Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties

Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO₄.7H₂O, 0.05% KCl, 0.2% K₂HPO₄ and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme retained more than 90% of its activity. Enzyme activity was inhibited by Hg²⁺ and K⁺, whereas Ca²⁺ and Mn²⁺ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity.     

Alternansucrase mutants of Leuconostoc mesenteroides strain NRRL B-21138

Alternan is a unique α-D-glucan of potential commercial interest, produced by rare strains of Leuconostoc mesenteroides. Natural isolates that produce alternan, such as NRRL B-1355, also produce dextran as a troublesome contaminant. We previously isolated mutants of strain NRRL B-1355 that are deficient in dextran production, including the highly stable strain NRRL B-21138. In the current work, we mutagenized strain NRRL B-21138 and screened survivors for further alterations in production of alternansucrase, the enzyme that catalyzes the synthesis of alternan from sucrose. Second generation mutants included highly stable strain NRRL B-21297, which produced four-fold elevated levels of alternansucrase without an increase in the proportion of dextransucrase activity. Such alternansucrase overproducing strains will facilitate studies of this enzyme, and may become valuable for the enzymatic production of alternan. Another highly stable mutant strain, NRRL B-21414, grew slowly on sucrose with negligible production of glucan or extracellular glucansucrase activity. This strain may prove useful as an expression host for glucansucrase genes. Received 30 July 1996/ Accepted in revised form 15 December 1996     

Production of L-leucine aminopeptidase by selected Streptomyces isolates

Aims: To screen various Streptomyces cultures producing l ‐leucine aminopeptidase (LAP). Methods and Results: Twenty‐one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B‐3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8·0–8·5 and the temperature optima varied between 50 and 65°C. LAP of S. mobaraensis was stable at 60°C and pH 8·5 for 60 min. Metal ion salts, CoCl₂.6H₂O and ZnSO₄.7H₂O in 0·7 mmol l^?1 concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. Conclusions: Streptomyces mobaraensis NRRL B‐3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. Significance and Impact of the Study: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N‐terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Role of the pectinolytic enzyme in the lactic acid fermentation of potato pulp by<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis>

Rhizopus oryzae strain NBRC 4707 produced lactic acid and ethanol more efficiently than strain NRRL 395 in potato pulp, an agricultural by-product of the starch industry. The two strains developed comparable activities of xylanase, cellulase, -amylase, and glucoamylase, while the polygalacturonase activity of strain NBRC 4707 was double that of strain NRRL 395. The addition of commercial pectinase enhanced the formation of metabolites, suggesting that the degradation of pectic substances determines the fermentation of potato pulp by R. oryzae. Orange and apple peel were more effective in the induction of polygalacturonase activity than potato pulp, sugarbeet pulp, or wheat bran when used as a principal carbon source for fungal growth in a solid-state culture. The fungal cells in both types of fruit peel stimulated the fermentation of potato pulp and increased the quantity of lactic acid and ethanol to higher levels than those in other agricultural by-products.     

Two New Native β-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast

Yeast strain Clavispora NRRL Y-50464 is able to produce cellulosic ethanol from lignocellulosic materials without addition of external β-glucosidase by simultaneous saccharification and fermentation. A β-glucosidase BGL1 protein from this strain was recently reported supporting its cellobiose utilization capability. Here, we report two additional new β-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain NRRL Y-50464. Quantitative gene expression was analyzed and the gene function of BGL2 and BGL3 was confirmed by heterologous expression using cellobiose as a sole carbon source. Each gene was cloned and partially purified protein obtained separately for direct enzyme assay using varied substrates. Both proteins showed the highest specific activity at pH 5 and relatively strong affinity with a K_m of 0.08 and 0.18 mM for BGL2 and BGL3, respectively. The optimum temperature was found to be 50°C for BGL2 and 55°C for BGL3. Both proteins were able to hydrolyze 1,4 oligosaccharides evaluated in this study. They also showed a strong resistance to glucose product inhibition with a K_i of 61.97 and 38.33 mM for BGL2 and BGL3, respectively. While BGL3 was sensitive showing a significantly reduced activity to 4% ethanol, BGL2 demonstrated tolerance to ethanol. Its activity was enhanced in the presence of ethanol but reduced at concentrations greater than 16%. The presence of the fermentation inhibitors furfural and HMF did not affect the enzyme activity. Our results suggest that a β-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group that validate its cellobiose hydrolysis functions for lower-cost cellulosic ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464, and further supported this dual functional yeast as a candidate for lower-cost cellulosic ethanol production and next-generation biocatalyst development in potential industrial applications.     

