WO2011101339A2

WO2011101339A2 - Fermentation broth and filtration filter cake and uses thereof

Info

Publication number: WO2011101339A2
Application number: PCT/EP2011/052200
Authority: WO
Inventors: Albert Schaap; Gabriel Marinus Henricus Meesters; Henriëtte Maria Wilhelmina Jacoba Catharina UIJEN
Original assignee: Dsm Ip Assets B.V.
Priority date: 2010-02-18
Filing date: 2011-02-15
Publication date: 2011-08-25
Also published as: AR080212A1; UY33232A; WO2011101339A3

Abstract

The present invention relates to the field of enzymes. More specific, the present invention relates to lipases, more specifically phospholipases. The invention provides a fermentation broth or a filter cake comprising non-viable micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing an enzyme, preferably a lipase and more preferably a phospholipase during fermentation and wherein at least part of the produced enzyme adheres to the extracellular part of said micro organisms.

Description

FERMENTATION BROTH AND FILTRATION FILTER CAKE AND USES THEREOF

Field of the invention

The present invention relates to the field of enzymes. More specific, the present invention relates to lipases, more specifically phospholipases.

Background of the invention

Enzymes in general, lipases in specific and phospholipases more specific, are used in multiple (industrial) applications. Enzymes are often used as an alternative for chemical processes. The use of enzymes offers a cleaner solution for chemical processes.

However, some enzymes tend to be difficult to produce and as a consequence such enzymes are expensive and can not be considered to be a fair alternative for a chemical process.

The use of expensive enzymes can be made more economical by using the same batch of enzymes m u lti ple ti mes. Th is is for exam ple accom pl ished by immobilising said enzymes. However, using immobilised enzymes results in higher changes of contamination in the treated product which is undesirable as well, and the process of immobilisation adds to the costs of the overall process as well.

The present invention intends to overcome some of the current drawbacks of using enzymes and immobilisation techniques.

Description of the Figures

Figure 1 : Analysis on lipase activity in phospholipase compositions.

Summary of the invention

Surprisingly, the inventors of the current invention have recognised that enzymes immobilised to their host cells (i.e. cells that are capable of expressing the relevant enzyme) can be obtained in a rather cheap way and more surprisingly said enzymes are firmly adhered to said host cells and -important as well- are functional, without artificially designing such adhesive property (i.e. without adding an adhesive property to the enzymes).

The invention will be explained in more detail with a phospholipase as a non-limiting example. However, the invention can - mutadis mutandis- equally well be applied with other enzymes such as lipases.

Detailed description of the invention

Generally, the invention provides a fermentation broth comprising non-viable micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing an enzyme, preferably a lipase and more preferably a phospholipase, during fermentation and wherein at least part of the produced enzyme, preferably a lipase and more preferably a phospholipase, adheres to the extracellular part of said micro organisms.

In one of its embodiments, the invention provides a fermentation broth comprising non-viable micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms.

Fermentation h as m any i m porta nt uses i n i n d u stry. Thou gh the word fermentation can have stricter definitions, when speaking of it in industrial fermentation it more loosely refers to the breakdown of organic substances and re-assembly into other substances. Fermenter cultures in industrial capacity often refer to h ighly oxygenated and aerobic growth conditions. Some examples of commercially interesting fermentations are (i) microbial cells or biomass as the product, e.g. single cell protein, bakers yeast, lactobacillus, E. coli, (ii) microbial enzymes, such as but not limited to catalase, amylase, protease, pectinase, glucose isomerase, cellulase, hemicellulase, lipase, lactase and streptokinase, (iii) microbial metabolites and (iv) recombinant products such as insulin, HBV, interferon, GCSF or streptokinase. Growth media are required for industrial fermentation, since any microbe requires water, oxygen, an energy source, a carbon source, a nitrogen source and micronutrients for growth.

A fermentation process can be schematically depicted by:

Micro-organism + carbon & energy source + nitrogen source + 0₂ + other requirements→ Biomass + Product + byproducts + C0₂ + H₂0 + heat The term "fermentation broth" is used herein to refer to the overall product as present after fermentation has been allowed to proceed to or near its endpoint and typically comprises a large amount of microbial biomass, growth medium (and/or remains thereof), as well as products secreted by the micro organism into the medium.

A typical next step in an industrial fermentation is a killing off step to make the micro organisms, i.e. the biomass, non-viable. Examples of a suitable killing step will be provided later on.

A non-viable fermentation broth as described herein comprises free (unbound or non-adhered) enzymes, specifically lipase or phospholipase (i.e. lipase or phospholipase as present in the growth medium) as well as lipase or phospholipase adhered to the microorganisms. Typical ratios (at least for phospholipase A2) of free versus adhered phospholipase as present in a non-viable fermentation broth (after fermentation has been allowed to proceed to or near its endpoint) are within the range of 80% free versus 20% adhered to 20% free to 80% adhered. As will be explained in more detail later on, in a typical embodiment, at least 20% of the produced phospholipase (after fermentation and killing off) adheres to the micro organisms. The percentage free and/or adhered lipase or phospholipase is determi ned based on the amou nt of activity of said l ipase or phospholipase. For example, the enzymatic activity (for example phospholipase activity) is determined in broth (total amount of enzymatic activity) as well as in corresponding filter cake (bound amount of activity) and/or filtrate (non adhered amount of activity). Upon comparing these values, the amount of adhered and free enzyme is established.

A fermentation broth according to the invention typically comprises of biomass (cells, cell walls, cell contents, for example proteins, carbohydrates and nucleic acids) and (remains of) growth medium as well as products secreted by the used micro organisms into the medium.

A further embodiment is separating the soluble fraction from the fraction that is adhered and using that fraction immobilised on biomass and/or filteraids in specific applications.

In a second embodiment, the invention provides a filter cake comprising nonviable micro organisms which micro-organisms before subjecting the corresponding fermentation broth to a killing off step are capable of producing a lipase in general and a phospholipase in specific during fermentation and wherein at least part of the produced lipase or phospholipase adheres to the extracellular part of said m icro organisms, i.e. the invention also provides a non-viable filter cake comprising (nonviable) micro organisms which micro-organisms before subjecting the corresponding fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms.

After a killing off step (performed on fermentation broth) the resultant (i.e. the non-viable or killed micro organisms) is subjected to a solid/liquid separation step optionally in combination with a washing step. This results in a liquid fraction and a solid fraction. The solid fraction is called filter cake. The liquid fraction is called the filtrate.

A filter cake as described herein typically comprises (all in weight percentages) 30 - 60% (preferably 30-40%) dry matter and has a pH of 6-8 (preferably between 6.5- 7.5). Typically a filter cake as described herein comprises:

- organic matter (Kg/t gross weight), 50 - 100, preferably around 65

- N total (N Kjeldahl) (Kg/t gross weight), 3 - 12, preferably around 4.8

- N ammonia (NH₄) (Kg/t gross weight), 1 - 6, preferably around 1 .1

- P total (P2O₅) (Kg/t gross weight), 5 - 14, preferably around 7.5

- potassium total (K₂0) (Kg/t gross weight), 5 - 15, preferably around 10.6

- magnesium Total (MgO) (Kg/t gross weight), 0.5 - 2, preferably around 1 .2; and

- calcium total (CaO) (Kg/t gross weight), 5 - 30, preferably around 12.5.

