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GRAS Notice 891, Cellulase Produced by Trichoderma Reesei

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GRAS Notice 891, Cellulase Produced by Trichoderma Reesei GRAS Notice (GRN) No. 891 https://www.fda.gov/food/generally-recognized-safe-gras/gras-notice-inventory Vo,, m-\\ Cellulase Enzyme Preparation Produced by Trichoderma reesei Janet Oesterling, Regulatory Affairs, Novozymes North America, Inc., USA November 2019 PART 2 - IDENTITY, METHOD OF MANUFACTURE, SPECIFICATIONS AND PHYSICAL OR TECHNICAL EFFECT OF THE NOTIFIED SUBSTANCE ............................................ 4 2.1 IDENTITY OF THE NOTIFIED SUBSTANCE ................................................................................. 4 2.2 IDENTITY OF THE SOURCE ........................................................................................................ 4 2.2(a) Trichoderma reesei Production Strain ................................................................................. 4 2.2(b) Recipient Strain ......................................................................................................................... 5 2.2(c) Expression Plasmids .................................................................................................................. 5 2.2(d) Construction of the Recombinant Microorganism ...................................................................... 5 2.2(e) Stability of the Introduced Genetic Sequences ...................................................................... 6 2.2(f) Antibiotic Resistance Gene .................................................................................................. 6 2.2(g) Absence of Production Organism ......................................................................................... 6 2.3 METHOD OF MANUFACTURE ..................................................................................................... 6 2.3(a) Raw Materials ..................................................................................................................... 6 2.3(b) Fermentation Process.......................................................................................................... 7 2.3(c) Production Organism .......................................................................................................... 7 2.3(d) Criteria for the Rejection of Fermentation Batches ............................................................... 7 2.3(e) Recovery Process ................................................................................................................ 8 2.3(f) Purification Process............................................................................................................. 8 2.4 COMPOSITION AND SPECIFICATIONS........................................................................................ 8 2.4(a) Quantitative Composition .................................................................................................... 8 2.4(b) Specifications ...................................................................................................................... 9 2.5 PHYSICAL OR TECHNICAL EFFECT ............................................................................................ 9 2.5(a) Mode of Action .................................................................................................................... 9 2.5(b) Intended Use ....................................................................................................................... 9 2.5(c) Use Levels ......................................................................................................................... 11 2.5(d) Enzymes Residues in the Final Food .................................................................................. 11 PART 3 - DIETARY EXPOSURE .................................................................................. 12 3(a) Assumptions in Dietary Exposure .......................................................................................... 12 3(b) Food Consumption Data ......................................................................................................... 13 PART 4 - SELF-LIMITING LEVELS OF USE ................................................................. 17 PART 5 - COMMON USE IN FOOD BEFORE 1958 ....................................................... 18 PART 6 - NARRATIVE ON THE CONCLUSION OF GRAS STATUS ............................... 19 6(a) Safety of the Production Organism ......................................................................................... 19 6(b) Safe Strain Lineage................................................................................................................. 21 6(c) Safety of the Donor Organism ................................................................................................ 22 6(d) Safety of the Cellulase Enzymes Within the Complex .............................................................. 23 6(e) Allergenic/Toxigenic Potential of the Cellulase Enzymes ........................................................ 25 6(f) Safety of the Manufacturing Process ...................................................................................... 27 6(g) Safety Studies Conducted ....................................................................................................... 27 6(h) Description of the Test Article................................................................................................ 27 6(i) Results and Conclusion .......................................................................................................... 27 2 Cellulase Enzymes Produced by Trichoderma reesei PART 7 – SUPPORTING DATA AND INFORMATION ................................................. 29 APPENDICES ............................................................................................................. 29 REFERENCES............................................................................................................. 30 3 Cellulase Enzymes Produced by Trichoderma reesei PART 2 - IDENTITY, METHOD OF MANUFACTURE, SPECIFICATIONS AND PHYSICAL OR TECHNICAL EFFECT OF THE NOTIFIED SUBSTANCE 2.1 IDENTITY OF THE NOTIFIED SUBSTANCE The subject of this notification is a multi-component cellulase enzyme preparation, produced by submerged fermentation of a genetically modified Trichoderma reesei. The cellulase enzyme preparation consists of cellobiohydrolase 1, beta-glucosidase and endo-glucanase 1 enzymes. It is well established that the Trichoderma reesei complex is multi-enzymatic, there are at least three enzyme components that are physically and enzymatically distinct and that all three components are essential for the conversion of cellulose to glucose (1). The characterizing activity of the preparation is cellulase (E.C. 3.2.1.4; systematic name: 1,4-(1,3:1,4)-β-D-glucan 4-glucanohydrolase), an enzyme responsible for the endohydrolysis of 1,4-β-D-glucosidic linkages in cellulose, lichenin and cereal β-D- glucans. Key enzyme and protein chemical characteristics of the enzymes are given below. Table 1. Key enzyme characteristics CHARACTERISTICS Classification Cellulase Cellulase Cellulase Systematic name 4-beta-D-glucan beta-D-glucoside 4-beta-D-glucan- cellobiohydrolase glucohydrolase glucanohydrolase Accepted name Cellobiohydrolase 1 Beta-glucosidase Endo-glucanase 1 EC No. 3.2.1.176 3.2.1.21 3.2.1.4 CAS No. 37329-65-0 9001-22-3 9012-54-8 Specificity Hydrolysis of (1->4)-beta- Hydrolysis of terminal, hydrolysis of (1->3)-beta-D- D-glucosidic linkages in non-reducing beta-D- glucosidic linkages in (1->3)- cellulose glucosyl beta-D-glucans Molecular Weight 54 kDa 91 kDa 48 kDa Amino Acid Count 506 844 437 2.2 IDENTITY OF THE SOURCE 2.2(a) Trichoderma reesei Production Strain The Trichoderma reesei production strain, designated AyGm61-2C-2, was constructed via the intermediate strain, BTR213. Strain BTR213 is derived from a natural isolate of Trichoderma reesei parental strain RUT-C30 (ATCC 56765). RUTC30 is derived from the well-known wild type strain QM6a. QM6a is the parent of practically all Trichoderma reesei industrial production strains (2). Trichoderma reesei is classified as a Biosafety Level 1 microorganism by the American Type Culture Collection (ATCC) based on risk assessment from U.S. department of Public Health guidelines (3). The expression plasmids used in the strain construction contain well defined chromosomal DNA fragments and synthetic DNA linker sequences. The DNA sequence 4 Cellulase Enzymes Produced by Trichoderma reesei for the introduced genes are based on the sequences encoding the cellobiohydrolase 1 and beta-glucosidase variant from Aspergillus fumigatus and the endo-glucanase 1 from Trichoderma reesei. This genetically modified Trichoderma reesei production strain complies with the OECD (Organization for Economic Co-operation and Development) criteria for GILSP (Good Industrial Large-Scale Practice) microorganisms (4). It also meets the criteria for a safe production microorganism as described by Pariza and Foster (5) and later Pariza and Johnson (6) and several expert groups (7) (8) (9) (4) (10) (11) (12). 2.2(b) Recipient Strain The recipient strain used in the construction of the Trichoderma reesei production strain AyGm61-2C-2, was modified by several rounds of classical mutagenesis and genetic modifications of RUTC30. Furthermore, the paracelsin gene (parS) has been deleted in the recipient strain to eliminate the potential production of paracelsin. The recipient strain was prepared for targeted homologous recombination in two loci. 2.2(c) Expression Plasmids The expression plasmids used to transform the recipient strain
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