NOVEL HALOTOLERANT AND HALOPHILIC ENZYMES AND THE USE OF HALOTOLERANT AND HALOPHILIC ENZYMES
FIELD OF INVENTION
The present invention relates to the field of enzymes capable of having enzymatic activity at high salinities. Specifically there is provided novel xylanolytically, mannanolytically, cellulolytically, lipolytically and amylolytically active halotolerant or halophilic enzymes which i.a. are useful in the manufacturing of food products, animal feedstuffs, paper prod- ucts and other polysaccharide-containing materials and oligosaccharides.
TECHNICAL BACKGROUND AND PRIOR ART
In several conventional industrial processes where enzymes are used such as in the manufacturing of salt containing products and/or products having a low water activity including food products such as meat products, dairy products and bakery products, high salinities or low water activity are frequently factors which limit the activity of the enzymes. This problem is currently being solved by adding a high amount of enzyme or by desalting the starting materials.
However, it has now been found that enzymes derivable from halophilic or halotolerant microorganisms are effective in the manufacturing of products having high salinity i.e. low water activity.
Hypersaline environments constitute typical examples of "extreme" environments in which a relatively low species diversity can be found. Microorganisms, which are specialised to live only in such environments are designated halophiles, whereas those from hypersaline environments that are capable of growth in freshwater media, but tolerant of higher salt concentrations, are referred to as halotolerant.
To date, a limited number of extra- and intracellular enzymes have been isolated from halotolerant, moderately and extremely halophilic microorganisms. Such enzymes, referred to as halophilic or halotolerant enzymes, are adapted to be catalytically active under conditions where the salinity is high and thus under conditions with low water activity.
Only a limited number of halotolerant/halophilic extracellular enzymes have been reported as originating from aerobic, extremely halophilic Archaea For example amylases produced by Halobacteπum halobium and Halobacteπum sodomense, proteases from Halo- bacterium salinaπum and Halobactenum halobium, and hpases from several halobactena (Ventosa et al , 1995, World Journal of Microbiology & Biotechnology 11 85-94) Halo- bacterium salinaπum and Halobactenum halobium have recently been reclassified as Halobactenum salmarum whereas Halobactenum sodomense has been reclassified to Halorubrum sodomense (Kamekura, 1998, Extremophiles 2 289-295) Halophilic enzymes have also been isolated from moderately halophilic bacteria and characterised These m- elude amylases, nucleases, proteases and 5'-nucleotιdases (Ventosa et al , 1995) Johnson et al (Arch Microbiol , 1986, 143.370-378) have isolated and characterised several enzymes (e g xylanases and β-xylosidases) from the halophilic gram-positive actmomy- cete Actmopolyspora halophila and Winterhalter et al (Appl Environ Microbiol , 1995, 61 1810-1815) have isolated two types of xylanases from the gram-negative hyper- thermophilic marine bacterium Thermotoga mantima
To the best of our knowledge, there have been no reports on industrial application of halophilic or halotolerant enzymes e g for the preparation of food products, animal feedstuff and other polysacchande-containing materials
The present inventors have now discovered a variety of novel halophilic and halotolerant enzymes having xylanolytic, mannanolytic, amylolytic or hpolytic activity of microbial origin which are particularly useful in industrial manufacturing of products having high salinities and/or low water activities
SUMMARY OF THE INVENTION
Accordingly, the invention relates in one aspect to a polypeptide having hemicellulolytic, cellulolytic or starch degrading activity that is derivable from a gram-negative bacterium, the polypeptide having at least one of the following characteristics (i) it is active at any NaCI concentration up to and including 30% (w/v) or higher, (n) it has at least 10% of its maximum activity at a NaCI concentration of about 28% (w/v) or higher, (in) it has a maximum activity at a NaCI concentration of about 4% (w/v) or higher and (iv) it is active at a NaCI concentration of about 20% (w/v) or higher, and in other aspects to an isolated gram-negative halophilic or halotolerant bacterial strain capable of producing such a poly-
peptide, a recombinant microorganism comprising a gene encoding the above polypeptide and/or a regulatory nucleotide sequence modifying the expression of the gene coding for said polypeptide, a method of producing such polypeptide, comprising cultivating a gram- negative bacterium naturally producing the polypeptide under conditions permitting the production of the polypeptide, optionally followed by a recovery of the polypeptide, a method of producing the polypeptide, comprising isolating from the gram-negative bacterium a DNA sequence coding for the polypeptide, forming a vector containing the DNA sequence, transferring the obtained vector to a host cell, cultivating the host cell under conditions where the cell is capable of expressing the polypeptide, and recovering the polypeptide, and use of such a polypeptide including its use in the preparation of an animal feedstuff, a food product or a carbohydrate.
In further aspects the invention relates to the use of the above polypeptide in the preparation of a paper product, a food or feed improving composition comprising the polypeptide, and a fermentation medium composition having xylanolytic, hemicellulolytic or starch degrading activity obtained by cultivating the above cells of a bacterial strain in a medium and optionally separating the cells from the medium.
There is also provided a polypeptide having xylanolytic or cellulolytic activity that is deriv- able from a gram-positive bacterium, the polypeptide having at least one of the following characteristics (i) it is active at any NaCI concentration up to and including 30% (w/v) or higher, (ii) it has at least 9% of its maximum activity at a NaCI concentration of about 25% (w/v) or higher, (iii) it has a maximum activity at a NaCI concentration of about 1% (w/v) or higher, (iv) it is active at a NaCI concentration of about 15% (w/v) or higher, and in other aspects to an isolated gram-positive halotolerant or halophilic bacterial strain capable of producing such a polypeptide, a recombinant microorganism comprising a gene encoding the above polypeptide and/or a regulatory nucleotide sequence modifying the expression of the gene coding for this polypeptide, a method of producing such a polypeptide comprising cultivating a gram-positive bacterium naturally producing the polypeptide under conditions permitting the production of the polypeptide, optionally followed by recovering the polypeptide, a method of producing the polypeptide comprising isolating from a gram- positive bacterium a DNA sequence coding for the polypeptide, forming a vector containing the DNA sequence, transferring the obtained vector to a host cell, cultivating the host cell under conditions where the cell is capable of expressing the polypeptide, and recov-
ering the polypeptide, and use of such a polypeptide including its use in the preparation of an animal feedstuff, a food product, a paper product or a carbohydrate
In further aspects the invention relates to the use of the above polypeptide in the prepara- tion of a food or feed improving composition comprising the polypeptide, and a fermentation medium composition having xylanolytic or cellulolytic activity obtained by cultivating the above cells of a bacterial strain in a medium and optionally separating the cells from the medium
The invention pertains in a still further aspect to a mannan hydrolysing polypeptide that is derivable from a gram-positive bacterium, the polypeptide having at least one of the following characteristics (i) it is active at any NaCI concentration up to and including 28% (w/v) or higher, (n) it has at least 5% of its maximum activity at a NaCI concentration of about 25% (w/v) or higher, (in) it has a maximum activity at a NaCI concentration of about 1 % (w/v) or higher, (iv) it is active at a NaCI concentration of about 15% (w/v) or higher, and in other aspects to an isolated gram-positive bacterial strain which is capable of producing this polypeptide, a recombinant microorganism comprising a gene encoding the above polypeptide and/or a regulatory nucleotide sequence modifying the expression of the gene coding for said polypeptide, a method of producing such polypeptide comprising cultivating a gram-positive bacterium naturally producing the polypeptide under conditions permitting the production of the polypeptide, optionally followed by recovering the polypeptide, a method of producing a polypeptide, comprising isolating from the gram-positive bacterium a DNA sequence coding for the polypeptide, forming a vector containing the DNA sequence, transferring the obtained vector to a host cell, cultivating the host cell un- der conditions in which the cell is capable of expressing the polypeptide, and recovering the produced polypeptide, and to the use of such a polypeptide including its use in the preparation of an animal feedstuff a food product or a carbohydrate
In yet other aspects the invention relates to the use of the above polypeptide in the prepa- ration of a paper product, a food or feed improving composition comprising the polypeptide, and a fermentation medium composition having mannan hydrolysing activity obtained by cultivating the above cells of a bacterial strain in a medium and optionally separating the cells from the medium
The invention provides in another aspect a polypeptide that is derivable from a halotolerant or a halophilic Archaea, which is a polypeptide having hemicellulolytic, cellulo- lytic or starch degrading activity, and in other aspects to an isolated halophilic or halotolerant archaeal strain capable of producing such a polypeptide, a recombinant microor- ganism comprising an inserted gene encoding the above polypeptide and/or a regulatory nucleotide sequence modifying the expression of the gene coding for said polypeptide, a method of producing the polypeptide comprising cultivating an Archaea naturally producing the polypeptide under conditions permitting the production of the polypeptide, optionally followed by recovering the polypeptide, a method of producing such polypeptide com- prising isolating from an Archaea a DNA sequence coding for the polypeptide, forming a vector containing the DNA sequence, transferring the obtained vector to a host cell, cultivating the host cell under conditions where the cell is capable of expressing the polypeptide, and recovering the produced polypeptide, and use of such a polypeptide including its use in the preparation of an animal feedstuff, a food product or a carbohydrate.