Moreover, such a filter cake comprises at least 20% of the phospholipase activity which was present in the fermentation broth. This phospholipase activity is bound to the non-viable biomass present in the filter cake.

The term "non-viable" refers to the fact that the cells and/or micro-organisms are no longer capable of living, multiplication or protein production. The terms "non-viable" and "killed" are used interchangeably herein.

Preferably, essentially all cells in a fermentation broth or a filter cake according to the invention are non-viable, i.e. such as a log reduction of at least 4, preferably a log reduction of at least 5, more preferably a log reduction of at least 6 and most preferably a log reduction of at least 7. In a preferred embodiment, the invention provides a fermentation broth or a filter cake essentially comprising non-viable micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms. Or in other words, the invention provides a non-viable fermentation broth or a non-viable filter cake comprising (non-viable) micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms.

Independent of the particular embodiment (i.e. fermentation broth or filter cake) at least part of the micro-organisms are capable of producing a phospholipase during fermentation . The phospholipase m ay be p rod u ced d u ri n g a n y sta g e of t h e fermentation, for example during the log phase. As explained above, at or near the end of the fermentation the micro organisms are subjected to a killing off step.

The produced phospholipase is preferably secreted into the fermentation medium. This is for example accomplished by fusing the gene encoding said phospholipase with a nucleic acid sequence encoding a secretion signal. The secretion signal is preferably cleaved off during secretion. An artificial cleavage site (such as a KEX site) located between the secretion signal and the phospholipase can be used for such purpose. During or after secretion at least part (for example 20 - 60% (based on activity) of the total amount of produced phospholipase (i.e. as present within the fermentation broth or as present in the filter cake and the filtrate) adheres to the extracellular part of the micro organisms. Without being bound by theory, the inventors believe that the phospholipase binds non-covalently to the extracellular part of the micro organisms, through hydrophobic and/or electrostatic interactions. Experiments have shown that this binding is firm as the phospholipase can not be washed from the biomass by extensive rinsing.

The adhered phospholipase is very stable. Experiments have shown that particularly adhered phospholipase A2 remains active even after heat treatments up to 90°C and at various pH values. Moreover, the phospholipase activity is not affected by the killing of the micro organisms.

As will be shown in the experimental part, the phospholipase adhered to the non-viable micro organism is - surprisingly- still functional. As described above a solid-liquid separation is typically performed to obtain a liquid phase and a filter cake as solid phase. This solid-liquid separation can be performed via multiple techniques. Non-limiting examples are a membrane filterpress or a vacuum beltfilter, vacuum drum filter, centrifuges like disc stack and sedicanter, microfiltration. I n case a membrane filterpress is used, filter aids such as perlites of different grade eg. Dicalite 4108 and DBF are used. In that case the resultant filter cake will comprise filter aids as well. In a preferred embodiment the invention provides a filter cake comprising non-viable micro organisms which micro-organisms before subjecting the corresponding fermentation broth to a killing off step are capable of producing a phospholi pase d u ri ng fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms, wherein said cake further comprises filter aids. A beltfilter could typically be used without adding any auxiliary compound.

The killed fermentation broth or filter cake could be used as such in any of the herein mentioned applications (for example oil degumming), i.e. non-dried or non-formulated.

The fermentation broth or the filter cake according to the invention is preferably dried which allows easy handling of the products. The skilled person is capable of subjecting a fermentation broth or a filter cake (both comprising non-viable micro organisms) to a drying step by using conventional industrial methods known in the art, such as spray drying or drum drying. In a preferred embodiment, the invention provides a killed fermentation broth as described above or a killed filter cake as described above which is dried. In other words, the invention also provides a dried fermentation broth comprising non-viable micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms or the invention provides a dried filter cake comprising non-viable micro organisms which micro-organisms before subjecting the corresponding fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms. I n yet another preferred embodiment the micro organisms in the (optionally dried) fermentation broth or filter cake are genetically modified (i.e. are recombinant) such as to express a non endogenous (i.e. a heterologous) phospholipase and/or an additional endogenous phospholipase. The micro organisms are typically provided with a nucleic acid sequence encoding a lipase or phospholipase. For this purpose a nucleic acid sequence encoding said lipase or phospholipase is operatively linked to one or more regu latory seq uences selected on the basis of the host cells (i .e. m icro organisms) to be used for expression. With the term "operatively linked" is intended to mean that the nucleic acid encoding the particular protein is linked to regulatory sequence(s) in a manner which allows for expression of the protein involved. The term "regulatory sequences" is typically used to include promoters, enhancers and other expression control elements (e.g. polyadenylation signal). Such regulatory sequences are described extensively within the prior art. In a preferred embodiment, the used regulatory sequences are non-endogenous with respect to the involved protein, i.e. the regulatory sequence does not naturally regulate said protein. As described above, the nucleic acid encoding said phospholipase is preferably linked to a secretion signal allowing the phospholipase to be secreted in the medium. The secretion signal is preferably cleaved off during secretion. An artificial cleavage site (such as a KEX site) located between the secretion signal and the phospholipase can be used for such purpose. The secreted phospholipase is - by itself - capable of bindi ng to the extracellular parts of the micro organism. It is therefore not necessary (and moreover not desired) to provide the nucleic acid encoding a phospholipase with a nucleic acid encoding a, for example heterologous (with respect to the phospholipase), so-called cell wall anchor (in literature also referred to as anchor attachment signal sequence, cell wall binding protein or artificially designed adhesive property etc.) In other words, the (preferably heterologous, with respect to the used micro organism) phospholipase is produced as such and is not functionally linked to a cell wall anchor. Surprisingly, the phospholipase as such is capable of binding to the extracellular part of the used micro organisms, is moreover firmly attached, and is active.

I n o n e of i t s preferred embodiments, the produced phospholipase is heterologous with respect to the used micro organisms. Heterologous (meaning 'derived from a different organism') refers to the fact that the phospholipase was initially cloned from or derived from a different cell type or a different species when compared to the micro organisms used for expressing the phospholipase. The terms heterologous a n d n o n-endogenous are used interchangeable herein. For example, the phospholipase is porcine phospholipase, preferably porcine phospholipase A2, and the used micro organism is a fungus, such as but not limited to Aspergillus. The invention therefore provides a fermentation broth or a filter cake comprising non viable micro organisms which micro organisms before subjecting the (corresponding) fermentation broth to a killing off step are capable of producing a heterologous phospholipase during fermentation and wherein at least part of the produced heterologous phospholipase adheres to the extracellular part of said micro organisms.

In yet another embodiment, the invention provides a fermentation broth or a filter cake comprising non viable micro organisms which micro organisms before subjecting the (corresponding) fermentation broth to a killing off step are capable of producing a heterologous phospholipase during fermentation and wherein at least part of the produced heterologous phospholipase adheres to the extracellular part of said micro organisms, and wherein said phospholipase is not linked to a separate cell wall anchor protein.

