Expression of β-mannanase in chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis

Patent metadata

Field	Value
Publication number	US-10689633-B2
Application number	US-201213659396-A
Country	US
Kind code	B2
Filing date	Oct 24, 2012
Priority date	Feb 29, 2008
Publication date	Jun 23, 2020
Grant date	Jun 23, 2020

Abstract

Disclosed herein are materials useful for degrading plant biomass material. In exemplary embodiments, the plant material comprises one or more enzymes that are expressed in plants and/or bacteria. Specifically exemplified herein are plant degrading enzymes expressed in chloroplasts. The chloroplast expressed enzymes may be provided as cocktails for use in conjunction with conventional methods of converting biomass into biofuels, such as cellulosic ethanol. In other exemplary embodiments, methods and materials are disclosed for degrading mannans.

First claim

What is claimed is:

1. A method for producing plant degrading cocktail consisting of extracts of chloroplast produced β-mannanase, xylanase encoded by sequences obtained from Trichoderma reesei , acetyl xylan esterase encoded by sequences obtained from Trichoderma reesei , recombinant CelD endoglucanase encoded by sequences obtained from Clostridium termocellum, Eg1 endoglucanase, CelO exoglucanase encoded by sequences obtained from Clostridium thermocellum , recombinant Beta glucosidase encoded by sequences obtained from Trichoderma reesei , PelA pectate lyase, recombinant PelB pectate lyase encoded by sequences obtained from Fusarium solani , and PelD pectate lyase encoded by sequences obtained from Fusarium solani plant degrading enzymes and swollenin encoded by sequences obtained from Trichoderma reesei , wherein nucleic acids encoding said plant degrading enzymes and swollenin are introduced into chloroplasts of at least one plant, thereby producing transplastomic plants; said method comprising
a) grinding said transplastomic plants in liquid nitrogen;
b) suspending ground transplastomic plant material of step a) in a buffer in the presence of a protease inhibitor cocktail, thereby forming a suspension;
c) subjecting said suspension to centrifugation and harvesting said enzymes in the resultant supernatant; and
d) filtering said supernatant to remove any sugars present, thereby producing a crude plant enzyme extract for said plant degrading cocktail.

2. The method of claim 1 , wherein said plant is tobacco.

3. A plant degrading enzyme cocktail useful in digesting a wood-based biomass sample consisting of extracts of chloroplast expressed degrading enzymes β-Mannanase, xylanase encoded by sequences obtained from Trichoderma reesei , acetyl xylan esterase encoded by sequences obtained from Trichoderma reesei , CelD endoglucanase encoded by sequences obtained from Clostridium termocellum, Eg1 endoglucanase encoded by sequences obtained from Trichoderma ressei , recombinant beta-glucosidase encoded by sequences obtained from Trichoderma reesei , CelO exoglucanase encoded by sequences obtained from Clostridium thermocellum , swollenin encoded by sequences obtained from Trichoderma reesei , PelA pectate lyase, PelB pectate lyase, and PelD pectate lyase or consisting of chloroplast produced β-Mannanase, xylanase, acetyl xylan esterase encoded by sequences obtained from Trichoderma reesei , CelD endoglucanase encoded by sequences obtained from Clostridium termocellum, Eg1 endoglucanase encoded by sequences obtained from Trichoderma ressei , CelO exoglucanase encoded by sequences obtained from Clostridium thermocellum , swollenin encoded by sequences obtained from Trichoderma reesei , recombinant PelA, and PelB pectate lyase encoded by sequences obtained from Fusarium solani , PelD pectate lyase, cellulase, recombinant beta-glucosidase encoded by sequences obtained from Trichoderma reesei , recombinant alpha amylase encoded by sequences obtained from Bacillus ssp., amyloglucosidase, and rubisco, said cocktail being effective to hydrolyze 100% of a wood biomass sample.

4. A method of producing the plant degrading enzyme cocktail of claim 3 comprising the steps of producing plants comprising chloroplasts that express said plant degrading enzymes, harvesting said plant; and processing said plant to produce plant degrading material suitable for mixing with and degrading a biomass sample.

5. The method of claim 3 , wherein said wood-based biomass sample is pinewood.

6. The method of claim 3 , wherein said extracts are in powdered form.

Assignees

• Univ Pennsylvania

Inventors

• Daniell Henry

Classifications

• C12N15/8257

  for the production of primary gene products, e.g. pharmaceutical products, interferon · CPC title

• C12N9/2402 Primary

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C12P7/12

  substrate containing sulfite waste liquor or citrus waste · CPC title

• C12N9/2494

  Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase · CPC title

• C12N15/8214

  Plastid transformation · CPC title