Kevin A. Gray

Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement

ABSTRACT The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62°C for 1 h and 27% of its initial activity after 10 min at 85°C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.

Soil-Based Gene Discovery: A New Technology to Accelerate and Broaden Biocatalytic Applications

Publisher Summary This chapter discusses the general schemes for the discovery and application of biocatalysis. Various stages of a biocatalytic process are illustrated in the chapter. The first step is the discovery of a suitable biocatalyst by screening against a target reaction. Once the biocatalyst is found, the subsequent steps include rigorous characterization in terms of specificity, productivity, bioprocess development, and manufacturing. If necessary, the biocatalyst can be optimized by directed evolution or traditional techniques of mutation and selection. The chapter examines the limitations in developing a biocatalytic process. The ability of soil-based gene discovery to provide libraries of enzymes for industrial applications is explained in the chapter. It is shown that the soil-based generation of enzyme libraries and screening can help in the rapid discovery of an appropriate enzyme to enable biocatalytic applications. The chapter focuses on an overall scheme for the discovery of dehalogenases from soil using biopanning. The chapter also illustrates the power of expression screening to discover lipases and esterases that differ significantly from known enzymes in both sequence and activities.

Gene Site Saturation Mutagenesis: A Comprehensive Mutagenesis Approach

Publisher Summary This chapter describes the various aspects of gene site saturation mutagenesis (GSSM). GSSM systematically explores minimally all possible single amino acid substitutions along a protein sequence. This comprehensive technique introduces point mutations into every position within a target gene using degenerate primer sets containing 32 or 64 codons to generate a complete library of variants. It is found that unlike rational mutagenesis, GSSM does not require prior knowledge of the structure, or mechanism of the target protein due to its ability to generate all mutations at all positions within the protein. GSSM has been used to improve the thermostability of a haloalkane dehalogenase by 30,000-fold. It is found that when the mutations were analyzed at the eight single sites that individually improved thermostability, three of the substitutions would likely never have been accessed by routine procedures, such as error-prone polymerase chain reaction independent of sampling scale. Single nucleotide substitutions result in a theoretical maximum of 5–6 amino acid changes per codon with only 2–3 amino acid substitutions generally accessed with standard protocols. It is found that GSSM enables a complete analysis of every position in a given gene.

Session 6: Advances in Enzyme Science and Technology

As ethanol from lignocellulosic materials progresses to commercial fruition, finer details of the hydrolysis process are being investigated. In this session, papers were presented that dealt with several aspects of biomass conversion including substrate effects, additives to the saccharification reaction, and high performance enzymes. The influence of each one of these aspects on hydrolysis performance was analyzed by different authors. Rajeev Kumar from Dartmouth College described how the choice of pretreatment technology influences cellulose accessibility. He investigated how cellulose accessibility in poplar solids changed depending on the pretreatment method. The pretreatment methods used were ammonia fiber expansion (AFEX), ammonia recycle percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide. The main conclusion was that cellulase enzyme site accessibility is influenced by the type of pretreatment as well as by the type and composition of the substrate. Maobing Tu from the Pulp and Paper Research Institute of Canada investigated the influence of added surfactant (Tween 80) on cellulose conversion of steam-exploded lodgepole pine and ethanol-pretreated lodgepole pine. He showed that the presence of Tween 80 resulted in a significant increase in the cellulose-to-glucose yield for the steamexploded substrate, but had no effect for the ethanol-treated substrate. In addition, the presence of surfactant increased the amount of free enzyme for both substrates implying that surfactant might be minimizing non-productive binding of cellulases to the surface. Lastly, the presence of surfactant did not affect final ethanol yields in SSF reactions with yeast. Feng Xu from Novozymes observed that cellulose oxidation had a profound impact on the rate and extent of cellulose hydrolysis by single enzymes (cellobiohydrolases) and complete cellulase mixtures (Trichoderma reesei-secreted proteins). Thermochemical pretreatments may negatively affect enzymatic hydrolysis by producing inhibitory Appl Biochem Biotechnol (2009) 154:302–303 DOI 10.1007/s12010-009-8613-0

Introduction to Session 1B

Enzymes are clearly recognized as a keystone technology for the production of fuels and chemicals from renewable feedstocks. Their specificity, performance under mild reaction conditions, and biodegradability make them ideally suited to widespread use in biorefineries around the world, and as the world puts greater and greater value on sustainable processes and environmentally friendly production methods, the further the development of enzyme technology grows in importance. This session focuses on the discovery, production, modification, and use of enzymes by bringing together 6 oral and 64 poster presentations describing the state of the art in enzyme technology.

Improved Dechlorination of Industrial Waste Streams Utilizing Molecular Evolution

A number of biochemical mechanisms for dehalogenation have been identified in microbial flora. Removal of halogen substituents and ultimate mineralization of the available carbon is performed by individual organisms and by consortia in soil and marine environments. Discovery of the genes coding for the individual dehalogenating reactions and laboratory evolution to optimize the phenotypes of the enzymes holds significant promise for the use of these activities in targeted remediation efforts. A collaborative effort by Dow Chemical Company and Diversa Corporation focused on the dechlorination of a process byproduct, trichloropropane (TCP), and its conversion to a valuable synthetic starting material using a haloalkane dehalogenase. The gene for this enzyme, isolated from a Rhodococcus rhodochrous strain, catalyzed the hydrolytic dehalogenation of TCP to dichlorohalohydrin (DCH). However, the activity of this enzyme was too low for recovery of DCH from TCP (turnover of 0.15/second). In addition, the enzyme was competitively inhibited by DCH. Using laboratory evolution techniques the thermal stability of this enzyme was improved over 30,000-fold, enabling the use of this catalyst at elevated temperatures and, thus, at significantly higher rates. Additionally, new haloalkane dehalogenases were discovered using a combination of environmental biopanning and ultrahigh throughput expression screening. These enzymes differed from each other with respect to substrate specificity, rate, stability, and, most importantly for the process, in degree of product inhibition.