In another embodiment, the produced phospholipase is endogenous to the used micro organism. In such a case the micro organism does already comprise a nucleic acid sequence encoding said phospholipase but is provided with additional nucleic acid seq uences for exam ple to i mprove the expression levels of said endogenous phospholipase.

I n a preferred em bod iment, the micro organ ism is provided with a non- endogenous nucleic acid sequence encoding said phospholipase and hence the invention provides a (optionally dried) fermentation broth or a filter cake comprising non-viable micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms, wherein said micro organism is genetically modified to express said phospholipase and even more preferred said phospholipase is non endogenous with respect to the micro organism. To avoid any misunderstanding, the produced phospholipase which adheres to the extracellular part of the micro organisms is the non-endogeneous (heterologous) phospholipase. This applies to all embodiments disclosed herein.

Preferably, the produced heterologous phospholipase is porcine phospholipase A2 and the micro organism is a fungus, such as - but not limited to- Aspergillus. Said micro organism (preferably Aspergillus and more preferably Aspergillus niger) has been provided with a nucleic acid encoding the porcine phospholipase A2 such that the micro organism is capable of producing said porcine phospholipase A2. This is typically accomplished by functionally linking a nucleic acid encoding porcine phospholipase A2 with regulatory sequences as described above.

In case of a genetically modified micro organism the fermentation broth or the filter cake can be treated to remove (foreign, i.e. non-endogenous) nucleic acid. This is for example accomplished by thorough washing optionally followed by enzymatic breakdown of the remaining fractions of polynucleotides by incubation with for instance DNAses or RNAses that breakdown the polymeric structure of the polynucleotide and thereby destroying the genetic information encrypted in the polynucleotide.

Several classes of phospholipases exist. These classes will be explained in more detail.

Phospholipase A1 , PLA1 , hydrolyses the fatty acid on the sn1 position of lecithin, giving the 2-lysophospholipid that is not thermodynamically stable, the fatty acid on the 2 position migrates to the 1 position, after which it can be hydrolysed once more to yield a glycerophospholipid.

Phospholipase A2, PLA2, hydrolyses the fatty acid on the sn2 position of lecithin yielding a thermodynamically stable 1 -lysophospholipid and a free fatty acid. Maxapal A2 is a DSM PLA2 enzyme; Lecitase 10L, Rohalase MPL and Nagase PLA2 are known alternatives of other companies. In an application like enzymatic oil degumming, the free fatty acid, , that is split off can be removed in the alkali washing step, giving a soap solution from which the FFA can be extracted and used for different purposes.

Phospholipase B, PLB, is said to remove both fatty acids on the sn1 and the sn2 positions.

Phospholipase C, PLC, removes the phosphatidyl group from the phospholipid, giving a diglyceride and an easily removable phosphatidyl moiety. With this the oil yield should be even higher as the diglyceride stays behind in the triglyceride fraction, counting as oil. Purifine is a commercial PLC available from Verenium Corporation that is promoted for enzymatic oil degumming.

Phospholipase D, PLD, removes the groups on the phosphate group, giving phosphatidic acid and choline, ethanolamine or inositol. Phosphatidic acid however is difficult to remove from crude oil; it associates easily with multivalent cations and is non- hydratable. PLD activity is therefore usually unwanted.

The inventors started their experiments with a phospholipase A2 (which adheres particularly well to the extracellular part of the used micro organisms) but have also expressed a phospholipase A1 in Aspergillus, but surprisingly this phospholipase A1 did not adhere to the extracellular part of the used micro organisms.

Although a(n) (optionally dried) fermentation broth or filter cake according to the invention could comprise either type of phospholipase, a preferred embodiment is with phospholipase A2. The invention thus also provides a fermentation broth or a filter cake as described above, wherein said phosphol ipase is a phospholi pase A2. The phospholipase A2 is preferably heterologous in respect to the micro organism of the fermentation broth or filter cake. The heterologous phospholipase adheres to the exterior of the used micro organisms.

As described above the phospholipase produced by the micro organisms adheres to the outside of said micro organism. Upon comparing the amount of phospholipase adhered to the micro organisms to the total amount of phospholipase produced (i.e. the total activity as present in the medium and adhered to the cells; typically the activity present in the fermentation broth), at least 20% of the overall produced phospholipase adheres to the outside of said micro organisms during fermentation. The amount of adhered phospholipase increases during the killing off after which at least 40 % of the produced phospholipase adheres to the outside of the used host cell. The invention therefore provides in one of its embodiments a fermentation broth or a filter cake as described herein, wherein at least 40% of the produced phospholipase adheres to the outside of said micro organisms. Depending on the exact fermentation conditions and killing off conditions this percentage may increase to at least 50 % or 60%. Although the amount of phospholipase can be determined based on the amount of protein as such, in a preferred embodiment the percentage free or ad hered enzyme is determined by testing for the amou nt of enzymatic activity.

For example, the phospholipase A2 activity can be expressed in 'egg-yolk Units' (EYU), which corresponds to the amount of enzyme that liberates Ι μηηοΙ of fatty acid per minute from egg yolk at 40°C (104°F) and pH 8.0. MAXAPAL A2 is a liquid PLA2 enzyme solution standardized on phospholipase activity (CPU/g). Here CPU stands for Chromogenic Phospholipase Unit measured spectrophotometrically at 405 nm using the synthetic substrate rac 1 ,2-dioctanoyldithio-phosphatidylcholine. For Maxapal A2 CPU corresponds 1 :1 with EYU.

Suitable microorganisms are preferably prokaryotic microorganisms such as bacteria, or more preferably eukaryotic organisms, for example fungi, such as yeasts or filamentous fungi, or plant cells. In general, yeast cells are preferred over filamentous fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from yeasts, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a filamentous fungal host organism should be selected.

Bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas. A preferred yeast host cell for the expression of the DNA sequence encoding a phospholipase is one of the genus Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, or Schizosaccharomyces. More preferably, a yeast host cell is selected from the group consisting of the species Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var. lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica,and Schizosaccharomyces pombe.

Most preferred for the expression of the DNA sequence encoding a phospholipase are, however, filamentous fungal host cells. Preferred filamentous fungal host cells are selected from the group consisting of the genera Aspergillus, Trichoderma, Fusarium, Disporotrichum, Penicillium, Acremonium, Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia, and Talaromyces. More preferably a filamentous fungal micro organism is of the species Aspergillus oyzae, Aspergillus sojae or Aspergillus nidulans or is of a species from the Aspergillus niger Group (as defined by Raper and Fennell, The Genus Aspergillus, The Williams & Wilkins Company, Baltimore, pp 293-344, 1965). These include but are not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans, Aspergillus japonicus, Aspergillus oryzae and Aspergillus ficuum, and also those of the species Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium alabamense, Neurospora crassa, Myceliophtora thermophilum, Sporotrichum cellulophilum, Disporotrichum dimorphosporum and Thielavia terrestris.

