INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

Comparative Studies on Lipase Production and Optimization using

Bacillus Subtilis and PseudomonasAeruginosa

Dr. C. Vinothini¹, Dr. A. Chanthru²

¹Assistant Professor,Department of Biotechnology,E.G.S.Pillay Arts and Science College,Nagapatinam.

²Head of the Department of Microbiology, E.G.S.Pillay Arts and Science College,Nagapatinam

DOI: https://doi.org/10.51583/IJLTEMAS.2026.150500209

Received: 14 May 2026; Accepted: 19 May 2026; Published: 15 June 2026

ABSTRACT

The presents study deals with screening of lipase producers from the lipase rich environment like petrol bank,

Auto mobile shops from soil sample. The micro organisms have the ability to produce the lot amount of lipase.

The sample was collected from (petrol bank, Auto mobile shops). Tributyrin agar medium was prepared 14

strains were isolated from that 2 strains able to produce lipase. The bacterial strain confirmed by biochemical

method. The isolated bacterial strain are Bacillus sp. and Pseudomonas sp. The bacterial strains need temperature

around 35⁰c, PH 7, olive oil as a carbon source and Incubation for 48hr. If the temperature is higher the enzyme

productivity is also high. Thin layer chromatography of lipids revealed that bacillus sp has higher lipase activity

than pseudomonas sp.

The present study revealed that extracellular lipase production by

Bacillus sp. and Pseudomonas sp. isolated from soil samples was found to be accelerated at optimized culture

conditions such as medium pit, temperature and carbon source. From the result, it could be concluded that the

medium pH of 7.0 and temperature 35⁰c were optimum for maximizing lipase production by Bacillus sp. and

Pseudomonas sp. Inferred that the optimum contain based on this study lipase activity is higher in Bacillus sp.

and Pseudomonas sp. than compare other 12 strains.

Keywords: Lipase,Enzyme productivity,Optimization,Pseudomonas sp

INTRODUCTION

Enzymes are biomolecules that catalyze (i.e. Increase that rate of) chemical reactions. Almost all enzymes are

proteins in nature. A very important property of an enzyme in its specificity and selectivity.The biological

relevance and variability of lipids described above has led to the development of a great variety by lipid –

degrading enzymes throughout all kingdoms of life. Among them, lipase are hydrolytic enzymes that catalyses

the cleavage of ester bonds in triglycerides and producing glycerol and free fatty acids, are considered to be

some of the most important biocatalysts due to their widespread biological functions and due to their

biotechnological potential(Bornscheuer, 2002).

Lipases are widely distributed among bacteria, fungi, plants and animal , although they are more frequently

found in microorganisms .Their physiological functions are not yet clear for many of them, although they seem

to be involved, in general, in the bioconversions of lipids (mainly TAGs) between different organism or into the

same organism (Pandey et al., 1999).If bacteria source bacteria is the most important source for lipase. They are

many lipase productions bacteria are Bacillus, Bacillus subtitis, Bacillus pumilus, Bacillus termoleavorans,

Bacillus stearothermophilus, Bacillus sphaericus, aerogenesa, Staphylococcus epidermidis, Preuclomonase sp.

etc.Lipolytic activity of difficult to determine due to the fact that lipases are water soluble enzymes acting on

water insoluble substrate concentration at the interface or the use of different detergents must be taken into

account to interpret the activity and the enzyme kinetics obtained.

Chromatographic techniques can be used to detect or quantity FAs released from TAGs and other lipids. These

techniques are very sensitive, although, they are time – consuming. The most common ones are: silicic acid

Page 2608

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

columns, thin – layer chromatography, gas – liquid chromatography of FA methyl esters, and high – performance

liquid chromatography (HPLC) .The latest is usually performed by detecting b – naphtol or P – nitro phenol

released as a result of lipolytic activity on their corresponding FAs ester derivatives. Chromatographic assays

are also used for detecting the reaction products of lipases in reactions of synthesis or acyl exchange, as well as

for assaying the enantioselectivity of these enzymes.(Jaeges et al., 1999).Bacterial lipases are gaining an

increasing interest due to their potential as biotechnological catalysts, as well as by their role as virulence factors

in some pathogenic bacteria(Rosenau& Jaeger, 2000).