In further aspects the invention relates to the use of the above polypeptide in the preparation of a paper product, a food or feed improving composition comprising the polypeptide, and a fermentation medium composition having cellulolytic and/or hemicellulolytic activity obtained by cultivating the above cells of an archaeal strain in a medium and optionally separating the cells from the medium.
Another aspect of the present invention is the use of a halotolerant and/or a halophilic enzyme in the preparation of a food product, a feedstuff and a paper product.
DETAILED DISCLOSURE OF THE INVENTION
The present invention provides a range of novel halotolerant or halophilic enzymes. Thus, there is provided such polypeptides having hemicellulolytic, starch degrading, lipolytic or cellulolytic activity that are derivable from halotolerant or halophilic gram-negative bacte- ria, gram-positive bacteria or Archaea.
In the present context, the term "hemicellulolytic activity" relates to any enzyme having the capability to degrade at least one substance belonging to the group of compounds generally referred to as hemicellulose or enzymatically or chemically modified derivatives thereof. Hemicellulose is a large group of polysaccharides found in the primary and sec-
ondary cell walls of plants. The most ubiquitous and abundant hemicelluloses are the xylans and the mannans
Xylans are usually heteropolymers, composed of a backbone of 1 ,4-lιnked β-D- xylopyranose residues and branches of L-arabinofuranose, D-glucuronic acid, or 4-O- methyl-D-glucuromc acid. Hydrolysis of the xylose backbone of xylan involves \_- xylanases (1 ,4-β-D-xylan xylanohydrolase: EC 3 2.1 8) and β-xylosidases (1 ,4-β-D-xylan xylohydrolase: EC 3.2 1 37). β-xylanases hydrolyses internal xylosidic linkages on the backbone while β-xylosidases release xylose residues by endwise attack of xylooligosac- chandes. The degradation of xylan is further enhanced by the action of side-group cleaving enzymes such as β-L-arabinofuranosidases, acetyl esterases, and α-glucuronidases.
Mannans (1 ,4-β-D-mannans), the other major constituent of hemicellulose are made up of polymerised mannose residues The mannose units of some mannans are substituted to varying degrees with galactose, glucose and O-acetyl groups, e g in galactomannan and galactoglucomannan. Examples of mannan hydrolysing enzymes are β-mannanase (1 ,4- β-D-mannan mannano-hydrolase; EC 3.2.1.78) and β-mannosidase (β-D-mannoside mannohydrolase, EC 3 2.1.25) which hydrolyse the β-D-mannoside linkages in β-1 ,4-D- mannan producing small manno-oligosacchaπdes and D-mannose, respectively
Enzymes having starch degrading activities are also referred to as amylolytically active enzymes. Such enzymes include amylases including α- and β-amylases, amyloglucosi- dases, pullulanases, α-1 ,6-endoglucanases, α-1 ,4-exoglucanases and isoamylases
Enzymes having lipolytic activity are also generally referred to as lipases As used herein the term "lipase" is used to designate any tnacylglycerol hydrolysing enzyme, including such enzymes that are capable of splitting of fatty acids having short, medium and long chain lengths. Other enzymes having lipolytic activity which are encompassed by the present invention include phospholipases, lysophospho pases, acylglycerol lipases and ga- lactolipases
Enzymes having cellulolytic activity are also generally referred to as cellulases As used herein the term "cellulase" is used to designate any cellulose hydrolysing enzyme.
As mentioned above, microorganisms specialised to live in hypersaline environments are generally referred to as halophiles, whereas those capable of growth in freshwater media, but tolerant of higher salt concentrations, are generally referred to as halotolerant In the present context, microorganisms are defined as non-halophihc when they exhibit optimal growth at < 1 % (w/v) NaCI and halophilic when optimal growth is above 1 % (w/v) NaCI Furthermore, halophilic microorganisms, as defined herein, are divided into slightly halophilic having optimal growth at 1-5 % (w/v) NaCI, moderately halophilic with optimal growth at 5-18% (w/v) NaCI and extremely halophilic with optimal growth above 18% (w/v) NaCI Also in the present context, halotolerant microorganisms are defined as microor- ganisms selected from the following types slightly halotolerant which grow at NaCI concentrations up to 6-8% (w/v) NaCI, moderately halotolerant which grow at NaCI concentrations up to 18-20% (w/v) NaCI, and extremely halotolerant growing at NaCI concentrations up to and above 20% (w/v) and occasionally to the point of saturation of NaCI (ap- prox 36% (w/v) NaCI)
As mentioned above, the polypeptide having hemicellulolytic, cellulolytic or starch degrading activity that is derivable from a gram-negative bacterium has at least 10% of its maximum activity at a NaCI concentration of about 28% (w/v) or higher In a preferred embodiment the polypeptide has at least 20% of its maximum activity, such as at least 30% including at least 40% of its maximum activity In even more preferred embodiments, the polypeptide has at least 50% of the maximum activity under the above conditions such as at least 60%
It has been found that the above enzyme has a high stability when it is kept at high salt concentrations Thus, in preferred embodiments the polypeptide retains at least 20% of its maximum activity, such as at least 30% including at least 40% of its maximum activity after being kept at a NaCI concentration of about 28% (w/v) or higher for 24 h In even more preferred embodiments, the polypeptide retains at least 50% of the maximum activity under the above conditions such as at least 60%
In one embodiment the polypeptide having hemicellulolytic, cellulolytic or starch degrading activity that is derivable from a gram-negative bacterium is an α-amylase (EC 3 2 1 1), including such an enzyme which is active at a pH of about 9 or higher
In accordance with the invention the above polypeptide is derivable from any gram- negative bacterial species naturally producing an enzyme having the above characteristics. One suitable source of such an enzyme is a gram-negative bacterial strain selected from a proteobacterial species, including a strain selected from the gamma sub- class of Proteobacteria. Specific examples of such strains are the strains XYL15 (DSM 12620), A5 (DSM 12618) and CL8 (DSM 12619) as described in details in the following examples. Other suitable sources for the above polypeptide are bacterial species showing >93 % 16S rRNA sequence similarity with any of the strains XYL15, CL8 or A5, i.e. bacterial species within the same genera as any of said strains.