Examples of preferred microorganisms within the scope of the present invention are fungi such as Aspergillus species (in particular those described in EP-A-184,438 and EP-A-284,603) and Trichoderma species; bacteria such as Bacillus species (in particular those described in EP-A-134,048 and EP-A-253,455), especially Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Pseudomonas species; and yeasts such as Kluyveromyces species (in particular those described in EP-A-096,430 such as Kluyveromyces lactis and in EP-A-301 ,670) and Saccharomyces species, such as Saccharomyces cerevisiae.

In one of its preferred embodiments, the invention provides a fermentation broth or a filter cake as described herein wherein said micro organisms are filamentous fungi. More preferably are an Aspergillus micro organism and even more preferred are Aspergillus niger micro organisms.

Other preferred embodiments are those wherein:

the phospholipase is a phospholipase A2 and the micro organisms are Aspergillus or

the phospholipase is a phospholipase A2 and the micro organisms are Aspergillus niger or

the phospholipase is a phospholipase A2 identical to the enzyme extracted from porcine pancreas and the micro organisms are Aspergillus or

the phospholipase is a phospholipase A2 identical to the enzyme extracted from porcine pancreas and the micro organisms are Aspergillus niger The "phospholipase A2 identical to the enzyme extracted from porcine pancreas" was obtained by extracting the mRNA coding for phospholipase A2 from pig pancreas tissue and by using the obtained mRNA as a template to produce cDNA in vitro. The amino acid sequence of the enzyme expressed by Aspergillus n/^'ger is exactly the same as the phospholipase obtained from pig pancreas. The amino acid sequence of the pig pancreas enzyme has been published in literature (Verheij et al. (1981 ) Rev. Physiol. Biochem. Pharmacol. 91 :91 -203). More detailed information in respect of the production of this porcine phospholipase A2 can be obtained from GRAS Notice 183 (Phospholipase A2 enzyme preparation from Aspergillus niger expressing a gene encoding a porcine phospholipase A2). The examples described herein use this particular phospholipase A2.

Preferably, the used micro organism is fermented on large or industrial scale, for example at least 10 m³ (gross), more preferably at least 30 m³ (gross, i.e. approximately 20 m³ net), even more preferably at least 50 m³ (gross) and most preferred at least 100 m³ (gross).

Surprisingly, this phospholipase producing fermentation broth or phospholipase containing filter cake as described herein is very low in tri-acyl lipase activity. That is, no lipase acitivity is seen in a sensitive lipase assay. Tri-acyl lipase activity is unwanted in many applications of phospholipases such as in oil degumming as it leads to generation of monoglycerides and diglycerides and more free fatty acids that all need to be removed by subsequent washing steps and lead to yield reduction of refined oil . Whether or not an enzyme preparation (for example a fermentation broth or filter cake as described herein) is low in lipase (alternatively free from detectable tri-acyl lipase) activity can be determi ned as described herein below. Preferably, an enzyme preparation as claimed herein does not (or at least not significantly) produce diglycerides and/or monoglycerides when compared to a positive and/or negative control sample. Triglycerides present in the starting composition stay intact. Lipases are known for their propensity to adhere to many surfaces and biomass, and the fact that here phospholipases are found and no tri-acyl lipases are found on the biomass is unique. In one of its embodiments the invention provides a fermentation broth or a filter cake comprising non-viable micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms, wherein lipase activity is not detectable. Whether or nor lipase activity is detectable is for example determined by exposing triglyceride rich lipids to a broth or filter cake as described herein. Prolonged exposure to biomass of triglyceride rich lipids showed no detectable (or significant) decrease of triglyceride levels and no detectable increase of di- and monoglyceride levels by current sensitive analysis techniques such as an analysis described by the American Oil Chemists' Society, AOCS Official Method Cd 1 1 d-96 Mono- and Diglycerides Determination by HPLC-ELSD, or AOCS method Cd 1 1 c-93 Quantitative Separation of Monoglycerides, Diglycerides, and Triglycerides by Silica Gel Column Chromatography, see www.aocs.org .

In yet another embodiment, the invention provides a method for obtaining a fermentation broth or a filter cake as described herein, said method comprising

optionally providing a micro organism with a nucleic sequence encoding phospholipase A2 (preferably porcine phospholipase A2) fermenting microorganisms capable of producing a phospholipase during growth

allowing said micro organisms to produce said phospholipase and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms resulting in a fermentation broth

subjecting the obtained fermentation broth to a killing off step for obtaining a fermentation broth as described herein

optionally subjecting the non-viable fermentation broth to a solid-liquid separation and obtaining a filter cake as described herein optionally subjecting said non-viable fermentation broth or said filter cake to a drying step

As described above, said phospholipase is preferably heterologous with respect to the used micro organism and said heterologous phospholipase (preferably phospholipase A2) adheres to the exterior of the used micro organism. The invention also provides a fermentation broth or a filter cake obtainable by the method described above.

The terms and phrases "filter cake", "fermentation broth", "phospholipase", "wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms" and "solid-liquid separation" have been discussed above and have the same meaning as described above.

Standard Aspergillus {niger) fermentation protocols are used.

Killing off of a fermentation broth is well known by the skilled person and can be performed in various ways. One non-limiting example is lowering the pH to around 4 and incubating the fermentation broth in the presence of 0.5% (w/w) sodium benzoate with decreased pH of 4.0 at a temperature of around 30 degrees Celsius for a period of approximately 4 hours. Other well known killing off methods use methyl ethyl parabens (MEP), heat, potassiumsorbate or octanol or combinations thereof. In the case of a heat stable enzyme such as the here discussed PLA2 even pasteurisation is possible.

Drying (such as spray drying) are also techniques which are well known to the skilled person and do not need any elaborate discussion.

In a preferred embodiment, the non-viable fermentation broth is directly subjected to a drying step, such as spray drying, drum drying. The heat involved in many drying processes will kill off micro organisms too, whilst the activity of this thermostable enzyme remains intact in such a process.

The fermentation broth or the filter cake as described herein can be formulated to for example improve their ease of handling or their stability.

In a preferred embodiment, the invention provides a filter cake comprising nonviable micro organisms which micro-organisms before subjecting the corresponding fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms and wherein said (optionally dried) filter cake is formulated in glycerol and/or salts and/or oil and or carbohydrates such as maltodextrins or glucose syrups. For degumming applications, a formulation in oil has certain advantages because this allows an easy distribution of the phospholipase in the oil. The invention therefore also provides a formulation comprising a filter cake as described herein together with glycerol and/or salt and/or oil and or carbohydrates, maltodextrines or glucose syrups. Additionally one can add non adhered phospholipase to such a formulation. Said phospholipase may be the same as the adhered enzyme or may be different. Moreover other enzymes can be co-formulated with the fermentation broth or the filter cake, preferably enzymes that assist in the particular industrial process, such as carbohydrases in enzymatic oil degumming.

Formulation of a fermentation broth or a filter cake as described herein in oil is particularly advantageous. This is particularly true for subsequent oil degumming processes (see later on). Preferably the fermentation broth or cake is formulated in oil which corresponds to the oil that will be degummed with said fermentation broth or cake. If one for example wants to degum soybean oil the fermentation broth or cake is preferably formulated in soybean oil to avoid contaminations with other kinds of oil as much as possible. Moreover, the filter cake is better dispersed in oil compared to water.