Understanding the mechanisms of gene expression, folding, and secretion of bacteria lipases, as well as their

enzymatic properties is essential for improving the biotechnological application of these enzymes, and for the

treatment of lipase – related diseases (Rosenau & Jaeger, 2000).

Lipases are used in situ, and sometimes together with other enzymes, during the elaboration of bread, cheese,

and other foods to improve their shelf – life and their rheological properties, or to produce armas or emulgents.

Moreover, they are used ex situ to produce flavors, and to modify the structure or composition of AGS by inter

– or transesterification, in order to obtain AGs with an increased nutritional value, or suitable for parenteral

feeding (Gunstone,1999; Reetz, 2002).Bacteria (and fungal) lipases the most versatile and widely used class of

enzymes in biotechnological applications and organic chemistry.

This is reflected by more than 1000 original articles and reviews on lipase and lipase applications that appear

each year, and by a the fact that the use of lipase in biotechnology was a business of more than1.5 billion U.S.

dollar in year 2000( Jaeger & Eggert, 2002; Gupta et al., 2004).

MATERIALS AND METHODS

Sample Collection and Isolation of Bacteria

The soil was collected from auto mobile shop in Thanjavur, collected in a sterile container and it was brought to

the laboratory for further processing. The collected sample was serially diluted upon 10⁷dilution using sterile

saline as a blank and the dilutes samples were plated into the sterile nutrient agar using spread plate method.

Screening for Lipase Producing Organism

The isolated pure strain were screened for the production of extracellular lipase production using screening

medium contain Tributyrine Substrate. The pure culture were streaked at the center of the sterile screening plates

were incubated at 375°C for 24 hours only positive andbetter zone formed strain was taken for further study.

Identification of Organisms

The positive strain that produces maximum lipase enzyme was selected and identification by biochemical.

Lipase Production Medium

Composition of Production Medium

Component

Quantity (g/L)

Peptone

10

Yeast extract 5

NaCl

5

Olive oil

10 mL

Page 2609

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

CaCl₂

0.1

Distilled water 1000 mL

pH adjusted to 7.0 before sterilization.

Screening for Lipase Production

Tributyrin Agar Method

Bacterial isolates were streaked on tributyrin agar plates and incubated at 37°C for 24–48 hours.

Observation

Clear zones around colonies indicated lipase production.

Enzyme Production

The enzyme production was carried out by shake flask fermentation using production medium which comprising

of GW cose as a carbon source and amended with peptone as a protein acids substrate with pH₇. Five hundred

ml of sterile production broth was prepared in one liter conical flask an 5% inculum was transferred aseptically

into the production medium. The inoculated medium was incubated at 37°C for 48hours. The medium was

agitated at 200rpm for better aeration and growth of the organism.

Parameter Optimization Studies

Temperature

Sterile production media was prepared and inoculated with 5% inoculums in different test tube each flask was

incubated at different temperature such as 35, 40, 45, watch 24hrs. The OD value was observed.

pH

Production media was prepared and inoculums with 5% inoculums in different test tube and pH and medium

was adjusted to different pH such as 6, 7, and 8. Using 0.1 N Noah. The flask was incubated at 32°C for 24hrs.

The OD was measured.

Carbon Source

Growth and lipase production were determined at 12h interval up to 42h using different carbon source. The

growth and lipase production by the organism were highest in olive oil and least in coconut oil (Kamini et al.,

and Bhushan and Hoon Dail) have also reported olive oil as the carbon source.

Lipase Assay (Confirmation Test)

Titrimetric Lipase Assay

Principle

Lipase hydrolyzes

الزيت

/olive oil into fatty acids. Released fatty acids are titrated with NaOH.