In this context, the 93% 16S rRNA sequence similarity value is to be understood as the value currently accepted as the criterion for establishing new prokaryotic genera as defined by Fry et al. (Journal of General Microbiology, 137:1215-1222, 1991 ), and the 16S rRNA sequence similarity is analysed by currently established methods e.g. by DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Braunschweig, Germany).
The enzymes according to the invention may for certain applications be used in the form of crude enzyme preparations e.g. a fermentation medium composition having the pre- selected enzymatic activity that is obtained by cultivating cells of a bacterial strain producing the enzyme in a medium and optionally separating the cells from the medium. However, it may for other applications be advantageous to use the enzyme in a substantially pure form. Such a pure enzyme preparation may be provided by separating the enzyme from a crude enzyme preparation using protein separation techniques.
The enzymes according to the invention can be provided by cultivating a microbial strain naturally producing the enzymes under conditions permitting the production of the polypeptide, optionally followed by a recovery of the polypeptide.
However, it will be understood that the enzymes can also be provided by using a recombinant microorganism comprising a gene encoding the enzyme polypeptide. Accordingly, the invention provides methods of producing the enzymes according to the invention that comprise isolating from the selected source organism a DNA sequence coding for the polypeptide, forming a vector containing the DNA sequence, transferring the
obtained vector to a host cell, cultivating the host cell under conditions where the cell is capable of expressing the polypeptide, and recovering the polypeptide.
The halotolerant or halophilic enzymes according to the invention are useful for a variety 5 of applications where it is desired to degrade a substance serving as a substrate for the particular enzymes. Thus, enzymes having cellulolytic, hemicellulolytic, starch degrading or lipolytic activity are highly useful in the preparation of animal feedstuff compositions as a means of improving the conversion of the feedstuff in the gastro-intestinal tract, resulting in a improved feed conversion rate and hence, a growth promoting effect. 0
In interesting embodiments, the enzymes according to the invention are used in the preparation of food products, in particular food products where the water activity is low such as at the most 0.98 including at the most 0.95 e.g. at the most 0.90 or even lower such as at the most 0.85 including at the most 0.80. Typical examples of such food prod- 5 ucts include dairy products such as cheese, bakery products such as bread and other flour dough based products and meat or vegetable products having relatively high salt content such as cured products.
In addition to use in food or feedstuff products, the enzymes according to the invention 0 are useful in any other industrial applications where it is desired to achieve a high enzymatic activity at low water activity levels, e.g. in the preparation of a paper product, including the processing of cellulosic pulps. Another example is the use of the enzymes according to the invention in the synthesis of oligosaccharides and polysaccharides that takes place under low water activity conditions. 5
In one further aspect of the invention, the polypeptide according to the invention is a polypeptide having xylanolytic or cellulolytic activity that is derivable from a gram-positive bacterium, including halophilic or halotolerant species. A significant feature of this polypeptide is that it has at least 9% of its maximum activity at a NaCI concentration of about 30 25% (w/v) or higher. In a preferred embodiment the polypeptide has at least 20% of its maximum activity, such as at least 30% including at least 40% of its maximum activity. In even more preferred embodiments, the polypeptide has at least 50% of the maximum activity under the above conditions such as at least 60%.
It has been found that the above enzyme has a high stability when it is kept at high salt concentrations Thus, in preferred embodiments the polypeptide retains at least 20% of its maximum activity, such as at least 30% including at least 40% of its maximum activity after being kept at a NaCI concentration of about 28% (w/v) or higher for 24 h In even more preferred embodiments, the polypeptide retains at least 50% of the maximum activity under the above conditions such as at least 60%
The above xylanolytic or cellulolytic enzyme can be derived from any gram-positive bacterial species naturally producing the enzyme A suitable source organism is a strain se- lected from Bacillaceae including Bacillus spp and "Gracilibacillus spp" , including
"Gracilibacillus halotolerans" A typical example of the latter organism is the strain referred to herein as strain NN (DSM 12617) and described in further details in the following examples Other suitable source organisms for the above enzymes are any gram-positive strain showing >93 % 16S rRNA sequence similarity with strain NN, i e a bacterial spe- cies within the same genus as strain NN
In addition to the above xylanolytic activity the above source strains may be capable of producing further enzymes such as a mannanolytic, amylolytic, cellulolytic, lipolytic or proteolytic enzyme
In one further aspect of the invention, the polypeptide according to the invention is a polypeptide having mannan hydrolysing activity that is derivable from a gram-positive bacterium, the polypeptide having at least one of the following characteristics (i) it is active at any NaCI concentration up to and including 30% (w/v) or higher, (n) it has at least 10% of its maximum activity at a NaCI concentration of about 28% (w/v) or higher, (in) it has a maximum activity at a NaCI concentration of about 1% (w/v) or higher, (iv) it is active at a NaCI concentration of about 15% (w/v) or higher In a preferred embodiment the polypeptide has at least 20% of its maximum activity at a NaCI concentration of about 25% (w/v) or higher, such as at least 30% including at least 40% of its maximum activity In even more preferred embodiment, the polypeptide has at least 50% of the maximum activity under the above conditions such as at least 60%
It has been found that the above enzyme has a high stability when it is kept at high salt concentrations Thus, in preferred embodiments the polypeptide retains at least 20% of its maximum activity, such as at least 30% including at least 40% of its maximum activity af-
ter being kept at a NaCI concentration of about 20% (w/v) or higher for 24 h. In even more preferred embodiments, the polypeptide retains at least 50% of the maximum activity under the above conditions such as at least 60%.
The above mannan hydrolysing enzyme can be derived from any gram-positive bacterial species naturally producing the enzyme. A suitable source organism is a strain selected from Bacillaceae including Bacillus spp. and "Gracilibacillus spp"., including "Gracilibacillus halotolerans". A typical example of the latter organism is the strain referred to herein as strain NN (DSM 12617) as described in further details in the following examples. Other suitable source organisms for the above enzymes are any gram-positive strain showing >93 % 16S rRNA sequence similarity with strain NN, i.e. a bacterial species within the same genus as strain NN. In addition to the above mannanolytic activity the above source strains may be capable of producing further enzymes such as a xylanolytic, amylolytic, cellulolytic, lipolytic or proteolytic enzyme.
As mentioned above, the invention pertains in a still further aspect to a polypeptide that is derivable from a halotolerant or a halophilic Archaea, which is selected from a polypeptide having hemicellulolytic, cellulolytic or starch degrading activity including a polypeptide having xylanolytic activity and a lipase. In one preferred embodiment such a polypeptide has at least one of the following characteristics: (i) it is active at any NaCI concentration up to and including 30% (w/v) or higher, (ii) it has at least 10% of its maximum activity at a NaCI concentration of about 28% (w/v) or higher, (iii) it has a maximum activity at a NaCI concentration of about 5-15% (w/v) or higher, (iv) it is active at a NaCI concentration of about 15% (w/v) or higher. In a preferred embodiment the polypeptide has at least 20% of its maximum activity at a NaCI concentration of about 28% (w/v) or higher, such as at least 30% including at least 40% of its maximum activity. In even more preferred embodiment, the polypeptide has at least 50% of the maximum activity under the above conditions such as at least 60%.
It has been found that the above enzyme has a high stability when it is kept at high salt concentrations. Thus, in preferred embodiments the polypeptide retains at least 20% of its maximum activity, such as at least 30% including at least 40% of its maximum activity after being kept at a NaCI concentration of about 28% (w/v) or higher for 24 h. In even more preferred embodiments, the polypeptide retains at least 50% of the maximum activity un- der the above conditions such as at least 60%.
