Harnessing the Power of Thermophilic

Anoxybacillus sp. For High-Yield

Thermophilic Lipase Production and
Characterization
A Project-I report (20314406) submitted in partial fulfillment
of the requirements for the degree of

B. TECH
IN
BIOTECHNOLOGY

By

PATEL ADITIBEN KALPESHBHAI (210305144008)

KAPIL SINGH (210305144027)

ADITHYA P.R (210305144025)

DIKSHITA GAJJAR (210305144046)

Department Of Biotechnology

Parul Institute of Technology,

Parul University, Vadodara, Gujarat

(India)

October, 2024

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 DECLARATION
We thus certify that the project we submitted, Harnessing the Power of
Thermophilic Anoxybacillus sp. For High-Yield Thermophilic Lipase
Production and Characterization" satisfies the academic requirements for the
Parul Institute of Technology, Parul University's (Bachelor of Tech) in
Biotechnology degree. This document documents the work we completed under the
guidance of Dr. Mitun Chakraborty, Professor, Parul Institute of Technology.

Furthermore, we affirm that the work described in this project has not been
submitted—in whole or in part—for the granting of any other degree or diploma at
this institution or any other institution or university.

Place:
Date: Signature of the candidate

___________________
Patel Aditiben KalpeshBhai

____________________

Kapil Singh

_____________________

Adithya P. R

_____________________

Dikshita Gajjar

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 CERTIFICATE

It is certified that the project entitled “Harnessing the Power of Thermophilic

Anoxybacillus sp. For High-Yield Thermophilic Lipase Production and
Characterization” submitted by “PATEL ADITIBEN KALPESHBHAI
(210305144008), KAPIL SINGH (210305144027), ADITHYA
P.R(210305144025), DIKSHITA GAJJAR (210305144046), Parul Institute of
Technology” Is record of work carried out by them under my supervision, as per
the requirement of academics and ethics.

The contents of this report have not been submitted and will not be submitted
either in part or in full, for the award of any other degree or diploma in this
institute or any other institute or university.

_______________________

Signature of the Guide

Prof. (Dr.) Mitun Chakraborty
Professor, Dept. Of Biotechnology,
PIT, Parul University

_________________________
Signature of the HoD
Dr Rajan Kumar Singh
HOD, Department of Biotechnology,
Parul University and the entire
Department of Biotechnology

Place: PIT
Date: 21/10/2024

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 ACKNOWLEDGEMENT

We would like to express our sincere gratitude to everyone who helped make this
project a success. First and foremost, we would want to express our profound
gratitude to our assignment supervisor, Dr. Mitun Chakraborty, for his
unwavering guidance, insightful advice, and support throughout the study process.
Their knowledge and assistance had been essential to making this project a success.
We express our sincere gratitude to the Parul Institute of Technology's
Department of Biotechnology for providing the infrastructure and the resources
needed to carry out this investigation. We also acknowledge the support of the
laboratory staff for their timely and technical assistance throughout the
experimental phase.

Place: PIT
Date: 21/10/2024

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 ABSTRACT

This study examined thermophilic Anoxybacillus sp. for high yield thermostable
lipase enzymes production. Bacterial strains were isolated from hot springs
Manikarna, Himachal Pradesh, India, and cultured under thermophilic
conditions. Lipase production was induced in the bacterial strains using olive oil as
a substrate. The extracts were prepared through precipitation by ammonium sulfate.
The protein bands of lipase at around 31 kDa were confirmed through SDS-PAGE
analysis indicating its successful isolation. The protein concentration was evaluated
by Bradford assay, and the result was averagely at 1.338 mg/ml with high linear
relationship of R2 = 0.9978, which proved that efficient extraction and
measurement occurred. Further characterization is needed, but this study
demonstrates Anoxybacillus sp. potential for thermostable lipase production that
may be exploited in industrial processes under high temperature. This research
contributes to sustainable development of biocatalysts and leads towards optimizing
lipase production from the species of Anoxybacillus.

Keywords: Thermophilic bacteria, Anoxybacillus, thermostable lipase,

enzyme production, ammonium sulfate precipitation, SDS-PAGE, Bradford
assay, biocatalysts, industrial enzymes

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 TABLE OF CONTENTS

Contents Page No.

ACKNOWLEDGEMENT…………………………………………… 4
ABSTRACT…………………………………………………………… 5
LIST OF FIGURES………………………………………………… 8
LIST OF TABLES……………………………………………………… 9
LIST OF TERMS AND ABBREVIATIONS…………………………. 10

1 Chapter 1: Introduction……………………………………… . 12

2 Chapter 2: Review of literature.................................................. 16

2.1: Thermophiles..................................................................................... 16

2.1.1: Classification of thermophiles............................................... 16

2.2: Thermophilic enzymes....................................................................... 17

2.2.1: Thermophilic lipase enzyme ................................................... 17

2.3: Anoxybacillus species........................................................................ 20

2.3.1: Importance of enzymes and whole cell.................................. 22

2.3.2: Pathogenicity of bacteria ...................................................... 24

2.4: Objective of the study ....................................................................... 26

2.5: Gap analysis .................................................................................... 27

3 Chapter 3: Materials and Methods…………………………… 29

3.1: Sample collection and isolation of thermophilic bacteria.................... 30

3.2: Culture revival....................................................................................... 31

3.3: Primary culture................................................................................... 29

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3.4: Secondary culture.............................................................................. 29

3.5: Salting-out of protein ......................................................................... 30

3.6: SDS-PAGE.......................................................................................... 31

3.7: Bradford’s Assay.................................................................................. 32

4 Chapter 4: Results....…………………………………… ............... 33

4.1: culture Revival................................................................................. 33

4.2: Salting out........................................................................................... 34

4.3 SDS-PAGE.......................................................................................... 36

4.4 Bradford’s Assay Test...................................................................... 38

5 Chapter 5: Discussion....………………………………….............. 40

6 Chapter 6: Conclusion ....……………………………………...........43

7 Chapter 7: References....…………………………………............... 47

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 LIST OF FIGURES

Fig 1. Thermostable L2 lipase crystallization and 3D structural clarification of

thermophilic locally isolated Bacillus sp. L2 (.pdb database)

Fig 2. Isolate the lipase enzyme from Manikarna hot spring archaea bacteria

Fig 3. Ref. Recent discoveries and applications of Anoxybacillus

Fig. 4.1 Culture Revived
Fig. 4.2 Precipitate formed after salting out method
Fig. 4.3 SDS PAGE Gels Images showing bands at approx. 31kDa
Fig. 4.4 Graph for Bradford Assay Test

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 LIST OF TABLES

Table 1: Volume of sample obtained and stored after salting out

Table 2: Showcasing data for the preparation of standard curve

Table 3: showcasing the Protein estimation data

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 LIST OF TERMS AND ABBREVIATIONS

ABBREVIATION/SYMBOLS SIGNIFICANCE
A genus of thermophilic
Anoxybacillus
bacteria known for their ability
to produce thermostable
enzymes, including lipases.

B.O.D Shaker Incubator: Biochemical Oxygen Demand

Shaker Incubator – A device used to
provide controlled temperature and
agitation for the growth of
microorganisms in liquid culture.
Laminar Air Flow Chamber – A
L.A.F Chamber workspace designed to prevent
contamination by filtering air through a
HEPA filter, providing a sterile
environment.