Procedure

 Prepare substrate emulsion using olive oil and gum acacia.

Page 2610

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

 Add crude enzyme extract.

 Incubate at 37°C for 30 min.

 Stop reaction using acetone-ethanol mixture.

 Titrate liberated fatty acids with 0.05 N NaOH using phenolphthalein.

Formula

Lipase Activity (U/mL)=V×N×1000T×Vs\text{Lipase Activity (U/mL)} = \frac{V \times N \times 1000}{T

\times V_s}Lipase Activity (U/mL)=T×VsV×N×1000

Where:



V = Volume of NaOH used

N = Normality of NaOH

T = Incubation time

VsV_sVs = Volume of enzyme sample

Thin Layer Chromotographic

Preliminary characterization of the lipase was done by TLC method and was separated on a silica gel plating

using CHCl:H₂O (70:10:0.5^-V/V/V)as developing solvent system with different colour developing reagents.

Ninydrin reagent (0.5g inhydrin in 100 ml anhydrous acetone) was used to detect lipopeptide, asyellow spots

and lipase are generally identified as yellow spots.(yin.h.,j.Qiang 2008) .

RESULT AND DISCUSSION

Naturally occurring microorganisms’ are having ability to produce the various enzymes. Now a day’s most of

the enzyme is industrially important and human welfare. Lipase is one of the important enzymes, which can be

produced from microorganisms.

In this study, the bacterial strain was isolated from auto mobile shops and petrol bank soil from Thanjavur. The

samples were inoculated on tributyrin agar plate by serial dilution using sterile distilled water. 14 bacterial strains

were isolated from the sample. Then screening medium was prepared from 14bactria strain are produce lipase

(Plate: 1). The samples contained 2.06 x 10⁶ CFU/ml (plate: 1).

Then the dominant organism were isolated and individually streaked on tributyrin (Hi media 0>1), agar plates

and the formation at halo zone around the colony on tributyrin agar was considered as the positive colony for

lipase production

Biochemical characters:

Enrichment culture technique enabled the isolation of strain with lipolytic activity in tributyrin media plates.

The lipolytic microbes were further and reactions and then identified as Gram positive, rod shaped motile

organism. (Table: 1). Finally the morphological and biochemical test indicated that the suspected organism were

Bacillus sp. and the lipolytic microbes were further and reactions and then identified as Gram negative, rod

shaped, motile organism. The morphological and biochemical test indicated that the suspected organism were

Pseudomonas sp.

The isolated organism were B₁(Auto mobile shop) identified Bacillus sp. based on biochemical characters.

The isolated organism was B₂(petrol bauk) identified as Pseudomonas sp. based on biochemical characters.

Page 2611

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

Effect of temperature:

Fig: 3 Effects of different incubation temperature on the production of lipase by bacillus sp. The optimum

temperature for lipase production by bacillus sp was observed to be 35°C.The effects of different incubation

temperature on the production of lipase by Pseudomonas sp. The optimum temperature for lipase production by

Pseudomonas sp. was observed to be 35°C.

Effect of pH:

Fig:4 In our study the lipase production by Bacillus sp. produced the maximum lipase at pH 7and lipase was

minimum at pH4.

In our study the lipase production by Pseudomonas sp. production the maximum lipase at pH 7and lipase was

minimum at pH4.

Effect of carbon source:

Fig:5

Growth and lipase production were determined at 12h interval up to 42h using different carbon source.

The growth and lipase production by the organism were higher in olive oil and lower in coconut oil. It have also

reported olive as the best carbon source.

Analysis of lipase:

Lipase isolated by acid precipitation method at pH was identified by TLC method. Bacillus sp. and Pseudomonas

sp. was identified by yellow colour spot. The volue of Rhamo lipid are 0.78,0.64,oil and petrol. Bacillus sp.

have higher lipase activity. The Pseudomonas sp. has low lipase activity.

