Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

Mutagenic Effect during Combined Exposure to Ionizing and Non-Ionizing Electromagnetic Radiation.

Using the method of dominant lethal mutations, we assessed the frequency of the death of Drosophila melanogaster embryos under combined exposure to ionizing γ-radiation and non-ionizing pulsed magnetic field at various doses and modes of exposure. Mutagenic effect of combined exposure is antagonistic in nature. The antagonism is more pronounced when the following mode of exposure was used: exposure to non-ionizing pulsed magnetic field for 5 h followed by exposure to γ-radiation at doses of 3, 10, and 60 Gy. In case of reverse sequence of exposures, the antagonistic effect was statistically significant after exposure to γ-radiation at doses of 3 and 10 Gy, whereas at a dose of 20 Gy, a synergistic interaction was noted.

Asymmetric repair of UV damage in nucleosomes imposes a DNA strand polarity on somatic mutations in skin cancer.

Nucleosomes inhibit excision repair of DNA damage caused by ultraviolet (UV) light, and it has been generally assumed that repair inhibition is equivalent on both sides of the nucleosome dyad. Here, we use genome-wide repair data to show that repair of UV damage in nucleosomes is asymmetric. In yeast, nucleosomes inhibit nucleotide excision repair (NER) of the nontranscribed strand (NTS) of genes in an asymmetric manner, with faster repair of UV damage occurring on the 5' side of the nucleosomal DNA. Analysis of genomic repair data from UV-irradiated human cells indicates that NER activity along the NTS is also elevated on the 5' side of nucleosomes, consistent with the repair asymmetry observed in yeast nucleosomes. Among intergenic nucleosomes, repair activity is elevated on the 5' side of both DNA strands. The distribution of somatic mutations in nucleosomes shows the opposite asymmetry in NER-proficient skin cancers, but not in NER-deficient cancers, indicating that asymmetric repair of nucleosomal DNA imposes a strand polarity on UV mutagenesis. Somatic mutations are enriched on the relatively slow-repairing 3' side of the nucleosomal DNA, particularly at positions where the DNA minor groove faces away from the histone octamer. Asymmetric repair and mutagenesis are likely caused by differential accessibility of the nucleosomal DNA, a consequence of its left-handed wrapping around the histone octamer.

PKA-mediated phosphorylation of ATR promotes recruitment of XPA to UV-induced DNA damage.

The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals.

Whole-exome sequencing reveals the impact of UVA light mutagenesis in xeroderma pigmentosum variant human cells.

UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-exome sequencing. In UVA-irradiated cells, the increase in the mutation frequency in deficient cells included a remarkable contribution of C>T transitions, mainly at potential pyrimidine dimer sites. A strong contribution of C>A transversions, potentially due to oxidized bases, was also observed in non-irradiated XP-V cells, indicating that basal mutagenesis caused by oxidative stress may be related to internal tumours in XP-V patients. The low levels of mutations involving T induced by UVA indicate that pol eta is not responsible for correctly replicating T-containing pyrimidine dimers, a phenomenon known as the 'A-rule'. Moreover, the mutation signature profile of UVA-irradiated XP-V cells is highly similar to the human skin cancer profile, revealing how studies involving cells deficient in DNA damage processing may be useful to understand the mechanisms of environmentally induced carcinogenesis.

The cold - resistance mechanism of a mutagenic Volvariella volvacea strain VH3 with outstanding traits revealed by transcriptome profiling.

BACKGROUND: The straw mushroom (Volvariella volvacea) is one of the important vegetables that is popular for its delicious taste. However, the straw mushroom is sensitive to low temperature, resulting in economic loss during transportation and storage. We obtained a novel straw mushroom strain, named VH3, via ultraviolet mutagenesis. RESULTS: Our study revealed that VH3 exhibited high cold resistance compared to an ordinary straw mushroom cultivar, V23. We found that the electrolyte leakages of VH3 were always significantly lower than that of V23 treated with 4 °C for 0 h, 2 h,4 h, 8 h, 16 h, and 24 h. Before cold treatment (0 h), there were no difference of MDA contents, SOD activities, and CAT activities between VH3 and V23. At the late stage (8 h, 26 h, and 24 h) of cold treatment, the MDA contents of VH3 were lower while both the SOD and CAT activities were higher than those of V23. To investigate the potential mechanisms of VH3 cold resistance, we performed transcriptome sequencing to detect the transcriptome profiling of VH3 and V23 after 0 h and 4 h cold treatment. Transcriptome sequencing revealed that 111 differentially expressed genes (DEG) between V23 (0 h) and VH3 (0 h) (V23-0_vs_VH3-0), consisting 50 up-regulated and 61 down-regulated DEGs. A total of 117 DEGs were obtained between V23 (4 h) and VH3(4 h) (V23-4_vs_VH3-4), containing 94 up-regulated and 23 down-regulated DEGs. Among these DEGs, VVO_00021 and VVO_00017 were up-regulated while VVO_00003, VVO_00004, VVO_00010, and VVO_00030 were down-regulated in V23-0_vs_VH3-0 and VH3-4_vs_V23-4. KEGG and GO analysis revealed that the 6 DEGs were annotated to pathways related to cold stress. Besides, the GA3 content was also decreased in VH3. CONCLUSIONS: Collectively, our study first revealed that the increased cold resistance of VH3 might be caused by the expression change of VVO_00003, VVO_00004, VVO_00017, VVO_00021, and VVO_00030, and decreased GA3.