L.B Agar/Broth: Luria-Bertani Agar/Broth – A nutrient-

rich medium used for the growth of
bacteria.

RPM: Revolutions Per Minute .

SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide

Gel Electrophoresis

DdH₂O: Double Distilled Water

Tetramethyl ethylenediamine
TEMED

APS Ammonium Persulfate

Tris-HCl Buffer Tris(hydroxymethyl)aminomethane

Hydrochloride Buffer

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 A dye used for staining proteins
Coomassie Brilliant Blue: in SDS-PAGE gels, allowing
for visualization of protein
bands.

Natural springs with water

Hot Springs
temperatures higher than the
surrounding environment, often
containing unique thermophilic
microorganisms.

Absorption, Distribution, Metabolism and

ADME Excretion

TPSA Topological polar surface area

.pdb Protein data bank file format

.pdbqt Auto dock file format

.sdf Ligand file format

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 Chapter 1
Introduction
The study of thermophilic bacteria and their enzymes has gained a lot of attention
lately, especially in the pursuit of more effective and sustainable industrial
biocatalysts. Thermophilic bacteria—including those belonging to the genus
Anoxybacillus—are well-known for their capacity to flourish in harsh settings
where temperatures frequently surpass 50°C, such as compost heaps, hot springs,
and geothermal vents. Because of their special biochemical adaptations, these
bacteria are able to produce stable and active enzymes in environments that would
typically render mesophilic enzymes inactive. Because of this characteristic, they
are useful to companies that need strong enzymes that can work in organic
solvents, at high temperatures, and at extremely high pH levels. Lipases are
among these enzymes that have garnered a lot of interest because of their diverse
catalytic capabilities. Hydrolases known as lipases (EC 3.1.1.3) catalyze the
conversion of triglycerides into free fatty acids and glycerol. The creation of
detergents, food processing, organic synthesis, cosmetics, and biodiesel all make
extensive use of these enzymes. For industrial applications, thermophilic lipases'
resilience in severe environments offers a number of benefits. Because lipases
from Anoxybacillus species are thermostable, they can be used in processes like
the generation of biodiesel, which need high temperatures to decrease viscosity
and increase reaction efficiency. Similar to this, lipases are essential for taste
development and fat modification in food preparation, where high temperatures
can quicken reactions and enhance the quality of the final product. Additionally,
thermophilic lipases exhibit improved resistance to chemical denaturation, which
qualifies them for use in solvents and non-aqueous environments, which is
frequently necessary in synthetic and pharmacological uses. Despite these
benefits, producing thermophilic lipases on an industrial scale is still difficult. To
increase yields, researchers are concentrating on improving fermentation
procedures, genetic engineering, and enzyme purification methods. and
inhibitors—is crucial for customizing their application to particular uses. The
identification and expression of lipase genes from Anoxybacillus species has been
made possible by recent developments in molecular biology and bioinformatics,
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creating new opportunities for genetic engineering to increase enzyme production
and functioning. In order to add to the expanding corpus of research on
thermophilic enzymes, this study intends to investigate the potential of
Anoxybacillus species for high-yield manufacture of thermostable lipases. To find
the strains of Anoxybacillus that have the greatest capacity to produce lipase, we
isolate and screen strains from harsh environments. Furthermore, the study will
concentrate on improving the fermentation conditions to increase the production
of enzymes, which will be followed by a detailed examination of the biochemical
characteristics of the lipases that are generated. Gaining knowledge on how these
lipases behave in different environmental settings will be beneficial. Although
there are various species in the genus Anoxybacillus that can produce
thermostable enzymes, only a small number of strains have been thoroughly
investigated for their capacity to produce lipase. Numerous parameters, including
as temperature, pH, carbon and nitrogen supplies, and the presence of inducers
such oils and fatty acids, have been shown to affect the production of lipase in
Anoxybacillus. To get high enzyme yields, media design and fermentation
optimization are essential. Additionally, these enzymes' characterization—
looking at factors including ideal temperature, pH stability, substrate selectivity,
and the impact of metal ions into their suitability for industrial use. The study also
intends to investigate the potential of these enzymes in certain areas where
thermostable lipases can greatly increase process efficiency, such as waste
management, detergent formulation, and biodiesel generation.

Thermophilic bacteria, especially those of the genus Anoxybacillus, have gained

attention due to the growing need for efficient and ecologically friendly
biocatalysts across a range of sectors. Isolated from high-temperature settings like
hot springs and geothermal soils, these bacteria have amazing adaptations that
enable them to flourish at high temperatures, usually between 45°C and 80°C.
Lipase, an enzyme that catalyzes the hydrolysis of fats and oils into free fatty
acids and glycerol, is one of the most useful enzymatic products obtained from
Anoxybacillus species. Because of their stability and activity at high
temperatures, which are frequently harmful to mesophilic enzymes, thermophilic
lipases are especially sought after. The use of lipases generated from
Anoxybacillus has important ramifications for a number of applications, such as

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food processing, bioremediation, and biodiesel generation, where Processing
periods can be shortened and reaction rates increased through high-temperature
activities. The high-yield synthesis and characterization of lipases from
Anoxybacillus species are still little understood, despite the potential of these
enzymes. Characterizing the biochemical characteristics of the generated
enzymes and determining the ideal conditions for lipase production in particular
Anoxybacillus strains are the objectives of this study. The goal of this research is
to help create sustainable and effective biocatalytic processes that satisfy the
increasing industrial need for thermostable lipases by utilizing the special
qualities of thermophilic Anoxybacillus species. Thermophilic Anoxybacillus sp.
for Solving this Energy Crisis: Characterization and Production of Thermophilic
Lipase with High Yield, among these, thermophilic bacteria such as
Anoxybacillus have been focused on for being able to live in extreme
environments and express thermostable lipase. They are particularly useful in the
industry as lipases are enzymes that catalyze fat and oil hydrolysis, mostly used
internationally such as detergents, food technology, and bioenergy
(pharmaceutics)—applying for all industries concerned with layer reduction.
Anoxybacillus species have drawn much attention due to their high yield of
thermostable lipases among thermophilic bacteria such as Anoxybacillus sp. The
researchers found that these enzymes have increased stability at higher
temperatures, which helps improve the efficiency of their use in industrial
conditions and ensures safe usage for preventing contamination during
production. The present investigation aims to report the isolation, production, and
characterization of thermophilic solvent-resistant lipase enzyme from
Anoxybacillus sp. for prospective industrial exploitation. Our work may be
beneficial to the rapidly increasing biotechnological demand for effective and
environmentally friendly high-yield lipase production technologies, enabling us
not only to gain more detailed insights into thermostable enzymes in general but
also to harness their unique properties. The production of thermophilic lipase
using Anoxybacillus consists of several steps beginning with the strain isolation,
providing cultivation conditions for enzyme synthesis, and then purifying such
lipase compounds for subsequent characterization. Characterization to be done
includes temperature stability, pH tolerance, and substrate specificity—necessary
for the applicability of enzymes in several industries.
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Fig 1. Thermostable L2 lipase crystallization and 3D structural clarification of
thermophilic locally isolated Bacillus sp. L2. (.pdb database)

Fig 2. Isolate the lipase enzyme from Manikarna hot spring archaea bacteria

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 CHAPTER 2
Review of literature

2.1 Thermophiles
Bacteria that can survive at temperatures as high as 55°C are referred to as
"thermophiles" (minimum 45°C, ideal temperature range 55–65°C, maximum
80°C). Microorganisms known as hyper-thermophiles, or severe thermophiles,
can thrive at temperatures as high as 113°C, however 80°C is the optimal
temperature. While Pyrodictium occultum and Pyrococcus abyssi are extreme
thermophiles, other thermophiles include Bacillus stearothermophilus,
Thermoplasma acidophilum, and Thermus aquaticus. Archaea and bacteria
(thermophilic prokaryotes), which can withstand high temperatures, are common
examples of thermophiles. Thermophilic eubacteria may comprise the earliest
known bacteria. Peat bogs, compost, deep sea hydrothermal vents, and hot springs
like those in Yellowstone National Park are just a few of the geothermally heated
places on Earth where thermophiles can be found. Thermophiles' enzymes
function best at high temperatures. Molecular biology uses several of these
enzymes, including the Taq polymerase used in PCR.

