)

CTAB (1%)

Triton X-100 (1%)

SDS (1%)

Tween-60 (1%)

Relative activity (%±SE)

Control

100 ± 1.46

102.75 ± 0.08

103.36 ± 0.89

97.66 ± 2.18

75.76 ± 2.91

91.34 ± 1.54

80.45 ± 0.65

74.75 ± 0.65

79.02 ± 1.29

83.60 ± 0.57

93.48 ± 0.32

92.57 ± 3.16

100 ± 2.07

91.84 ± 1.60

93.26 ± 1.97

77.30 ± 0.19

39.48 ± 1.31

68.91 ± 2.35

39.83 ± 4.79

67.85 ± 3.19

36.10 ± 1.22

41.73 ± 0.47

40.54 ± 0.66

53.31 ± 0.09

100 ± 1.46

137.48 ± 0.79

101.28 ± 1.37

122.79 ± 3.18

86.40 ± 1.63

128.69 ± 2.89

91.47 ± 0.56

þ

K

þ

Na

2

Cu

Cd

2

2þ

Mg

2

þ

Ca

Sr

Zn

Co

Al

2

3

þ

Fe

Figure 6. Effect of organic solvents on GBPI_508 lipase stability (a) and its substrate specificity (b). pNPO, 4-Nitrophenyl octanoate;

pNPD, 4-Nitrophenyl decanoate, pNPL, 4-Nitrophenyl dodecanoate; pNPM, 4-Nitrophenyl myristate; pNPP, 4-Nitrophenyl palmitate.

well as tolerance to extreme physiological conditions

pH and salt) makes it an ideal candidate having

in lipase production by GBPI_508. Triton X-100

increased the availability of substrate to the bacteria

by forming an emulsion, enhancing the lipase produc-

tion in the medium. Also, detergents increase lipase

production by destabilizing the bacterial membranes

and, thereby, releasing the enzyme (Boekema et al.

2007); although this was not true in case of tweens in

the present study.

Lipases due to their hydrophobic nature are well

known to form aggregates (Velu et al. 2012; de Lima

et al. 2013); similar results were observed in this study

as well. The GBPI_508 lipase showed strong aggrega-

tion in its native form and, therefore, during purifica-

tion attempts using gel filtration chromatography, the

enzyme was eluted in void volume and was not able

to move through the stacking gel in native PAGE.

Variable degree of aggregation was observed probably

due to surface hydrophobic domains in the enzyme

(Sugimura et al. 2000). The solubilisation to a partial

level could be achieved using NP-40 detergent as con-

firmed by DLS as well as native PAGE, where lipase

band was able to migrate in the resolving gel. DLS

has been adopted by various researchers to study

(

potential applications. The lipase secretion was

ꢀ

exhausted at 35 C while slow but continuous release

of enzyme was observed at low temperatures. These

ꢀ

results suggest that at 35 C bacterium attained early

stationary growth phase that provided short duration

for enzyme secretion as compared to low temperature.

GPPI_508 secreted more lipase in alkaline condition

(pH 9.0) in comparison to pH 7.0. Kiran et al. (2008)

has reported maximum production of psychrophilic

alkaline lipase at pH 9.0 by a marine endosymbiotic

Pseudomonas sp. (MSI057). Similarly, Bisht et al. (2013)

and Thakur et al. (2014) reported maximum lipase pro-

duction at pH 9.0 by different Pseudomonas spp.