Alternatively the fermentation broth can be used as such - even without further treatments (i.e. even without a killing off step) - directly in an oil degumming process, by adding the fermentation broth directly into the crude oil, or alternatively adding it to the crude oil after pasteurisation. In a preferred embodiment, the fermentation broth production and the oil degumming facilities are located on the same location (i.e. close to each other), avoiding transport costs of fermentation broth to an oil degumming plant. The invention therefore also provides an oil degumming process comprising adding fermentation broth to vegetable (optionally partly purified) oil, wherein said fermentation broth comprises (optionally non viable) micro organisms which micro organisms (optionally before subjecting the fermentation broth to a killing off step) are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms.

A fermentation broth or a filter cake as described herein can have multiple industrial applications.

One example of an industrial application is oil degumming.

Common seed oils are soy, rape, canola, or sunflower oil. Typically, these are first pressed from the dehulled and pretreated (e.g. heated to stop endogenous enzyme activity) seed (rape and sunflower, oil content -40%) or directly extracted from this seed by hexane (soy, oil content -20%). The crude seed oil contains impurities that need to be removed; a major impurity is formed by phospholipids (PL) or lecithins (the terms are used interchangeably herein). Depending on the seed, grade and pretreatment this PL level can be as high as 3% or 1200 ppm P.

Phopholipids from these sources are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA) and traces of phosphatidylserine (PS). In addition the oils contain a certain amount of glycolipids or galactolipids (GL). Typical composition of oil free lecithin for soy oil is 75% PL, 15% GL and 10% complex carbohydrates + traces of triglyceride (TG) oil, and within the group of PL the variation for soy is 29% PC, 29% PE, 32% PI and 10% others or 33% PC, 22% PE, 22% PI, 9% PA.

Part of this group of phospholipids occurs as non-hydratable phospholipids which are believed to be complexes of phospholipids with polyvalent cations like Ca and Mg specifically PI and PA are susceptible to complexation with such ions for its primarily negative charge. The level of non-hydratables also depends on seed source and process.

Degumming is the name of the process by which the phospholipids are removed. Traditionally this is done by the addition of water, by which the phospholipid is hydrated. Usually just a bit more water is added than the phospholipids level in the crude oil - up to 3-4%. In this hydrated state the phospholipids can be removed as a (slimy) gum phase by centrifugation. With this process the remaining level of phospholipids goes down to 100-200 ppm of phosphorus, corresponding to approximately 0.23 - 0.46% of phospholipids. This is still too high for refined oil where levels should be as low as 10 ppm, preferably 5 ppm. The remaining phospholipids are further removed in the last steps with aqueous alkali neutralisation, and bleaching earth treatment. This causes however use of higher amounts of bleaching earth and presumably the soapstock contaminated with phospholipids.

Phospholipids in refined oil are unwanted because it leads to unwanted effects as poor taste, turbid oil. For biodiesel, phospholipid leads to faltering of the combustion process and dirt in the engines.

With normal water degumming especially the non-hydratable phospholipids are poorly removed. For this reason the super degumming or acid degumming process was devised, by which instead of water citric acid (or other acid) rich water phases were used. The process is not optimal, too much P left, and the degummed phospholipids phase sti ll contai ns about 50-60% of triglyceride oil that is dragged into the phospholipids phase, which is then sold for a lower value than if it would remain in the refined oil.

Most of the phospholipids coming from the degumming is sprayed on the soy meal and further used as animal feed. Triglyceride oil left in that lecithin phase represents no value. A part of the removed phospholipids is sold as emulsifiers for the food industry, as crude lecithin or as worked up fractions, such as de-oiled, alcohol extracted or enzymatically hydrolysed (PLA2). Generally the value of this stream is not high: the market price of hydrolysed lecithin is only 30-40% higher than of normal lecithins.

Already for some 20 years the use of phospholipases to improve degumming is promoted. The main advantage of enzymatic degumming is said to be that

a) the degummed phospholipids phase contains low levels of triglyceride oil, b) the refined oil phase contains low levels of phospholipids (<10 ppm of P , preferably <5 ppm), and

c) the oil refining process is improved by reducing the viscosity of the phospholipid rich gum phase.

Typical reaction / processing conditions are such that about 3-4% of water is added to the crude oil for degumming, at 55°C. Lower water levels, such as 1 -4%, are also preferred for costs and environmental reasons. Reaction times vary and depend for exa m pl e on th e specific enzyme used. On industrial scale these are often continuous processes.

The main disadvantage of enzymes is their high cost in use.

I n early days of enzymatic degumming also processes were developed that recycled (part) of the enzyme. This is not applied anymore. Further processing on the gum stream involves heating steps and usually the enzyme is inactivated in this way. Even when sprayed on meal it is preferred that the enzyme is inactivated . An advantage of a broth or filter cake of the invention is that the enzyme is adhered and that such an inactivation step can be omitted.

The inventors of the present invention have recognized that enzymatic oil degumming is hampered by the high cost in use of the used enzyme. Surprisingly, phospholipase adhered to the extracellular part of (production) micro organisms is functional. Moreover, said phospholipase is very stable and as a consequence pasteurisation, drying steps or formulation steps can be performed without noticeably affecting the activity of the phospholipase. As an alternative to purified phospholipase one can now use a (optionally dried and/or formulated) fermentation broth and/or filter cake during enzymatic oil degumming. Another advantage is that after the process the biomass can be removed by e.g. filtration of centrifugation and thereby the residual enzyme activity in the refined oil can be reduced to fully absent.

I n a yet another embodiment the invention therefore provides a method for vegetable oil degumming comprising incubating crude oil with a fermentation broth as claimed and decribed herein or a filter cake as claimed and decribed herein or a formulation as claimed and described herein and allowing the phospholipase as present in said fermentation broth or said filter cake or said formulation to optimise the removal of phospholipids form crude oil especially including the removal of non-hydratable phospholipids. Preferably, the used phospholipase is heterologous with respect to the used micro organism. More preferably, said phospholipase is porcine phospholipase A2 and the used micro organism is Aspergillus niger, the produced heterologous phospholipase adheres to the exterior of Aspergillus niger and the fermentaion broth or filter cake is free from detectable lipase activity.

Surprisingly, the phospholipase as present in a fermentation broth or filter cake or a formulation according to the invention does not - at least not detectable- comprise any lipase activity which is an important need for an oil degumming processes.

The invention further provides the use of fermentation broth or a filter cake or a formulation as described herein for hydrolysis of phospholipids in particular in applications like vegetable oil degumming. Preferably, the used phospholipase is heterologous with respect to the used micro organism. More preferably, said phospholipase is porcine phospholipase A2 and the used micro organism is Aspergillus niger, the produced heterologous phospholipase adheres to the exterior of Aspergillus niger and the fermentaion broth or filter cake is free from detectable lipase activity.

