DE19531944A1 - New endo-xylanase enzymes - Google Patents

Abstract

New endo-xylanases consist of a xylanase-active catalytic domain (B), opt. a N-terminal thermostability-mediating domain (A) and opt. a C-terminal cellulose-binding domain (C).

Description

The invention relates to new extreme thermo-xylanases stability. Fields of application of the invention are inter alia. the Enzyme, pulp, paper and animal feed Industry.

Microbial cellulases and xylanases have been used for grafting vegetable, d. H. renewable raw materials naturally great importance: the structural polysaccharides cellulose and Hemicellulose (xylan is its main component) 50% of the total plant biomass and are therefore the the most common organic molecules on earth. There are already numerous possible uses for o. g. known microbial enzymes:

• - Enzymatic treatment (β-glucanases, xylanases) of vegetable feed (digestibility, improved efficiency)
  Enzyme preparations are increasingly being used to improve the accessibility of hexosans and pentosans for feed purposes from grains and processing residues of vegetable raw materials. These are added to the feed and develop their polysaccharide-splitting effect directly in the intestinal tract of the animals. This results in an advantageous change in both the chemical composition (increasing content of monosaccharides and thus better feed value) and the physical properties (lower viscosity and thus better digestibility) of the intestinal contents.
• - Improvement of the ensiling process.
• - Enzymatic saccharification of agricultural and industrial waste products.
• - Xylanase pretreatment of cellulose for papermaking lung (removal of interfering xylan).

Xylanases play due to the high pentosan content in Ge cereals and other vegetable feedstuffs play a major role in improving digestibility and efficiency. They are often deficient in enzyme complexes of fungal origin. The increase in their share in industrial enzyme preparations is a current task. The main goals of the research are on an increase in the specific activity, improved application stability and correspondingly efficient and cost-effective Manufacturing directed. During the increased use of Cellulases due to the low specific activity than The interest in the significantly more efficient xylanases, which are 100-1000 times higher Show activity, steadily increased. Especially the extraction extremely thermostable xylanases could be used for industrial purposes Use mean an immense advantage.

Use of xylanase in pulp production - the current one State of the art

Every year around 56-70 million tons of bleached pulp are produced worldwide manufactured according to the KRAFT process. This is done by alkaline Sodium sulfite boiling obtained a colored raw pulp and this then over five stages with chlorine or chlorine oxide bleached. Dioxins and organo- Chlorine compounds that pollute the environment.

To avoid higher environmental pollution, the insert is micro bieller xylanases are interesting because they bleach pulp promote and thus to the partial use of chlorine-containing Bleach contribute. As a good bleach only at high Temperatures and under alkaline conditions, the xylanases used should ideally be among these Conditions to be effective.

Xylanases from thermophilic organisms are increasingly selective that between 70-90 ° is still relatively good Have stability. Unfortunately, the enzymes only work in a great deal small amount formed, so that only method of recombinant The production of corresponding enzyme preparations is promising seem. This is the cloning of the relevant enzyme genes unavoidable first step for their later efficient Manufacturing. In addition, the be did not write xylanases with regard to their thermostability, because an insert during the cooking process is still out is closed (G. Schulz and W. Hirte: Cellulose- und Hemi cellulose-splitting enzymes. In H. Ruttloff (ed.): Industrielle Enzymes. Behrs Verl. Hamburg. 1994).

The invention aims to provide a complex of new Endo-xylanases with a wide range of properties, tailor-made for each purpose, available too place. The main properties are said to be high thermal stability and specificity. Derived from this is the Object of the invention inter alia in this

• - new endo-xylanases with a temperature optimum of at least 100 ° C, suitable for use at Cooking processes,

and

• - new endo-xylanases with low temperature stability, but with specific degradability for disruptive ones Components in animal nutrition,

to develop.

The object of the invention is achieved according to claim 1, which Subclaims are preferred for each specific one Case variants to be used.

The core of the invention is an enzyme system that consists of 3 domains exists and can be represented schematically as follows:

These domains responsible for the respective properties can be recombined in various ways according to the invention will.

A preferred endo-xylanase is that from Thermotoga neapolitana recovered endo-xylanase A (xynA), which consists of 3264 bp and Coded 1055 amino acids. 340 amino acids belong to the N-terminal domain A, 316 amino acids to the catalytic domain B and 367 amino acids to the C-terminal domain C. Is also suitable another endo-xylanase from Thermotoga neapolitana (xynB), which have strong sequence homology to the catalytic domain B of xynA, but only has a molecular weight of 40 kDa. (corresponds to 350 amino acids) The domains have the following Number of amino acids: A 340 aa, B1 317 aa, B2 317 aa, C 368 AS.

The elimination of the cellulose binding domain (i.e. the combination A-B according to claim 3) leads to an increase in the specific Activity without a drop in thermostability. In the case of xynA the activity is increased 3 times what a more efficient use of the enzyme allowed.

The combination B-C has a lower thermal stability, shows however, there are special activities for this. So in the case of xynA obtained an enzyme that is responsible for the digestion of materials can be used advantageously for animal nutrition (degradation viscous hemicelluloses).

The catalytic domain B alone is sufficient in cases in which pure xylans are broken down under relatively mild conditions should be.

Further combinations according to the invention arise when one the domain A is shortened. Removing a repeat with the With xynA, a size of 168 AS means that the high Thermostability of the enzyme continues to be guaranteed.

The invention is to be described in more detail below by means of exemplary embodiments explained.

1. Subcloning and DNA sequencing

The plasmid pTT17 with a 4.8 kb DNA insert from Thermotoga neapolitana chromosomal DNA encodes activities that xylan, Degradation of lichenan and MUC (Dakhova et al., Biochim Biophys Res Commun 194: 1359-1364 (1993)). The complete nucleotide sequence was determined in both strands, the sequence appears as an ORF of 3264 bp corresponding to 1055 amino acids. The molecular weight of the enzyme derived from DNA was 119,323 Da.

As with other cellulases and xylanases, this has been derived primary translation product of xynA three different Domains A, B and C, which have a signal peptide of 29 amino acids goes ahead. A significant similarity regarding the whole deduced amino acid sequence of the 1055 residues with two representatives of Glycosylhydrolase XynX from Clostridium thermocellum (PROT SWISS input P38853) and XynA from Thermoanaerobium saccharolyticum (Lee et al., J Bacteriol 175: 5890-5898 (1993)) was found. Both enzymes show 27.4% identity and 27.9% Resemblance to the Thermotoga enzyme. Recently has Winterhalter et al., Mol Microbiol 15: 431-444 (1995) via the Cloning and sequencing of a multidomain xylanase from Tt. maritima reports that 88.7% identity with XynA from Tt. having neapolitana.

Examination of the primary structure of xylanase A shows a significant similarity of its central domain "B", which comprises residues 371 to 687, with enzymes of the glycosyl hydrolase family 10 (Henrissat, Biochem J 280: 309-316 (1991), Fig. 3). Surprisingly, the next to the Tt. maritima enzyme greatest similarity (58.8%) found in the N-terminal catalytic domain of CelB from Caldocellum saccharolyticum (Saul et al, Appl Environ Microbiol 56: 3117-3124 (1990)). The active center in numerous family 10 glycosyl hydrolases

[GT] -x (2) - [IVN] -x- [LIVMF] - [ST] -E- [LIVMF] - [DN] - [LIVMF]

was found within the core domain around the putative catalytic Glu-604 ( Fig. 2).

The N-terminal non-catalytic domain A (residues 30-367) shows a significant similarity to the N-terminal domains of XynX from thermocellum and XynA from Tt. maritima and T. saccharolyticum.

The C-terminal non-catalytic domain C is over 80% identical to the cellulose binding domain, which is found in Tt. maritima xylanase A was found. The sequence analysis by means of "dot plot" reveals the presence of 2 repeating sequences C1 (position 720-851) and C2 (895-1040, see Fig. 2). The 2 direct repeats have 29.8% identical and 8.4% similar residues.

2. Expression in E. coli and purification of the recombinant XynA

Ultrasonically treated extracts of recombinant E. coli cells transformed with pTT17 contain various isozymes with sizes 116 kD, 91 kD and 72 kD. It is very likely that proteolytic processes are responsible for the various forms of xylanase that arise in E. coli cells. The M _{R of} the largest isozyme is comparable to the molecular weight derived for the xynA sequence. Approximately 10% of the total enzyme activity was found in the periplasmic fraction, indicating that the putative signal peptide can also function in E. coli (results not shown).

In order to characterize the function of the various domains, suitable restriction sites were used to remove some C-terminal regions of the gene ( FIG. 4). The expressed truncated proteins AB '' and AB 'with either 572 or 668 N-terminal amino acids showed no xylanase activity. The other protein AB with 775 N-terminal amino acids was completely active, which shows that the catalytic domain in this protein is intact.

Sequences which encode 6 consecutive histidine residues (His tags) adjacent to the N- and / or C-terminus were introduced into suitable locations of the gene by the pQE system (DIAGEN GmbH, Düsseldorf). Constructs containing either the B domain with residues 248 to 775 (6xHis-B) or the B and C domains with residues 248 to 1055 (6xHis-BC-6xHis) are active. This in turn confirms that the N- and C-terminal parts of the multidomain enzyme are unnecessary for the enzymatic activity ( Fig. 1).

The characteristics of the xylanase were purified in Preparations determined by ion exchange and by Molecular Sieve Chromatography of Recombinant E. coli (pTT17) Extracts were obtained. Furthermore, there was an affinity chromatography of xylanase with N- and C-terminal 6xHis tags performed on NiNTA agarose, only the 116 kD protein was obtain. The biochemical properties of the by various Protein samples obtained from cleaning operations were identical, the data shown here were mainly from protein samples obtained after the first cleaning scheme.

The specific xylanase activity of 111.3 µ / mg des purified protein was relatively low. However, there was also one clear activity (2.3 µ / mg and 4.0 µ / mg) against lichenan and barley glucan was found to have 1,3-1,4-β-glucanase activity indicates. In addition, the enzyme shows activity against Methyl umbiferryl cellobioside (2.8 µ / mg) and p-nitrophenyl-β-D-xyloside (0.47 µ / mg), indicating cellobiohydrolase and xylosidase activity indicates. The xylanase shows an optimal activity at a pH of 5.5-6 and only low activity (less than 30%) below pH 4.5 or above 7.5. The temperature optimum is at least 102 ° C, with 50% activity at 70 ° C and only 20% Activity at 50 ° C. The enzyme was at an incubation of 200 Stable min. At 90 ° C, its half-life is 120 min. At 100 ° C.

Different constructs with the domains ABC, AB, BC or B (see Fig. 1) were used to characterize the effect of the different domains on the thermostability of XynA. The results shown in Table 1 show the influence of the N-terminal A domain on the thermal behavior of Thermotoga xylanase A. The removal of the N-terminal A domain in the constructs 6xHis-BC-6xHis and 6xHis-B resulted in 86 % Loss of activity when incubated for 30 minutes at 80 ° C. The removal of the C-terminal c-domain in XynA AB did not affect the thermostability.

The cellulose binding activities of proteins with and without C-terminal domains were obtained by mixing the extracts of recombinant E. coli cells that contain the entire XynA (ABC) or the truncated XynA proteins AB, 6xHis-BC-6xHis and 6xHis-B express, tested with microcrystalline avicel cellulose. the Supernatants from these mixtures were determined for their Enzyme activity investigated. 97.7% of the protein without the C-terminal domain (XynA-AB) remained in the supernatant. Proteins with however, the C-terminal domains were attached to the cellulose absorbed. The xylanase activity of the protein 6xHis-BC was predominantly bound to cellulose (58.8%), which shows that the C domain acts as a cellulose binding domain (CBD).

Claims (11)

1. New endo-xylanases, consisting of

• - a catalytic domain with xylanase activity (B)
• - possibly an N-terminal, thermostability-mediating domain (A) and
• - possibly a C-terminal cellulose binding domain (C).

2. New endo-xylanases according to claim 1, consisting solely of the domain B. 3. New endo-xylanases according to claim 1, consisting of the Domains A-B. 4. New endo-xylanases according to claim 1, consisting of the Domains B-C. 5. New endo-xylanases according to claim 1, consisting of the Domains A-B-C. 6. New endo-xylanases according to claims 1-5, characterized by the catalytic domain xynA. 7. New endo-xylanases according to claims 1-5, characterized by the catalytic domain xynB. 8. New endo-xylanases according to claims 1-5, characterized by the catalytic domain xynA of the sequence. Domain B (catalytic domain of XynA 371-687) 9. New endo-xylanases according to claims 1-5, characterized by the catalytic domain xynB of the sequence. XYNB, MATURE PROTEIN (29-347) 10. New endo-xylanases according to claims 1-5, characterized by the N-terminal domain of the sequence which mediates the thermostability. Domain A (N-terminal domain of XynA 30-370) 11. New endo-xylanases according to claims 1-5, characterized by the C-terminal cellulose binding domain of the sequence. Domain C (C-terminal domain of XynA 688-1055)