The above enzymes can derived from any archaeal species naturally producing the enzymes A suitable source organism is a strain selected from Euryarchaeota including Ha- lobactenaceae, such as a "Halorhabdus" including "Halorhabdus utahensis" A typical example of the latter organism is the strain referred to herein as strain AX-2 (DSM 12616) as described in further details in the following examples Other suitable source organisms for the above enzymes are any archaeal species showing >93 % 16S rRNA sequence similarity with AX-2, i e an archaeal species within the same genus as AX-2 In addition to the above enzyme activities the above source strains may be capable of producing further enzymes such as an amylolytic, mannan hydrolysing or proteolytic enzyme
As mentioned above, the invention pertains in a still further aspect to the use of a halotolerant and/or a halophilic enzyme in the preparation of a food product, a feedstuff or a paper product In such use, the enzymes according to the invention are highly suitable but any halotolerant or halophilic enzyme can be used In this connection, the term "halotoler- ant" refers to an enzyme that is active in the range from 0 to at least 20% (w/v) NaCI and which has optimum activity at a NaCI concentration < 1% (w/v) The term "halophilic" refers to an enzyme which is active in the range from 0 to at least 30% NaCI (w/v) and has optimum activity at a NaCI concentration > 1 % (w/v)
The invention will now be further illustrated in the following non-limiting examples and the drawing wherein
Fig 1 shows the effect of NaCI concentration on the activities of β-xylanase from strain XYL15,
Fig 2 shows the effect of NaCI concentration on β-xylanase from strain CL8 grown in the presence of 10% NaCI NaCI (O) and 20% NaCI (•),
Fig 3 shows the effects of NaCI concentrations on β-mannanase (O) and β-mannosidase (•) activities from strain NN,
Fig 4 shows the halostability of β-mannanase and β-mannosidase from strain NN at 20% NaCI (30°C, pH 7 0), the β-mannanase was dialysed against either 10 mM NaP buffer (•) or Tris medium (A) and β-mannosidase was tested using either culture broth (Δ) or washed cells suspended in 10 mM NaP buffer (O),
Fig. 5 shows the effect of NaCI concentration on β-xylanase activity from strain NN,
Fig. 6 shows the effect of NaCI concentration on the activities of β-xylanase (•) and β- xylosidase (O) from strain AX2, and
Fig. 7 shows the halostability of β-xylanase at 0.05% NaCI (•); 27% NaCI (A) and β- xylosidase at 0.5% (O) and 25% (Δ) activities from strain AX2.
EXAMPLES
A. GRAM-NEGATIVE HEMICELLULOLYTICALLY AND AMYLOLYTICALLY ACTIVE HALOTOLERANT AND HALOPHILIC BACTERIA.
EXAMPLE 1
Isolation and cultivation of gram-negative halotolerant and halophilic bacteria producing β-xylanase, β-xylosidase, α-amylase β-mannanase and cellulase
1 .1 . Enrichment and isolation
Xylan and amylose degrading gram-negative bacteria were isolated from water and sediment samples obtained from different hypersaline environments, by enrichment culture in a liquid isolation medium containing either xylan or amylose as the main carbon and energy source. The enrichment medium (GSL-2 medium) had the following composition (amounts per litre of Milli-Q water): NaCI, 50-200 g; MgSO4-7H2O, 10 g; KCI, 5 g; CaCI2-6H2O, 0.2 g; citric acid, 0.5 g; NH4CI, 2 g; NaHCO3, 1 g; KH2PO4, 0.5 g; FeCI2-4H2O, 0.04g; MnCI2-4H2O, 0.04 g; Yeast extract (Difco Laboratories), 2 g; Poly- peptone peptone (Becton Dickinson), 2 g; trace element solution TMS 3 (Ingvorsen and Jørgensen, 1984, Arch. Microbiol. 139:61-66), 2 ml. The pH of the final medium was 7.4. The enrichments were initiated by adding various source materials to GSL-2 medium containing either 0.3 % (w/v) xylan or 0.3 % amylose. Enrichment cultures were incubated aerobically at 30°C and 200 rpm using a rotary shaker.
Three gram-negative strains were isolated and designated XYL15, CL8 and A5. The bacteria were isolated from the enrichments by standard serial-dilution techniques on
solid GSL-2 medium. Strains XYL15, CL8 and A5 were deposited under the Budapest Treaty on 7 January 1999 with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Braunschweig, Germany) under the accession Nos. 12620, 12619, 12618, respectively.
1 .2. Phylogenetic characterisation of strain XYL1 5
Strain XYL 15 was found to be gram-negative, and according to partial 16S rDNA sequence analysis carried out at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Braunschweig, Germany), strain XYL15 belongs to the gamma subclass of the Proteobacteria. Strain XYL15 shows highest sequence similarity values of 89.9 % to Oceanospirillum linum and 90.3 % to Marinobacter hydrocarbonoclasticus. Thus, strain XYL15 at least represents a novel species and possibly a novel genus. Strain XYL15 shows about 93 % (93.3) and about 93 % (93.5) sequence similarities with strains A5 and CL8, respectively.
1 .3. Phylogenetic characterisation of strain CL8
Strain CL8 was found to be gram-negative, and comparison of 16S rRNA sequences sug- gest that strain CL8 represents a new genus within the gamma subclass of the Proteobacteria. It shows about 99 % (99.3 %) 16S rRNA gene sequence similarity with strain A5 and about 93 % (93.5) sequence similarity with strain XYL15.
1 .4. Phylogenetic characterisation of strain A5
Strain A5 was found to be gram-negative, and comparison of 16S rRNA sequences suggest that strain A5 represents a new genus within the gamma subclass of the Proteobacteria. It shows about 93 % (93.3 %) 16S rRNA gene sequence similarity with strain XYL15 and about 99 % (99.3) sequence similarity with strain CL8.
1 .5. Cultivation of strain XYL1 5
Strain XYL15 was routinely cultivated aerobically at 30°C on a shaker in GSL-2 medium (20 % w/v NaCI, 0.3 % birchwood xylan, Roth 7500).
1 .6. Cultivation of strain CL8
Strain CL8 was cultivated aerobically at 30°C in TRIS 11 medium containing in grams per liter of demineralized water: MgSO4-7H2O, 20.0; KCI, 5.0; NH4CI, 2.0; NaBr, 0.1 ; yeast extract (Difco), 2.0; trypticase peptone (BBL), 0.5; Tris-HCI, 12.0; birchwood xylan (Roth, Karlsruhe - Germany), 2.0; trace metal solution (TMS 3) (Ingvorsen & Jørgensen, 1984), 2 ml. NaCI was added to a final concentration of 10 or 20% (w/v) NaCI. The pH was adjusted to 7.8. After sterilization and cooling to 5°C, 2.5 ml of a sterile phosphate solution (KH2PO4, 50 g/l), 0.5 ml of a sterile CaCI2 solution (CaCI2-2H2O, 100 g/l), and 0.5 ml of a sterile FeCI2/MnCI2 solution (FeCI2-4H2O, 20 g/l + MnCI2-4H2O, 20 g/l) were added. The final pH of the medium was approx. 7.6.
1 .7. Cultivation of strain A5
Strain A5 was cultivated in Tris-M medium which had the following composition (amounts per litre of Milli-Q water): NaCI, 100 g; MgSO4-7H2O, 20 g; KCI, 5 g; (NH4)2S04, 2 g; NaBr, 0.1g; yeast extract (Difco Laboratories), 0.2 g; Trypticase peptone (BBL), 0.2 g; Trizma (TRIS-hydroxymethyl aminomethan, hydrocloride), 12 g; and trace elements (TMS3), 2 ml. The pH of the solution (970 ml) was adjusted to 7.8 with NaOH and autoclaved. After sterilisation, 2 g amylose and 10 ml of the following three stock solutions (autoclaved separately) were added to obtain the final medium. Solution 1 : (25 g KHP0 , 25 g K2HPO4 per litre); Solution 2: (50 g CaCI2-2H2O per litre) and Solution 3 (0.2 g FeCI2-4H2O, 0.2 g MnCI2-4H20 per litre). The final pH of the medium was 7.5.
