TECHNICAL FIELD

This invention relates to novel enzymes. More specifically, the invention provides novel xylanases obtainable from microorganisms of the genus Thermotoga. The invention also relates to a process the for preparation of these xylanases, an agent containing these xylanases, and the use of these xylanases for treatment of lignocelluiosic pulp.

BACKGROUND ART

Xylan, a major component of plant hemicellulose, is a polymer of D- xylose linked by β-Λ ,4-xylosidic bonds. Xylan can be degraded to xylose and xylo- oligomers by acid or enzymatic hydrolysis. Enzymatic hydrolysis of xylan produces free sugars without the by-products formed with acid (e.g. furans).

The pulp and paper industry is using xylanase compositions in the bleaching process to enhance the brightness of bleached pulps, to decrease the amount of bleaching chemicals, e.g. chlorine, used in the bleaching stages, and to increase the freeness of pulps in the recycled paper process [Eriksson K.E.L. (1990), Wood Science and Technology 2479 -101.; Paice M.G., BernierR., and Jurasek L (1988), Biotechnol. and Bioeng., 32 235 - 239.; Pommier J.C., Fuentes J.L., and Goma G. (1989), Tappi Journal, 187 - 191.] Kraft pulping, a process widely used in the pulp and paper industry, involves the alkaline sulfate cooking of pulp to remove most of the lignin. The remaining pulp contains 2 - 5% of lignin, which gives the pulp a dark brown colour that has the tendency to darken in UV light or by oxidation. In order to obtain a white pulp for high quality paper, the brown colour is removed by a multi-stage bleaching process using bleaching chemicals, e.g. oxygen, ozone, hydrogenperoxide, chlorine and/or chlorine dioxide.

Presently, there is much concern about the environmental impact of the toxic compounds generated from the bleaching process. Enzymes can aid in the removal

of lignin from the pulp without any harmful side products. Reports show that lignin in wood is linked to xylan [Eriksson 0. et al. (1980), Wood Sci. Technol., 14 267.; TakashiN., and Koshijiima T. (1988), Wood Sci. Technol., 22177 - 189]. By a limited hydrolysis of the xylan a greater release of lignin occurs during bleaching. Thus, by

5 enzymatic treatment of the pulp prior to bleaching the amount of chemicals needed would in turn decrease [Viikari L et al. (1986), Proceedings of the 3rd International Symposium on Biotechnology in the Pulp and Paper Industry, 67].

Characteristic of the above processes is the need for xylanases capable of exerting hydrolytic activity at high temperatures and at alkaline conditions. o The xylanase should exert a substantial amount of its activity at pH values above pH 7.

Recently a new thermostable xylanase has been disclosed, vide Simpson H.D.. Haufler U.R, and Daniel R.M. (1991), Biochem J., 277(2) 413 - 418. The xylanase is obtained from the strain Thermotoga sp. FJSS3-B.1 , a strain collected s at Fiji, but, however, not available to the public [HuserBΛ.. Patel B.K.C. Daniel R.M. and Morgan H.W. (1986), FEMS Mikrobiol. Lett., 37 121 - 127]. The enzyme has a pH optimum at 5.4, with 50% of activity limits at pH 4.2 and 6.7, respectively.

SUMMARY OF THE INVENTION

We have now found that three species of the genus Thermotoga, T. o maritima, T. neapolitana, and T. thermarum, are able to produce novel thermostable xylanases, being improved in respect to activity at alkaline pH values. T. maritima and T. neapolitana are marine organisms, collected near Italy. T. thermarum is not a marine organism, and it is collected in Africa.

Three strains representative of T. maritima, T. neapolitana, and T. 5 thermarum, respectively, have been deposited as type cultures and hence are publicly available from Deutsche Sammlung von Mirkoorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-3300 Braunschweig, Germany. The strains and accession numbers are T. maritima, DSM 3109, T. neapolitana, DSM 5068, and T. thermarum, DSM 5069.

