Rosalind A. Reeves

Multidomain and Multifunctional Glycosyl Hydrolases from the Extreme Thermophile Caldicellulosiruptor Isolate Tok7B.1

Abstract. DNA sequencing techniques have revealed widespread molecular diversity of the genomic organization of apparently closely related bacteria (as judged from SSU rDNA sequence similarity). We have previously described the extreme thermophile Caldicellulosiruptor saccharolyticus, which is unusual in possessing multi-catalytic, multidomain arrangements for the majority of its glycosyl hydrolases. We report here the sequencing of three gene clusters of glycosyl hydrolases from Caldicellulosiruptor sp. strain Tok7B.1. These clusters are not closely linked, and each is different in its organization from any described for Cs. saccharolyticus. The catalytic domains of the enzymes belong to glycosyl hydrolase families 5, 9, 10, 43, 44, and 48. The cellulose binding domains (CBDs) of these enzymes from Caldicellulosiruptor sp. Tok7B.1 are types IIIb, IIIc, or VI. A number of individual catalytic and binding domains have been expressed in Escherichia coli, and biochemical data are reported on the purified enzymes for cellulose degradation encoded by engineered derivatives of celB and celE.

Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a β-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant β-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (kcat/KM) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (kcat) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (Ki) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.

Sequencing and Expression of a β-Mannanase Gene from the Extreme Thermophile Dictyoglomus thermophilum Rt46B.1, and Characteristics of the Recombinant Enzyme

Abstract. A β-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.1. ManA is a single-domain enzyme related to one group of β-mannanases (glycosyl hydrolase family 26). The manA gene was expressed in the heat-inducible vector pJLA602 and the expression product, ManA, purified to homogeneity. The recombinant ManA is a monomeric enzyme with a molecular mass of 40 kDa and an optimal temperature and pH for activity of 80°C and 5.0. In the absence of substrate, the enzyme showed no loss of activity at 80°C over 16 h, while at 90°C the enzyme had a half-life of 5.4 min. Hydrolysis of the galactomannan locust bean gum (LBG) by purified ManA released mainly mannose, mannobiose, and mannotriose, confirming that ManA is an endo-acting β-mannanase. Sequence comparisons with related β-mannanases has allowed the design of consensus PCR primers for the identification and isolation of related genes.

Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1.

ABSTRACT Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking–PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87°C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity toxynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.

Heterogeneity of SSU and LSU rDNA sequences of Alexandrium species

With the growth of aquaculture, toxic phytoplankton have become a significant problem. At present, the identification of specific algal species within blooms requires considerable expertise, and the methods are both costly and time consuming. It is clear that alternative diagnostic tests must be developed that allow rapid and simple assessments to be made on site. We report on the identification of unique sequences within the SSU and LSU rDNA genes of Alexandrium species that provide data for the design of species- and genus-specific molecular probes. In addition we examine some aspects of the phylogeny of Alexandrium isolates from New Zealand waters.

A gene encoding a thermophilic alkaline serine proteinase from Thermus sp. strain Rt41A and its expression in Escherichia coli

The extreme thermophile Thermus sp. strain Rt41A produces an extracellular alkaline serine proteinase during growth. This enzyme is stable for more than 24 h at 70 degrees C and has a pH optimum of 8.0. The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene. The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino-terminal sequencing of purified Rt41A proteinase. Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from Thermus aquaticus YT-1 and with exoprotease A from Vibrio alginolyticus. The Rt41A proteinase was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein. The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present.

Alteration of the pH optimum of a family 11 xylanase, XynB6 of Dictyoglomus thermophilum

We reported previously that the activities of several glycosyl hydrolase family 11 xylanases claimed to be active under alkaline conditions, were found to have optima in the pH 5-6 range when assayed under optimal conditions. One enzyme, BadX, had enhanced activity at pHs greater than 7 compared to other family 11 xylanases. Gene shuffling between badX and Dictyoglomus thermophilum xynB6 was performed in an attempt to elucidate regions conferring alkaline activity to BadX, and potentially, to increase the alkaline activity of the highly thermophilic XynB6. Segment substitution using degenerate oligonucleotide gene shuffling (DOGS) experiments with combinations of input parental gene fragments from xynB6 and badX was not able to improve the activity of XynB6 at alkaline pH. With one exception, the replacement of a single segment of BadX with the equivalent segment from XynB6 reduced the alkaline activity BadX. The results indicate that it might not be possible to alter significantly the alkaline pH characteristics of family 11 xylanases by one or a few mutations and that family 11 xylanases showing enhanced activity at alkaline pHs require multiple sequence adaptations across the protein.

The activity of family 11 xylanases at alkaline pH

Xylanases have several industrial uses, particularly in baking, modification of animal feed and in pulp bleaching in the paper industry. Process conditions in kraft pulp bleaching generally favour an enzyme that is active at high pH values. The activities of several glycosyl hydrolase family 11 xylanases reported to be active under alkaline conditions were determined under optimal conditions and found to have optima in the pH 5-6 range. Only one enzyme tested, BadX, was shown to have an alkaline pH optimum. Significant activity at pH values higher than 8 appears often to be the result of excess enzyme added to the reaction mixtures so that substrate is limiting. The different nature of laboratory and industrial substrates needs to be taken into consideration in designing assay conditions. In some cases, significant differences were observed in pH profiles generated using a small-molecule substrate when compared to those generated using xylan. We conclude that small-molecule substrates are not a suitable proxy for determining the pH profiles of family 11 xylanases.

Molecular diversity and catalytic activity of Thermus DNA polymerases

Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s. We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22 representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5′–3′ exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction.