Industrial applications of microbial lipases
Introduction
Enzymes are considered as nature's catalysts. Most enzymes today (and probably nearly all in the future) are produced by the fermentation of biobased materials [1]. Lipids constitute a large part of the earth's biomass, and lipolytic enzymes play an important role in the turnover of these water-insoluble compounds. Lipolytic enzymes are involved in the breakdown and thus in the mobilization of lipids within the cells of individual organisms as well as in the transfer of lipids from one organism to another [2]. Microorganisms have earlier been found to produce emulsifying agents or biosurfactants to help solubilize lipids [3]. Several thousand enzymes possessing different substrate specificities are known, however only comparatively few enzymes have been isolated in a pure form and crystallized, and little has been known about their structure and function. The advent of protein engineering techniques makes their application to important industrial enzymes, such as proteases and lipases used in detergents, amylases and glucose isomerase used in starch processing and in the bioprocessing of raw materials or in the synthesis of organic chemicals are very efficient [4].
The particular benefits offered by enzymes are specificity, mild conditions and reduced waste. It may be possible, by choosing the right enzyme, to control which products are produced, and unwanted side reactions are minimized due to specificity of enzymes that appear in the waste stream. The plant using enzymatic reactions can be built and operated at much lower capital and energy cost. Enzyme-based processes tend to have lower waste treatment costs. Enzymes however are biodegradable, and since they usually are dosed at 0.1–1.0% of the substrate, the contribution of the enzyme to the BOD in the waste stream is negligible [5].
Microbial enzymes are often more useful than enzymes derived from plants or animals because of the great variety of catalytic activities available, the high yields possible, ease of genetic manipulation, regular supply due to absence of seasonal fluctuations and rapid growth of microorganisms on inexpensive media. Microbial enzymes are also more stable than their corresponding plant and animal enzymes and their production is more convenient and safer [6].
Only about 2% of the world's microorganisms have been tested as enzyme sources. Bacterial strains are generally more used as they offer higher activities compared to yeasts [7] and tend to have neutral or alkaline pH optima and are often thermostable. Genetic and environmental manipulation to increase the yield of cells [8], to increase the enzyme activity of the cells by making the enzyme of interest constitutive, or by inducing it, or to produce altered enzymes [9], may be employed easily using microbial cells because of their short generation times, their relatively simple nutritional requirements, and since screening procedures for the desired characteristic are easier.
Some industrially important enzymes have been isolated from plants. Hydroxynitrile lyases (HNLs) have been purified from various species of higher plants. Release of HCN from cyanogenic glycosides is due to the cleavage of the carbohydrate moiety by beta-glucosidases to yield the corresponding alpha-hydroxynitrile, which dissociates spontaneously into HCN and a carbonyl compound, or by action of an alpha-hydroxynitrile lyase (HNL). HNLs have great potential to be used as biocatalysts for the synthesis of optically active alpha-hydroxynitriles which are important building blocks in the fine chemical and pharmaceutical industries [10]. (R)- as well as (S)-cyanohydrins are now easily available as a result of the excellent accessibility, the relatively high stability and the easy handling of hydroxynitrile lyases (HNLs) [11].
Many plant lipases have been isolated now which can be used for the production of important lipases. A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity [12]. Many companies market digestive enzymes prepared from plant and fungal lipases. Doctor's Holistic Market manufactures Chiro-Zyme, the digestive plant enzymes formula containing lipase from Aspergillus niger and Rhizopus oryzae [13].
Bacillus species have been found to produce a number of enzymes other than lipases that may be utilized industrially. Some of the industrially important enzymes produced by Bacillus sp. are listed in Table 1.
Section snippets
Historical background
The presence of lipases has been observed as early as in 1901 for Bacillus prodigiosus, B. pyocyaneus and B. fluorescens [14] which represent today's best studied lipase producing bacteria now named Serratia marcescens, Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively. Enzymes hydrolyzing triglycerides have been studied for well over 300 years and the ability of the lipases to catalyse the hydrolysis and also the synthesis of esters has been recognized nearly 70 years ago [15].
Industrially used enzymes
Lipolytic enzymes are currently attracting an enormous attention because of their biotechnological potential [16]. They constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimization of industrially relevant lipase properties can be
Use of thermostable/alkalophilic enzymes
The importance of thermostable lipases for different applications has been growing rapidly. Most of the studies realized so far have been carried out with mesophilic producers. Many lipases from mesophiles are stable at elevated temperatures [35]. Proteins from thermophilic organisms have been proved to be more useful for biotechnological applications than similar proteins from thermophiles due to their stability [36].
Biocatalyst thermostability allows a higher operation temperature, which is
Applications of lipases
Microbial lipases constitute an important group of biotechnologically valuable enzymes, mainly because of the versatility of their applied properties and ease of mass production. Microbial lipases are widely diversified in their enzymatic properties and substrate specificity, which make them very attractive for industrial applications. In the industrial segment, lipases and cellulases are anticipated to post the best gains. It is expected that in the next few years lipases will benefit from
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