EXAMPLE 2
Enzyme production
2.1 . Production of β-xylanase and β-xylosidase
For β-xylanase production, strain CL8 was grown at 30°C in Erlenmeyer flasks containing 100 ml TRIS 11 medium shaken at 180 rpm. for approx. 70 h. β-xylanase activity was determined using either culture supernatant or dialysate. Supernatant was prepared by centrifugation of culture broth for 3 min. at 11.000 * g, while dialysate was made by dia-
lyzing the supernatant for approx. 12 h against a 10 mM sodium phosphate (NaP) buffer (10% NaCI, pH 7.0) at 4°C.
For β-xylanase and β-xylosidase production, strain XYL15 was grown for 96 h in 100 ml of GSL-2 medium or Tris-M medium containing 0.5 % w/v birchwood xylan (Roth 7500) and 10 % NaCI. The culture was centrifuged at 11.400 x g in a refrigerated centrifuge for 5 min. to remove cells and other particulate matter. The supernatant liquid - hereinafter referred to as "enzyme solution" - was used as crude β-xylanase or stored at -20°C until used for experiments. The cell pellet obtained by centrifugation was resuspended in assay buffer and used as crude β-xylosidase.
The following assays for β-xylanase and β-xylosidase activities from strain XYL 15 were used:
The β-xylanase assay was carried out using a substrate solution containing 0.1 % w/v AZCL-birchwood glucuronoxylan (Megazyme) in sterile GSL-2 medium (20 % NaCI; pH 7) at 30°C with slow shaking in an Eppendorf thermomixer. The reaction was started by adding 0.2 ml of suitably diluted enzyme solution to 1.8 ml substrate solution in a 2.0 ml Eppendorf safe-lock tube. The degree of hydrolysis was measured spectrophotometrically at 590 nm (OD590) in a supernatant sample obtained by centrifugation of the reaction mixture. Several time points were measured in order to assure linearity of the hydrolytic reaction.
β-xylosidase (1 ,4-β-D-xylan xylohydrolase, EC 3.2.1.37) activity was assayed using p- nitrophenyl-β-D-xylopyranoside (PNXP, Sigma Co., N-2132) as substrate as described by Poutanen and Puls (Appl. Microbiol. Biotechnol. 28:425-432,1988).
The following assays for β-xylanase activities from strain CL 8 were used:
The effect of NaCI on β-xylanase activity was determined using a substrate solution containing 0.1% (w/v) AZCI-birchwood glucuronoxylan (Megazyme, Ireland) and 0.1 ml supernatant of a culture grown in 10% or 20% TRIS 11 medium in 10 mM NaP buffer (2% MgS0 -7H2O, pH 7.0) containing varying amounts of NaCI. Total volume of the reaction mixture was 1 ml and this was incubated in a thermomixer at 30°C for 4 h.
The effect of NaCI on β-xylanase stability was tested by modification of the above- mentioned assay as follows; 0.05 ml dialysate in a total assay volume of 1 ml was incubated in a thermomixer for 24 h at 30°C. Centrifugation of the reaction mixtures at 11.000 x g for 3 min. was followed by spectrophotometric measurement of the dye-release from AZCI-xylan at 595 nm.
2.2. Production of α-amylase
For production of α-amylase, strain A5 was cultivated at 30°C in Tris-M containing 10% w/v NaCI, 0.05% w/v CaCI2 and 0.2% w/v amylose. After approx. 48h (stationary phase), the cells were removed by centrifugation (11 ,340 * g, 5 min). The cell-free supernatant was used as a crude enzyme preparation.
The following assay for α-amylase (E.C. 3.2.1.1 ) activity was applied:
α-amylase activity was routinely assayed by mixing 200 ml of enzyme solution with 1.8 ml of a 50 mM phosphate assay buffer containing 0.88mg/ml AZCL-amylose (Megazyme, Ireland) and 10% w/v NaCI, (pH 7.3). The samples were incubated for an appropriate period of time at 30°C in an Eppendorf thermomixer. After centrifugation (10,000 rpm, 3 min), the release of dye from AZCL-amylose was measured spectrophotometncally at 590 nm.
2.3. Production of cellulase
Strain CL8 produces cellulase when it is grown in TRIS 11 medium containing the following carbon sources: CMC (carboxymethylcellulose), Whatman no.1 filter paper and Avicel. When grown on the latter two substrates both endo-cellulase and exo-cullulase activities were detectable in cell-free supernatants using a range of dyed substrates (e.g. RBB-CMC, RBB-Avicel, AZCL-HE-Cellulose(RBB = Remazol Brilliant Blue)).
When grown on solid TRIS 11 medium containing 0.03 % (w/v) finely ground (ball-milled) Whatman no.1 filter paper, cellulolytic activity was clearly evidenced through the formation of distinct clearing zones in the agar medium around individual colonies.
The cellulase system of Strain CL8 exhibited activity at salinities ranging from 0.1 % to 30 % NaCI."
2.4. Production of β-mannanase
Strain CL8 produces β-mannanase when grown on TRIS 11 medium containing birchwood xylan and mannan. This was evidenced with the dyed substrate AZCL- galactomannan using the same general assay procedures as described for other AZCL substrates (cf. above sections 2.1 ; 2.2 and 7.1). The β-mannanase activity produced by strain CL8 was active at salinities ranging from 0.02 % to 30 % NaCI.
EXAMPLE 3
Characterisation of β-xylanase and β-xylosidase from XYL15
3.1 . Effect of NaCI concentration on β-xylanase activity from strain XYL15.
The effect of salinity (NaCI concentration) on β-xylanase activity of strain XYL15 was investigated using reaction mixtures containing the desired concentrations of sodium chlo- ride and 0.25 % w/v AZCL-birchwood xylan (Megazyme). The rate of AZCL-xylan hydrolysis was monitored by the standard β-xylanase assay as described above. The results of this experiment are summarised in Fig. 1.
As can be seen from Fig. 1 , rates of xylan hydrolysis were low yet detectable at 1 and 2 % NaCI. Maximum activity occurred between 8 and 10 % NaCI and at 30 % NaCI the activity was still about 40 % of the maximum. The β-xylanase of strain XYL15 was thus active over an extremely large salinity interval ranging from 1 to 30 % NaCI. Time course studies showed that β-xylanase was stable and active for at least 25 h at salinities between 1 and 30 % NaCI since xylan hydrolysis proceeded linearly throughout this period.
3.2. Temperature optimum and thermostability of XYL1 5 β-xylanase
The temperature optimum for β-xylanase activity was determined at 10% NaCI in reaction mixtures incubated at different temperatures for 1.0 h using AZCL-xylan as substrate. The 5 maximal activity was found around 40°C with almost none at 55°C.
The thermostability of β-xylanase activity was determined in enzyme solutions at 10% NaCI incubated at different temperatures in the absence of substrate and subsequently assayed at 30°C using the standard assay described in 2.1. 0 β-xylanase activity of XYL15 was stable for at least 24 h at 40°C. At 50°C only 30% of the initial activity remained after 4 h of incubation, β-xylanase activity was completely lost within 1 h when incubated at 60°C and 70°C.
5 3.3. Effect of pH on β-xylanase activity
The effect of pH on β-xylanase activity was studied by incubating enzyme samples at different pH values using AZCL-xylan as a substrate. The reaction was carried out as described above except for using a Britton-Robinson buffer (Rauen, H.M., Biochemisches 0 Taschenbook, Springer Verlag, 1964) containing 11% NaCI. The β-xylanase of strain XYL15 was most active within a neutral pH range of between pH 6 and pH 8. There was only low activity below pH 5 and above pH 9.5.
