Environmental DNA as a source of a novel epoxide hydrolase reacting with aliphatic terminal epoxides

We describe a convenient method for amplification of novel epoxide hydrolase-encoding genes directly from the metagenome. In a first step, small specific regions of putative epoxide hydrolase genes were amplified by using PCR with degenerate consensus primers specific for prokaryotic epoxide hydrolases, and environmental DNA as template. In a second step, the sequence obtained from one randomly selected epoxide hydrolase gene fragment served as the starting point for genome-walking PCR. This technique enabled us to recover a complete novel epoxide hydrolase gene with a GC content of 64.7%. A database search revealed that this novel gene was 44% and 43% identical to two putative epoxide hydrolases from Ralstonia metallidurans and Ralstonia eutropha, respectively, at the amino acid level, the highest among all orthologs searched. The gene, which encodes a polypeptide with a molecular mass of 34 kDa, was cloned and overexpressed in Escherichia coli. The recombinant enzyme showed hydrolyzing activity toward aliphatic terminal epoxides with chain lengths ranging from 6 to 10 carbon atoms. In all cases, the enantioselectivity of the enzyme was low. Determination of the regioselectivity coefficients α_R and α_S revealed that the oxirane ring was attacked almost exclusively at the non-substituted carbon of the R-epoxide. The preference for attack at the non-substituted ring carbon of the S-epoxide was dependent on the chain length of the substrate and ranged from 55% to 78%, resulting in a partially enantioconvergent reaction.     

Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis

Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative alpha/beta-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.     

High Diversity of Diazotrophs in the Forefield of a Receding Alpine Glacier

Forefields of receding glaciers are unique and sensitive environments representing natural chronosequences. In such habitats, microbial nitrogen fixation is of particular interest since the low concentration of bioavailable nitrogen is one of the key limitations for growth of plants and soil microorganisms. Asymbiotic nitrogen fixation in the Damma glacier (Swiss Central Alps) forefield soils was assessed using the acetylene reduction assay. Free-living diazotrophic diversity and population structure were resolved by assembling four NifH sequence libraries for bulk and rhizosphere soils at two soil age classes (8- and 70-year ice-free forefield). A total of 318 NifH sequences were analyzed and grouped into 45 unique phylotypes. Phylogenetic analyses revealed a higher diversity as well as a broader distribution of NifH sequences among phylogenetic clusters than formerly observed in other environments. This illustrates the importance of free-living diazotrophs and their potential contribution to the global nitrogen input in this nutrient-poor environment. NifH diversity in bulk soils was higher than in rhizosphere soils. Moreover, the four libraries displayed low similarity values. This indicated that both soil age and the presence of pioneer plants influence diversification and population structure of free-living diazotrophs.     

Preliminary Study on Effects of Glacial Retreat on the Dominant Glacial Snow Bacteria in Laohugou Glacier No. 12

One impact of climate change is the rapid shrinking of glaciers, resulting in microorganisms deposited into glacial snow or ice being exposed to new environments such as glacier foreland. A pyrosequencing analysis based on the bacterial 16S rRNA gene showed that bacterial diversity was the highest in proglacial soil, followed by that of glacial snow in ablation zone, then by that of glacial snow in the accumulation area, finally by that of glacial snow in glacier terminus, with the combination of Chao1, ACE, Shannon and Simpson analysis. Eighteen phyla were detected from the 7 samples, but mainly composed of Proteobacteria, Actinobacteria and Bacteroidetes. Flavobacterium, Massilia, Pedobacter, Polaromonas were more abundant in glacial snow samples than in glacial soil sample. Massilia was rarely reported in other environments, implying the necessity for its conservation under scenarios of glacier and snowpack loss induced by climate change.     

来源于嗜热氢化杆菌的新型对苯二甲酸双(羟乙)酯水解酶的表达纯化与酶学性质

石化来源的聚对苯二甲酸乙二酯(polyethylene terephthalate,PET)被广泛用于矿泉水瓶、食品包装和纺织品等领域,因其在自然界中不易分解,大量使用后的PET废弃物造成了严重的环境污染与资源浪费。使用生物酶法对PET废弃物进行解聚,并对解聚产物进行升级循环利用是进行塑料污染治理的重要方向之一,其中关键的是PET水解酶的解聚效率。对苯二甲酸双(羟乙基)酯(bis(hydroxyethyl)terephthalate,BHET)是PET生物酶解的中间产物,其累积是限制PET水解酶催化效率的一个重要因素,BHET水解酶和PET水解酶的联用能提升PET的整体水解效率。来源于嗜热氢化杆菌(Hydrogenobacter thermophilus)的双烯内酯酶(HtBHETase)对BHET有显著水解效果,将该酶在大肠杆菌(Escherichia coli)中进行重组表达并纯化后,对其酶学性质进行了研究。结果显示,HtBHETase对短碳链的酯类如对硝基苯酚乙酸酯催化活性较高,HtBHETase以BHET为底物时的最适反应pH值和最适反应温度分别为5.0和55℃;该酶有较好的热稳定性,经80℃的条件处理1 h仍能保持80%以上活性,显示出了良好的热稳定性,HtBHETase有在PET塑料生物解聚中使用的潜力,本研究为推动生物酶法降解PET提供了新的参考。     

A mono-2-ethylhexyl phthalate hydrolase from a Gordonia sp. that is able to dissimilate di-2-ethylhexyl phthalate

Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K(m) and V(max) values for mono-2-ethylhexyl phthalate were 26.9 +/- 4.3 microM and 18.1 +/- 0.9 micromol/min . mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.     

