Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations

Abstract

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.

Figure 1

Enzyme proportions for a 16-component synthetic mixture optimized for (a) Glc or (b) Xyl release from AFEX-corn stover. Numbers are percentages of each enzyme in the optimized mixture. Enzyme loading was 15 mg/g glucan. Yields are shown in Figure 4. γ-Amylase is also known as amyloglucosidase.

Figure 2

Enzyme proportions for a 16-component mixture optimized for (a) Glc or (b) Xyl release from AP-corn stover. Numbers are percentages of each enzyme in the optimized mixture. Enzyme loading was 15 mg/g glucan. Yields are shown in Figure 4.

Figure 3

Enzyme proportions for a 16-component mixture optimized for (a) Glc or (b) Xyl release from AFEX-DDGS. Numbers are percentages of each enzyme in the optimized mixture. Enzyme loading was 15 mg/g glucan. Yields are shown in Figure 4.

Figure 4

Yields of Glc (a) and Xyl (b) for Spezyme CP, Accellerase 1000, or the 16-component optimized mixture on three pretreatment/substrate combinations. Enzyme loadings were fixed at 15 mg/g glucan and digestions were for 48 h. Yields (as a percentage of total Glc or Xyl) are shown above the data bars. Values for Spezyme CP and Accellerase 1000 are taken from Table 1. Error bars indicate ±1 SD of the mean (n = 8).

Figure 5

Ternary diagrams of optimization of mixtures of four commercial enzyme preparations for release of Glc (left) or Xyl (right) from AP-treated DDG. In these graphical representations, Multifect Xylanase was kept constant. Loadings were constant at 15 mg/g glucan. The experimental data on which the models were based are shown in Additional file 1, Table S3.

Figure 6

Glc release from AP-corn stover as a function of enzyme loading. (a) 6 h, (b) 12 h, (c) 48 h. The "core set" and "core set + accessory" mixtures were identical to those used in Figures 2 and 3, i.e., optimized for 15 mg/g glucan loading and 48-h digestion. Error bars, which are sometimes obscured by the data symbols, represent ± 1 SD of the mean (n = 8).

Figure 7

Xyl release from AP-corn stover as a function of enzyme loading. (a) 6 h, (b) 12 h, (c) 48 h. The "core set" and "core set + accessory" mixtures were identical to those used in Figures 2 and 3, i.e., optimized for 15 mg/g glucan loading and 48-h digestion.

Figure 8

Time course of release of Glc and Xyl from AP-corn stover. (a) Glc, (b) Xyl. All enzyme loadings were 15 mg/g glucan. Data are from the same experiment shown in Figures 6 and 7. The "core set" and "core set + accessory" mixtures were identical to those used in Figures 2 and 3, i.e., optimized for 15 mg/g glucan loading and 48-h digestion.