3.4. Stability of β-xylanase activity 5
In order to study the stability of β-xylanase activity, enzyme solutions were dialysed and subsequently assayed in 10% NaCI. The enzyme solutions (10 - 20 ml) were dialysed against 2 x 2 litre 10 mM NaH2PO4/Na2HPO4 buffer (NaP-buffer, pH = 7.0) for a minimum of 24 h at 5°C. The dialysed enzyme samples were stored without further treatment at -
30 20°C. The dialysed enzyme samples retained >90% of the initial xylan hydrolysing activity upon assay in 10% NaCI in GSL-2 medium after storage for 2 months at -20°C.
3.5._β-Xylosidase activity
When grown on xylans, strain XYL15 also produced β-xylosidase activity, β-xylosidase activity was almost entirely associated with the cell. The effect of NaCI concentration on β- xylosidase activity was investigated using intact cells. Relative enzyme activity was at a maximum at 5-10 % NaCI but more than 50 % of maximum activity was obtained at NaCI concentrations ranging between 1 and 30 %.
EXAMPLE 4
Characterisation of β-xylanase from strain CL8
4.1 . Effect of salinity on β-xylanase activity
In order to study the effect of different NaCI concentrations on the activity of β-xylanase strain CL8 was grown in TRIS 11 medium containing either 10 or 20% (w/v) NaCI and the supernatant was tested for β-xylanase activity at various NaCI concentrations and 2% (w/v) MgSO4. The results of this experiment are summarised in Fig. 2.
As can be seen from Fig. 2, β-xylanase activity of strain CL8 was present over a broad salinity range from 2% up to 28% NaCI. It exhibited maximum activity at both 2-5% NaCI and 15% NaCI. When strain CL8 was grown at 10% NaCI, the β-xylanase activity of the supernatant at 28% NaCI retained 30% of the activity present at 2% NaCI.
4.2. Halostability of CL8 β-xylanase activity
In order to test the stability of the β-xylanase from strain CL8 when exposed to different salinities, an assay was performed using a dialysate of a culture grown in a medium containing 10% NaCI. The dialysate was incubated for a period of 24 h at NaCI concentra- tions of 2, 10, or 28%, respectively.
The results revealed that the β-xylanase activity of strain CL8 was extremely stable at all salinities. Less than 40% of the initial activity was lost during the 24 h incubation irrespective of the salinity tested and approx. 12% of the activity measured at 2% NaCI was pres- ent in the assay at 28% NaCI.
4.3. Isoelectric points of xylanolytic enzymes from strain CL8
Strain CL8 produces several xylanolytic enzymes when grown on xylan substrates. Thus as evidenced by standard agarose lEF/zymogram technique, strain CL8 produces at least 3 xylanolytic peptides having isoelectric points (pi's) of approx. 8, approx. 3.7 and approx. 2.7, respectively.
EXAMPLE 5
Characterisation of α-amylase from strain A5
5.1 . Effect of salinity on α-amylase activity
The effect of NaCI on α-amylase activity was investigated at NaCI concentrations ranging from 0 to 25% w/v NaCI using enzyme solutions prepared from cultures grown in the absence of added NaCI. The assay was performed as described in Example 2.2 adjusting the salinity of the buffer to the above salinity range.
α-amylase activity was maximal at 0% w/v NaCI, at 20% NaCI the activity was approx. 25% of maximum and at 25% NaCI the activity was 3% of the maximal activity.
5.2. Effect of temperature on A5 amylase activity
The effect of temperature on amylase activity of strain A5 was highly dependent on the salinity. Using assay buffers with no added NaCI, maximum activity was observed at 40°C. At 10% NaCI, maximal activity was detected in the temperature range from 40 to 60°C.
5.3. Determination of pH dependency
For testing the pH dependency of strain A5 α-amylase activity, enzyme samples were assayed as described in Example 2.2 with the exception that a Britton-Robinson buffer containing 10% w/v NaCI was applied.
The pH optimum for α-amylase activity was around pH 7.5 with approx. 25 % of this activity remaining at pH 10.0 and only very low activity remaining at pH 11.
B. GRAM-POSITIVE XYLANOLYTIC AND MANNANOLYTIC ACTIVE HALOTOLERANT BACTERIA
EXAMPLE 6
Cultivation of a gram-positive halotolerant and halophilic bacterium producing β- xylanase, β-xylosidase, β-mannanase and β-mannosidase
6.1 . Cultivation of strain NN
For β-xylanase and β-xylosidase production, strain NN was grown in TRIS 11 medium containing 10% (w/v) NaCI as described in Example 1.6. For β-mannanase and β- mannosidase production the strain was grown in the same medium, except that birchwood xylan was replaced by the same amount of locust bean gum (Sigma).
Strain NN was deposited under the Budapest Treaty on 7 January 1999 with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Braunschweig, Germany) under the accession No. DSM 12617.
6.2. Phylogenetic characterisation of strain NN
Strain NN grew in TRIS 11 medium containing 0 to 20 % (w/v) NaCI. Phenotypic, physiologic, biochemical, chemotaxonomic data and 16S rRNA sequence analysis suggest that strain NN belongs to Bacteria within the family Bacilliaceae and is a novel species within a novel genus for which the name Gracilibacillus halotolerans has been proposed (Wainø et al., 1999, International Journal of Systematic Bacteriology, vol. 49 pp 821-831 ).
EXAMPLE 7
Production and assay of β-mannanase, β-mannosidase, β-xylanase and β- xyiosidase from strain NN
7.1 . Production and assay of β-mannanase and β-mannosidase
β-mannanase and β-mannosidase was produced by cultivating strain NN aerobically in 500 ml Erlenmeyer flasks containing 100 ml TRIS 11 medium and locust bean gum as carbon substrate at 30°C for approx. 50 h with shaking (180 rpm.).
The enzymatic activities were assayed using either culture broth, washed cells or a dialysate obtained by the following procedure; cell-free culture supernatant was prepared by centrifugation of culture broth for 3 min. at 11.000*g and subsequently dialysed overnight against either 10 mM NaP buffer (0.05% NaCI, pH 7.0) or TRIS 11 medium (devoid of C sources, pH 7.0) at 4°C.
The following assays for β-mannanase (Mannan endo-1 ,4-β-mannanase, EC 3.2.1.78) activity were used:
The effect of NaCI on β-mannanase activity was assayed by incubation of 1 ml 0.25 % (w/v) AZCL-galactomannan (Megazyme, Ireland) in 50 mM NaP buffer (containing different amounts of NaCI, pH 7.0) and 0.25 mi dialysate (10 mM NaP buffer, 0.05% NaCI, pH 7.0) in a thermomixer at 30°C for 3 h. The effect of NaCI on β-xylanase stability was tested by incubation of 1 ml 0.25 % (w/v) AZCL-galactomannan (Megazyme, Ireland) in 50 mM NaP buffer (25% NaCI, pH 7.0) and 0.25 ml NaP-dialysate (10 mM NaP buffer, 0.05% NaCI, pH 7.0) or TRIS-dialysate (TRIS 11 medium devoid of C sources, pH 7.0) in a thermomixer at 30°C for a period of up to approx. 45 h. The effects of temperature and pH on β-mannanase activities were likewise investigated at 10% (w/v) NaCI by incubating cell-free culture supernatant at different temperatures. pH-activity profiles were determined in Britton-Robinson buffer. The thermostability of the β-mannanase activity was determined by preincubation of cell-free culture supernatant for up to 24 h at 60, 65, and 70°C (pH 7.0, 10% (w/v) NaCI) and subsequently assaying at 10% (w/v) NaCI. Centrifugation of the reaction mixtures at 11.000*g for 3 min. was followed by spectrophotometric measurement of the dye-release from AZCI-galactomannan at 595 nm.