A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample

Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100 degrees C and pH 6.0, and was extremely thermostable at 90 degrees C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.     

Compositional profile of α / β‐hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil‐contaminated sites

The occurrence of genes encoding biotechnologically relevant α/β‐hydrolases in mangrove soil microbial communities was assessed using data obtained by whole‐metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β‐hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ～ 23%), microsomal hydrolases (abH09; ～ 12%) and Moraxella lipase‐like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01‐02‐03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus‐Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already‐described genes opens perspectives for both production in an expression host and genetic screening of metagenomes.     

Relative Incidence of Ascomycetous Yeasts in Arctic Coastal Environments

Previous studies of fungi in polar environments have revealed a prevalence of basidiomycetous yeasts in soil and in subglacial environments of polythermal glaciers. Ascomycetous yeasts have rarely been reported from extremely cold natural environments, even though they are known contaminants of frozen foods. Using media with low water activity, we have isolated various yeast species from the subglacial ice of four glaciers from the coastal Arctic environment of Kongsfjorden, Spitzbergen, including Debaryomyces hansenii and Pichia guillermondii, with counts reaching 10⁴ CFU L⁻¹. Together with the basidiomycetes Cryptococcus liquefaciens and Rhodotorula mucilaginosa, these yeasts represent the stable core of the subglacial yeast communities. Other glacial ascomycetous species isolated included Candida parapsilosis and a putative new species that resembles Candida pseudorugosa. The archiascomycete Protomyces inouyei has seldom been detected anywhere in the world but was here recovered from ice in a glacier cave. The glacier meltwater contained only D. hansenii, whereas the seawater contained D. hansenii, Debaryomyces maramus, Pichia guilliermondii, what appears to represent a novel species resembling Candida galli and Metschnikowia bicuspidata. Only P. guilliermondii was isolated from sea ice, while snow/ice in the fjord tidal zone included C. parapsilosis, D. hansenii, P. guilliermondii and Metschnikowia zobellii. All of these isolated strains were characterized as psychrotolerant and xero/halotolerant, with the exception of P. inouyei.     

A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films

TfCut2 from Thermobifida fusca KW3 and the metagenome‐derived LC‐cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono‐(2‐hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC‐cutinase produced a 2.4‐fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.     

Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica

Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50% residual activity). Its K_m value was 256 μM and its catalytic efficiency was 81.7 s⁻¹. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.

     

Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).     

Structural and functional characterization of a novel α/β hydrolase from cariogenic pathogen Streptococcus mutans

The protein Smu.1393c from Streptococcus mutans is annotated as a putative α/β hydrolase, but it has low sequence identity to the structure‐known α/β hydrolases. Here we present the crystal structure of Smu.1393c at 2.0 Å resolution. Smu.1393c has a fully open alkaline substrate pocket, whose conformation is unique among other similar hydrolase structures. Three residues, Ser101, His251, and Glu125, were identified as the active center of Smu.1393c. By screening a series of artificial hydrolase substrates, we demonstrated Smu.1393c had low carboxylesterase activity towards short‐chain carboxyl esters, which provided a clue for exploring the in vivo function of Smu.1393c. Proteins 2014; 82:695–700. © 2013 Wiley Periodicals, Inc.     

Influence of Deglaciation on Microbial Communities in Marine Sediments Off the Coast of Svalbard,Arctic Circle

Increases in global temperatures have been shown to enhance glacier melting in the Arctic region. Here, we have evaluated the effects of meltwater runoff on the microbial communities of coastal marine sediment located along a transect of Temelfjorden, in Svalbard. As close to the glacier front, the sediment properties were clearly influenced by deglaciation. Denaturing gradient gel electrophoresis profiles showed that the sediment microbial communities of the stations of glacier front (stations 188–178) were distinguishable from that of outer fjord region (station 176). Canonical correspondence analysis indicated that total carbon and calcium carbonate in sediment and chlorophyll a in bottom water were key factors driving the change of microbial communities. Analysis of 16S rRNA gene clone libraries suggested that microbial diversity was higher within the glacier–proximal zone (station 188) directly affected by the runoffs than in the outer fjord region. While the crenarchaeotal group I.1a dominated at station 176 (62%), Marine Benthic Group-B and other Crenarchaeota groups were proportionally abundant. With regard to the bacterial community, alpha-Proteobacteria and Flavobacteria lineages prevailed (60%) at station 188, whereas delta-Proteobacteria (largely sulfate-reducers) predominated (32%) at station 176. Considering no clone sequences related to sulfate-reducers, station 188 may be more oxic compared to station 176. The distance-wise compositional variation in the microbial communities is attributable to their adaptations to the sediment environments which are differentially affected by melting glaciers.     