Evaluation of strains of Geotrichum candidum for lipase production and fatty acid specificity

Summary Three strains of Geotrichum candidum (ATCC 34614, NRRL Y-552 and NRRL Y-553) were examined for lipase production and activity. Variables including medium, pH, temperature, agitation rate and incubation time were examined to define the optimal culture conditions. Growth on oil in complex medium at 30°C, 300 rpm, and pH 7 produced maximal lipase activity. Fatty acid specificity of these strains and of two crude G. candidum enzyme preparations (lipase 26557 RP, Rhône Poulenc and lipase GC-4, Amano) was measured using equimolar mixtures of methyl or butyl esters of palmitic and oleic acids. The lipase from NRRL Y-553 and lipase 26557 RP displayed preferential specificity for hydrolyzing oleic acid esters, while the lipases from ATCC 34614, NRRL Y-552 and lipase GC-4 failed to discriminate between plamitic and oleic acids.     

Optimization of the Process for the Production of L(+)‐Lactic Acid from Cull Potato by Rhizopus oryzae

Cull potato is currently an under‐utilized biomass in the potato processing states of the USA. L(+)‐Lactic acid production by three Rhizopus strains and one homofermentative, facultative anaerobic Lactobacillus amylophilus strain was investigated using potatoes as the sole nutrient supply in the culture medium. Rhizopus oryzae NRRL 395 was chosen as the strain for further studies because it showed the highest lactate yield. The fermentation conditions for seed cultures were studied for three treatment structures using a completely randomized design. Optimum conditions for the seed culture were determined to be 2 % potato medium, 10⁴ spores/mL concentration, and 24 h of fermentation. Plackett‐Burman and central composite designs were used to screen and optimize the factors for lactic acid production. Substrate (potato) concentration, fermentation temperature, and shaking speed were found to be the most significant factors affecting both the yield and concentration of lactate. Optimum values for substrate concentration, fermentation temperature, and shaking speed were 10 %, 27 °C, and 170 rpm, respectively. Under these optimum conditions, the lactate concentration was predicted by the model to be 35.5 g/L, which was verified by the experimental data (33.3 g/L). The results indicate that cull potato can be an effective feedstock for R. ryzae NRRL 395 in the production of lactic acid.     

Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates

The relevance of oxidative stress in the production of aflatoxin and its precursors was examined in different mutants of Aspergillus parasiticus, which produce aflatoxin or its precursor intermediates, and compared with results obtained from a non-toxigenic strain. In comparison to the non-toxigenic strain (SRRC 255), an aflatoxin producing strain (NRRL 2999) or mutants that accumulate aflatoxin precursors such as norsolorinic acid (by SRRC 162) or versicolorin (by NRRL 6196) or O-methyl sterigmatocystin (by SRRC 2043) had greater oxygen requirements and higher contents of reactive oxygen species. These changes were in the graded order of NRRL 2999 > SRRC 2043 > NRRL 6196 > SRRC 162 > SRRC 255, indicating incremental accumulation of reactive oxygen species, being least in the non-toxigenic strain and increasing progressively during the ternary steps of aflatoxin formation. Oxidative stress in these strains was evident by increased activities of xanthine oxidase and free radical scavenging enzymes (superoxide dismutase and glutathione peroxidase) as compared to the non-toxigenic strain (SRRC 255). Culturing the toxigenic strain in presence of 0.1–10 μM H₂O₂ in the medium resulted in enhanced aflatoxin production, which could be related to dose-dependent increase in [¹⁴C]-acetate incorporation into aflatoxin B₁ and increased acetyl CoA carboxylase activity. The combined results suggest that formation of secondary metabolites such as aflatoxin and its precursors by A. parasiticus may occur as a compensatory response to reactive oxygen species accumulation.     

Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii (“Clostridium butylicum”)

Clostridium beijerinckii (“Clostridium butylicum”) NRRL B592 and NRRL B593 were grown in batch cultures without pH control. The use of more sensitive and accurate procedures for the determination of solvents in cultures led to the recognition of the onset of solvent production about 2 h earlier than the previously assigned point and at a higher culture pH for both strains. Reliable assays for solvent-forming enzyme activities in cell extracts have also been developed. The results showed that activities of solvent-forming enzymes in strain NRRL B592 started to increase about 1 h before the measured onset of solvent production and that the increase in activities of solvent-forming enzymes was not simultaneous. The degree of increase of these enzyme activities for both strains ranged from 2- to 165-fold, with acetoacetate decarboxylase and butanol-isopropanol dehydrogenase showing the largest activity increases. However, the pattern of increase of enzyme activities differed significantly in the two strains of C. beijerinckii. When an increase in solvent-forming enzyme activities was first detected in strain NRRL B592, the culture pH was at 5.7 and the concentrations of total acetic and butyric acids were 5.2 and 3.6 mM, respectively. For strain NRRL B593, the corresponding pH was 5.5. Thus, the culture conditions immediately preceding the expression of solvent-forming enzyme activities differed significantly from those that have been correlated with the production of solvents at later stages of growth.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

The Comparative Discriminating Abilities of Lipases in Different Media and Their Application in Fatty Acid Enrichment

The generally held belief that the selectivity of lipase can be changed by changing the media from aqueous to non-aqueous was tested by monitoring the rates of hydrolysis, ester synthesis and transesterification with a range of fatty acid mono-esters. Although the absolute rates of reaction varied, hydrolysis was by far the most rapid of the three, the relative rates for the fatty acids used were similar in all three reaction types. The selectivity of the five enzymes used appeared to remain unchanged irrespective of the type of reaction, i.e. hydrolysis of p-nitrophenyl esters, direct ester synthesis with butanol and fatty acid or transesterification with butyl butyrate and fatty acid, and could not be changed by changing water activity. This principle was applied to screen for suitable lipases which could be used to increase the gamma linolenic acid content of a fatty acid mixture. Enzymes could be selected by measuring the rate of hydrolysis of a range of P-nitrophenyl esters.     

Diversity of Oleic Acid,Ricinoleic Acid and Linoleic Acid Conversions Among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strains*

Sixteen Pseudomonas aeruginosa strains, including patent strain NRRL B-18602, three recent isolates from composted materials amended with ricinoleic acid, and 12 randomly selected from the holdings of the ARS Culture Collection, were examined for their fatty acid converting abilities. The study examined the bioconversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and ricinoleic acid to 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD). A new DOD-like compound from linoleic acid was observed. All strains except NRRL B-247 exhibited varying levels of DOD production. NRRL B-1000, NRRL B-18602 and NRRL B-23258 with yields up to 84% were among the best DOD producers. TOD production generally paralleled DOD production at a relatively lower yield of up to 15%. Strains NRRL B-1000 and NRRL B-23260 were the best TOD producers. A DOD-like product in low yields was obtained from linoleic acid. The fatty acid bioconversion capability was related neither to growth rate nor to variation in the greenish pigmentation of the strains. Production of significant quantities of DOD and TOD from oleic and ricinoleic acids, respectively, appeared to be a characteristic trait of P. aeruginosa strains. A number of highly effective strains for DOD production were identified.     