While examining the effect of various media com-

ponents, it was observed that presence of oil source

as substrate was necessary to induce lipase produc-

tion, while inorganic N-source didn’t influence the

enzyme production as significant lipase production

was observed in its absence. In contrast, organic nitro-

gen similar to other studies (Sharma et al. 2002;

Mobarak-Qamsari et al. 2011) acted as limiting factor

BIOCATALYSIS AND BIOTRANSFORMATION

9

lipase aggregation. Rashno et al. (2018) made

attempts to solubilize lipase from Pseudomonas sp.

through mutation in aggregation promoting regions

frigidimarina NCIMB 400, isolated from Antarctic sea-

water, when expressed in Escherichia coli BL21(DE3)

ꢀ

retained 35% activity at 10 C with high activity in the

ꢀ

(APR) of lipase which are the major driver of protein

range 15–25 C (Parra et al. 2015). Two pH optima (pH

aggregation. DLS as well as other techniques revealed

smaller aggregates of F171E mutated lipase forms in

comparison to wild type lipase. Acharya and Rao

7.0 and 11.0) for lipase stability were observed prob-

ably due to presence of two iso-enzymes in the par-

tially purified preparation. The lipases produced by

GBPI_508 were alkaline lipase as the activity reduced

sharply below pH 7.0 but consistent up to pH 12.0.

Alkaline lipases have been extensively studied to have

various biotechnological applications (Rathi et al. 2001;

Patel et al. 2014; Hu et al. 2018). Generally, lipase does

not require cofactor for its activity (Gupta et al. 2004).

Results suggest that the activity of lipase produced by

GBPI_508 significantly decreased in the presence of

5 mM metal ions, although the activity enhanced in

the presence of chelating agent EDTA. This suggest

that the GBPI_508 lipase does not require metal ions

as cofactor at its active sites. Similar results on a

recombinant lipase from Yarrowia lipolytica (LIPY8)

were also observed by Li et al. (2019).

(2003) investigated to unfold the lipase of Bacillus sub-

tilis in guanidinium chloride (GdmCl). The enzyme

showed drastic reduction in aggregation at 0.5 M con-

centration of GdmCl as confirmed by DLS with mar-

ginal loss of enzyme activity. Aggregation of lipases in

solutions is still a major hindrance in their purification.

Therefore, to uncover their full potential in biotechno-

logical processes new improved methods to prevent

aggregation of lipases is still a major research area.

In =-gel detection of lipase for molecular weight

determination was possible only in the presence of

strong anionic detergent i.e. SDS, although it has been

reported to often denature the enzyme and inactivate

the biochemical activity of proteins (Ne’eman et al.

1

971). SDS-PAGE under denaturing as well as reducing

Enhancement of activity in presence of strong

detergents can be accounted to disaggregation of

enzyme in their presence making substrate more

accessible to the enzyme. In contrast to this study,

SDS and CTAB has been reported to negatively influ-

ence the lipase activity (Peng et al. 2010). The

GBPI_508 lipase preparation retained >50% activity

in the presence of tested organic solvents except tolu-

ene and dichloromethane. Activity of lipase in non-

aqueous solvents is desirable in various synthetic

applications (Sarethy et al. 2011). A possible stability

in non-aqueous solvents can be because of aggrega-

tion properties of enzyme which tend to disaggregate

when incubated in organic solvents, although overall

activity decreased possibly because of removal of

water molecule necessary for catalysis (Patel et al.

2014) and change in the conformation of the protein

(Kumar et al. 2016). Similar to our previous report on

P. proteolytica, GBPI_508 lipases also had the max-

imum substrate specificity for moderate chain fatty

acid ester with maximum specificity for pNP dec-

anoate/caprate (C10). Lipase from Pseudomonas spp.

as well as other bacteria are reported to prefer small

or medium chain esters (Kojima and Shimizu 2003;

Parra et al. 2015). Overall, the lipase enzymes pro-

duced by GBPI_508 is ideal enzyme for use in condi-

tions of low temperature, wide pH and in the

presence of organic solvents. Aggregation behaviour

of GBPI_508 lipase can be further investigated for its

applications in various commercial industries. Cold tol-

erance along with stability in organic solvents by

conditions showed two lipase bands (isoenzymes I

and II) having molecular weight of ꢁ50 kDa and

54 kDa. Our previous study also showed presence of

two lipase isoenzymes in P. protelolytica with approxi-

mately same molecular weight of 50 kDa which could

not be resolved in SDS-PAGE (Jain et al. 2017). Lipases

from Candida rugosa (CRL) (L oꢀ pez et al. 2008) and

Aspergillus niger (H o€ felmann et al. 1985) have been

reported earlier to present in isoforms. Similar molecu-

lar weight of lipase from Pseudomonas aeruginosa

strains has been reported by Hu et al. (2018) (51 kDa),

Ji et al. (2010) (56 kDa), and Karadzic et al. (2006)

(54 kDa). Furthermore, a cold adapted lipase of 64 kDa

from Pseudomonas sp. 7323, a psychrotroph isolated

from Antarctic sea, has been reported by Jin-wei and

Run-ying (2008).