An enzymatic oil degumming process utilising fermentation broth or filter cake would be similar to a process where regular phospholipase would be used. In such a process the crude oil is mixed with 1 -4% of water and in this case fermentation broth or filter cake containing PLA2. The amount of fermentation broth or filter cake depends on its phospholipase activity. A useful dose is 10 CPU / gram phospholipid, ranging from 1 to 100 CPU / gram phospholipid. The fermentation broth or filter cake can be added as such , or after a killing off step and/or after a pasteurisation step, wet or dry and rehydrated. The crude oil, water and the fermentation broth or filter cake is then mixed by a high shear mixer and then held in a retention tank, typically for 1 to 4 hours at 60°C. Thereafter the gums (hydrated phospholipids) and the fermentation broth or filter cake are separated from the degummed oil by centrifugation.

Proof of phospolipase action applicable in oil degumming can readily be obtained by subjecting vegetable lecithins (obtained from certain crude seed oils such as soy bean oil, sunflower oil or rape seed oil in a water-degumming process) to an incubation by phospholipase on biomass or filtercake and following the hydrolysis process of this material by measuring the levels of unhydrolysed phospholipids and the lysophospholipids that result from the phospholipase action. This can for instance be done by 31 P N MR. Phospholipid hydrolysis seen in this process is representative for what happens in an enzymatic oil degumming process.

A fermentation broth or a filter cake as claimed and described herein can (optionally dried and/or formulated) be formulated in granules, to be used to fill a column. This column can then subsequently be used in an enzymatic oil degumming process by passsing the oil through the column. Alternatively larger granules can easily be removed from the liquid process stream and reused.

Possi ble leakage of enzymes or other u nwanted compon ents from the fermentation broth or the filter cake can be prevented by further immobilisation of the broth or cake by known immobilisation techniques.

The amount of broth or filter cake needed in an oil degumming process can easi ly be d eterm i n ed by th e skilled person and does not need any elaborate discussions.

The invention further provides a degummed oil obtainable from:

- a method for vegetable oil degumming comprising incubating said oil with a fermentation broth as claimed and decribed herein or a filter cake as claimed and decribed herein or a formulation as claimed and described herein and allowing the phospholipase as present in said fermentation broth or said filter cake or said formulation to reduce the P-level in the vegetable oil.

- use of fermentation broth or a filter cake or a formulation as described herein for vegetable oil degumming.

The refined oil resulting from a process using fermentation broth or a filter cake will be essentially free from extra generated monoglycerides and diglycerides unlike some already known enzymatic degumming proceses.

The use of fermentation broth or a filter cake as described herein to hydrolyse phosphoplipids can also be employed in other industrial processes, such as the modification of egg yolk or the formation of high lysophospholipid containing lecithin compositions used for instance as emulsifier in spreads and margarines.

In a similar way it is envisioned that general (triacylglycerol)lipases immobilised on a fermentation broth or a filter cake can be employed to modify triglycerides or other lipids e.g. for use in enzymatic interesterification of triglycerides; or to generate mono- and diglycerides specifically by enzymatic means, either from hydrolysing triglycerides or by esterification of glycerol with fatty acids.

The invention further provides a method for increasing the value of a non-viable filter cake wherein the micro organisms present in said filter cake before subjecting the fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms, said method comprising using said filter cake in an industrial process instead of or next to non-adhered phospholipases.

As will be explained in more detail in the experimental part, the invention further provides a method for determining the presence of lipolytic activity in a phospholipase sample comprising incubating at least part of said phospholipase sample (in water or buffer) with refined (triglyceride) oil and determining the presence and/or amount of diglycerides and/or monoglycerides and/or free fatty acids, preferably by comparing said amounts of dyglycerides, monoglycerides or free fatty acids with the amounts of diglycerides, monoglycerides or free fatty acids as obtained with a positive and/or negative control. An example of a suitable positive control is a lipase. Alternatively one can after incubation of the refined (triglyceride) oil with at least part of a phospholipase sample review the macroscopic appearance.

The term "refined oil" is typically used to refer to an oil fit for human consumption or fit for use in food products. More in detail, a refined oil is an oil which has been subjected to a refining process which removes undesirable materials (phospholipids, monoacylglycerols, diacylglycerols, free acids, colour and pigments, oxidised materials, flavour components, trace metals and sulfur compounds) but which process may also remove valuable minor components which are antioxidants and vitamins such as carotenes and tocopherols.

The invention is hereby illustrated with the following non-limiting examples.

Example 1 Obtaining fermentation broth and filter cake

A fermentation broth and/or filter cake is produced by a controlled submerged fermentation of an Aspergillus niger culture.

The living microorganisms in the fermentation broth at the end of fermentation were killed at 30 °C during 6 hours at pH 4 using 0.5 % sodium benzoate at production scale. After the broth was killed the broth was prepared to be filtered. To the killed broth 7 % of filter aid (such as dicalite 4108 or dicalite BF) was added and the broth was filtered at pH 4 at 5 bars using a membrane filterpress. The cake was washed with 2.2 cake volumes process water, squeezed and discharged from the membrane filterpress. In the resulting cake and filtrate the PLA2 activity was analyzed. Based on the activity results it turned out that a substantial amount of PLA2 was adhered to the biomass. The cake of the membrane filterpress was collected and samples were used for conversion soy lecithin phospholipids.

The phospholipase activity was measured as the Chromogenic Phospholipase Unit (CPU/g) and determined spectrophotometrically at 405 nm using the synthetic substrate rac 1 ,2-dioctanoyldithio-phosphatidylcholine.

Example 2 Activity of filter cake on soy lecithin

This experiment describes the conversion of soy lecithin phospholipids hydrolysed with PLA Aspergillus niger filter cake as obtained in example 1 . Materials

- Crude soy lecithin, I MCOSOY LEC F010 obtained from I MCOPA (Den Bosch, the Netherlands); the phospholipids composition is given in table 1 .

- Maxapal A2 batch PLA6148 NZ, with activity of 10.400CPU CPU / gram, DSM Food Specialties, Delft, the Netherlands

- Biomass filter cake of Aspergillus niger after PLA processing (the left-over) is obtained from DFS/DSP; batch 7159, activity is 1300 CPU/gram biomass

Table 1 : The content of phospholipids varieties in soy lecithin and egg yolk

^*- not observed

Methods

The hydrolysis of soy lecithin

A 70/30 o/w dispersion of soy lecithin was hydrolysed at 60°C for 72 hours with 180 CPU PLA Aspergillus niger biomass / gram phospholipids.

The amount of phosphatidyl choline PC, Lyso-PC, phosphatidyl ethanolamine PE, Lyso-PE, PI, lyso-PI, PA and lyso-PA in soy lecithin were determined with P31 NMR (see Diehl, B.W.K., High resolution NMR spectroscopy Eur. J. Lipid Sci. Technol. 103 (2001 ) 830-834).

