Characterization of alcohol dehydrogenase 1 and 3 from <Emphasis Type="Italic">Neurospora crassa</Emphasis> FGSC2489

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.     

Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5

BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.     

Characterization of four Rhodococcus alcohol dehydrogenase genes responsible for the oxidation of aromatic alcohols

Four genes were isolated and characterized for alcohol dehydrogenases (ADHs) catalyzing the oxidation of aromatic alcohols such as benzyl alcohol to their corresponding aldehydes, one from o-xylene-degrading Rhodococcus opacus TKN14 and the other three from n-alkane-degrading Rhodococcus erythropolis PR4. Various aromatic alcohols were bioconverted to their corresponding carboxylic acids using Escherichia coli cells expressing each of the four ADH genes together with an aromatic aldehyde dehydrogenase gene (phnN) from Sphingomonas sp. strain 14DN61. The ADH gene (designated adhA) from strain TKN14 had the ability to biotransform a wide variety of aromatic alcohols, i.e., 2-hydroxymethyl-6-methylnaphthalene, 2-hydroxymethylnaphthalene, xylene-α,α’-diol, 3-chlorobenzyl alcohol, and vanillyl alcohol, in addition to benzyl alcohol with or without a hydroxyl, methyl, or methoxy substitution. In contrast, the three ADH genes of strain PR4 (designated adhA, adhB, and adhC) exhibited lower ability to degrade these alcohols: these genes stimulated the conversion of the alcohol substrates by only threefold or less of the control value. One exception was the conversion of 3-methoxybenzyl alcohol, which was stimulated sevenfold by adhB. A phylogenetic analysis of the amino acid sequences of these four enzymes indicated that they differed from other Zn-dependent ADHs.The first two authors contributed equally to this work     

Evolution of the alcohol dehydrogenase (ADH) genes in yeast: characterization of a fourth ADH in Kluyveromyces lactis

Summary Three alcohol dehydrogenase (ADH) genes have recently been characterized in the yeast Kluyveromyces lactis. We report on a fourth ADH in K. lactis (KADH II: KADH2 gene) which is highly similar to other ADHs in K. lactis and Saccharomyces cerevisiae. KADH II appears to be a cytoplasmic enzyme, and after expression of KADH2 in S. cerevisiae enzyme activity comigrated with a K. lactis ADH present in cells grown in glucose or in ethanol. KADH I was also expressed in S. cerevisiae and it comigrated with a major ADH species expressed under glucose growth conditions in K. lactis. The substrate specificities for KADH I and KADH II were shown to be more similar to that of SADH II than to SADH I. SADH I cannot efficiently utilize long chain alcohols, in contrast to other cytoplasmic yeast ADHs, presumably because of the presence of a methionine (residue 271) in its substrate binding cleft. A comparison of the DNA sequences of ADHs among K. lactis, S. cerevisiae and Schizosaccharomyces pombe suggests that the ancestral yeast species contained one cytoplasmic ADH. After divergence from S. pombe, the ADH in the ancestor to K. lactis and S. cerevisiae was duplicated, and one ADH became localized to the mitochondrion, presumably for the oxidative use of ethanol. Following the speciation of S. cerevisiae and K. lactis, the gene encoding the cytoplasmic ADH in S. cerevisiae duplicated, which resulted in the development of the SADH II protein as the primary oxidative enzyme in place of SADH III. In contrast, the K. lactis mitochondrial ADH duplicated to give rise to the highly expressed KADH3 and KADH4 genes, both of which may still play primary roles in oxidative metabolism. These data suggest that K. lactis and S. cerevisiae use different compartments for their metabolism of ethanol. Our results also indicate that the complex regulatory circuits controlling the glucose-repressible SADH2 in S. cerevisiae are a recent acquisition from regulatory networks used for the control of genes other than SADH2.