GBPI_508 lipase stability in low temperature and

wide pH makes it suitable enzyme for various com-

mercial applications where low temperature and/or pH

variation can affect the enzyme reaction. The lipases

in the partially purified fraction showed drastic

ꢀ

decrease in its activity beyond 40 C and retained

>

ꢀ

60% activity at 10 C, featuring these enzymes to be

a cold-active lipase rather than a mesophilic lipase.

Kiran et al. (2008) reported extracellular lipase from

Pseudomonas sp. (MSI057) to have 80% activity at

ꢀ

20 C. Psychrobacter sp. ArcL13 produced a typical

cold-adapted enzyme possessing 39% and 70% activ-

ity at 10 and 20 C, respectively (Kim et al. 2015).

Similarly, a lipase (lipE5) gene from Shewanella

ꢀ

1

0

R. JAIN ET AL.

GBPI_508 lipase could be useful for its applicability in

isoenzymes from a technical Aspergillus niger enzyme. J

Food Science. 50(6):1721–1726.

detergent

industries

and

environmental

Hu J, Cai W, Wang C, Du X, Lin J, Cai J. 2018. Purification

and characterization of alkaline lipase production by

Pseudomonas aeruginosa HFE733 and application for bio-

degradation in food wastewater treatment. Biotechnol

Equip. 32(3):583–590.

Jain R, Pandey A, Pasupuleti M, Pande V. 2017. Prolonged

production and aggregation complexity of cold-active lip-

ase from Pseudomonas proteolytica (GBPI_Hb61) isolated

from cold desert Himalaya. Mol Biotechnol. 59(1):34–45.

Jain R, Pandey A. 2016. A phenazine-1-carboxylic acid pro-

ducing polyextremophilic Pseudomonas chlororaphis

bioremediation.

Acknowledgements

The authors are thankful to Director (GBPNIHESD) for

extending facilities.

Disclosure statement

No potential conflict of interest was reported by the authors.

(MCC2693) strain, isolated from mountain ecosystem, pos-

sesses biocontrol and plant growth promotion abilities.

Microbiol Res. 190:63–71.

Funding

Javed S, Azeem F, Hussain S, Rasul I, Siddique MH, Riaz M,

Afzal M, Kouser A, Nadeem H. 2018. Bacterial lipases: a

review on purification and characterization. Prog Biophys

Mol Bio. 132:23–34.

This study was financially supported by the Ministry of

Environment, Forest and Climate Change, Govt. of India,

New Delhi.

Ji Q, Xiao S, He B, Liu X. 2010. Purification and characteriza-

tion of an organic solvent-tolerant lipase from

Pseudomonas aeruginosa LX1 and its application for bio-

diesel production. J Mol Catalysis B: Enzymatic. 66(3-4):

ORCID

Rahul Jain

Anita Pandey

http://orcid.org/0000-0002-7554-6599

http://orcid.org/0000-0003-0592-0220

2

64–269.

Jin-Wei Z, Run-Ying Z. 2008. Molecular cloning and expres-

sion of a cold-adapted lipase gene from an antarctic deep

sea psychrotrophic bacterium Pseudomonas sp. 7323. Mar

Biotechnol. 10:612–621.

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Article Doi

Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

DOI: 10.1080/10242422.2019.1666829

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Article abstract of DOI:10.1080/10242422.2019.1666829

Full text of DOI:10.1080/10242422.2019.1666829

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