The conversion is expressed in percentage of the PC, PE, PI and PA that is converted. The conversion is expressed in two ways: 1 ) relative to the general blank at the beginning "Ref" and 2) as the total amount of the individual phospholipids "Indiv", based on the total amount PL + LPL at a specific time. The latter because of incomplete extraction pretreatment for P31 NMR measurement, by which the total amount of phospholipids measured with the P31 NMR is too low in these samples. First, the % of the phospholipid class (PC, PE, PI , or PA) was converted to mmol using the specific molecular weight; calculation: (grams phospholipid / mol weight) ^* 1000 = mmol phospholipid and

(1 ) subsequently the amount of LPC, LPE, LPA and LPI was compared to a blank (the amount of phospholipids in the not hydrolysed lecithin); calculation: (mmol (LPL) sample /mmol (PL blank) ^* 100 = % conversion Ref, and

(2) the amount of LPC, LPE, LPA and LPI was compared to the individual amount of phospholipids PL + LPL at each specific time; calculation: (mmol (LPL) sample /mmol PL + LPL) ^* 100 = % conversion 'Indiv'

Table 2 The molecular weight data of the phospholipids that were used for the conversion calculation.

Phospholipid Mol weight

PC - phosphatidylcholine 779

LPC - lyso phosphatidylcholine 524

PE - phosphatidylethanolamine 737

LPE - lyso phosphatidylethanolamine 482

PI - phosphatidyl 862

LPI - lysophosphatidyl 580

PA - phosphatidyl 700

LPA - lysophosphatidyl 437

The amount of PC, Lyso-PC, PE, Lyso-PE, PI, lyso-PI, PA and lyso-PA were determined with P31 NMR to calculate the conversion as described above.

The conversion of the soy lecithin phospholipids.

A soy lecithin 70/30 (lecithin/water) dispersion is hydrolysed at 60°C for 72 hours with 180 CPU Aspergillus niger biomass / gram phospholipids. The PC, PE, PI and PA conversions are calculated from the P31 NMR results.

Table 3. The PC, PE, PI and PA soy conversion after hydrolysis at 60°C with 180 CPU Aspergillus niger biomass

Incubation Conversion % of the phospholipids

time [h] PC PE PI PA

Ref^* Indiv^** Ref Indiv Ref Indiv Ref Indiv

0 1 1 2 2 2 2 7 7

2 45 86 54 97 52 47 100 95

24 51 94 34 95 45 97 94 94

48 38 98 27 95 35 96 80 94

72 43 96 32 95 36 96 100 95

Ref^* = conversion is calculated with a reference blank phospholipids.

Indiv^** = conversion is calculated with the individual total amount phospholipid class at that particular time point. In table 3 the degree of the soy phospholipids, PC, PE, PI and PA conversion is given. Clearly Soy lecithin is hydrolysed by PLA2 in the Aspergillus niger biomass at 60°C.

Table 4 70/30 lecithin/ water with 50 CPU / gram PL at 60°C

It is obvious that the PLA adhered to Aspergillus niger is active in soy lecithin and functional for four phospholipids types, PC, PE, PI and PA. The degree of conversion is > 80 % for PC, PE and PA within 2 hours hydrolysis and for PI after 24 hours at 60°C with 180 u PLA Aspergillus niger biomass/ g PL. For comparison also the conversion of vegetable lecithin (70% crude lecithin and 30% water) by commercial Maxapal A2 (50 units / gram of phospholipid) at 60°C is given in Table 4. It shows that more time is needed to reach a corresponding conversion. Taking into account that in the latter experiment a lower dose (on unit level) was used, it shows that about comparable conversion was obtained in both cases, and that the residual filter cake was approximately equally active in conversion.

Being able to hydrolyze phospholipids in crude lecithin means that such conversion also takes place in crude oil in an oil degumming process if biomass or filter cake residue and water are added.

Example 3 Activity of filter cake on egg yolk

This experiment describes the conversion of egg yolk phospholipids hydrolysed with PLA Aspergillus niger filter cake obtained in example 1 .

Materials

- Egg yolk liquid included 8 % salt was obtained from Bouwhuis-Enthoven, Raalte, the Netherlands ; the phospholipids composition is given in table 1 . - Maxapal A2 batch PLA6148 NZ, with activity of 10.400CPU CPU / gram, DSM Food Specialties, Delft, the Netherlands

Methods

The hydrolysis of egg yolk:

- Egg yolk was hydrolysed with residual PLA Aspergillus niger biomass at 50°C for 3 hours with 100 u CPU Aspergillus niger biomass / gram of phospholipid in the egg yolk and at a similar activity level of commercial Maxapal A2.

- The amount of phosphatidyl choline PC, Lyso-PC, phosphatidyl ethanolamine PE, Lyso-PE in egg yolk was determined with P31 NMR.

- The conversion is expressed in percentage of the PC and PE that is converted. The conversion is calculated in the following way: the % of the phospholipids was converted to mmol using the specific molecular weight (see table 2) and subsequently the amount of LPC, and LPE, was compared to a blank (the amount of phospholipids in not hydrolysed egg yolk); calculation: (mmol (LPC or LPE) sample /mmol (PC or PE) blank) ^* 100 = % conversion

The conversion of the egg yolk phospholipids.

The egg yolk phospholipids are hydrolysed at 50°C for three hours with 100 CPU Aspergillus niger biomass / gram PL. The PC and PE conversion is calculated from the P31 NMR results, as given in table 5.

Table 5. The PC and PE conversion in egg yolk after hydrolysis at 50°C with 100 CPU Aspergillus niger biomass / gram phospholipidand 100 CPU Maxapal A2 / gram phospholipid PC & PE conversion % with PC & PE conversion % with PLA Aspergillus niger Maxapal A2

biomass

PC PE PC PE

Egg yolk standard 0.6 7.7 3.8 8.5

Hydrolysis after 30 93.8 87.4 61 .2 64.9 min.

Hydrolysis after 180 95.6 92.7 82.6 81 .9 min.

In table 5 the PC and PE egg yolk conversion is shown after 30 minutes and 3 hours incubation with PLA Aspergillus niger biomass and the reference, PLA2 in Maxapal A2. The degree of conversion of PC and PE after 30 minutes incubation is even better with the PLA adhered to the Aspergillus niger biomass. After three hours incubation the conversion is 10 % higher compared to the Maxapal incubation.

Conclusion Example 2 and 3

The PLA adhered to Aspergillus niger biomass shows hydrolysing activity for phospholipids in egg yolk and soy lecithin comparable to or even better than Maxapal A2. It can be concluded that the phospholipids can easily reach the active side of the enzyme or the active side of the enzyme is free to move into or to the phospholipids in egg yolk and soy lecithin to hydrolyse the second fatty acid, without the need to design the enzyme such that it has a specific anchor to be attached to the biomass, as well that there is no need to design the enzyme such that there is a spacer between anchor and the active site of the enzyme.

The phospholipase adhered to Aspergillus niger biomass is

- active in a soy lecithin dispersion and functional for PC, PE, PI and PA soy phospholipids to convert these in lyso-phospholipids.

- active in egg yolk and functional for egg yolk phospholipids PC and PE as PLA2 in Maxapal A2. Example 4 Test on lipase activity

Enzyme preparations were incubated with commercial refined soy bean oil during 4 hours (comparing to process conditions in present industrial degumming process) and 24 hours at a pH of 4.5 (in buffered systems) and a temperature of approximately 55 °C under vigorously mixing. The oil to water ratio was 90/10 w/w. After incubation of the samples at 2 different enzyme concentrations the samples were separated using a centrifuge during 10 minutes at 5000 rpm.