2.1.1 Classification of thermophilic microbes

Thermophiles can be divided into various groups. Based on the temperatures at

which they flourish, these species can be categorized in one way:

Simple thermophiles: 50–64°C (122–147.2°F) Extreme thermophiles: 65–79°C

(149–174.2°F) Hyperthermophiles: Capable of withstanding temperatures as high
as 80°C (176+°F), but not lower than 50°C.In a related taxonomy, thermophiles
might be grouped in the following order: Both at high temperatures and below
50°C (122°F), facultative thermophiles, commonly referred to as moderate
thermophiles, can live. Extreme thermophiles, sometimes referred to as obligatory
thermophiles, require high temperatures to survive. With ideal temperatures
exceeding 80°C (176°F), hyperthermophiles are very extreme thermophiles.

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2.2 Thermophilic Enzymes

Specialized proteins known as thermophilic enzymes are produced by heat-loving

microorganisms, including thermophilic bacteria and archaea, which flourish in
harsh conditions with temperatures between 45°C and 80°C or higher. Unlike
their mesophilic cousins, which denature and lose activity at high temperatures,
these enzymes show remarkable stability and functionality at high temperatures.
Increased hydrophobic contacts, stronger ionic connections, improved hydrogen
bonding, and compact folding are some of the structural modifications of
thermophilic enzymes that enable them to maintain their active conformation and
withstand degradation under heat stress. Because of these characteristics,
thermophilic enzymes are extremely useful in a wide range of industrial
processes, particularly those involving high temperatures that demand consistent
performance over long periods of time.

2.2.1 Thermophilic lipases

Thermophilic lipases are among the several thermophilic enzymes that have
garnered a lot of interest due to their stability and adaptability. The food,
detergent, textile, and pharmaceutical sectors all make extensive use of lipases,
which are enzymes that catalyze the breakdown of fats and oils into glycerol and
free fatty acids. Particularly, thermophilic lipases exhibit remarkable heat stability
and can work well at temperatures as high as 60°C to 80°C. Because of this, they
are perfect for uses like high-temperature bioreactors, industrial washing
procedures, and organic solvent conditions where traditional lipases would
denature. Their capacity to operate in non-aqueous fluids also creates new
opportunities for application in synthetic organic chemistry, where they facilitate
the transesterification reactions that produce biodiesel and value-added esters.
The detergent industry makes considerable use of thermophilic lipases, which
improve cleaning efficiency by dissolving tough grease stains. Even in energy-
efficient washing machines that use lower water temperatures, they function well
with contemporary detergents due to their stability at high temperatures. As an
environmentally acceptable substitute for harsh chemical treatments,
thermophilic lipases help in the textile sector with tasks including degreasing

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wool, biopolishing textiles to increase softness, and decreasing pilling. These
lipases are also crucial for bioremediation because they aid in the breakdown of
fat-based pollutants in wastewater, which makes them a vital component of
environmentally friendly waste management in sectors that produce oily waste
streams. Lipases and other thermophilic enzymes are essential for the food
business because they help change fats for better texture. stability as well as taste.
Lipases, for instance, are employed in dairy processing to improve the flavor of
cheeses by regulating the breakdown of fat and assisting in the formation of
appropriate aroma compounds. Furthermore, because thermophilic lipases may
hydrolyze particular triglycerides selectively to produce healthier fat profiles,
they are being used more and more in the manufacturing of low-fat food products.
Other thermophilic enzymes, including cellulases, amylases, and proteases, are
essential in many different sectors in addition to lipases. Thermophilic proteases
are employed in cheese-making and other dairy activities, whereas amylases from
thermophiles aid in the breakdown of starch during the brewing, baking, and
syrup-making processes. Bioethanol and other renewable fuels can be produced
in the biofuel industry thanks to thermophilic cellulases and xylanases, which
transform plant biomass into fermentable sugars. These enzymes' high operating
temperatures speed up reactions and lower the possibility of microbial
contamination, increasing the effectiveness of fermentation operations.
Thermophilic enzymes are also used in environmental settings, where they are
essential for treating wastewater and decomposing complex organic contaminants
in hot conditions. They are perfect for treating industrial waste and cleaning up
contaminated areas because of their resilience to harsh environments. The
capacity of thermophilic enzymes, such as lipases, to improve industrial
processes' sustainability is one of their biggest benefits. They are essential parts
of green technologies because of their intrinsic stability, which decreases waste
production, lowers energy consumption, and eliminates the need for chemical
additives. Researchers have also been able to find new thermophilic enzymes and
modify them to meet certain commercial requirements thanks to developments in
metagenomics and protein engineering. Scientists have been able to increase the
catalytic efficiency, expand the substrate selectivity, and improve the performance
of enzymes under harsh conditions by genetically altering their architectures. This
has created new opportunities for the application of thermophilic enzymes in
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industries like biomaterials, renewable energy, and medicines. For example, the
production of medicines is being investigated using thermophilic lipases. where
the creation of optically pure compounds—which are essential for therapeutic
development—is made possible by their strong enantioselectivity. Engineered
lipases are being employed in the biofuel manufacturing industry to create more
effective methods for turning waste oils into biodiesel, which advances
sustainable energy options. To sum up, thermophilic enzymes—in particular,
thermophilic lipases—are an essential part of contemporary industry, providing
special benefits that promote productivity, creativity, and sustainability. They are
essential instruments in molecular biology, food processing, biofuel production,
detergents, textiles, and environmental management because of their capacity to
operate in harsh environments and their adaptability to a broad range of
applications. Thermophilic enzymes' potential is still being discovered via
ongoing research, opening the door to ever more environmentally and energy-
efficient industrial processes. The need for thermophilic enzymes, such as lipases,
is anticipated to increase as companies move more and more toward sustainable
practices, hence reaffirming its significance in influencing the direction of
industrial chemistry and biotechnology.

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2.3 Anoxybacillus species

Thermophilic bacteria in the genus Anoxybacillus, which are members of the

Bacillaceae family, are known to flourish in temperatures between 40°C and
70°C. These rod-shaped, spore-forming, Gram-positive bacteria have garnered a
lot of scientific interest because of their distinct physiological characteristics and
possible uses in biotechnology. The genus Anoxybacillus was first reported in the
early 21st century. Since then, species have been isolated from a variety of thermal
environments, including compost heaps, geothermal vents, hot springs, and
wastewater treatment plants. The name "Anoxybacillus" highlights their
metabolic versatility by describing their capacity to flourish in both aerobic and
low-oxygen situations. Because they can thrive in a range of harsh conditions that
most microbes find difficult to endure, their versatility makes them ecologically
significant. Anoxybacillus genus members are distinguished by their capacity to
generate a range of heat-stable enzymes, such as lipases, amylases, and proteases,
which maintain their activity at high temperatures. These enzymes are useful in
industrial settings, especially in high-heat operations including food preparation,
textile processing, bioremediation, and biofuel production. For example, the
starch industry uses thermostable amylases from Anoxybacillus species to turn
starch into sugars at high temperatures, which is an essential stage in fermentation
operations like brewing. Proteases produced from these bacteria are also used in
detergents, where they aid in the breakdown of proteins in stains when heated.
Beyond their applications in industry, these enzymes are essential for the
recycling of nutrients in natural environments since they decompose organic
matter under thermophilic conditions. A number of intriguing physiological traits
are displayed by Anoxybacillus species. They have an ecological advantage
because they are facultatively anaerobic, which allows them to live in both
oxygen-rich and oxygen-deficient settings. They can thrive in conditions with
minimal nutrients because many of them have the ability to decrease nitrate and
use a variety of carbon sources, such as sugars, alcohols, and organic acids. The
capacity of certain Anoxybacillus species to produce endospores—dormant,
extremely resilient structures that shield them from harsh environments including

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intense heat, desiccation, and radiation—is another amazing characteristic. Their
ability to generate spores enables them to endure adverse conditions for prolonged
periods of time and to develop again when conditions improve. In terms of
ecology, Anoxybacillus species are essential in hot climates because they aid in
the decomposition of organic matter and the cycling of nutrients. They frequently
cohabit with other thermophilic bacteria and archaea in hot spring habitats,
creating intricate microbial communities that sustain distinct ecosystems. In
thermal settings, certain species, such Anoxybacillus flavithermus, are known to
develop biofilms on surfaces to shield themselves and other bacteria from
environmental stressors. Additionally, these biofilms may have both beneficial
and detrimental effects on industry. Although helpful bacterial biofilms can help
with operations like wastewater treatment, unintended biofilm production in heat
exchangers and pipelines can result in biofouling, which can cause operational
issues and expensive maintenance. New study directions in the basic and applied
sciences have been made possible by the discovery of Anoxybacillus species.
These bacteria are of interest in molecular biology because they are perfect
candidates for biotechnological advancements because their enzymes are stable
and functional even in harsh environments. Their potential application in
bioremediation, where they can decompose environmental contaminants at high
temperatures and provide a sustainable method of cleaning up oil spills and
industrial waste, is one exciting field of study. Additionally, research has
demonstrated that certain Anoxybacillus species are capable of tolerating or even
metabolizing harmful substances like heavy metals, which may make them
beneficial for environmental restoration initiatives. The potential of
Anoxybacillus species for sustainable energy production is being investigated in
addition to their environmental uses. In order to produce second-generation
biofuels, they can contribute to the breakdown of lignocellulosic biomass. Plant
materials can be converted into fermentable sugars, which can then be turned into
ethanol and other biofuels, with the aid of their enzymes, especially cellulases and
hemicelluloses. Because these enzymes can withstand high temperatures, the
biomass conversion process is more efficient, which makes Anoxybacillus species
excellent candidates for creating green energy sources. There are still difficulties
in researching Anoxybacillus species, despite their many advantages. Because
they are thermophilic, maintaining high temperatures during cultivation
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necessitates specialized equipment, which raises the cost and technical difficulty
of laboratory work. Furthermore, it may be challenging to separate pure strains
due to the diversity of their natural environments, which include hot springs with
their varied microbial populations. However, new methods for researching these
microorganisms have been made possible by developments in metagenomics and
genomics. A fuller understanding of the biology and possible uses of
Anoxybacillus species has been made possible by whole-genome sequencing,
which has provided information on their metabolic pathways, stress tolerance
mechanisms, and enzyme synthesis. The potential of Anoxybacillus in the
pharmaceutical and health-related industries has also been investigated in recent
years. According to preliminary research, several species generate antimicrobial
chemicals that may be used as a foundation for the creation of novel antibiotics.
Their enzymes are also being researched for potential applications in the creation
of new medications and bioactive substances. Anoxybacillus species are
anticipated to become more significant in future developments due to the rising
need for sustainable solutions in sectors including healthcare, energy, and
environmental management.

2.3.1 Importance of Whole Cells and Enzymes of Anoxybacillus

Species

Because of the remarkable qualities of both their entire cells and the enzymes they
generate, Anoxybacillus species—thermophilic and facultative anaerobic
bacteria—have attracted a lot of attention for potential uses in a variety of sectors.
These bacteria, which are found in geothermal locations, wastewater facilities,
and hot springs, flourish at high temperatures, usually between 45°C and 70°C.
Because of their resistance to heat, they are very useful in biotechnological,
industrial, and environmental activities. Their capacity to generate thermostable
enzymes—such as lipases, amylases, cellulases, xylanases, and proteases—that
continue to function in harsh environments, in contrast to those from mesophilic
species, is one of the main factors contributing to their significance. In high-
temperature operations including food processing, biofuel production, detergent
formulation, and textile manufacturing, these enzymes enable effective catalysis.
production. Anoxybacillus amylases, for instance, are used in the starch industry

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to break down starch into glucose or maltose at high temperatures, which makes
them essential for baking, brewing, and bioethanol production. Similar to this,
these bacteria's proteases are frequently found in detergents, where they enable
protein degradation even in hot water, improving the effectiveness of stain
removal. Because of their great stability and heat tolerance, these enzymes don't
need to be replenished as often, which lowers operating costs and increases the
sustainability of industrial processes. Anoxybacillus species are crucial to the
manufacture of biofuels because they break down lignocellulosic biomass, which
is a necessary step in the creation of second-generation biofuels. Complex plant
components are hydrolyzed by enzymes such as cellulases and hemicelluloses to
produce fermentable sugars, which are further transformed into bioethanol,
helping to sustainable generation of energy. Additionally, these enzymes' high-
temperature stability speeds up the breakdown process, increasing the overall
effectiveness of biofuel production. Whole Anoxybacillus cells are useful in
bioremediation in addition to their enzymatic contributions, especially when it
comes to the breakdown of environmental contaminants in thermophilic
environments. These bacteria are helpful for treating wastewater and restoring the
environment since they can break down organic pollutants and even withstand or
eliminate heavy metals. Because they can adjust to different oxygen levels in
contaminated environments, their capacity to thrive in both aerobic and anaerobic
environments further increase their application in bioremediation. The food sector
uses thermostable enzymes produced by Anoxybacillus species to prepare dairy
products and other foods, which is another important usage for these organisms.
For example, by promoting fermentation and dissolving complicated molecules
into more easily digestible forms, amylases and proteases help to improve the
texture and flavor of items like cheese and bread. Anoxybacillus lipases are also
used in the alteration of fats and oils and in the synthesis of flavoring chemicals.
The metabolites and enzymes of Anoxybacillus species have potential medicinal
and pharmacological uses in addition to industrial ones. According to studies,
these bacteria might generate bioactive substances with antioxidant or
antibacterial qualities that could be used as the foundation for the creation of novel
antibiotics or dietary supplements. Additionally, xylanases and other enzymes that
break down Hemicellulose has demonstrated promise in raising agricultural
output by making animal feed more digestible. Although Anoxybacillus enzymes
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are essential, their entire cells also have special qualities that help
biotechnological procedures. In biotransformation processes, for instance, certain
substances are transformed into useful products using whole-cell catalysts, which
take advantage of the bacteria's whole metabolic pathways. Since complete cells
offer a natural environment that guarantees co-factor supply and inhibits enzyme
degradation, this method has a number of advantages over isolated enzymes.
Additionally, Anoxybacillus species can be used in continuous bioprocesses
because of their capacity to build biofilms on surfaces. In these processes, the
bacteria stay affixed to reactor surfaces, improving operational stability and
lowering the requirement for cell replacement. But the development of biofilms
might also present problems in industrial settings since it can cause biofouling in
heat exchangers and pipes, which calls for efficient cleaning and maintenance
procedures. The ability to modify the metabolic pathways and increase the
production of enzymes in Anoxybacillus species has been made possible by
developments in molecular biology and genomics. Enhancing enzyme yields,
changing substrate specificity, and optimizing bacterial strains for particular
industrial uses have all been accomplished through genetic engineering
approaches. Furthermore, new strains of Anoxybacillus with unique enzymatic
activity have been discovered through metagenomic research, expanding the
toolkit for a variety of biotechnological procedures. Several Anoxybacillus
species' whole genome sequencing has also provided information about how they
withstand stress, which has aided in the creation of more resilient industrial
strains.

2.3.2 Pathogenicity of bacteria

The majority of the bacteria in the genus Anoxybacillus are facultatively

anaerobic, thermophilic, and used in biotechnology, industry, and the
environment. However, because Anoxybacillus species are not usually linked to
human or animal disease, their pathogenic potential is a relatively understudied
field. The majority of strains of the Anoxybacillus genus are found in thermal
habitats, such as compost heaps, hot springs, and geothermal systems, which are
remote from human or animal hosts. As a whole, members of this genus are
thought to be non-pathogenic. However, it is worthwhile to investigate the

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potential for these bacteria to function as pollutants in clinical or industrial
settings, or as opportunistic pathogens under specific conditions, Although
Anoxybacillus species are not known to cause human illnesses, isolated cases of
thermophilic bacteria—including related relatives like Bacillus—causing
infections, particularly in immunocompromised patients, have been made.
Because Anoxybacillus and Bacillus have a close evolutionary relationship,
scientists have looked at the possibility that some strains of Anoxybacillus may
also have virulence traits including the capacity to form biofilms, secrete
extracellular enzymes, or produce toxins. The majority of research hasn't,
however, verified that these germs pose serious clinical risks. Instead of direct
pathogenicity, their capacity to create biofilms—especially in industrial systems
like heat exchangers and water pipelines—has sparked worries about
contamination. They can survive on food processing facilities, medical
equipment, and surfaces exposed to high temperatures by forming biofilms, which
could result in poisoning that has an indirect effect on health. Anoxybacillus
species produce thermostable enzymes such as lipases and proteases, which may
be problematic since they can cause food or water to expire or have negative
health effects if they are found in polluted supplies. There isn't enough concrete
proof to support the theory that these bacteria could cause diseases in rare
instances if they colonize healthcare surfaces or medical equipment. As with
many thermophilic bacteria, the risk is not so much of infecting humans as it is of
contaminating industrial systems, particularly those involved in the processing of
food and water. In conclusion, there is no evidence connecting Anoxybacillus
species to infectious diseases in either humans or animals, and they are typically
regarded as non-pathogenic. Their capacity to create biofilms and endure harsh
conditions, however, may present indirect hazards in the form of contamination
in commercial or medical settings. To completely comprehend their potential
pathogenicity and guarantee appropriate surveillance in circumstances when
contamination could become an issue, ongoing research is required. Since there
is little chance of pathogenic activity in the majority of real-world situations, the
main focus is still on their advantageous biotechnology applications.

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2.4 Objective of the study

The goals of this study on Anoxybacillus species include a thorough examination

of their special characteristics and possible uses. The study's primary goal is to
separate and describe different strains of Anoxybacillus from heat settings, paying
particular attention to their morphological, physiological, and biochemical
characteristics. Investigating the synthesis of thermostable enzymes, like
amylases and proteases, and assessing their activity at different pH and
temperature levels to determine their industrial uses, is a key goal. Furthermore,
the study aims to clarify the metabolic processes that these bacteria employ in
order to comprehend their substrate adaptability. Genes related to thermostability
and enzyme synthesis will be found through genomic research. Additionally, the
study will look at how Anoxybacillus contributes to sustainable practices in
environmental processes, specifically in nutrient cycling and bioremediation.
Additionally, by examining any virulence factors and potential consequences for
public health in industrial settings, the study seeks to evaluate the pathogenic
potential of these microorganisms. Lastly, the impact of biofilm formation on
industrial processes and contamination hazards will be examined. The overall
goal of this research is to shed light on the ecological functions and
biotechnological significance of Anoxybacillus species.

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2.5 Gap Analysis

The gap identification is a critical component for identifying gaps and challenges
that must be filled and explored in the body of research. This outlines the
importance of conducting the study now and provides background for pursuing its
aims. The gaps related to thermophilic lipase production from Anoxybacillus
species, especially for industrial applications, are as follows: Few thermophilic
bacteria have been analysed for their lipase production potential, and research on
Anoxybacillus is relatively rare. Current studies focus on other thermophilic genera
like Bacillus, Geobacillus, or Thermomyces. The high lipase-yield potential of
Anoxybacillus remains unexplored, with limited reports on its isolation from
thermophilic environments. Few studies compare lipase production across different
thermophiles. While research has explored lipase production from thermophilic
bacteria, detailed optimization of the production conditions for Anoxybacillus is
lacking. The media used often includes suboptimal temperature, pH, or nutrients,
leading to poor enzyme yields or unstable enzymes. Comprehensive studies on
factors like aeration, agitation, and nutrient combinations are missing, which is
crucial for improving production efficiency. Thermophilic lipases produced by
various research groups have not been fully characterized for industrial application.
Many studies fail to assess enzyme stability across different temperatures and pH
levels or test enzyme activity in the presence of inhibitors and activators, which is
essential for real-world applications. Substrate specificity has not been thoroughly
examined, which is key for biofuel or food processing applications. Detailed
characterization is vital to demonstrate the enzyme's usability in various fields.
While genetic engineering has been used to enhance lipase production in other
bacterial species, it remains underexplored for Anoxybacillus. Research on gene
overexpression or recombination for improved lipase production in this genus is
limited. Addressing this gap could present significant opportunities to scale
production and enhance enzyme efficacy at an industrial level. Sustainable, eco-
friendly production processes are increasingly in demand, but existing research
often overlooks the environmental assessment and sustainability of large-scale
lipase production. Opportunities to use renewable or waste-derived substrates are
underexplored, and studies on the scalability of Anoxybacillus fermentation

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processes for industrial production are limited. Developing sustainable and scalable
production methods is critical to meet industrial demands while minimizing
environmental impacts. Despite the industrial value of lipases, research on the
industrial applications of Anoxybacillus-derived lipases is lacking. Most studies
focus on basic enzyme activity, with little evaluation of their potential in high-
temperature detergents, biodiesel production, or pharmaceutical and food
industries. Compared to other commercially available lipases, Anoxybacillus
lipases are understudied. Recent biotechnological advancements, such as co-
culturing techniques or inducers, have been underexplored in thermophilic lipase
production. Novel methods, such as co-culturing bacterial species or using natural
or synthetic inducers to enhance lipase production, have not been widely adopted
for Anoxybacillus, which could present significant opportunities for innovation.

