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Background: Although a-linked xylose is a major constituent of the hemicelluloses of land plants, few secreted a-xylosidases have been described from fungi or bacteria. AxlA of Aspergillus niger is a secreted α-xylosidase that was earlier shown to promote the release of free glucose (Glc) and xylose (Xyl) from substrates containing α-linked xylose, including isoprimeverose (IP), the heptasaccharide subunit of pea xyloglucan (XG), and tamarind XG. Results: The utility of AxlA for enhancing release of free Glc and Xyl in combination with commercial enzyme cocktails from dicotyledonous and monocotyledonous plants was examined. Without AxlA supplementation, a mixture of CTec2 and HTec2 (both of which are derived from T. reesei) did not release significant levels of Glc from pea XG or tamarind XG. This is consistent with their lack of detectable α-xylosidase activity using model substrates. On alkaline hydrogen peroxide-pretreated corn stover, supplementation of CTec2/HTec2 (at a loading of 2.5 mg/g glucan) with AxlA (at a loading of 8 mg/g glucan) increased Glc yields from 82% to 88% of the total available Glc and increased Xyl yields from 55% to 60%. AxlA supplementation also improved Glc yields from corn stover treated with the commercial cellulase Accellerase 1000. The AxlA enhancement was not a general protein effect because bovine serum albumin or bovine gamma-globulin at similar concentrations did not enhance Glc yields from corn stover in response to CTec2/HTec2. Supplementation of CTec2/HTec2 with AxlA did not enhance Glc release from pretreated green or etiolated pea tissue. However, AxlA did enhance Glc and Xyl yields compared to CTec2/HTec2 alone from another dicotyledonous herbaceous plant, Chenopodium album (lamb's quarters). Conclusion: Supplementation of commercial cellulase cocktails with AxlA enhances yields of Glc and Xyl from some biomass substrates under some conditions, and may prove useful in industrial lignocellulose conversion. [ABSTRACT FROM AUTHOR]

Abstract: A high throughput enzyme assay platform, called GENPLAT, was used to guide the development of an optimized mixture of individual purified enzymes from ten “accessory” and six “core” enzymes. Enzyme mixtures were optimized for release of Glu, Xyl, or a combination of the two from corn stover pretreated by ammonia-fiber expansion (AFEX). Assay conditions were a fixed enzyme loading of 15mg/g glucan, 48h digestion, and 50°C. Five of the ten tested accessory proteins enhanced Glu or Xyl yield compared to the core set alone, and five did not. An 11-component mixture containing the core set and five accessory enzymes optimized for Glu released 52.1% of the available Glu, compared to 38.5% with the core set alone. A mixture optimized for Xyl released 39.9% of the Xyl, compared to 26.4% with the core set alone. We predict that there is still considerable opportunity for further improvement of synthetic mixtures. Furthermore, the strategy described here is applicable to the development of more efficient enzyme cocktails for any pretreatment/biomass combination and for detecting enzymes that make a heretofore unrecognized contribution to lignocellulose deconstruction. [ABSTRACT FROM AUTHOR]

Background: Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings. Results: When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h. Conclusion: The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes. [ABSTRACT FROM AUTHOR]