Recurrent Noncoding Mutations in Skin Cancers: UV Damage Susceptibility or Repair Inhibition as Primary Driver?

Somatic mutations arising in human skin cancers are heterogeneously distributed across the genome, meaning that certain genomic regions (e.g., heterochromatin or transcription factor binding sites) have much higher mutation densities than others. Regional variations in mutation rates are typically not a consequence of selection, as the vast majority of somatic mutations in skin cancers are passenger mutations that do not promote cell growth or transformation. Instead, variations in DNA repair activity, due to chromatin organization and transcription factor binding, have been proposed to be a primary driver of mutational heterogeneity in melanoma. However, as discussed in this review here, recent studies indicate that chromatin organization and transcription factor binding also significantly modulate the rate at which UV lesions form in DNA. The authors propose that local variations in lesion susceptibility may be an important driver of mutational hotspots in melanoma and other skin cancers, particularly at binding sites for ETS transcription factors.

Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2'-guanosine.

DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2'-guanosine (8OG) by Family X Polymerase µ (Pol µ) in steady-state kinetics and cell-based assays. Pol µ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol µ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol µ active site with none of the DNA substrate distortions observed for Family X siblings Pols ß or λ. Kinetic characterization of template 8OG bypass indicates that Pol µ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

Biochemical reconstitution of UV-induced mutational processes.

We reconstituted two biochemical processes that may contribute to UV-induced mutagenesis in vitro and analysed the mutational profiles in the products. One process is translesion synthesis (TLS) by DNA polymerases (Pol) δ, η and ζ, which creates C>T transitions at pyrimidine dimers by incorporating two dAMPs opposite of the dimers. The other process involves spontaneous deamination of cytosine, producing uracil in pyrimidine dimers, followed by monomerization of the dimers by secondary UV irradiation, and DNA synthesis by Pol δ. The mutational spectrum resulting from deamination without translesion synthesis is similar to a mutational signature found in melanomas, suggesting that cytosine deamination encountered by the replicative polymerase has a prominent role in melanoma development. However, CC>TT dinucleotide substitution, which is also commonly observed in melanomas, was produced almost exclusively by TLS. We propose that both TLS-dependent and deamination-dependent mutational processes are likely involved in UV-induced melanoma development.

Mutagenic Effects of a 2-Deoxyribonolactone-Thymine Glycol Tandem DNA Lesion in Human Cells.

Tandem DNA lesions containing two contiguously damaged nucleotides are commonly formed by ionizing radiation. Their effects on replication in mammalian cells are largely unknown. Replication of isolated 2-deoxyribonolactone (L), thymine glycol (Tg), and tandem lesion 5'-LTg was examined in human cells. Although nearly 100% of Tg was bypassed in HEK 293T cells, L was a significant replication block. 5'-LTg was an even stronger replication block with 5% TLS efficiency. The mutation frequency (MF) of Tg was 3.4%, which increased to 3.9% and 4.8% in pol ι- and pol κ-deficient cells, respectively. An even greater increase in the MF of Tg (to ∼5.5%) was observed in cells deficient in both pol κ and pol ζ, suggesting that they work together to bypass Tg in an error-free manner. Isolated L bypass generated 12-18% one-base deletions, which increased as much as 60% in TLS polymerase-deficient cells. The fraction of deletion products also increased in TLS polymerase-deficient cells upon 5'-LTg bypass. In full-length products and in all cell types, dA was preferentially incorporated opposite an isolated L as well as when it was part of a tandem lesion. However, misincorporation opposite Tg increased significantly when it was part of a tandem lesion. In wild type cells, targeted mutations increased about 3-fold to 9.7% and to 17.4, 15.9, and 28.8% in pol κ-, pol ζ-, and pol ι-deficient cells, respectively. Overall, Tg is significantly more miscoding as part of a tandem lesion, and error-free Tg replication in HEK 293T cells requires participation of the TLS polymerases.

Targeted exome sequencing of unselected heavy-ion beam-irradiated populations reveals less-biased mutation characteristics in the rice genome.

Heavy-ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost-efficient whole-exome sequencing procedure in rice, and its application to characterize an unselected population of heavy-ion beam-induced mutations. The bioinformatics pipeline identified single-nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy-ion beams at the population level. In total, 165 individual M2 lines derived from six irradiation conditions as well as eight pools from non-irradiated 'Nipponbare' controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon-ion beam-induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M2 lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing-based determination of the optimal irradiation condition for induction of mutations.

Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.

Ultraviolet radiation accelerates BRAF-driven melanomagenesis by targeting TP53.