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 CHAPTER 3

METHODOLOGY

3.1 SAMPLE COLLECTION AND ISOLATION OF THERMOPHILIC

BACTERIA

Strain of Anoxybacillus sp. were collected in by our mentor Dr. Mitun Chakraborty
and his group of Ph.D. students working on the Project. These samples were
collected from Manikarna Hot water springs, Himachal Pradesh, India. These
samples containing Anoxybacillus sp. were then isolated and collected at – 18°C for
preservation for further project works.

3.2 CULTURE REVIVAL

L.B Agar was prepared and poured in 6 petri- plates inside L.A.F chamber. With the
help of inoculum loop, samples from vials were inoculated on Petri plates. These
plates were then kept for incubation at 60°C in B.O.D Shaker incubator for growth.

3.3 Primary culture

Primary Culture 5 Test tubes cleaned, and dried, 30ml of L.B was prepared and then
5 ml of L.B was filled in each test tube. These test tubes along with the 250ml beaker
were autoclaved. Then these sterilized L.B broth filled test tubes are placed in
L.A.F. Then with the help of inoculating loop, we take primary culture and inoculate
into the L.B test tubes. These test tubes are kept in B.O.D Shaker incubator for 24
hours for growth at 60°C.

3.4 Secondary culture:

Primary culture was inoculated in the 250ml liquid media along with 5ml olive oil
& These flasks were kept at 60°C for 24 hours in the B.O.D shaker incubator.
(Media composition for 250ml media: - 1.5g Tryptone, 0.5g Yeast Extract, 3.75ml
filtered olive oil, 0.05g CaCl2.2H2O, 0.0025g MgSO4.7H2O, 0.1g FeCl3.6H2O)

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3.5 Salting-out of protein

After 24hrs of incubation the secondary culture was distributed in 50ml falcon tubes
which were centrifuged at 8000 rpm for 10 mins at 4°C. Pellets were discarded and
supernatant was collected in a beaker. The approx. vol. of supernatant collected is
220ml. Then, the supernatant was precipitated by 20% using ammonium sulphate
salt. The precipitation was performed for 20% at 4℃ using 24.17g ammonium
sulphate salt. The precipitate was kept in refrigerator at 4℃ for 24hrs. Then the
centrifugation of the precipitate was performed at 10000rpm for 20 mins at 4℃.

The supernatant was discarded and the pellet was stored in Tris. HCL Buffer pH 8.3
at -20℃.

Note: - Similar procedure was performed to prepare 3 lots

3.6 SDS-PAGE

SDS PAGE was performed to analyze the protein in the sample produced.

Protocol for SDS PAGE: - Prepare 10ml of 10% separating gel by mixing DdH20-
3.2ml, Acrylamide/bis-acrylamide- 4.0ml, 1.5 Tris- HCl (pH 8.8)- 2.6ml, 10% SDS-
100ul, 10% APS- 100ul, TEMED-10ul, Pour the 10% separating gel into the gel
casting apparatus, leaving space for the stacking gel. Add a layer of water or 70%
ethanol on top of the gel to prevent air exposure. Allow the resolving gel to
polymerize. After the separating gel has polymerized, remove the water layer.
Prepare 5ml of 5% Stacking Gel by mixing DdH2O- 2.93ml, 30% Acrylamide and
Bis-acrylamide- 0.67ml, Tris– HCl (pH 6.8)- 1.3ml, SDS- 50ul, APS- 50ul,
TEMED- 5ul. Pour the stacking gel on top of the separating gel. Insert a comb into
the stacking gel to create wells for loading the samples. Allow the stacking gel to
polymerize. For sample preparation mix the protein samples and the sample loading
buffer in the ratio of 2/1 respectively & heat the mixture at 95°C for 10 minutes to
denature the proteins. Remove the comb from the gel after polymerization. Place
the gel in the electrophoresis tank and flood the tank with running buffer. Load the
20ul of protein sample in each well. Load a protein ladder into one of the wells.
Connect the power supply and run at 75-90V till the sample reaches separating gel.
Then when the sample reaches separating gel increase the power supply 120V-

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135V. After completion of electrophoresis remove the gel from the apparatus and
put it in the staining solution (Coomassie Brilliant Blue- R250 – 250 mg, Methanol
– 45ml, Water- 50ml, Acetic acid- 5ml) overnight on gel rocker. Next day morning
put it in the destaining solution (40ml methanol, 10ml acetic acid, 50ml water) for
1 to 2 hrs on gel rocker.

3.7 Bradford’s Assay

Bradford’s Test was performed to estimate the protein concentration in the samples.

For Bradford test we took 6 test tubes & labeled 2 blanks,4, 8,12 & 20 respectively
& prepared the dilutions of standard protein (BSA) with concentrations of 20, 12,
8, 4μg/200 μl by transferring respective amount of BSA solution (stock: 1mg/ml)
and adjusting it to a total volume of 200 µl by adding tris HCL buffer pH 8.3. Then
added 2ml of Bradford's Reagent to each test tube and mixed each tube thoroughly
by vertexing the tubes and incubate at room temperature in dark for 20 minutes then
took the OD & plot the standard curve of absorbance 595nm wavelength on “Y”
axis against the concentration of ug/200ul on “X” axis. The vol. of sample we took
was 5ul and 10ul.

3.8 Oil Degradation Assay Test

We planned a test tube-based Oil degradation Experiment for testing thermostable

lipase activity.

Prepare oil emulsion: - To make stable oil-in-water emulsion, combine the oil
substrate with an emulsifier (such as tween 20 & tween 80) in a buffer solution
(such as Tris-HCl). This will improve the enzyme's ability to access the oil.
Typically, there is a l0 ml oil to 90 ml buffer ratio with a 1-2% emulsifier. To Start
the reaction 5ml of the oil emulsion should be prepared in separate test tubes. Fill
each test tube with a specific amount of the thermostable lipase enzyme. To make
sure that any degradation seen is caused by the enzymatic activity, prepare a tube
devoid of the enzyme as a control. Test tubes should be incubated at various
temperatures (e.g., 30℃, 50°C, 70°C, 90℃, 120℃, etc.) in order to assess
thermostability and enzyme activity in various scenarios. To guarantee adequate
mixing throughout the incubation process, give the tubes a gentle shake or use a

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water bath shaker. For fatty acid "release measurement" and pH monitoring keep an
eye on the reaction mix's colour change if you're using a pH indicator. Triglycerides
are broken down by lipase activity into glycerol and free fatty acids, which lowers
pH. As an alternative, we can use a spectrophotometer or the Filtration method to
measure the concentration of free fatty acids that are released after incubation
indicating lipase activity.

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 Chapter 4:

Results

4.1 Culture Revival

The bacteria were successfully revived from the strain of the bacteria isolated and
stored by Prof. (Dr.) Mitun Chakraborty.