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Association between common alcohol dehydrogenase gene (ADH) variants and schizophrenia and autism

Humans express at least seven alcohol dehydrogenase (ADH) isoforms that are encoded by ADH gene cluster (ADH7–ADH1C–ADH1B–ADH1A–ADH6–ADH4–ADH5) at chromosome 4. ADHs are key catabolic enzymes for retinol and ethanol. The functional ADH variants (mostly rare) have been implicated in alcoholism risk. In addition to catalyzing the oxidation of retinol and ethanol, ADHs may be involved in the metabolic pathways of several neurotransmitters that are implicated in the neurobiology of neuropsychiatric disorders. In the present study, we comprehensively examined the associations between common ADH variants [minor allele frequency (MAF) >0.05] and 11 neuropsychiatric and neurological disorders. A total of 50,063 subjects in 25 independent cohorts were analyzed. The entire ADH gene cluster was imputed across these 25 cohorts using the same reference panels. Association analyses were conducted, adjusting for multiple comparisons. We found 28 and 15 single nucleotide polymorphisms (SNPs), respectively, that were significantly associated with schizophrenia in African-Americans and autism in European-Americans after correction by false discovery rate (FDR) (q < 0.05); and 19 and 6 SNPs, respectively, that were significantly associated with these two disorders after region-wide correction by SNPSpD (8.9 × 10^?5 ≤ p  ≤ 0.0003 and 2.4 × 10^?5 ≤ p ≤ 0.0003, respectively). No variants were significantly associated with the other nine neuropsychiatric disorders, including alcohol dependence. We concluded that common ADH variants conferred risk for both schizophrenia in African-Americans and autism in European-Americans.     

Detoxification of promutagenic aldehydes derived from methylpyrenes by human aldehyde dehydrogenases ALDH2 and ALDH3A1

Methylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulpho conjugation to reactive esters, which can induce mutations and tumours. Yet, further oxidation of the alcohol may compete with this toxification. We previously demonstrated that several human alcohol dehydrogenases (ADH1C, 2, 3 and 4) oxidise various benzylic alcohols (derived from alkylated pyrenes) to their aldehydes with high catalytic efficiency. However, all these ADHs also catalysed the reverse reaction, the reduction of the aldehydes to the alcohols, with comparable or higher efficiency. Thus, final detoxification requires elimination of the aldehydes by further biotransformation. We have expressed two human aldehyde dehydrogenases (ALDH2 and 3A1) in bacteria. All pyrene aldehydes studied (1-, 2- and 4-formylpyrene, 1-formyl-6-methylpyrene and 1-formyl-8-methylpyrene) were high-affinity substrates for ALDH2 (K_m = 0.027–0.9 μM) as well as ALDH3A1 (K_m = 0.78–11 μM). Catalytic efficiencies (k_cat/K_m) were higher for ALDH2 than ALDH3A1 by a moderate to a very large margin depending on the substrate. Most important, they were also substantially higher than the catalytic efficiencies of the various ADHs for the reduction the aldehydes to the alcohols. These kinetic properties ensure that ALDHs, and particularly ALDH2, can complete the ADH-mediated detoxification.     

Genome-wide identification and comparative analysis of the ADH gene family in Chinese white pear (Pyrus bretschneideri) and other Rosaceae species

Alcohol dehydrogenase (ADH) is essential to the formation of aromatic compounds in fruits. However, the evolutionary history and characteristics of ADH gene expression remain largely unclear in Rosaceae fruit species. In this study, 464 ADH genes were identified in eight Rosaceae fruit species, 68 of the genes were from pear and which were classified into four subgroups. Frequent single gene duplication events were found to have contributed to the formation of ADH gene clusters and the expansion of the ADH gene family in these eight Rosaceae species. Purifying selection was the major force in ADH gene evolution. The younger genes derived from tandem and proximal duplications had evolved faster than those derived from other types of duplication. RNA-Seq and qRT-PCR analysis revealed that the expression levels of three ADH genes were closely correlated with the content of aromatic compounds detected during fruit development.     

Functional Characterization of a Melon Alcohol Acyl-transferase Gene Family Involved in the Biosynthesis of Ester Volatiles. Identification of the Crucial Role of a Threonine Residue for Enzyme Activity*

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon. *Accession numbers Cm-AAT1 (CAA94432), Cm-AAT2 (AAL77060), Cm-AAT3 (AAW51125), and Cm-AAT4 (AAW51126). Islam El-Sharkawy, Daniel Manriquez, Francisco B. Flores: These authors contributed equally to the work     

Role of ethylene in the biosynthetic pathway of aliphatic ester aroma volatiles in Charentais Cantaloupe melons.

Compared to other melon types, Cantaloupe Charentais melons are highly aromatic with a major contribution to the aroma being made by aliphatic and branched esters. Using a transgenic line in which the synthesis of the plant hormone ethylene has been considerably lowered by antisense ACC oxidase mRNA (AS), the aliphatic ester pathway steps at which ethylene exerts its regulatory role were found. The data show that the production of aliphatic esters such as hexyl and butyl acetate was blocked in AS fruit and could be reversed by ethylene. Using fruit discs incubated in the presence of various precursors, the steps at which ester formation was inhibited in AS fruit was shown to be the reduction of fatty acids and aldehydes, the last step of acetyl transfer to alcohols being unaffected. However, treating AS fruit with the ethylene antagonist 1-methylcyclopropene resulted in about 50% inhibition of acetyl transfer activity, indicating that this portion of activity was ethylene-dependent and this was supported by the low residual ethylene concentration of AS fruit discs (around 2 microl l(-1)). In conclusion, the reduction of fatty acids and aldehydes appears essentially to be ethylene-dependent, whilst the last step of alcohol acetylation has ethylene-dependent and ethylene-independent components, probably corresponding to differentially regulated alcohol acetyltransferases.     

Molecular and biochemical characteristics of a gene encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening.

Two genes (CM-AAT1 and CM-AAT2) with strong sequence homology (87% identity at the protein level) putatively involved in the formation of aroma volatile esters have been isolated from Charentais melon fruit. They belong to a large and highly divergent family of multifunctional plant acyl-transferases and show at most 21% identity to the only other fruit acyl-transferase characterized so far in strawberry. RT-PCR studies indicated that both genes were specifically expressed in fruit at increasing rates in the early and mid phases of ripening. Expression was severely reduced in ethylene-suppressed antisense ACC oxidase (AS) fruit and in wild-type (WT) fruit treated with the ethylene antagonist 1-MCP. Cloning of the two genes in yeast revealed that the CM-AAT1 protein exhibited alcohol acyl-transferase activity while no such activity could be detected for CM-AAT2 despite the strong homology between the two sequences. CM-AAT1 was capable of producing esters from a wide range of combinations of alcohols and acyl-CoAs. The higher the carbon chain of aliphatic alcohols, the higher the activity. Branched alcohols were esterified at differential rates depending on the position of the methyl group and the nature of the acyl donor. Phenyl and benzoyl alcohols were also good substrates, but activity varied with the position and size of the aromatic residue. The cis/trans configuration influenced activity either positively (2-hexenol) or negatively (3-hexenol). Because ripening melons evolve the whole range of esters generated by the recombinant CM-AAT1 protein, we conclude that CM-AAT1 plays a major role in aroma volatiles formation in the melon.     

Alcohol dehydrogenase (ADH) isozymes in the AdhN/AdhN strain ofPeromyscus maniculatus (ADH− deermouse) and a possible role of class III ADH in alcohol metabolism

Although the Adh^N/Adh^N strain ofPeromyscus maniculatus (so-called ADH^? deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the Adh^F/Adh^F strain (so-called ADH⁺ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH^? strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (<100 mM) greatly increased (about sevenfold), to more than 50% that of ADH⁺ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH^? deermouse. It was also found that even the ADH⁺ strain has low liver ADH activity (<40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH^? and ADH⁺ deermouse were different not only from each other but also from that in the ddY mouse; the ADH^? strain was deficient in only Class I ADH, and the ADH⁺ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH^? strain.     

Purification and characterization of two NAD-dependent alcohol dehydrogenases (ADHs) induced in the quinoprotein ADH-deficient mutant of Acetobacter pasteurianus SKU1108

High NAD-dependent alcohol dehydrogenase (ADH) activity was found in the cytoplasm when a membrane-bound, quinoprotein, ADH-deficient mutant strain of Acetobacter pasteurianus SKU1108 was grown on ethanol. Two NAD-dependent ADHs were separated and purified from the supernatant fraction of the cells. One (ADH I) is a trimer, consisting of an identical subunit of 42 kDa, while the other (ADH II) is a homodimer, having a subunit of 31 kDa. One of the two ADHs, ADH II, easily lost the activity during the column chromatographies, which could be stabilized by the addition of DTT and MgCl2 in the column buffer. ADH I but not ADH II contained approximately one zinc atom per subunit. The N-terminal amino acid analysis indicated that ADH I and ADH II have homology to the long-chain and short-chain ADH families, respectively. ADH I showed a preference for primary alcohols, while ADH II had a preference for secondary alcohols. The two ADHs showed clear difference in their kinetics on ethanol, acetaldehyde, NAD, and NADH. The physiological function of both ADH I and ADH II are also discussed.     