The dominant organism were isolated by streaking on tributyrin agar plates and the formation of halo zone

around the colony on tributryrin agar was considered as the positive colony. Enrichment culture technique

enabled the isolation of strain with lipolytic activity in tributrin media plates. The lipolytic microbes were further

screened and characterized.

The optimum temperature for lipase production by Bacillus coagulans was observed to be 35°C, However the

organism showed on growth below 20°C and 40°C. Similar the same result were also reported by [A. Thomas

et al., 2003].

Similar result were obtained, maximum growth was achieved after 44h of incubation at 35°C. The lipase

production was detected in the late logarithmic phase (after 20h), and increased until optimum production was

achieved, after 48h of incubation. The production of degradative enzymes tends to occur during the late

logarithmic phase of growth, when all density is high [Rahman et al., 2005].

Bacillus coagulans produced by the maximum lipase at pH₄, Jose and Kurup have also reported pH₇, for the

optimal lipase production in Bacillus pumilus.

The pH of the culture dropped from the initial neutral pH to a pH of 5.3 during the first 12h of incubation. This

might be attributed to the production of acids during bacterial growth. However, the pH of the culture medium

increased gradually to a pH of 8.5 after 24h. The pH then gradually dropped, and remained unchanged at pH

7.8 after 48h. The rise in pH after 12h of incubation may be due to the utilization of organic acids or the

production of alkaline compounds during this period.

The growth and lipase production by the organism were high in olive oil and least in coconut oil (Kamine et al.,

and Bhushan and Hoon dial) have also reported olive oil and the best carbon source.

Page 2612

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

Interpredation

As reported by (Nakashima et al., 1988) the presence of olive oil as growth medium greatly enhanced the lipase

activity of bacillus strain (B₁) in the present study. As reported by (Tsuzuki et al., 1999). The presence of olive

oil as growth medium greatly enhanced the lipase activity of Pseudomonas sp. strain (B₂) in the present study.

Thin layer chromatography of lipids revealed that Bacillus sp. has higher lipase activity when compared to

pseudomonas sp.

Lipase production increased with incubation time up to 48 hours in both organisms.

Maximum enzyme activity was observed at 48 hours:

Bacillus subtilis = 65 U/mL

Pseudomonas aeruginosa = 100 U/mL

After 48 hours, lipase production gradually decreased.

The reduction in enzyme activity at longer incubation periods may be due to:

nutrient depletion,

accumulation of toxic metabolites,

changes in pH,

enzyme degradation The paper conclutes

Pseudomonas aeruginosa produced higher lipase activity than Bacillus subtilis at all incubation periods,

indicating its greater efficiency as a lipase-producing organism

Table :1

Biochemical Characterization of Lipase production from Bacillus and Pseudomonas

Biochemical Tests for Identification

Biochemical Test Table

Test

Bacillus subtilis Pseudomonas aeruginosa

Gram staining

Catalase test

Oxidase test

Positive rods

Positive

Negative rods

Positive

Negative/Variable Positive

Citrate utilization Positive

Positive

Indole test

Negative

Page 2613

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

Methyl Red

Negative

Positive

Motile

Voges-Proskauer Positive

Urease test

Negative

Motile

Motility test

Starch hydrolysis Positive

Gelatin hydrolysis Positive.

Negative

Positive

EFFECT OF PH

B1-Bacillus

B2 = Pseudomonas

TABLE-2-Effect of pH

Sample1&2

PH VALUE

OD VALUE

Bacillus subtilis

5

6

7

8

5

6

7

8

0.4,

0.6.