Due to their improved activity at alkaline pH, the novel enzymes of this invention are especially well suited for use in the manufacture of paper pulp.

Accordingly, in its first aspect, the invention provides xylanases having more than 50% of residual activity in the a pH range 4.0 - 7.5, more preferred the pH 5 range 4.5 - 7.5, most preferred the pH range 5.5 - 7.5, when determined after 20 minutes at 90°C, more than 50% of residual activity in a pH range 4.5 - 8.0, more preferred pH range pH 4.5 - 7.5, most preferred the pH range 5.5 - 7.5, when determined after 20 minutes at 70°C, a temperature optimum in the range 80°C - 100°C, more preferred the range 85°C - 95°C, when determined after 20 minutes at o pH 6.0, and being obtainable from a strain of T. maritima, T. neapolitana, or T. thermarum.

In a more specific aspect, the invention provides xylanases having pH optimum in the pH range 5.5 - 6.5, around pH 6.0, when determined at 90°C, at least

50% of relative residual activity at pH 7.5, when determined at 90°C, at least 50% of s relative residual activity at pH 8.0, when determined at 70°C, and being obtainable from a strain of T. maritima, T. neapolitana, or T. thermarum.

In its second aspect, the invention provides a process for the preparation of the xylanase comprising cultivation of a xylanase producing strain of

T. maritima, T. neapolitana, or 7. thermarum, in a suitable nutrient medium, 0 containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.

In its third aspect, the invention provides an agent containing the xylanase, provided in the form of a granulate, preferably a non-dusting granulate, a liquid, in particular a stabilized liquid, a slurry, or a protected enzyme. 5 In its fourth aspect, the invention relates to a process for treatment of lignocellulosic pulp, in which the lignocellulosic pulp is treated with an enzyme of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to the o accompanying drawings, in which:

Fig. 1A shows the relation between temperature and residual activity (% relative) of the xylanase obtained from T. maritima, determined after 20 minutes at pH 6.0;

Fig. 1 B shows the relation between pH and residual activity (% relative) of the xylanase obtained from T. maritima, determined after 20 minutes at 90°C;

Fig 2A shows the relation between temperature and residual activity (% relative) of the xylanase obtained from T. neapolitana, determined after 20 minutes at pH 6.0;

Fig.2B shows the relation between pH and residual activity (% relative) of the xylanase obtained from T. neapolitana, determined after 20 minutes at 90°C;

Fig 3A shows the relation between temperature and residual activity (% relative) of the xylanase obtained from T. thermarum, determined after 20 minutes at pH 6.0;

Fig.3B shows the relation between pH and residual activity (% relative) of the xylanase obtained from T. thermarum, determined after 20 minutes at 90°C;

Fig. 4A shows the relation between pH and residual activity (% relative) of the xylanase obtained from T. neapolitana, determined after 20 minutes at 70°C; and

Fig. 4B shows the relation between pH and residual activity (% relative) of the xylanase obtained from 7. thermarum, determined after 20 minutes at 70°C.

DETAILED DISCLOSURE OF THE INVENTION

The xylanases of this invention are obtainable from and may be produced by cultivation of a strain of T. maritima, T. neapolitana, or T. thermarum, preferably the strain T. maritima, DSM 3109, T. neapolitana, DSM 5068, or T. thermarum, DSM 5069, or mutants or variants thereof, in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme. The enzyme may also be obtained by recombinant DNA- technology.

When determined after 20 minutes at 90°C, the xylanases obtainable from T. neapolitana and T. thermarum have xylanolytic activity in the range of from

approximately pH 4 to approximately pH 10. Optimum pH for activity was detected in the pH range 5.0 - 7.5, more specifically the pH range 5.5 - 6.5, around pH 6.0, as presented in Figs. 2B and 3B. 7. neapolitana showed 50% of relative activity at pH 4.5 and pH 7.5, respectively. T. thermarum showed 50% of relative activity at pH 5.5 and pH 7.5, respectively.