The following assays for β-mannosidase (EC 3.2.1.25) activity were used:
The effect of NaCI on β-mannosidase activity was assayed by incubation of 0.9 ml 0.56 mM p-nitrophenyl-β-D-mannopyranoside (Sigma) in 10 mM NaP buffer (containing differ- ent amounts of NaCI, pH 7.0) and 0.1 ml culture broth in a thermomixer at 30°C for 2 h. The effect of NaCI on β-mannosidase stability was tested using culture broth or washed cells suspended in 10 mM NaP buffer (pH 7.0) in the above-mentioned assay with incubation times of up to about 45 h. The effects of temperature and pH on β-mannosidase activities were investigated at 10% NaCI by incubating culture broth samples as described above at different temperatures or in Britton-Robinson buffer, respectively. Centrifugation of the reaction mixtures at 11.000χg for 3 min. was followed by spectrophotometric measurement of the liberation of p-nitrophenol at 405 nm.
7.2. Production and assay of β-xylanase and β-xylosidase
β-xylanase and β-xylosidase was produced by cultivating strain NN aerobically in 500 ml Erlenmeyer flasks containing 100 ml TRIS 11 medium at 30°C for approx. 50 h with shaking (180 rpm.).
β-xylanase activity was determined using either culture supernatant or dialysate. Supernatant was prepared by centrifugation of culture broth for 3 min. at 11.000*g, while dialysate was made by dialyzing the supernatant for approx. 12 h against a 10 mM sodium phosphate (NaP) buffer (10% NaCI, pH 7.0) at 4°C. β-xylosidase activity was assayed as described in Example 2.1.
The effects of NaCI on β-xylanase activity and stability were tested as described in Example 2.1 The effects of temperature and pH on β-xylanase activities were investigated as described in example 7.1 for β-mannanase assay, though AZCI-galactomannan was replaced with AZCI-xylan.
β-xylosidase activity was assayed as described in Example 2.1.
EXAMPLE 8
Characterisation of β-mannanase and β-mannosidase from strain NN
8.1 . Effect of salinity on β-mannanase and β-mannosidase activity
The effects of NaCI on β-mannanase and β-mannosidase activities were tested using dialysate and culture broth, respectively. As illustrated in Fig. 3, β-mannanase and β- mannosidase activities were at a maximum at 1 % and 0.5% NaCI, respectively and de- creased with increasing salinity. However, 3% of maximum β-mannanase activity and 13% of maximum β-mannosidase activity remained at 20% NaCI, implying significant ha- lotolerance.
8.2. Halostability of β-mannanase and β-mannosidase
The halostability of β-mannanase was investigated using supernatant dialysed against either NaP buffer or TRIS 11 medium (devoid of C sources) while halostability of β- mannosidase was tested using either culture broth or washed cells suspended in NaP buffer. As can be seen from Fig. 4, β-mannosidase activity was extremely halostable, since no loss of activity was observed after approx. 45 h of incubation at 20% NaCI. The β-mannanase activity was also very halostable, with approximately 50 % of the initial activity remaining after incubation for about 45 h at 20% NaCI. β-mannanase activity showed slightly lower halostability when dialysed against TRIS 11 medium. Nevertheless, when assayed at 10 % NaCI, β-mannanase activities determined in TRIS 1 1 medium were about twice as high as those obtained with NaP buffer (data not shown).
8.3. Effect of temperature on β-mannanase and β-mannosidase activity
The β-mannanase exhibited activity between 20 and 80°C with optimum at 70°C, while β- mannosidase activity existed between 15 and 50°C with optimum at 25°C.
8.4. Effect of pH on β-mannanase and β-mannosidase activity
The β-mannanase exhibited activity in the pH range 4.0 to pH 10.2; optimal activity at pH 7.7. The β-mannosidase was found active between pH 5.2 to pH 9.5 with optimal activity at pH 6.9.
8.5. Thermostability of β-mannanase
The β-mannanase activity was stable at 60°C for at least 24 h, whilst 70% of the initial ac- tivity remained after incubation for 8 h and 1 h at 65°C and 70°C, respectively.
EXAMPLE 9
Characterisation of β-xylanase and β-xylosidase from strain NN
9.1 . Effect of salinity on β-xylanase activity
To investigate the effect of salinity on β-xylanase from strain NN, cell-free supernatant from cultures grown in 10% NaCI were incubated at different salinities. The results are summarised in Fig. 5.
As can be seen from Fig. 5, β-xylanase activity was maximal at 2% NaCI and decreased with increasing salinity. Approx. 5% of the maximum activity was retained at 28% NaCI.
9.2. Halostability of β-xylanase
In order to test the stability of the β-xylanase from strain NN when exposed to different salinities, an assay was performed using a dialysate of a culture grown in a medium containing 10% NaCI. The dialysate was incubated for a period of 24 h at NaCI concentra- tions of 2, 10, or 28%, respectively.
The results revealed that the β-xylanase activity of strain NN was stable at all salinities. Less than 45% of the initial activity was lost during the 24 hour incubation period irrespective of the salinity tested and approx. 5% of the activity measured at 2% NaCI was present in the assay at 28% NaCI.
9.3. Effect of temperature on β-xylanase activity
The β-xylanase exhibited activity between 20 and 80°C with optimum at 70°C.
9.4. Effect of pH on β-xylanase activity
The β-xylanase exhibited activity at a range of pH 4.0 to pH 9.6; optimal activity at pH 8.0.
9.5. Presence of β-xylosidase activity
Presence of β-xylosidase activity was investigated using intact cells grown in 10% NaCI. β-xylosidase activity was detected at 10% NaCI.
C. XYLANOLYTIC ACTIVE HALOPHILIC ARCHAEA
EXAMPLE 10
Cultivation of a halophilic Archaea producing β-xylanase, β-xylosidase, β- mannannase and β-amylase
10.1 . Cultivation of strain AX-2 and LBG-1
Strain AX-2 was cultivated at 30°C in TRIS 10 medium containing in grams per litre of demineralised water: NaCI, 270.0; MgSO4-7H20, 20.0; KCI, 5.0; NH4CI, 2.0; NaBr, 0.1 ; yeast extract (Difco), 1.0; Tris-HCI, 12.0; birchwood xylan (Roth, Karlsruhe - Germany), 2.0; trace metal solution (TMS 3) (Ingvorsen & Jørgensen, 1984), 2 ml. The pH was adjusted to 7.8. After sterilisation and cooling to 5°C, 2.5 ml of a sterile phosphate solution (KH2P04, 50 g/l), 0.5 ml of a sterile CaCI2 solution (CaCI2-2H2O, 100 g/l), and 0.5 ml of a sterile FeCI2/MnCI2 solution (FeCI2-4H2O, 20 g/l + MnCI2-4H2O, 20 g/l) were added. The final pH of the medium was approx. 7.6.
Strain AX-2 was deposited under the Budapest Treaty on 7 January 1999 with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Braunschweig, Germany) under the accession No. DSM 12616.
Strain LBG-1 was cultivated at 30°C in TRIS 10 medium as described above, though birchwood xylan was replaced with the same amount of locust bean gum (Sigma).
10.2. Phylogenetic characterisation of strain AX-2 and strain LBG-1 5
Strain AX-2 grew in TRIS 11 medium containing from 10 to 35 % (w/v) NaCI. Phenotypic, physiologic, biochemical, chemotaxonomic data and16S rRNA sequence analysis suggest that strain AX-2 belongs to Archaea within the family Halobacteriaceae and is a novel species within a novel genus for which the name Halorhabdus utahensis has been pro- 0 posed (Wainø et al., 2000, International Journal of Systematic and Evolutionary Microbiology, vol 50, pp 183-190).
