High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.     

Stereoselective lipases from Burkholderia sp., cloning and their application to preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for (R)-Metalaxyl

Two microbial strains (referred to as MC 16-3 and 99-2-1) that produce extracellular lipases were isolated from soil samples and identified as Burkholderia species. The lipases were partially purified by isopropyl alcohol precipitation and gave molecular weight of 33kDa. The lipases were characterized in terms of stereoselectivity with racemic methoxyethyl (R,S)-N-(2,6-dimethylphenyl)alaninate and the genes encoding the proteins have been identified by homology alignment of lipases reported belonging to I.2 subfamily and their complete DNA sequences were determined. The lipases will be useful for the preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for the synthesis of (R)-Metalaxyl, which is one of the best-selling fungicides.     

Cloning, purification and characterisation of Staphylococcus warneri lipase 2

A gene encoding an extracellular lipase was identified in Staphylococcus warneri 863. The deduced lipase is organised as a prepro-protein and has significant similarity to other staphylococcal lipases. The mature part of the lipase was expressed with an N-terminal histidine tag in Escherichia coli, purified and biochemically characterised. The results show that the purified lipase (named SWL2) combines the properties of the staphylococcal lipases characterised so far. It has both a high preference for short chain substrates and surprisingly, it also displays phospholipase activity. Homology alignment was used to analyse sequence-function relationships of the staphylococcal lipase family with the aim to identify the structural basis underlying the different properties of the staphylococcal lipases.     

Technical methods to improve yield,activity and stability in the development of microbial lipases

Lipases are ubiquitous biocatalysts that catalyze various reactions in organic solvents or in solvent-free systems and are increasingly applied in various industrial fields. In view of the excellent catalytic activities and the huge application potential, more than 20 microbial lipases have been realized in large-scale commercial production. The potential for commercial exploitation of a microbial lipase is determined by its yield, activity, stability and other characteristics. This review will survey the various technical methods that have been developed to enhance yield, activity and stability of microbial lipases from four aspects, including improvements in lipase-producing strains, modification of lipase genes, fermentation engineering of lipases and downstream processing technology of lipase products.     

Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase

The bacterial thermoalkalophilic lipases optimally hydrolyze saturated fatty acids at elevated temperatures. They also have significant sequence homology with staphylococcal lipases, and both the thermoalkalophilic and staphylococcal lipases are grouped as the lipase family I.5. We report here the first crystal structure of the lipase family I.5, the structure of a thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) determined at 2.0-A resolution. The structure is in a closed conformation, and the active site is buried under a long lid helix. Unexpectedly, the structure exhibits a zinc-binding site in an extra domain that accounts for the larger molecular size of the family I.5 enzymes in comparison to other microbial lipases. The zinc-coordinated extra domain makes tight interactions with the loop extended from the C terminus of the lid helix, suggesting that the activation of the family I.5 lipases may be regulated by the strength of the interactions. The unusually long lid helix makes strong hydrophobic interactions with its neighbors. The structural information together with previous biochemical observations indicate that the temperature-mediated lid opening is triggered by the thermal dissociation of the hydrophobic interactions.     

Purification of lipases.

Interest on lipases from different sources (microorganisms, animals and plants) has markedly increased in the last decade due to the potential applications of lipases in industry and in medicine. Microbial and mammalian lipases have been purified to homogeneity, allowing the successful determination of their primary aminoacid sequence and, more recently, of the three-dimensional structure. The X-ray studies of pure lipases will enable the establishment of the structure-function relationships and contribute for a better understanding of the kinetic mechanisms of lipase action on hydrolysis, synthesis and group exchange of esters. This article reviews the separation and purification techniques that were used in the recovery of microbial, mammalian and plant lipases. Several purification procedures are analysed taking into account the sequence of the methods and the number of times each method is used. Novel purification methods based on liquid-liquid extraction, membrane processes and immunopurification are also reviewed.     

微生物脂肪酶稳定性研究进展

脂肪酶广泛应用于食品、药物、生物燃料、诊断、生物修复、化学品、化妆品、清洁剂、饲料、皮革和生物传感器等工业领域,微生物脂肪酶是商品化脂肪酶的重要来源。高温、酸性、碱性和有机溶剂等恶劣的工业生产环境使得脂肪酶的进一步工业应用受到限制,获取稳定性好的脂肪酶成为打破这一限制的关键环节。本文重点对提高微生物脂肪酶稳定性的策略进行了综述:挖掘极端微生物脂肪酶资源;利用定向进化、理性设计和半理性设计等蛋白质工程策略改造脂肪酶;利用物理吸附、封装、共价结合和交联等酶的固定化技术提高脂肪酶的稳定性;利用物理/化学修饰、表面展示以及多种改良策略相结合提高脂肪酶的稳定性。结合作者前期对酶工程的研究发现,新型酶催化剂的获得应该基于明确的设计思路,结合多种改造方法,基于定向进化-理性设计、定向进化-半理性设计、蛋白质工程-酶的固定化、蛋白质工程-物理/化学修饰、酶的固定化-物理/化学修饰等组合改造,比单一的改造方法具有更高的效率。     

