Biochemical approaches to define biomass degrading communities

Extracellular enzymes catalyze the initial rate-limiting step of decomposition and are the primary means by which microbes degrade complex biomass into smaller molecules that can be assimilated. Therefore, it is reasonable to assume that determining the amount and type of hydrolytic activities associated with biomass can be an indicator of the hydrolytic po­tential of a given microbial community. Defining biomass degrading communities through the application of traditional biochemistry tools combined with new proteomic technolo­gies are advancing our understanding of how microorganisms attack biomass as well as our understanding of natural enzyme diversity. Proteomic analysis can be used to identify and validate hydrolytic enzyme targets and profile protein expression patterns in complex communities (71). By using two-dimensional PAGE it is possible to fingerprint the secreted proteins of cellulose-degrading microorganisms and obtain sufficient sequence informa­tion for cloning. Surveying the proteome of natural microbial communities can lead to the discovery and analysis of more diverse hydrolytic enzymes that can break down cellu­lose, hemicellulose, and lignin. New classes of ligninases and hemicellulases will likely be identified, their mechanisms of action understood, and their performance refined to allow introduction of enzymatic pretreatment that will free cellulose microfibrils for enzymatic saccharification (breakdown to sugars).

The traditional biochemical approach to understanding microbial biomass utilization is through kinetic assays designed to quantitatively determine the presence or absence of hydrolytic enzymes. Hydrolytic enzymes can be detected using either natural substrates such as cellulose or xylan by measuring sugar release or by using synthetic substrates that contain easily detected chromophores such as p-nitrophenol or 4-methyl umbelliferone (69). The detection of 4-methyl umbelliferone can be used as a sensitive, quantitative assay for endoglucanse or other enzymes that cleave substrates linked to 4-methyl umbelliferone (72). However, how tightly the enzymes are bound to the substrate or are associated with the microorganism is a limitation of direct enzymatic assays. A range of soil enzyme assays was developed by Lynch and coworkers as alternatives to population measurements (46). These included assays for determining chitobiosidase, N-acetyl glucosaminidase, p-glucosidase, p-galactosidase, acid phosphatase, alkaline phosphatase, phosphodiesterase, aryl sulfatase, and urease activities from small soil samples. Soil enzyme activities, therefore, can index changes in the microbial functioning in soil, and there is ample evidence in the literature of the importance of glycosyl hydrolases, and proteases to the soil’s performance (69, 73-77).

Monitoring many proteins simultaneously in a complex system can be best accom­plished for many hundreds of protein species across a large number of samples using mod­ern technology for two-dimensional differential gel electrophoresis in combination with sophisticated statistical algorithms for data analysis. Two-dimensional gel electrophoresis is capable of resolving several hundreds to several thousands of proteins on a single gel (78). This method utilizes independent properties of proteins (i. e., isoelectric point and molecular mass) to resolve proteins present in a biological extract in two dimensions. Methods for two­dimensional differential gel electrophoresis have been greatly improved over the last several years to enable quantitative analysis of relative protein abundance among a set of samples. The first major improvement involves derivatization of proteins in samples with spectrally distinct, covalently coupled, charge — and mass-balanced fluorescent dyes (79). These highly fluorescent tags allow for extremely sensitive detection limits [i. e., 1 ng; (80)] and a broad linear response range [i. e., 3-4 orders of magnitude; (81)]. Derivatization of two different protein extracts with spectrally distinct fluorescent tags enables multiplexing of two un­known samples on gels, which eliminates any question about which protein in one sample is co-migrating with what spot on another gel. A second major improvement of the ex­perimental design for two-dimensional differential gel electrophoresis is the inclusion of an internal standard labeled with a third, spectroscopically resolvable, fluorescent dye (82). This enables normalization of the data from a series of gels, which permits statistically valid comparison of protein amounts across a series of gels. Because of this improved experimen­tal design, it is now possible to detect changes in absolute protein abundance on the order of 10% with 95% confidence.