Cutaneous melanoma is epidemiologically linked to ultraviolet radiation (UVR), but the molecular mechanisms by which UVR drives melanomagenesis remain unclear. The most common somatic mutation in melanoma is a V600E substitution in BRAF, which is an early event. To investigate how UVR accelerates oncogenic BRAF-driven melanomagenesis, we used a BRAF(V600E) mouse model. In mice expressing BRAF(V600E) in their melanocytes, a single dose of UVR that mimicked mild sunburn in humans induced clonal expansion of the melanocytes, and repeated doses of UVR increased melanoma burden. Here we show that sunscreen (UVA superior, UVB sun protection factor (SPF) 50) delayed the onset of UVR-driven melanoma, but only provided partial protection. The UVR-exposed tumours showed increased numbers of single nucleotide variants and we observed mutations (H39Y, S124F, R245C, R270C, C272G) in the Trp53 tumour suppressor in approximately 40% of cases. TP53 is an accepted UVR target in human non-melanoma skin cancer, but is not thought to have a major role in melanoma. However, we show that, in mice, mutant Trp53 accelerated BRAF(V600E)-driven melanomagenesis, and that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma. Thus, we provide mechanistic insight into epidemiological data linking UVR to acquired naevi in humans. Furthermore, we identify TP53/Trp53 as a UVR-target gene that cooperates with BRAF(V600E) to induce melanoma, providing molecular insight into how UVR accelerates melanomagenesis. Our study validates public health campaigns that promote sunscreen protection for individuals at risk of melanoma.

Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli.

It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80-100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.

DNA Copy Number Variations as Markers of Mutagenic Impact.

DNA copy number variation (CNV) occurs due to deletion or duplication of DNA segments resulting in a different number of copies of a specific DNA-stretch on homologous chromosomes. Implications of CNVs in evolution and development of different diseases have been demonstrated although contribution of environmental factors, such as mutagens, in the origin of CNVs, is poorly understood. In this review, we summarize current knowledge about mutagen-induced CNVs in human, animal and plant cells. Differences in CNV frequencies induced by radiation and chemical mutagens, distribution of CNVs in the genome, as well as adaptive effects in plants, are discussed. Currently available information concerning impact of mutagens in induction of CNVs in germ cells is presented. Moreover, the potential of CNVs as a new endpoint in mutagenicity test-systems is discussed.

Overproduction of thermoalkalophilic lipase secreted by Bacillus atrophaeus FSHM2 using UV-induced mutagenesis and statistical optimization of medium components.

Microbial enzymes of extremophilic origin serve as a vital source of stable industrial enzymes. The present study focused on overproduction of a thermoalkalophilic lipase produced by Bacillus atrophaeus FSHM2 through UV-induced random mutagenesis (5-45 min exposure to UV light) and factorial experimental design augmented to response surface methodology. Firstly, a UV-induced mutant (designated as UV-45) was developed after the exposure of wild strain to UV irradiation for 45 min which was able to secrete 3484.8 U/L lipase. Afterward, Plackett-Burman experimental approach augmented to central composite design was employed to optimize medium components (olive oil, maltose, glucose, sucrose, yeast extract, tryptone, urea, (NH4)2SO4, NaCl, CaCl2, and ZnSO4) for lipase production by the UV-45 mutant strain. The maximum lipase production of 5505.3 U/L were predicted in medium containing 5% of olive oil, 0.69% of glucose, 0.69% of sucrose, 2.5% of maltose, yeast extract (0.7 g/L), urea (0.44 g/L), (NH4)2SO4 (2.44 g/L), tryptone (1.19 g/L), NaCl (1.61 g/L), CaCl2 (3.81 g/L), and ZnSO4 (1.42 g/L). A mean value of 5161.3 ± 83.3 U/L of lipolytic activity was acquired from real experiments. To sum up, the lipolytic activity of wild type strain (1720.4 U/L) increased by 3-fold after UV-induced mutagenesis and medium components optimization (5161.3 U/L).

Melanoma genome sequencing reveals frequent PREX2 mutations.

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5-55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)--a PTEN-interacting protein and negative regulator of PTEN in breast cancer--as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.

Heavy-ion beam mutagenesis of the ectomycorrhizal agaricomycete Tricholoma matsutake that produces the prized mushroom "matsutake" in conifer forests.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom "matsutake" in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

Enhancement of epoxide hydrolase production by 60 Co gamma and UV irradiation mutagenesis of Aspergillus niger ZJB-09103.

An effective epoxide hydrolase (EH) production strain was mutagenized using 60 Co gamma and UV irradiation. Among positive mutant strains, the EH activity of C2-44 reached 33.7 U/g, which was 267% as much as that of the original Aspergillus niger ZJB-09103. Compared with the wild type, there were significant changes in morphology for C2-44, including the color of mycelia on the slants and the shape of conidial head. In addition, glucose and soybean cake were the optimal carbon and nitrogen source in terms of EH activity for the mutant C2-44 instead of soluble starch and peptone for the wild-type strain. The reaction time required to reach 99% enantiomeric excesses of (S)-epichlorohydrin from racemic substrate was shortened significantly by the mutant C2-44. This phenomenon was probably explained by the higher Vmax for hydrolysis of racemic epichlorohydrin by C2-44 compared with Aspergillus niger ZJB-09103.