0.8

0.6

0.2

0.4

0.6

0.4

Pseudomonas

aeruginosa

Page 2614

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

Fig :4

pH Value

FIG.4

Page 2615

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

Tab: 5Effect of Incubation Time

Time (hrs) B. subtilis P. aeruginosa

24

48

72

96

30

65

55

40

50

100

85

60

Graph: Effect of Incubation Time on Lipase Production

Lipase Activity (U/mL)

100 |

90 |

●

80 |

●

70 |

60 |

■

50 |

● ■

40 |

■

30 | ■

20 |

10 |

------------------------------------------------

24 48 72 96

Incubation Time (hrs)

■ Bacillus subtilis

● Pseudomonas aeruginosa

Lipase Enzyme Production in Thin Layer Chromatography

Photo-1

The study demonstrated that both bacterial species produced extracellular lipase, but Pseudomonas aeruginosa

showed significantly higher enzyme activity under optimized conditions. Maximum production occurred at:

 pH 7

Page 2616

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

 Temperature 37°C

 Incubation time 48 hours

The higher lipase yield by Pseudomonas aeruginosa may be due to its strong extracellular enzyme secretion

system.

Both Bacillus subtilis and Pseudomonas aeruginosa are efficient lipase producers. However, Pseudomonas

aeruginosa exhibited superior lipase activity and better optimization response. These organisms can be exploited

for industrial lipase production.

REFERENCE

1. Akio Sugihara, Masahiko veshima, Yuji Shimad, Susumu Tsunasawa and Yoshio Tominaga, (1992).

Purification and characterization of a Novel Thermostable lipase from Pseudomonas capacia. Journal of

Biochemistry, Vol.112, 598-603.

2. An-Feihsu, Kerby C.Jones, Thomas A. Foglia and William N. Marmer (2003). Optimization of alkyl

ester production from grease using a phyllosilicate sol-gel immobilized lipase, Journal of Biotechnology

letters, Vol. 25, 1713-1716.

3. Angkhameen Buranorom, Khemika Lomthaisong and Hataichanoke Niamsuf (2005), Optimization of

Bacteria culture condition for maximal lipase activity.

4. Anjanashrma, Dipa Bardhan and Rashmi Patel (2009), Optimization of physical parameters for lipase

production from Arthrobacter sp. BGCC490, Indian Journal of Biochemistry Biophysics, Vol. 46, 178-

183.

5. Anurag Sekhon, Neetu Dahiya Ram P Tewari and Gurinder S Hoondal, Arthrobacter sp (2006),

Production of extracellular lipase by Bacillus megateruim AKG-1 in submerged fermentation. Indian

Journal of Biotechnology Vol.5, 179-183.

6. Aravindan Rajendran, Viruthagiri Thangavelu (2007), sequential optimization of culture medium

composition for extracellular lipase production by Bacillus sphaericus using statistical methods, Journal

of chemical technology and biotechnology Vol.82, (5), 460-470.

7. Benjamin.S, and A. Pandey, (1980), lipase production by Candida rugosa on copra waste extract, Indian

Journal of Microbiol, 20,302-309.

8. Bhushan.B, G.S.Hoondal, (1994), characterization of lipase from an alkalophilic yeast sp, Biotechnol

Lett, Vol. 16, 837-840.

9. Change-Soo Lee, Ju-Young Jung, Jae-seong so, and Eun-Ki Kim, 1997. Optimized production of a

thermostable lipase by recombinant Pseudomonas putida 1641. Journal of Biotechnol Bioprocess Eng.

Vol. 2, 11-14.

10. Gaur Ruchi, Gupta Anshu and S.K. Khare (2008). Lipase from solvent tolerant Pseudomonas aeruginosa

strain. Production optimization by response surface methodology and applications. Journal of

Bioresource technology Vol.99, (11) 4796-4802.

11. Ginalska.G, R. Bancerz, T. Korni owicz- Kowalsk (2000). A thermostable lipase produced by a newly

isolated Geotrichum-like strain, R59, Journal of Industrial microbiology and biotechnology, Vol. 31, (4)

177-182.

12. Grasian Immanuel, Palanichamy Esakiraj, Austin jebadhas, Palanisamy Iyapparaj and Arunachalam

Palavesam (2008). Investigations of lipase production by milk isolate serratia rubidaea, Journal of food

techno Biotechnol Vol.46, (1), 60-65.