When determined after 20 minutes at 90°C, the xylanase obtainable from T. maritima has xylanolytic activity from pH below 4 to pH approximately 11.

Optimum pH for activity was detected in the pH range 4.0 - 7.0, more specifically the pH range 4.5 - 6.5, around pH 5.0, as presented in Fig. 1 B. T. maritima has 50% of relative activity at pH below 4.0 and at pH 7.5, respectively.

When determined after 20 minutes at 70°C, T. neapolitana and T. thermarum have xylanolytic activity in the pH range from below pH 4 to above pH

11. Optimum pH for activity was detected in the pH range 5.0 - 7.5, more specifically the pH range 5.5 - 6.5, around pH 6.0, as presented in Figs. 4A and 4B. The xylanases both showed 50% of relative activity at pH 4.5 and pH 8.5, respectively.

The xylanases obtainable from T. neapolitana and T. thermarum have xylanolytic activity in the range of from below 20°C to above 115°C, as presented in Figs. 2A and 3A.

The xylanase obtainable from 7. maritima has xylanolytic activity from below 60°C to above 100°C, as presented in Fig. 1A.

All xylanases have temperature optimum in the temperature range 80°C - 100°C, more specifically 85°C - 95°C, around 90°C, when determined after 20 minutes at pH 6.0.

Industrial Applications Due to the excellent residual activity at alkaline pH values, the enzymes of this invention are well suited for treatment of lignocellulosic pulp, in order to improve delignification.

Due to its temperature stability, the enzyme of the invention may also be applied in a complexing stage of the pulp process, prior to hydrogen peroxide or ozone bleaching.

Therefore, in a further aspect, the invention relates to the use of the xylanases for delignification of lignocellulosic pulp.

Enzymatic treatment of lignocellulosic pulp improves the bleachability of the pulp and/or reduces the amount of chemicals necessary for obtaining a satisfactory bleaching.

For use of a xylanase of the invention for delignification of lignocellulosic pulp, the xylanase should preferably be provided in the form of a granulate, preferably a non-dusting granulate, a liquid, in particular a stabilized liquid, a slurry, or a protected enzyme. In a further preferred embodiment, the agent contains the xylanase in amounts of at least 20%, preferably at least 30%, of the total enzyme protein.

The xylanolytic activity can be measured in xylanase units. In this specification two kinds of units are used: FXU and EXU. By an analytical method a xylanase sample is incubated with remazol-xylan substrate. The background of non- degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzyme standard at standard reaction conditions.

This analytical method and standard reaction conditions are described in two folders, AF 293.6/1 (FXU) and AF 293.9/1 (EXU). FXU is determined at pH 6.0, and EXU is determined at pH 9.0. However, FXU and EXU express enzymatic activity in the same order of magnitude. The folders AF 293.6/1 and 293.9/1 are available upon request to Novo Nordisk A/S, Denmark, which folders are hereby included by reference.

A suitable xylanase dosage will usually correspond to a xylanase activity of 10 to 5000 FXU/kg or EXU/kg dry pulp, more preferred 100 to 5000 FXU/kg or EXU/kg dry pulp.

In many applications pH should be above pH 7.0 in order to prevent corrosion problems. In a preferred embodiment of the process of the invention, the enzymatic treatment is performed at a pH above 7.0, preferably above pH 8.0, more preferred above pH 9.0.

In another embodiment of the process of the invention, the enzymatic treatment is performed at temperatures between 50 and 100°C, preferably between 60 and 95°C, more preferred between 70 and 90°C.

In yet another preferred embodiment of the process of the invention, the enzymatic treatment is performed within a period of 5 minutes to 24 hours, preferably within a period of 15 minutes to 6 hours, more preferred within a period of 20 minutes to 3 hours.

In a further preferred embodiment of the process of the invention, the enzymatic treatment takes place at a consistency of 3 - 35%, preferably 5 - 25%, more preferred 8 -15%. The consistency is the dry matter content of the pulp. A pulp with a consistency above 35% is difficult to mix effectively with the enzyme preparation, and a pulp with a consistency below 3% carries too much water, which is a disadvantage from an economic point of view.