Glycoside hydrolases as components of putative carbohydrate biosensor proteins in <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The composition of the cellulase system in the cellulosome-producing bacterium, Clostridium thermocellum, has been reported to change in response to growth on different carbon sources. Recently, an extensive carbohydrate-sensing mechanism, purported to regulate the activation of genes coding for polysaccharide-degrading enzymes, was suggested. In this system, CBM modules, comprising extracellular components of RsgI-like anti-σ factors, were proposed to function as carbohydrate sensors, through which a set of cellulose utilization genes are activated by the associated σ^I-like factors. An extracellular module of one of these RsgI-like proteins (Cthe_2119) was annotated as a family 10 glycoside hydrolase, RsgI6-GH10, and a second putative anti-σ factor (Cthe_1471), related in sequence to Rsi24, was found to contain a module that resembles a family 5 glycoside hydrolase (termed herein Rsi24C-GH5). The present study examines the relevance of these two glycoside hydrolases as sensors in this signal-transmission system. The RsgI6-GH10 was found to bind xylan matrices but exhibited low enzymatic activity on this substrate. In addition, this glycoside hydrolase module was shown to interact with crystalline cellulose although no hydrolytic activity was detected on cellulosic substrates. Bioinformatic analysis of the Rsi24C-GH5 showed a glutamate-to-glutamine substitution that would presumably preclude catalytic activity. Indeed, the recombinant module was shown to bind to cellulose, but showed no hydrolytic activity. These observations suggest that these two glycoside hydrolases underwent an evolutionary adaptation to function as polysaccharide binding agents rather than enzymatic components and thus serve in the capacity of extracellular carbohydrate sensors.     

A Mono-2-Ethylhexyl Phthalate Hydrolase from a Gordonia sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate

Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K_m and V_max values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.     

Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367

Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon‐neutral biofuels from non‐food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9‐deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367.     

Genetic diversity detection and gene discovery of novel glycoside hydrolase family 48 from soil environmental genomic DNA

Sequence diversity within a family of functional enzymes provides a platform for novel gene development and protein engineering to improve the properties of these enzymes for further applications. Glycoside hydrolase family 48 (GH48) is an important group of microbial cellulases. However, the genetic diversity and gene discovery of GH48 enzyme in natural environments are rarely reported. In this study, the genetic diversity of GH48 from Changbai Mountain soil was evaluated by building a clone library via a culture-independent molecular method for the first time. Results showed that the genetic diversity of GH48 in Changbai Mountain soil was different from that in thermophilic compost and marine sediment libraries, and more than 80% of the sequences exhibited the highest identity with cellulase genes from Chloroflexi. Novel GH48 genes were also cloned, and the recombinants Cel48_hm01 and Cel48_hm02 were prokaryotically expressed, purified, and characterized. Characterization results suggested that they were probably endocellulases that adopted a catalytic mechanism similar to the GH48 cellulase from Clostridium. This study revealed the genetic distribution of glycoside hydrolases in soil environment, described Changbai Mountain soil as a valuable source for glycoside hydrolase gene screening, and presented supplementary property data on novel GH48 from natural soil environments.     

冰川微生物菌群分布的研究概况及其前景

冰川中以耐冷的生物为主,形成一个以微生物为主要生命形式的相对简单的生态系统.冰川中的微生物包括病毒、细菌、放线菌、丝状真菌、酵母菌和藻类.其中一些病毒对人类健康具有潜在的危害性.着重论述了不同区域和不同海拔高度的冰川微生物类群和数量分布特征以及冰芯（深冰川）细菌菌群分布与气候环境的关系.综述结果表明：一些微生物类群广泛存在于各地的冰川上,具有全球分布特性;另一些类群只出现在个别冰川上,为一些地方性冰川微生物.随着海拔高度的增加,冰川上呈现出冰、雪冰和雪环境明显不同的生态条件;微生物类群分布也具有明显的差异性,与冰川上的生态条件和盛行的风向有关.优势类群对冰、雪冰和雪环境具有一定的指示意义.冰川微生物数量分布不仅受到冰川上的水热、光照和营养状况的影响,还与降雪的沉积作用有关.冰芯中的细菌数量与矿物微粒含量具有密切的对应关系.最后指出了冰川微生物研究在基因多样性、气候环境变化、生物地球化学循环、微生物对环境变化的响应机制和星际生命探索中的重要性及其生态学和社会经济意义.     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.