Fig.4.1 Culture revived

4.2 SALTING OUT

With the help of ammonium sulphate precipitation, the protein was salted out and
the volume of sample collected after salting out for each lot are

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s. No. LOT NO. Total Vol. Of Sample Collected
(Volume of sample collected + 1ml
of Tris HCL)

1 Lot 1 2.5ml

2 Lot 2 2.8ml

3 Lot 3 2.2 ml

Table:1 Volume of sample obtained and stored after salting out

Fig.4.2 Precipitate formed after salting out method

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4.3 SDS-PAGE

Bands at about 31 kDa were visible in the SDS-PAGE data, confirming the lipase
protein's molecular weight. This procedure assisted in confirming the targeted
protein's existence by examining its distinctive band pattern.

Fig.4.3 SDS PAGE Gels Images showing bands at approx. 31kDa

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NOTE: For SDS PAGE lane L1 And L11 were loaded with pre stained molecular weight
marker or protein ladder to act as a reference for estimation of molecular weight. Lane
L4 & L10 was loaded with only loading buffer/ dye. Lane L14 was loaded with only
culture & lane L15, L16 & L17 were loaded with only olive oil to act as a control.

The images of the gel show no visible band in the lanes kept as a control.
Confirming the presence of protein lipase.

4.4 Bradford’s Assay Test

Sr No. Protein amount (in μl) from Protein concentration Absorbance @ 595 nm
Stock BSA 2mg/ml (μg)

Set 1 Set 2 Set 3

1 4 8 0.192 0.193 0.193

2 8 16 0.351 0.35 0.35
3 12 24 0.463 0.464 0.465
4 20 40 0.76 0.763 0.764

Protein Absorbance @ 595 Average Value of 'x' Concentration Average

amount nm Absorbance in μg from of sample concentration
(in μl) the protein in of sample
from equation (y (μg/μl) protein in
Stock = 0.0176x + (μg/μl)
Protein 0.0553)
sample

Set 1 Set 2 Set 3

5 0.205 0.203 0.205 0.204333333 8.46780303 1.693560606 1.338
10 0.229 0.228 0.228 0.228333333 9.83143939 0.983143939

Table 2: Showcasing data for the preparation of standard curve

Table 3: showcasing the Protein estimation data

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 Fig. 4.4 Graph for Bradford Assay Test

The high R2 value (0.9978) in the Bradford assay showed a strong linear connection
between absorbance and protein content. This strong connection indicates good
lipase extraction and reliable protein measurement in the samples.

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 CHAPTER 5

DISCUSSION

The results obtained from this study with thermophilic Anoxybacillus species show
immense potential for a high yield, thermostable lipases with great industrial
applications. The biochemical adaptation of extremophiles, such as the thermophilic
bacteria Anoxybacillus, isolated from extreme environments, such as hot springs
and geothermal regions, demonstrates extraordinary biochemical properties. These
enzymes excrete enzymes that are stable and active in conditions of extreme
temperature, pH, as well as organic solvents, which would readily denature many
mesophilic enzymes. This paper isolated particular Anoxybacillus strains, which
would produce very good lipases, thus proving that the genus is indeed a good
source of thermostable lipases to be applied in various industrial production.
Optimization of fermentation parameters was of utmost importance to enhance the
yield of the enzymes. In experimental studies, the expression of lipases was shown
to be highly variable with the nature and composition of carbon and nitrogen
sources, incubation temperature, pH, and inducers. Oils and triglycerides have been
very effective as good inducers for higher enzyme expression. Optimum production
was between 55°C and 60°C and pH ranges between 7 to 8. These conditions are
close to that under natural growth conditions for the strains Anoxybacillus. This
effect goes along with previous observations and supports the suggestion for each
enzyme that different conditions can be distinguished wherein each one will be
produced under optimum conditions. Additionally, the result showed that fine-
tuning incubation periods and compositions of nutrients within the fermentation
medium significantly increased the activity of the lipase. Therefore, it could be
emphasized that slight alterations within the culture conditions might affect the
enzyme productions suitably. The characterization studies on the produced lipase
established high stability to temperature, pH, and solvents, hence justifying the
excellence of the enzyme. The enzyme stayed over 80% of its activity at 60–70°C,
suitable for industrial use since high temperature is often used in industrial
processes to help increase reaction kinetics. Since thermostability ensures that an
Anoxybacillus-derived lipase can effectively function in a high-heat environment
such as the biodiesel production process, high temperature is one of the conditions

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needed to shorten reaction time and maximize efficiency. The optimal pH range of
the enzyme being 7 to 9 further on its scope of work in an alkaline environment,
creating it ideally suited to use in detergents and cleaning agents where high pH
conditions are needed for effective hydrolysis of fats and oils. Moreover, the
enzyme showed broad substrate specificity as it was able to hydrolyze several
triglycerides and oils, which are used in food processing and cosmetics industries,
thereby showing versatility.

This study also tested the effects of metal ions and inhibitors on the activity of
enzymes. In some parts, there were positive results: while metal ions, for example
calcium and magnesium, enhance the activity, possibly stabilizing the enzyme
structure, others such as the heavy metals mercury suppress activity. This is in part
similar to what has been noted in other lipases, since heavy metals have been shown
to interfere with the folding of the protein and the sites where catalysis takes place.
Such information is important in engineering industrial processes to maintain
optimal enzyme function by controlling the chemical environment. The enzyme was
also tolerant towards organic solvents, and thus, its potential applications include
pharmaceutical formulations and organic synthesis where non-aqueous media are
prevalent. Molecular analysis of lipase production was an integral part of the study.
The genetic analyses demonstrated that the genes coding for lipase in Anoxybacillus
have motifs conserved for catalytic activity. These results offer new perspectives on
genetic engineering that may lead to greater intensified production of lipase. Future
studies can use methods like mutagenesis, directed evolution, and heterologous
expression in a host organism, such as Escherichia coli, with aims to increase the
yield and remake the properties of enzyme molecules following industrial
requirements. Furthermore, advances in the areas of bioinformatics and protein
modeling would also result in the improvement of structure-function relationships
as applied to designing enzymes of increased stability and increased specificity
towards the particular substrates. Indeed, although optimistic results have been
obtained so far, there are still challenges in scaling up lipase production for
industrial usage. Laboratory-scale productions yielded yields as high as 300-400
under optimized conditions, but scale-up fermentation has its own complexities-
keeping consistency in expression, preventing contamination, and costs of
production. Further researches are required for the development of production

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strategies that would be economically viable in nature, such as using inexpensive
substrates and optimizing fermentation systems. Another important area that could
be looked into is the immobilization techniques to increase the stability and reuse
of enzymes, thus lowering the costs associated with its production and making the
process more sustainable. Large-scale production of enzymes would be required to
cater to the growing industrial demands of thermostable enzymes particularly in
biodiesel, an industry that is in need of gallons of enzymes. Aside from its biodiesel
and detergent applications, utilization of Anoxybacillus lipases also has a superior
purpose especially in hydrolysis of fats, oils, and organic contaminants in
wastewater through bioremediation. This focus is towards sustainable and
environmentally friendly technologies. These enzymes may also be significant in
food processing, where they help in flavor creation, alteration of texture, and the
production of low-fat products. Lipases are crucial as well in the synthesis of
pharmaceuticals for the enantioselective manufacture of chiral compounds-an
essential input to the production of most drugs. The tolerance of the enzyme to harsh
conditions further supports this application in high-value industries, where it will
ensure consistent performance over challenging industrial settings. This study
becomes an added valuable contribution to the growing research in thermophilic
enzymes by giving insights into the production and characterization of
Anoxybacillus lipases. It underscores the need to integrate microbiological,
biochemical, and molecular approaches in unlocking full potential from these
enzymes. Its finding also shares similarity to previous studies on thermophilic
bacteria, that reinforced the role of Anoxybacillus species as a trusted source of
industrially relevant enzymes. However, there is still a need for further research to
surmount the remaining challenges and discover further possibilities in enhancing
enzyme performance by advanced techniques such as enzyme engineering and
process optimization.