In several other preferred embodiments, the xylanases of this invention can be implemented in processes for treatment of lignocellulosic pulp essentially as described in e.g. International Patent Application PCT/DK91/00239, or International Patent Publication WO 91/02839.

The following examples further illustrate the present invention, and they are not intended to be in any way limiting to the scope of the invention as claimed.

EXAMPLE 1

Cultivation Example

Each of the strains, 7. maritima, DSM 3109, and 7. neapolitana, DSM 5068, respectively, were cultivated anaerobically at 80°C, using a 1 -litre glass fermentor under continuous gassing with N ₂ and constant pH 6.5 in a medium of the following composition (per litre):

Xylan 5.0 g

Yeast extract (Difco™) 0.5 g

KH ₂ P0 ₄ 0.5 g

NiCI ₂ ;6H ₂ 0 2.0 mg

NaCI 20.0 g

Sea salts (Sigma™) 12.5 g

Resazurin 1.0 mg

Na ₂ S;9H ₂ 0 0.5 g

Trace element solution ^* ' 15.0 ml

Distilled water pH 6.5

^" > See below

The strain, T. thermarum, DSM 5069, was cultivated anaerobically at 1 -litre glass fermentor under continuous gassing with N ₂ and constant edium of the following composition (per litre):

Xylan 5.0 g

Yeast extract (Difco™) 0.5 g

KH ₂ P0 ₄ 0.5 g

Trace element solution ^"1 15.0 ml

(NH ₄ ) ₂ Ni(S0 ₄ ) ₂ 3.0 mg

NaCI 3.46 g

MgS0 ₄ ;7H ₂ 0 0.88 g

MgCI ₂ ;6H ₂ 0 0.69 g

CaCI ₂ ;2H ₂ 0 0.14 g

KCI 0.08 g

NaBr 12.5 mg

H ₃ B0 ₃ 3.75 mg

SrCI ₂ ;6H ₂ 0 1.9 mg

Kl 0.006 mg

EDTA.Na ₂ 0.768 g

Resazurin 1.0 mg a^θ^O 0.5 g

Distilled water pH 7.0

^* * Trace element solution:

Nitriloacetic acid 1.5 g

MgS0 ₄ ;7H ₂ 0 3.0 g

MgS0 ₄ ;2H ₂ 0 0.5 g NaCI 1.0 g

FeS0 ₄ ;7H ₂ 0 0.1 g

CoS0 ₄ ;7H ₂ 0 0.18 g

CaCI ₂ ;2H ₂ 0 0.1 g

ZnS0 ₄ ;7H ₂ 0 0.18 g CuS0 ₄ ;5H ₂ 0 0.01 g

KAI(S0 ₄ ) ₂ ;12H ₂ 0 0.02 g

H ₃ B0 ₃ 0.01 g

Na ₂ Mo0 ₄ ;2H ₂ 0 0.01 g

NiCI ₂ ;6H ₂ 0 0.025 g Na ₂ Se0 ₃ ;5H ₂ 0 0.3 mg

Distilled water 1000.0 ml

First dissolve nitriloacetic acid and adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH).

The extracellular enzyme system was harvested at the late- exponential/early stationary phase of growth.

The supernatant was concentrated to 50 ml and used for further characterization.

EXAMPLE 2

Characterization Example Xylanase Activity

Xylanase is determined by assaying for reducing sugars released from oat spelt xylan (XU-method).

The assay is performed on using 0.5% of oat spelt xylan (Sigma-X- 0627) prepared in 40 mM Britton & Robinson buffer as substrate, heat treated 30 minutes at 100°C before use, and adjusted to the desired pH.

The assay is performed with 0.100 ml of enzyme solution and 0.100 ml 5 of substrate, both preheated to the desired temperature. The mixture is incubated for 20 minutes at the desired pH. Then 0.200 ml solution I (35.1 g Na ₂ HP0 ₄ ;2H ₂ 0; 40.0 g KNaC ₄ H ₄ 0 _β ;4H ₂ 0, suspended in 500 ml deionized H ₂ 0 add 110 ml 1N NaOH; 8.0 g CuS0 ₄ ,5H ₂ o; 180 g Na^O^ add deionized to a total volume of 1 litre) is added, and the solution is heated to 100°C for 20 minutes. ιo 0.200 ml solution II (50 g (NH ₄ ) ₆ Mo ₇ 0 ₂₄ ; ⁴ H>0 suspended in 900 ml deionized H ₂ 0; 42 ml Concentrated H ₂ S0 ₄ ; 6.0 g Na ₂ HAs0 ₄ ;7H ₂ 0 add deionized water to a total volume of 1 litre) is added. 2.0 ml deionized water are added, and the absorbance measured on a spectrophotometer (PYE UniCAM PU8600UV/Vls, Phillips) at 520 nm. is The reducing sugars are calculated from a standard curve prepared with xylose (40 - 400 μg/ml). One XU is equivalent to 1 nmol xylose released per second per millilitre or per gram of culture broth.

pH Activity

In a first test, the pH related activity of the enzymes was determined at 20 90°C in a pH range 4.0 - 11.0, using soluble xylan (Roth) in 40 mM Britton & Robinson buffer.

In this test, the xylanases obtained from 7. neapolitana and T. thermarum showed xylanolytic activity in the range of from approximately pH 4 to approximately pH 10. Optimum pH for activity was detected in the pH range 5.0 - 25 7.5, more specifically the pH range 5.5 - 6.5, around pH 6.0, as presented in Figs. 2B and 3B. 7. neapolitana showed 50% of relative activity at pH 4.5 and pH 7.5, respectively. T. thermarum showed 50% of relative activity at pH 5.5 and pH 7.5, respectively.

The xylanase obtained from 7. maritima showed xylanolytic activity from

30 pH below 4 to pH approximately 11. Optimum pH for activity was detected in the pH range 4.0 - 7.0, more specifically the pH range 4.5 - 6.5, around pH 5.0, as

presented in Fig. 1 B. 7. maritima has 50% of relative activity at pH below 4.0 and at pH 7.5, respectively.

In a second test, the pH related activity of the xylanases obtained from 7. neapolitana and 7. thermarum was determined at 7G°C in a pH range 4.0 - 11.0, using soluble xylan (Roth) in 40 mM Britton & Robinson buffer.

In this test, the xylanases both showed xylanolytic activity in the range of from below pH 4 to above pH 11. Optimum pH for activity was detected in the pH range 5.0 - 7.5, more specifically the pH range 5.5 - 6.5, around pH 6.0, as presented in Figs. 4A and 4B. The xylanases both showed 50% of relative activity at pH 4.5 and pH 8.5, respectively.

Temperature Activity

In a first test, the temperature related activity of the enzymes was determined using soluble xylan (Roth) in a 40 mM Britton & Robinson buffer, pH 6.0.

The xylanases obtained from 7. neapolitana and T. thermarum showed xylanolytic activity in the range of from below 20°C to above 115°C, as presented in Figs. 2A and 3A.

The xylanase obtained from 7. maritima showed xylanolytic activity from below 60°C to above 100°C, as presented in Fig. 1A.

All of the xylanases showed temperature optimum in the temperature range 80°C - 100°C, more specifically of from 85°C to 95°C, around 90°C.

Substrate Specificity

The substrate specificity was determined using the substrates listed in Table 1 below, and using the XU-Method described above.

Table 1

Substrate Specificity

Substrate (Xylan 0.5%) Residual Activity (%) T.maritima T.neapolitana T.thermarum

¹⁾ Not determined ² » Hydroxyethylcellulose

It appears from the table that the effects on the various xylans of the three enzymes of the invention are almost identical.