13. Gupta, R., N.Gupta and P.Rathi, 2004. Bacteria lipases: An overview of production, purification and

biochemical properties. Applied microbial. Biotechnol, Vol. 64, 763-781.

14. Gupta.A, R.Singh, S.K.Khare, and M.N Gupta, (2002) an extracellular alkaline lipase from Enterobacter

aeruginosa, Indian Journal of microbial, Vol.42, 263-265.

15. Gupta.R, N. Gupta, P. Rathi (2004), Bacterial lipases: an overview of production, purification and

biochemical properties, Journal of Appl microbial Biotechnol. Vol. 64, 763-781.

16. HaqI-U, Idress.S and M.I.Rajoka (2002) production of lipase by Rhizopes oligosporus by solid state

fermentation, process Biohem, Vol.37, 637-641.

Page 2617

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

17. Heravi.K.M, F.Eftekhar, B.Yakhchali and F. Tabandeh (2008), Isolation and Identification of a lipase

producing Bacillus sp, from soil, Pak Journal of biology science, Vol.11, 740-745.

18. J.L. Arpigny, K.E. Jaeger, Bacterial lipolytic enzymes classification and properties, Biochem. J. Vol.343

(1999) 177-183.

19. Jaeger, K.E.and Eggert, T. 1999. Lipase for biotechnology Curr. Opin. Biotechnol. Vol.13: 390-397.

20. Jane Gilbert. E, Alex Cornish and Colin W. Jones (1991), purification and properties of extra cellular

lipase from Pseudomonas aeruginosa EF2. Journal of general microbiology Vol.137, 2223-2229.

21. Jane Gilbert. E, Jan .W. Drozd and Colin W. Jones (1991), physiological regulation and optimization of

lipase activity in Pseudomonas aeruginosa EF2, Journal of General microbiology Vol.137, 2215-2221.

22. Jose J and M.G.Kurup (1999), purification and characterization of an extracellular lipase from a newly

isolated Bacillus pumillus, Indian Journal of exp Biol, 37, 1213-1217.

23. K.E. Jaeger, M.T. Reetz, Microbial lipase from versatile tools for biotechnology, Trends Biotechnol. 16

(1198) 396-403.

24. Kajao chakraborty and R. Paulraj (2009), purification and Biochemical characterization of an

extracellular lipase from Pseudomonas fluorescens MICC2421, Journal of Agriculture and food

chemistry 57, 3859-3866.

25. Kazlauskas, R.J., Bornscheuer, U.T. Biotransformation with lipases. Biotechnology.8. New York: VCH.

(2002) 37-192.

26. Kok RG, CB Nodel, RH Gonzatez, IM Nugteren-Roodzant and KJ Hellingwerf (1996). Physiological

factor affecting production of extracellular lipase (LipA) in Acinetobacter calcoaotiws BD413: fatty acids

repression of liPA expression and degradation of lipA, Journal of Bacteriol, Vol. 178 (20) 6025-6035.

27. Kombourova.M, E. Emanuilova, P. Dimitrov (1996). Influence of culture conditions on thermostable

lipase production by a thermophilic alkali tolerant strain of Bacillus sp, Journal of folia microbiologica,

Vol. 41 (2) 146-148.

28. Kouker, G. and E.E.Jaeger, 1987. Specific and sensitive plate assay for bacterial lipases. Appli. Environ.

Microbiol. Microbiol, Vol.53: 211-213.

29. Liangyu zheng, Suoginzhang, Lifang Zhao, Guangshan Zhu, xueying Yang, GuiGao, (2006). Resolution

of N- (2-ethyl- 6-methylphenyl) alaninevia free and immobilized lipase from pseudomonas capacia,

Journal of biocatalysis B: Enzymatic Vol.38, 119-125.

30. Limpon Bora, MC.Kalita (2009), Production of extracellular lipase from Bacillus sp. LBN4 by solid state

fermentation. The Internal Journal of microbiology, Vol.7,

31. Noureddini.H, X. Gao and R.S. Philkana (2005). Immobilized Pseudomans capacia lipase for biodiesel

fuel production from soybean oil, journal of Bioresource technology, Vol. 96, 769-777.