Mapping substrate selectivity of lipases from thermophilic fungi

New methods were adapted to screen, fast and easily, the lipase specificity (topo- or enantio-selectivity) on crude extracellular extracts from thermophilic fungi. Substrate acyl chain length specificity was tested using p-nitrophenyl esters and vinyl esters by the detection of released p-nitrophenolate anions in the first case and protonation of p-nitrophenolate anions (color diminution) in the second case. Enantioselectivity was tested using either the direct reaction rates on individual enantiomers of glycidyl butyrate or on competition between these enantiomers and resorufin esters (-butyrate or -acetate). Among a library of 44 thermophilic fungi, 10 strains were pre-selected (based on their capabilities to produce constitutively extracellular lipases) for further lipase specificity studies. The above methods were applied to lipases from these pre-selected fungi and also to other several lipases preparations from bacterial, fungal and mammalian origin. Remarkably, the method on competition allowed the accurate determination of the enantiomeric ratio (E), since experimental data fitted correctly with the E determined by classical chemical methods. Consequently, these methods can be applicable for screening selectivity in a high number of lipases or esterases from wild isolates or variants generated by directed evolution, using directly in the test, the substrate (i.e. esters) that will be worked out in a given process.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

嗜麦芽寡养单胞菌胞外蛋白酶功能研究进展

嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)是广泛分布于自然界的革兰氏阴性杆菌。作为一种新型、与高死亡率相关的条件致病菌,嗜麦芽寡养单胞菌能够导致人类或其他生物感染多种疾病。近年来,越来越多的研究结果显示来自于细菌的胞外蛋白酶是导致宿主发病的关键蛋白质。因此,探究嗜麦芽寡养单胞菌胞外蛋白酶的组成成分和功能将不仅有助于阐明其致病机制,更为今后以其为靶点进行临床治疗奠定基础。本文试图对嗜麦芽寡养单胞菌胞外蛋白酶的性质、功能及其应用进行归纳总结。     

Structural relationship between lipases and peptidases of the prolyl oligopeptidase family.

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three-dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10-residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.     

A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis

Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K(m) of 7.57 mM and V(max) of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37 degrees C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.     

Isolation and functional expression of a novel lipase gene isolated directly from oil-contaminated soil

A lipase gene SR1 encoding an extracellular lipase was isolated from oil-contaminated soil and expressed in Escherichia coli. The gene contained a 1845-bp reading frame and encoded a 615-amino-acid lipase protein. The mature part of the lipase was expressed with an N-terminal histidine tag in E. coli BL21, purified and characterized biochemically. The results showed that the purified lipase combines the properties of Pseudomonas chlororaphis and other Serratia lipases characterized so far. Its optimum pH and temperature for hydrolysis activity was pH 5.5-8.0 and 37°C respectively. The enzyme showed high preference for short chain substrates (556.3±2.8 U/μg for C10 fatty acid oil) and surprisingly it also displayed high activity for long-chain fatty acid. The deduced lipase SR1 protein is probably from Serratia, and is organized as a prepro-protein and belongs to the GXSXG lipase family.     

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.     

细菌生物被膜基质的研究进展

细菌生物被膜(biofilm)附着在生物或者非生物表面,由细菌及其分泌的糖、蛋白质和核酸等多种基质组成的细菌群落,是造成病原细菌持续性感染、毒力和耐药性的重要原因之一.细菌的生物被膜基质由复杂的胞外聚合物(extracellular polymeric substances,EPS)构成,影响生物被膜的结构和功能.本文...     

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">simulans</Emphasis> lipase expressed in <Emphasis Type="Italic">E. coli</Emphasis>

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.     

Inhibition of Candida rugosa lipase by berberine and structurally related alkaloids, evaluated by high-performance liquid chromatography.

It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478-1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with beta-naphthyl laurate, and beta-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of beta-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C10-16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.     

Acidic lipase Lip I.3 from a Pseudomonas fluorescens‐like strain displays unusual properties and shows activity on secondary alcohols

Aims

Identification, cloning, expression and characterization of a novel lipase – Lip I.3 – from strain Pseudomonas CR‐611.

Methods and Results

The corresponding gene was identified and isolated by PCR‐amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0‐1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C‐terminal type I secretion signal and several putative Ca²⁺ binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca²⁺ ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols.

Conclusions

A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts.

Significance and Impact of the Study

The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis.     

Recombinant microbial lipases for biotechnological applications

Lipases, mainly of microbial origin, represent the most widely used class of enzymes in biotechnological applications and organic chemistry. Modern methods of genetic engineering combined with an increasing knowledge of structure and function will allow further adaptation to industrial needs and exploration of novel applications. Production of such tailored lipases requires their functional overexpression in a suitable host. Hence, this article describes the functional heterologous production of commercially important microbial lipases. Based on the knowledge of different lipases' substrate binding sites, the most suitable lipase for a particular application may be selected.