甜瓜乙烯应答因子基因在果实发育成熟过程中的表达特性

根据已报道的甜瓜CMe-ERF1和CMe-ERF2基因cDNA序列设计合成特异性引物,应用RT-PCR技术从甜瓜品种‘河套蜜瓜’成熟果实中克隆得到CMe-ERF1和CMe-ERF2基因cDNA全长编码区序列,分别为498bp和822bp.序列比对分析表明,得到的cDNA序列与已报道的Andes甜瓜相应基因的cDNA序列完全一致.果实不同发育时期实时定量PCR检测结果表明,CMe-ERF1、CMe-ERF2基因表达与甜瓜果实成熟及乙烯生成量显著相关,表明该基因可能对果实成熟起重要作用.     

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives.     

Genetic Manipulation of Alcohol Dehydrogenase Levels in Ripening Tomato Fruit Affects the Balance of Some Flavor Aldehydes and Alcohols

Tomato (Lycopersicon esculentum) plants were transformed with gene constructs containing a tomato alcohol dehydrogenase (ADH) cDNA (ADH 2) coupled in a sense orientation with either the constitutive cauliflower mosaic virus 35S promoter or the fruit-specific tomato polygalacturonase promoter. Ripening fruit from plants transformed with the constitutively expressed transgene(s) had a range of ADH activities; some plants had no detectable activity, whereas others had significantly higher ADH activity, up to twice that of controls. Transformed plants with fruit-specific expression of the transgene(s) also displayed a range of enhanced ADH activities in the ripening fruit, but no suppression was observed. Modified ADH levels in the ripening fruit influenced the balance between some of the aldehydes and the corresponding alcohols associated with flavor production. Hexanol and Z-3-hexenol levels were increased in fruit with increased ADH activity and reduced in fruit with low ADH activity. Concentrations of the respective aldehydes were generally unaltered. The phenotypes of modified fruit ADH activity and volatile abundance were transmitted to second-generation plants in accordance with the patterns of inheritance of the transgenes. In a preliminary taste trial, fruit with elevated ADH activity and higher levels of alcohols were identified as having a more intense “ripe fruit” flavor.     

1-MCP对‘粉红女士’苹果冷藏期间品质变化和香气形成的影响

以'粉红女士'苹果为试验材料,研究了1 μL/L 1-MCP(1-甲基环丙烯)对苹果冷藏期间乙烯释放速率、呼吸速率、果实硬度以及香气成分和相对含量的影响.结果表明,1-MCP处理可显著抑制'粉红女士'苹果冷藏期间呼吸作用和乙烯释放,有效延缓果实硬度的下降.冷藏期内'粉红女士'苹果香气物质主要有醇类、醛类、酯类、烯类、酸类和烷烃类等,并以酯类香气为主(占46.15%);1-MCP能显著减少果实贮藏期间酯类、醇类和烷烃类香气成分种类和相对含量,处理果中酯类和醇类香气成分种类比同期对照分别减少了50%和78%,主要香气成分丁酸己酯在处理和对照果实的相对含量分别为1.12%～1.73%和1.87%～5.18%.可见,1-MCP处理对'粉红女士'苹果具有良好保鲜效果,也显著地抑制了贮藏期间香气的形成.     

Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea

Haloarchaeal alcohol dehydrogenases are of increasing interest as biocatalysts in the field of white biotechnology. In this study, the gene adh12 from the extreme halophile Haloarcula marismortui (HmADH12), encoding a 384 residue protein, was cloned into two vectors: pRV1 and pTA963. The resulting constructs were used to transform host strains Haloferax volcanii (DS70) and (H1209), respectively. Overexpressed His-tagged recombinant HmADH12 was purified by immobilized metal-affinity chromatography (IMAC). The His-tagged protein was visualized by SDS-PAGE, with a subunit molecular mass of 41.6 kDa, and its identity was confirmed by mass spectrometry. Purified HmADH12 catalyzed the interconversion between alcohols and aldehydes and ketones, being optimally active in the presence of 2 M KCl. It was thermoactive, with maximum activity registered at 60°C. The NADP(H) dependent enzyme was haloalkaliphilic for the oxidative reaction with optimum activity at pH 10.0. It favored a slightly acidic pH of 6.0 for catalysis of the reductive reaction. HmADH12 was significantly more tolerant than mesophilic ADHs to selected organic solvents, making it a much more suitable biocatalyst for industrial application.     

Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 °C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1 showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols. Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to 36.7 g l⁻¹. Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain.