FR2509749A1 - PROCESS FOR HYDROLYSIS OF CELLULOSIC SUBSTRATE AND CELLULOLYTIC PREPARATION - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR HYDROLYSIS OF A CELLULOSIC SUBSTRATE TO FORM MONO-ETOR DISACCHARIDES. ACCORDING TO THE INVENTION, THE CELLULOSIC SUBSTRATE IS TREATED WITH A PREPARATION OF CELLULOLYTIC ENZYMES FROM A MUTANT STRAIN OR DERIVATIVE OF A BACTERIA OF THE GENUS CELLULOMONAS OR PSEUDOMONAS, WHETHER COMPLETED WITH A CELLULASE DERIVED EITHER FROM A FUNGI OR OF AN ACTINOMYCETE. THE INVENTION APPLIES IN PARTICULAR TO THE PRODUCTION OF SUGARS.

Description

The present invention relates to a process for the hydrolysis of

  cellulosic substrates to produce mono and / or disaccharides using

cellulase enzymes.

  It is known that cellulose substrates can be hydrolysed to mono and / or disaccharides using the

  cellulase enzymes derived from fungi and actinomyces.

  However, it has been found that the rate of enzyme production

  and the time to produce maximum yields of -

  enzymes from fungi and actinomyces made the preparation of enzymes for these sources relatively expensive. It is also known that certain bacteria of the genus Cellulomonas or Pseudomonas can use cellulosic substrates and can grow at the expense of sugars that are produced as a result of the enzymatic hydrolysis of cellulosic substrates. Recently, it has been shown that these bacteria can be mutated to produce mutant strains capable of producing high levels of cellulase enzymes Culture or culture preparations of such strains of Cellulomonas or Pseudomonas can be used to hydrolyze cellulosic substrates by

  simple sugars Unfortunately, these bac-

  are limited in their range of saccharifica-

when used in vitro.

  The present invention relates to a process for the hydro-

  lysis of a cellulosic substrate to form mono and / or

  disaccharides, which consists in treating the cellulosic substrate

  with a cellulolytic enzyme preparation of a mutant strain or derivative of a bacterium of the genus Cellulomonas or Pseudomonas that is supplemented with a derived cellulase

  either a fungus or an actinomycete.

  The present inventors have found that a synergistic result could be achieved by adding the cellulase enzyme of a fungus or actinomycete to a culture of a bacterium of the genus Cellulomonas or Pseudomonas. hydrolyze the substrate much more extensively than a bacterial culture of itself

  much less enzyme protein for saccharifi

  given cation compared to an enzyme preparation

mushrooms as is.

  The present invention furthermore relates to a preparation

  cellulolytic material containing a cellulosic enzyme preparation

  lytic that is obtained from a bacterium of the genus Cellulomonas or Pseudomonas, and that one supplements a cellulase

  derived from either a fungus or an actinomycete.

  This synergistic effect is quite unexpected and an explanation

  total cation of this phenomenon is, at this stage, impossible.

  Cellulomonas and Pseudomonas strains have been found to show good growth rates and cellullose conversion rates in vivo, however, when used in vitro, the good initial levels rapidly reach a plateau. cellulase enzymes of a fungus or actinomycete that is added to the cell culture of Cellulomonas or Pseudomonas, gives greatly improved results It is possible that this is due to the high levels of activity that the

  mushroom cellulase shows against crystal cellulose

line in vitro.

  Bacterial preparations are much cheaper to produce than mushroom or actinomycete preparations, because enzyme production rates are considerably faster.

  mushroom cellulase quantities may, under conditions

  tions, give rise to hydrolysis levels that are five times higher than what can be expected

  if the mushroom cellulase is used on its own.

  The preparation of bacterial enzymes can be a complete culture preparation, a complete culture preparation where the cells have been disintegrated, or a

  extracellular preparation Fungi and actino-

  fungi excrete cellulase enzymes that can be separated from the producing organism and used in the present process if desired, whole cultures

  fungi or actinomycetes can be used.

  Any source of cellulosic material may be used

  as raw material for the process according to the invention.

  Among the various cellulosic materials available, and which are advantageously employed, mention may be made of sugar cane bagasse, sorgum bagasse, wheat straw, rice straw, stalks and corn forage, kenaff, sawdust, wood chips and other forest-derived materials, waste paper and

pulp.

  With materials that have not been previously treated, some form of chemical or physical pre-treatment is required. This pretreatment makes cellulose and hemicellulose more accessible to attack enzymes, breaking the bonds between cellulose and lignin, by decreasing the crystallinity of the cellulose and by removing the organic components The pre-treatment can take the form of a physical treatment such as grinding; a chemical form such as a treatment with a diluted alkali; or a combination of physical / chemical treatments such as heating at high temperatures and then rapid decompression As a result of pre-treatment,

  the material can be brought to a reactor for saccharification

  cation by the cellulase complex.

  Cellulomonas or Pseudomonas or mutant strains are preferably grown initially under conditions conducive to the growth and production of the enzymes, whereby bacterial cellulase must be obtained when a sufficient population of cellulase enzyme cells and enzyme complexes has been obtained. conditions that can be modified to favor the production of sugars These changed conditions that inhibit cell growth may be increased temperature, a limit on the availability of a major nutrient for growth, an operation under anaerobic conditions, or addition of inhibitors

  in the culture medium.

  Cellulase of fungi or actinomycetes is preferably obtained from a fungus that is a member of the species: Trichoderma Penicillium * Aspergillus Fusarium Schlerotium and Streptomyces Actinomycetes, and Thermoactinomyces

  Preferably, the fungus is Trichoderma reesei.

  Synergism has also been observed if the cellulase enzymes of more than one organism listed above

  are added to preparations of Cellulomonas or Pseudomonas.

  Fungi or actinomycetes cellulase should be present in an amount of at least 0.01 FPU / ml and more preferably between 0.1 FPU / ml and 5 FPU / ml above FPU / ml, there is no economic justification for further cellulase addition and yet it appears that the synergistic effect of the present invention is still observed above this level. The cell culture of Cellulomonas or Pseudomonas should be as concentrated as possible in order to To obtain the fastest possible conversion of cellulose to sugars In practice, preparations having a protein content of 0.5 to 20 mg / ml are suitable.

  In addition to the production of simple sugars by hydro-

  lysis of cellulosic materials, the hydrolysis of the cellulose can be carried out in the presence of bacteria or yeasts capable of directly fermenting the sugars produced, by hydrolysis, into ethanol In this way, it is possible to obtain

  a single stage conversion of cellulose to ethanol.

  Ethanol must be recovered from the reaction medium by

conventional techniques.

  We will give below a preferred embodiment

of the present invention.

Example 1

  Cellulomonas C 51-17 was grown for 24 hours on a balanced salt medium, yeast extract and bagasse pretreated with 2% alkali at a temperature of 32 C and a pH between 7.0 and 8.0.

  The bagasse of sugarcane had been pretreated in the heat

  With 0.1 g of NaOH / g, bagasse dry weight at 1000 ° C. for one hour, the culture temperature was then raised to 37 ° C., the culture was made anaerobic and

  75 g / l of bagasse pretreated with alkalis was added.

  The release of reducing sugar was followed by the deter-

  was found to be 18.5 mg / ml at 18 hours,

  22 mg / ml at 24 hours and 23 mg / ml at 48 hours.

  Cellulomonas C 51-17 was grown on alkali pre-treated bagasse as previously indicated. After 24 hours of growth, the alkali pretreated bagasse (75 g / l) was added as well as low levels of a Trichoderma reesei cellulase complex QM 9414 and saccharification was performed at 45 C and pH between 5 and 6 Control experiments were performed using only Cellulomonas enzymes and only 1 Trichoderma enzyme data

are indicated below: -

  BAGASSE DIGESTION PRETREATED AT 7.5% ALCALIS

BY TRICHODERMA REESEI QM 9414

  Concentra Sugar reducer Concentration (mg / ml) enzyme Concentrations 1 'enzyme CELLULASE protein 14 hours 24 hours 48 hours (mg / ml) 0.1 FPU / ml 0.25 7.0 8.4 11.2 0.25 FPU / ml 0.60 14.9 16.9 25.5 0.5 FPU / ml 1.45 21.8 26.0 34.6 1.0 FPU / ml 3.00 42.0 DIGESTION BAALE STUFFED AT 7.5% OF ALCALIS BY THE ULTRA C 51-17 CEL ULTRA CELL CETTLULASE AND THE ENZYME OF

  CEIL VUM 7 S, TRICHODERMA REESEI QM 9414 COMBINED

  Enzynme Sugar concentration reducer coplex protein 14 hours 24 hours 48 hours (mg / mi L) O, 1 FPU / ml Trichoderma Reesei + Celluloeonas

C 51-17 1.18 28.6 33.1 42.2

  O, 25 FPU / ml Trichoderma Reesei + Cellulomonas

C 51-17 1.53 35.1 41.2 49.2

  0.5 FPU / ml Trichoderma Reesei + Cellulcmonas

C 51-17 2.38 40.0 43.6 51.2

Celluloronas

C 51-17

only 0.93 16.9 20.1 23.2

Example 2

  Cellulomonas C 51-17 was grown on bagasse

  pretreated with alkalis as previously indicated.

  After 24 hours of growth, alkali pretreated bagasse (75 g / l) was added along with low levels of a Trichoderma reesei Rutgers C-30 cellulase complex and Novo Industry Denmark's β-glucosidase complex. , and the saccharification was carried out at 45 C and a p H

  between 5 and 6 The data is shown below.

  BAGASSE DIGESTION PRETREATED AT 7.5% ALCALIS

  BY CELLULOMONAS C 51-17, TRICHODERMA REESEI RUT C-30

AND NOVO X -GLUCOSIDASE

  Enzyme Sugar concentration Reducing complex (mg / ml) C 51-17 + glucosidase (2 IU / ml) + T reesei C-30 at 12 hours 24 hours 48 hours of 0.5 FPU / ml 50.7 56.5 60.4 0.25 FPU / ml 44.1 52.5 56.5 0.1 FPU / ml 33.4 40.6 48.3

Example 3

  A Pseudomonas P 52-1 strain was grown on

  bagasse pre-treated with alkalis as previously

  Specified for Cellulomonas During the saccharification conditions indicated for Cellulomonas, 12 mg / ml of reducing sugar was released from 7.5%

  bagasse pre-treated with alkalis that had been added.

  Saccharification in the presence of 0.1 FPU / ml of cellulase

  from Trichoderma reesei QM 9414 gave the liber-

  28 mg / ml reducing sugar No attempt has been made

  been made to improve Pseudomonas strains by mutagenesis

  or other means, however, the synergism observed for Cellulomonas also applies to the Pseudomonas strain

cellulolytic examined.

  In the above examples, the enzymes of Trichoderma reesei have been used with the enzymes of Cellulomonas or Pseudomonas. The present invention also covers the use of cellulases from other sources of fungi or actinomyces in conjunction with the preparations.

  of Cellulomonas or Pseudomonas enzymes.

  In the above example, FPU stands for "filter paper unit" (international unit), and it is a recognized measure of cellulolytic activity in enzymes

  cellulose derived from mushroom sources.

Claims (6)

  Process for the hydrolysis of a cellulosic substrate to form mono and / or disaccharides, characterized in that it consists in treating the cellulosic substrate with a cellulolytic enzyme preparation of a strain or a mutant derivative of a bacterium of the genus Cellulomonas or Pseudomonas, supplemented with a derived cellulase   either a fungus or an actinomycete.   Process according to Claim 1, characterized in   what cellulase derived from a fungus or an actino-   fungus is present in an amount of 0.01 FPU / ml to FPU / ml. Process according to claim 2, characterized in that the cellulase derived from a fungus or a   actinomycete is present in an amount of 0.1 to 5 FPU / ml.   Process according to any one of the claims   1 to 3, characterized in that the cellulase of the fungus is obtained from a fungus selected from the genus Trichoderma Penicillium Aspergillus Fusarium or Schlerotium Process according to Claim 4, characterized in that   what the mushroom is Trichoderma reesei.   Process according to any one of the claims   previous, characterized in that the preparation of bacterial cellulolytic enzymes is obtained from bacteria which have been grown under conditions favoring the production of enzymes.   Process according to any one of the claims   preceding, characterized in that the hydrolysis of the cellulose is carried out in the presence of a bacterium or a yeast capable of fermenting sugars to form ethanol. Cellulolytic preparation characterized in that it contains a cellulolytic enzyme preparation obtained from a bacterium of the genus Cellulomonas or Pseudomonas, which is supplemented with a cellulase derived either from a fungus or an actinomycete.