Details in respect of the amount of oil, temperature and amount of enzyme are presented in Table 6:

Table 6 dosing of the different PLA2 products on oil

Piccantase A (DSM Food Specialties, Delft, the Netherlands) is a lipase used as positive control in the experiment which has hardly phospholipase activity.

Maxapal A2 (DSM Food Specialties, Delft, the Netherlands) is a purified phospholipase

A2

Cakezyme L (DSM Food Specialties, Delft, the Netherlands) is a liquid formulation of non-purified phospholipase A2 in glycerol

Cake is biomass from solid liquid separation process of phospholipase A2

Acetate buffer is blanc without enzymatic activity

Amount of oil: 25 ml

Temperature: 52.2-54.9 degrees Celsius

The supernatant, the oil, was analyzed for mono-, di-, triglycerides and free fatty acids. If lipolysis of a triglyceride occurs, first a diglyceride and one free fatty acid is formed, this could be either a 1 ,2-diglyceride or a 1 ,3-diglyceride. Subsequently another fatty acid can be removed, yielding a monoglyceride. Finally the last fatty acid can be hydrolysed, giving a third free fatty acid and a glycerol. The lipid composition in levels of triglyceride, diglyceride, monoglyceride and free fatty acids can be analysed by high pressure liquid chromatography (H PLC), a method usually referred to as an M DT analysis. The oil/enzyme/water dispersions were frozen and freeze dried. The freeze dried samples were extracted: 20 mg homogenized sample was extracted with 1 ml solvent mixture (heptane chloroform 3:1 ). The solid material was then removed by filtration using 0.2 μηη PVDF membranes and 10 μΙ was injected into a normal phase HPLC (Agilent 1 100 liquid chromatograph, Agilent Technologies, Santa Clara, CA) equipped with a polar (cyano) column and an Evaporative Light Scattering Detection (ELSD; Sedex 75; Polymer Laboratories Ltd, Shropshire, UK). A gradient of heptane-t- butylmethylether was used as a running medium. Using the calibration line and the program Chromeleon Chromatography Management System (Dionex Corporation , Sunnyvale, CA), the amounts of lipid components were determined . Lipids were separated with a normal-phase chromatography system using a gradient of organic solvents. Retention times were determined with commercial samples of known composition.

From the results as presented in Figure 1 it is clear that the phospholipase form ulations, and the fi lter cake do not have any li pase activity (same glyceride distribution as the reference with only buffer), whereas a lipase (Piccantase) generates substantial amounts of free fatty acids, di- and monoglycerides.

Example 5

PLA2 filtercake VMJ1035-61 / FOP.VMJ.38, contains 80 gram filteraid / kg filter cake (dry matter), Dm = 38.1 %, with CPU activity 269CPU/g filter cake (dry matter)

Soy lecithin dispersion in water:

90 grams Soy lecithin Nutrilec (containing 40% phospholipids) and 250 grams water (26.5 wt% lecithin on dispersion) were mixed intensively using high shear Silverson mixer for 5 minutes at 5000 rpm. 0.4 gram Filtercake was added to 50 grams of lecithin dispersion, which corresponds to 20.3 CPU / gram PL, and incubated at 50°C, and samples were taken at 1 .5, 3, 5 and 24 hours. Samples were analysed for phospholipid content by P NMR

[Calculation: 269 CPU ^* 0.4 = 107.6 CPU in 50 ^* 0.265 ^* 0.40= 107.6 CPU / 5.3 g PL = 20.3 CPU / gram PL]

Soy lecithin dispersion with sunflower oil in water:

In a second experiment 90 grams Soy lecithin Nutrilec (40% phospholipids) + 60 grams sunflower oil + 140 grams water were combined and thoroughly mixed using a high shear Silverson mixer for 5 minutes at 5000 rpm.

0.4 gram Filtercake was added to 50 grams of lecithin/oil dispersion, which corresponds to 17.4 CPU / gram PL, and incubated at 50°C, and samples were taken at 1 .5, 3, 5 and 24 hours. Samples were analysed for phospholipid content by P NMR

[Calculation: 269 CPU ^* 0.4 = 107.6 CPU in 50 ^* 0.31 ^* 0.40 = 107.6 CPU / 6.2 = 17.4 CPU / gram PL]

Table 7

This proves that filtercake with PLA2 adhered to it is well capable of hydrolysing phospholipids to the lyso form. Also in an environment with a high amount of oil - representing the oil degumming situation even better - the conversion ru ns as expected.

Claims

1 . A fermentation broth comprising non viable micro organisms which micro organisms before subjecting the fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms.

2. A filter cake comprising non viable micro organisms which microorganisms before subjecting the corresponding fermentation broth to a killing off step are capable of producing a phospholipase during fermentation and wherein at least part of the produced phospholipase adheres to the extracellular part of said micro organisms.

3. A killed fermentation broth according to claim 1 or a killed filter cake according to claim 2 which is dried.

4. A killed fermentation broth according to claim 1 or 3 or a filter cake according to claim 2 or 3 wherein said phospholipase is non endogenous with respect to the micro organisms.

5. A killed fermentation broth according to claim 1 , 3 or 4 or a filter cake according to claim 2, 3 or 4 wherein said phospholipase is a phospholipase A2.

6. A killed fermentation broth according to claim 1 , 3-5 or a filter cake according to claim 2-5 wherein at least 40%, more preferably at least 50 %, of the produced phospholipase adheres to the outside of said micro organisms, preferably based on the enzyme activity of the material.

7. A formulation comprising a filter cake according to any one of claims 2-6 and glycerol and/or salt and/or oil and/or carbohydrates, glucose syrup or maltodextrins.

8. A formulation according to claim 7 further comprising non-adhered phospholipase.

9. A fermentation broth according to any one of claims 1 , 3-6 or a filter cake according to any one of claims 2-6 wherein said micro organisms are filamentous fungi.

10. A fermentation broth or a filter cake according to claim 9, wherein said filamentous fungi is Aspergillus.

1 1 . A method for hydrolysis of phospholipids comprising incubating said oil with a fermentation broth according to any one of claims 1 , 3-6 or 9-10 or a filter cake according to any one of claims 2-6 or 9-10 or a formulation according to claim 7 or 8.

12. A method for reducing the levels of phosphor-containing compounds in refined oil, such as vegetable oil degumming, comprising incubating said oil with a fermentation broth according to any one of claims 1 , 3-6 or 9-10 or a filter cake acccording to any one of claims 2-6 or 9-10 or a formulation according to claim 7 or 8.

13. Use of a fermentation broth according to any one of claims 1 , 3-6 or 9-10 or a filter cake acccording to any one of claims 2-6 or 9-10 or a formulation according to claim 7 or 8 for hydrolysing phospholipids.

14. Use of a fermentation broth according to any one of claimsl , 3-6 or 9-10 or a filter cake acccording to any one of claims 2-6 or 9-10 or a formulation according to claim 7 or 8 for vegetable oil degumming.

15. A method for determining the presence of lipolytic activity in a phospholipase sample comprising incubating at least part of said phospholipase sample with refined oil and determining the presence and/or amount of diglycerides, monoglycerides and/or free fatty acids.