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

Abstract

BackgroundEnzymes 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.ResultsWhen 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.ConclusionThe 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 influe...

Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw

“…Contrasting conclusions were drawn in the study of Banerjee et al [19]. They obtained two different optimal enzyme mixtures for AFEX corn stover (one having a much higher EG1/EG2 ratio than the other) and interpreted this finding by overlapping activities of the two endoglucanases.…”

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

“…Efforts in this area have incorporated one or more of the following properties also found in GENPLAT: robotic dispensing of enzymes and biomass slurries, statistical design of experiment, and/or automated determination of Glc and Xyl (Berlin et al, 2007;Decker et al, 2009;Kim et al, 1998;King et al, 2009). GENPLAT extends these earlier efforts, most significantly in the complexity of the enzyme mixtures that can be analyzed from at most 6 components in the earlier studies to more than 16 in our latest work (Banerjee et al, 2010c). Additional key features of GENPLAT are the use of a bead mixing chamber…”

“…Figure 3 shows the use of GENPLAT to optimize a mixture of four commercial enzyme preparations. The optimal proportions for release of Glc from AFEX-pretreated DDG were found to be 47% Accellerase1000, 27% Multifect Pectinase, 22% Novozyme 188, and 4% Multifect Xylanase (Banerjee et al, 2010c). Figure 3B shows the differences in Glc yield from AFEX-DDG with four enzyme preparations; the optimized commercial mixture gives higher Glc yield than Accellerase1000 alone, SpezymeCP alone, or a 16-component synthetic mixture.…”

Shotgun Approach to Increasing Enzymatic Saccharification Yields of Ammonia Fiber Expansion Pretreated Cellulosic Biomass

“…(2) Multicollinearity issue: though the regression models used here do not have direct biological significance, the model interaction terms are indicative of the relative degree of synergy between 2 and more enzyme cocktails or purified enzyme components. For simple individual enzyme mixtures (composed of 3-10 components) such models can more clearly provide a quantitative degree of synergy factor between individual enzyme components as also shown by our group and others (Banerjee et al, 2010a;Gao et al, 2010a). One of the major issues encountered, when attempting to delineate the relative contribution of individual enzyme families on hydrolysis yields for more complex enzyme cocktails (>50 components) explored in this study, was the high multicollinearity between relative enzyme abundance.…”