32. Marcin.C, L. Katz, R. Greasham and M. Chartrain. (1993), optimization of lipase production by

Pseudomonas aeruginosa MB 5001 in batch cultivation. Journal of Industrial microbiology and

Biotechnology Vol.12, (1) 29-34.

33. Ollist ol, Cheab E, Cyler M, Dijkstra B, Frolow P, Franken SM, Harel MM, Remingeton sj, Silman I,

Satrag J, Sussman JI, Verschueren K, Golman A (1992). The OC hydrolase fold problem Eng.S.PP.197-

211.

34. Pandey, A., Selvakumar, P., Soccol, CR., and Nigam P (1999) solid state fermentation for production of

Industrial enzymes. Curr sci., Vol.49, 51-61.

35. Pandey.A., Benjamin, S., Soccol.CR. Nigam, P,Krieger, N., and Soccol VT (1999). The realm of

microbial lipases in Biotechnology. Biotechnology Applied Biochemistry. Vol.29, 119-131.

36. Prasanth Kumar MP and AK valsa (2007), Optimization of culture media and cultural conditions for the

production of extracellular lipase by Bacillus coagulans. Indian Journal of Biotechnology, Vol. 6, 114-

117.

37. Raja Noor Zaliha Raja Abdul Rahman, Syarul Hatagain Baharum, Abu Bakar Salleh, Mahiran Basri

(2006, S5 Lipase: An organic solvent tolerant enzyme, Journal of microbiology, 583-590.

38. Rohit Sharma, Yusuf chisti and Chand Banerjee, (2006), production, purification characterization and

applications of lipases, African Journal of Biotechnology, Vol.5, (10), 878-882.

39. Rosenau F, Jaeger KE (2000) Bacterial lipases from Pseudomonas: regulation of gene expression and

mechanisms of secrelion. Biochemie, Vol. 82: 1023-1032.

40. Sai Fuddin. N, A.Z.raziah (2008), Enhancement of lipase enzyme activity in non-aqueous media through

a rapid three phase partitioning and microwave Irradiation, Journal chemistry, Vol.5, (4), 864-871.

Page 2618

www.rsisinternational.org

INTERNATIONAL JOURNAL OF LATEST TECHNOLOGY IN ENGINEERING,

MANAGEMENT & APPLIED SCIENCE (IJLTEMAS)

ISSN 2278-2540 | DOI: 10.51583/IJLTEMAS | Volume XV, Issue V, May 2026

41. Samad MYA, Salleh AB, Razak CAN, Ampon K Yunus WNZ, A lipase from a newly isolated

thermophilic Rhizopus rhizopodiformis, World Journal microbial Biolechnol, Vol. 6 (1990) 390-394.

42. Samia A. Ahmed (2008), optimization of production and extracellular parameters of Bacillus megateruim

levansucrase using solid-state fermentation Journal of Applied sciences Research, Vol. 4, 1199-1204.

43. Sangeetha.R, A.Geeth and I. Arulpandi, (2008), Optimization of protease and lipase production by

Bacillus pumillus SG2 isolated from an industrial effluent, the internet Journal of microbiology, Vol.5,

44. Sarada sarkar, B. Sreekanth, ShreyaKant, Rintu Banerjee, B.C Bhatla Charyya (H 1998). Production and

optimization of microbial lipase. The Journal of Bioprocess Engineering, Vol.19, 29-32.

45. Saravanan.A.N, N.Suchitra, K.Dhandayuthapani (2007). Role of saturated fatty acids in lipase

production-using Pseudomonas aeruginosa, Journal of food biochemistry Vol.31, (6), 748-756.