A preliminary characterisation of strain LBG-1 was carried out using 16S rDNA analysis. Approximately 75% of the 16S rDNA sequence was determined and compared to known 5 extremely halophilic archaea which revealed 99.0% similarity to Haloferax mediterranei. Strain LBG-1 is therefore considered a member of the genus Haloferax and most likely represent another Haloferax mediterranei strain consistent with phenotypic features such as pleomorphic cell morphology and pinkish, mucoid colony morphology.
20 EXAMPLE 1 1
Production and assay of β-xylanase and β-xylosidase from strain AX-2
In order to produce β-xylanase and β-xylosidase, strain AX-2 was grown aerobically in 25 500 ml Erienmeyer flasks containing 100 ml TRIS 10 medium (see Example 10.1) at 30°C for approx. 120 h with shaking (180 rpm.).
β-xyianase activity was determined using dialysate. Supernatant was prepared by centrifugation of culture broth for 3 min. at 11.000*g, and subsequently dialysed overnight 30 against a 10 mM NaP buffer (0.5% NaCI, pH 7.0) at 4°C. β-xylosidase activity was assayed using culture broth.
The following assay for β-xylanase (endo-1 ,4-β-D-xylan xylanohydrolase, EC 3.2.1.8) activity was used:
The effect of NaCI on β-xylanase activity was assayed by incubation of 0 9 ml 0 11% (w/v) AZCL-xylan (Megazyme, Ireland) in 10 mM NaP buffer (containing different amounts of NaCI, 1% MgSO4 7H2O, pH 7 0) and 0 1 ml dialysate in a thermomixer at 30°C for 2 h The effect of NaCI on β-xylanase stability was tested likewise by incubation at a total NaCI concentration of 0 05 or 27% (w/v) for a period of up to about 21 h The effects of temperature and pH on β-xylanase activities were investigated as described above by assaying cell-free culture supernatants at different temperatures or in Britton-Robinson buffer at 20% (w/v) or 10% (w/v) NaCI, respectively The thermostability of the β-xylanase activity was tested by incubating supernatants at 50, 55, and 60°C in the presence of 20% NaCI for 24 h Centrifugation of the reaction mixtures at 11 000*g for 3 mm was followed by spectrophotometric measurement of the dye-release from AZCI-xylan at 595 nm
The following assay for β-xylosidase (endo-1 ,4-β-D-xylan xylanohydrolase, EC 3 2 1 8) activity was used
β-xylosidase activity was determined using a reaction mixture containing 0 98 ml 0 51 mM p-nitrophenyl-β-D-xylopyranoside in 10 mM NaP-buffer (varying amounts of NaCI, 1% MgSO4 7H2O, pH 7 0) and 0 02 ml culture broth The reaction mixture was incubated in thermomixer at 30°C for 1 h The effects of temperature and pH on β-xylosidase activities were investigated as described above by assaying cell-free culture supernatants at different temperatures or in Britton-Robinson buffer at 20% (w/v) or 10% (w/v) NaCI, respectively Measurement of β-xyiosidase activity was done by centrifugation of the reaction mixture at 11 000*g for 3 mm , immediately followed by spectrophotometric monitoring of the released p-nitrophenol at 405 nm
EXAMPLE 1 2
Characterisation of β-xylanase and β-xylosidase from strain AX-2
12.1 . Effect of NaCI concentration on β-xylanase and β-xylosidase activity from strain AX-2
In order to investigate the effect of salinity on β-xylanase and β-xylosidase activity from strain AX-2, dialysed enzyme preparation of culture supernatant and culture broth, re-
spectively, were incubated at different salinities The results of this experiment are summarised in Fig 6
As can be seen from Fig 6, the β-xylanase and β-xylosidase exhibited activities over a very broad salinity range Both enzyme activities were present at all NaCI concentrations tested The β-xylanase had highest activity at 15% NaCI although a peak was also seen at about 5% NaCI The β-xylanase activity retained as much as 61 % and 49% of its maximum activity at 0 05% and 27% NaCI, respectively When using supernatant which had not been dialysed as the enzyme solution, about 32% of maximum activity remained at 30% NaCI (data not shown)
The β-xylosidase also retained high activities at the extremes of the salinity range tested Optimum activity was found at 5% NaCI, with 47% and 45% of maximum activity remaining at 0 5% and 30% NaCI, respectively
12.2. Halostability of β-xylanase and β-xylosidase
In order to test the stability of the β-xylanase and β-xylosidase from strain AX-2 enzyme samples were exposed to different salinities at 30°C for up to 24 h The results of this ex- penment are summarised in Fig 7
As can be seen from Fig 7, β-xylosidase activity was the most stable with 83% of initial activity remaining after 24 h at both 0 5% NaCI or 25% NaCI The β-xylanase retained 55 and 51% of the initial activity after about 21 h of incubation at 0 05% NaCI or 27% NaCI, respectively
12.3. Effect of temperature on β-xylanase and β-xylosidase activity
β-xylanase and β-xylosidase activities could be detected at temperatures up to 75°C The β-xylosidase exhibited optimum activity at 65°C, while the β-xylanase activity showed two peaks of optimum activity at 55°C and 70°C, respectively
12.4. Effect of pH on β-xylanase and β-xylosidase activity
The β-xylosidase was active over a broad pH range from pH 5 2 to pH 10 0 Optimum activity was at pH 7 6 with about 10% of this activity remaining at pH 10 β-xylanase was present between pH 5 1 to 9 0 with optimum at pH 6 9 There was no β-xylanase activity above pH 9 0, but 35% of maximum activity remained at pH 5 1
1 2.5. Thermostability of β-xylanase
The β-xylanase retained 100% activity after 8 h of incubation at 50°C, and 75% residual activity remained after 24 h After 8 h at 55°C only 16% of the initial β-xylanase activity was present, and after 1/2 h at 60°C approx 32% was remaining
EXAMPLE 13
Detection of β-xylanase, β-mannanase and β-amyiase from strain LBG-1
Assays were performed at 20% NaCI as described in Example 11 using whole culture broth as enzyme source and AZCI-xylan, AZCI-galactomannan, and AZCI-amylose to de- tect β-xylanase, β-mannanase and β-amylase activities, respectively Strain LBG-1 was found positive for all three enzyme activities
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
A. The indications made below relate to the microorganism referred to in the description on page 14 .line _~__
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | X |
Name of depositary institution DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
Address of depositary institution (including postal code and count )
Mascheroder eg IB D-38124 Braunschweig Germany
Date of deposit Accession Number
7 January 1999 DSM 12620
C ADDITIONAL. INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet ]
As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if Ac indications are not for all designated States)
E. SEPARATE FURNISHING o lNDICATIONS (leave blank if not appRcable)
The indications listed belowwillbesubmitted lo the International Bureau hlcτ(speάfylhegeneralnatureoflheindicationse.g^ 'Accession Number of Deposit")
For International Bureau use only
I j This sheet was received by the International Bureau on:
Authorized officer
INDICATIONS RELATING TO DEPOSITED MICROORGANISMS
(PCT Rule 12bis)
Additional sheet
In addition to the microorganism indicated on page 14 of the description, the following microorganisms have been deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
Mascheroder Weg 1 B D-38124 Braunschweig Germany on the dates and under the accession numbers as stated below:
Accession Date of Description Description number deposit Page No. Line No.
DSM 12618 7 January 1999 14 1-4
DSM 12619 7 January 1999 14 1-4
DSM 12617 7 January 1999 22 20-22 DSM 12616 7 January 1999 27 32-34
For all of the above-identified deposited microorganisms, the following additional indications apply:
As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.