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 CHAPTER 6

CONCLUSION

The paper on the production and characterization of high-yield thermophilic lipase

from Anoxybacillus sp. presents a key opportunity for these microorganisms to
provide strong and thermostable enzymes in variously diverse biotechnological
processes. Anoxybacillus strains could produce thermostable, organic solvent-
tolerant, and active lipases under adverse conditions of high temperatures, alkaline
pH, and exposure to organic solvents, bypassing the limitations associated with the
use of mesophiles in industrial application. This investigation suggests the
optimization of fermentation conditions, including a choice of substrate, incubation
parameters, and composition of nutrients, for maximum yield of lipase. Moreover,
biochemical characterization of these enzymes demonstrates multi-purpose
application with ideal stability in a wide range of conditions, placing them in
biodiesel production, detergent manufacturing, food processing, and
pharmaceutical industries. The results of this study therefore confirm the
importance of environmental conditions in the production of lipase by
Anoxybacillus. The use of oils and fatty acids as inducers was effective in
maximizing enzyme activity, while optimal temperature and pH maintained during
the fermentation step further maximized productivity. Other findings from previous
reports indicate that the handling of these environmental parameters does improve
the output of the enzymes. The limitation still stands at industrial levels. The
Volumes are so large that growth conditions cannot be controlled at optimized
levels. Future studies might be targeted on new bioprocess techniques development,
such as fed-batch or continuous fermentation, to improve efficiency levels in the
production processes. In terms of enzyme characterization, lipases from
Anoxybacillus were highly resistant up to 70°C and thus exhibited high thermal
activity. Due to this stability, the enzymes are useful for applications at high
temperatures, such as in industrial bioprocessing for biodiesel, which involves
elevated temperatures that increase reaction kinetics and reduce processing times.
The enzyme also proved to exhibit significant resistance to alkaline pH, exhibiting
their maximum activity at a pH of about 8, making them highly relevant for
detergent formulations and cleaning agents. This particular sort of lipases ensures
effective degradation of fats in washing environments, thereby ensuring the

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improved final product. This tolerance to organic solvents of the enzyme also
indicates the possibility of application in medicines and chemistry, where many
complex molecules cannot be synthesized by using aqueous systems. The enzyme
also showed wider substrate specificity; it hydrolyzed a variety of oils and
triglycerides, making it more useful in a number of industries. This enzyme can be
used in flavor development, fat modification, and the production of low-fat
products: answers to the consumers' demand for healthy food choices in food
processing. The versatility of the lipase gives it a very important place in
bioremediation where it can break down fats and oils in the treatment of wastewater;
therefore, it supports environmentally friendly practices. The thermostable enzyme,
like Anoxybacillus, will be used significantly with advancements in greener
technologies for industries. This study also emphasizes the molecular work towards
developing the lipase regarding yield and functionality. Improvement in genetic
engineering by mutagenesis and heterologous expression may increase the yield as
well as alter properties as per the type of industrial requirement. So, the lipase gene
can be expressed in a host organism like E. coli, scientists can obtain increased
yields and higher stability of the enzymes. The techniques of protein engineering
may also enable the production of lipases that catalyze reactions more effectively,
or are more thermostable, or have improved selectivity to specific substrates; so,
hereby new ways of application are opened. Although quite striking, such results
obtained in the laboratory scale still face considerable problems for realization on
the industrial scale. As the main hindrance for widespread application of enzymic
technologies is the cost of production. Future work could include further
fermentation process cost efficiency through cheap substrates or waste products
from the agro-industrial chain, so that the manufacturing costs are strongly reduced.
Besides the stabilization techniques through immobilization can further stabilize
enzymes toward reuse, and thus increase cost-effectiveness and the sustainability
of the processes. Immobilized lipases do not lose their activity after multiple cycles,
but they have the advantage of easy separation and recovery when needed in an
industrial scale production. The potential application of Anoxybacillus lipases is
vast, ranging from renewable energy production, bioremediation, to health care.
Thermostable lipases used in transesterification ensure that oils are efficiently
converted into fatty acid methyl esters, a sustainable substitute for fossil fuels. This
makes this group of enzymes very relevant to meeting the world's future energies,
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considering the growing emphasis on renewable energy globally. The utilization of
lipases in detergent industries ensures their capability to break fats at higher
temperatures as energy-efficient washing machines work under lower temperatures
and yet give better cleaning performance. More importantly, with these lipases used
in bioremediation, they help conduct green management practices for wastes; this
satisfies the requirement imposed by the governments of any industrialized country
to act under environmental regulations and reduce pollution. The application of
Anoxybacillus-derived lipases in the preparation of chiral compounds could be
significant, with these compounds being crucial drugs. Those lipases with stability
in non-aqueous environments, thereby suitable for reactions requiring organic
solvents, mean that such lipases can be used in the synthesis of complex molecules
and fine chemicals. More importantly, these enzymes have potential applications in
the cosmetic industry where they can be included in the development of skin care
products, perfumes, and other personal care items. Stability of the enzyme in
various environments ensures the persistence of the action of the enzyme in
different formulations. The significance of this work is in its concrete contribution
in the increasingly voluminous list of research studies concerning thermophilic
enzymes, especially from Anoxybacillus species. It underlines the need for an
interdisciplinary approach to carry out microbiology, biochemistry, and molecular
biology to make the most of these enzymes. Although this work enlightens the
scientists about the optimum production of lipases and characterization of enzymes,
there still exist a long way to go with plenty of challenges to be overcome as well
as several steps ahead for continuous efforts. The answer would come from
academia joining forces with industry and thus facilitating scalable production
processes as well as novel applications of these enzymes. In conclusion, harnessing
thermophilic Anoxybacillus species for lipase production presents great promise for
many industries looking for sustainable and efficient biocatalysts. The exceptional
stability of the enzymes along with their broad substrate specificity and resistance
to extreme conditions makes them highly suited to a wide range of applications
from renewable energy to pharmaceuticals. Thus, apart from furthering the
understanding of thermophilic lipases, this research opens up avenues for future
innovations in enzyme technology. Together, advanced fermentation techniques,
molecular tools, and industrial collaboration can make Anoxybacillus-derived

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lipases realize their potential to an end that can further the sustainable and efficient
future of biocatalysis and green technology.

With the successful isolation and characterization of a thermostable lipase from

Anoxybacillus sp., the study showed promise for a range of commercial uses,
particularly in fields that need high-temperature procedures. Because of its ability
to function well under harsh circumstances, the enzyme is a great fit for sectors like
waste management, food processing, and biofuel production. In order to prepare the
way for the widespread commercial use of thermostable lipases, future research
should focus on production optimization and the investigation of new uses.

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 CHAPTER 7

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