46. Satyendra kumar, Khyodano Kiran, Ashutosh upadhyay, Shamsher, S. Kanwar and Reena Gupta (2004),

production, purification and characterization of lipase. From thermophilic and alkaliphilic Bacillus

coagulant Bis- Journal of science Dire act, Vol.41, (1), 38-44.

47. Seghal Kiran.G, S. Shanmughapriya, J. Jayalakshmi, Joseph selvin, R. Gandhimathi, S.Sivarama

Krishnan, M.Arunkumar, T.Thangaveru, K.Natarajaseenivasan (2008). Optimization of extracellular

psychrophilic alkaline lipase produced by marine Pseudomonas sp. (MSI 057), Journal of Appl environ

microbial, Vol. 64, (7) 2644-2651.

48. Selvamohan.T, A.Palavesam and G.Immanvel, (2008), Isolation and characterization of lipase-producing

Bacillus strains from oil mill waste. African Journal of Biotechnology 7, (15), 2728-2735.

49. Shuen-Fuhlin (1996), production and stabilization of a solvent-tolerant alkaline lipase from

Pseudomonas pseudoalkaligenes F-111, Journal of fermentation and bioengineering Vol.82, (5), 448-

451.

50. Takeshi Ito, Hirokazukikula, Eiji nagamori, Hiroyuki Honda, Hivoyase ogino, Haruo Ishi (2001). Lipase

production in two-step fed-batch culture of organic solvent-tolerant Pseudomonas aeruginosa LJT-03,

Journal of Bioscience and Bioengineering, Vol.91, (3), 245-250.

51. Thomas A, M.Mathew, A.Valsa, S.Mohan and R.Manjula (2003), Optimization of growth conditions for

the production of extracellular lipase by Bacillus mycoides, Indian Journal of microbial, Vol.43, 67-69.

52. Uwe Bornscheuer.T, Cornelius Bussler, Ramisetti Srinivas, Sajja Harikrishna (20020. Optimizing lipases

and related enzymes for efficient applications, Journal of Trends in Biotechnology, Vol. 20, (10), 433-

437.

53. Vijay Gunasekaran, Shireen Meher Kotay, Debabrata Das (2006). Alkaline lipase production by

Citrobacter freundii 11T-BTL139, Indian. Journal of experimental Biology, Vol.44, 485-491.

54. Winteler HV, B. Schneidinger, KE Jaeger and D Hass (1996). Anaerobically controlled expression

system derived from the arc DABC operon of Pseudomonas aeruginosa: application to lipase

productions, APPL. Environ. Microbial, Vol. 62, (9) 3391-3398.

55. Wu. WH, T.AFoglia, W.N. Marmer, and J.G. Phillips (1999), Optimizing production of ethyl esters of

grease using 95% ethanol by response surface methodology, Journal of the American oil chemist’s

society, Vol.76, (4), 517-521.

56. Xiao yan Wu, Sanna jaaskelainen and yu-yen linko (2000). An investigation of crude lipase for

hydrolysis, esterfication, and trans esterification. Journal of Bacteriol, Vol. 182, 696-703.

57. Ycos, L. Robledo, C. Otero (1996), stability of a pseudomonas sp. Lipase: comparison between

solubilized enzyme in reverse micelles and suspended lipase in dry solvents, Biocatalysis and

Biotransformation, Vol.14, (4), 251-267.

58. Yu luo, Yitao Zheng, Zhengbing Jiang, Yujhu Ma, Dong Zhi wei, (2006), A novel psychrophilic lipase

from Pseudomonas fluorescence with unique property in Chiral resolution and biodiesel production via

transesterfication, APPl, microbial Biotechnol, Vol.73, 349-355.

59. Yuji Shilada, Akiosugihara, Hirofumi nakano, Tomomi Yokota, Toshihire Nagao, Sadao Komemushi,

Yashio Tominaga (1996). Production of structured lipids containing essential fatty acids by immobilized

Rhizopus delemar lipase, Journal of the American oil chemists’ society, Vol.73 (11) 1415-1420.

Page 2619

www.rsisinternational.org