Iso-migrastatin titer improvement in the engineered <Emphasis Type="Italic">Streptomyces lividans</Emphasis> SB11002 strain by optimization of fermentation conditions

The heterologous production of iso-migrastatin (iso-MGS) was successfully demonstrated in an engineered S. lividans SB11002 strain, which was derived from S. lividans K4-114, following introduction of pBS11001, which harbored the entire mgs biosynthetic gene cluster. However, under similar fermentation conditions, the iso-MGS titer in the engineered strain was significantly lower than that in the native producer — Streptomyces platensis NRRL 18993. To circumvent the problem of low iso-MGS titers and to expand the utility of this heterologous system for iso-MGS biosynthesis and engineering, systematic optimization of the fermentation medium was carried out. The effects of major components in the cultivation medium, including carbon, organic and inorganic nitrogen sources, were investigated using a single factor optimization method. As a result, sucrose and yeast extract were determined to be the best carbon and organic nitrogen sources, resulting in optimized iso-MGS production. Conversely, all other inorganic nitrogen sources evaluated produced various levels of inhibition of iso-MGS production. The final optimized R2YE production medium produced iso-MGS with a titer of 86.5 mg/L, about 3.6-fold higher than that in the original R2YE medium, and 1.5 fold higher than that found within the native S. platensis NRRL 18993 producer.     

Growth enhancement of black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis>) by a newly isolated <Emphasis Type="Italic">Bacillus tequilensis</Emphasis> NII-0943

A Gram positive, rod-shaped potential strain was selected from the pool of bacterial isolates obtained from the Western Ghats forest (India) on the basis of zone of P-solubilization activity. Identification based on 16S rRNA gene sequence revealed that the strain is of Bacillus species, sharing highest sequence similarity to Bacillus tequilensis NRRL B-41771^T (99.5%). Strain NII-0943 was able to produce good amount of indole acetic acid (IAA) and was positive for siderophore production. In addition to IAA and siderophore attributes, strain NII-0943 also possessed the characteristics like Ca₃(PO₄)₂ solubilization and growth in nitrogen-free medium. Seed inoculation with the strain NII-0943 resulted in significantly higher root initiation in black pepper cuttings grown under pots. The contents of nitrogen and phosphorus in both soil and plant were also enhanced significantly in treatments inoculated with these bacterial inocula. Hence, based on this evidence it is proposed that strain NII-0943 could be deployed as a plant growth-promoting inoculant to attain the desired results of bacterization.     

Four new Candida species from geographically diverse locations

Four new species of Candida are described based on their unique nucleotide sequences in the D1/D2 domain of large subunit (26S) ribosomal DNA. Candida peoriaensis (type strain NRRL YB-1497, CBS 8800) and C. ponderosae (type strain NRRL YB-2307, CBS 8801) are members of the Pichia anomala clade and were isolated in the U.S. from, respectively, the stump of an elm tree (Ulmus sp.) and from insect frass of a Ponderosa pine (Pinus ponderosa). Candida ghanaensis (type strain NRRL YB-1486, CBS 8798) is a phylogenetically divergent species from soil in Ghana and appears related to the Dipodascus/Geotrichum clade. Candida litsaeae (type strain NRRL YB-3246, CBS 8799) was isolated from the frass of an insect-infested Litsaea polyantha tree from India, and is a divergent species that is most closely related to Candida ontarioensis.     

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*

Aims: A Lactobacillus buchneri strain NRRL B‐30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B‐30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B‐30929 for fermenting mixed substrates from biomass hydrolysates. Methods and Results: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B‐30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B‐30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1·98–2·35 g l^?1 ethanol from corn stover hydrolysates and 2·92–3·01 g l^?1 ethanol from wheat straw hydrolysates when supplemented with either 0·25× MRS plus 1% corn steep liquor or 0·5× MRS. Conclusions: The L. buchneri NRRL B‐30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. Significance and Impact of the Study: These results are valuable for future research in engineering L. buchneri NRRL B‐30929 for fermentative production of ethanol and chemicals from biomass.     

Three new species of Candida from apple cider: C. anglica, C. cidri and C. pomicola

Three new anamorphic ascomycetous yeasts are described: Candida anglica (type strain NRRL Y-27079, CBS 4262), Candida cidri (type strain NRRL Y-27078, CBS 4241), and Candida pomicola (type strain NRRL Y-27083, CBS 4242). These three species were isolated from cider produced in the United Kingdom, and their identification was determined from unique nucleotide sequences in the species-specific D1/D2 domain of large subunit (26S) ribosomal DNA. Phylogenetic analysis of D1/D2 sequences placed C. anglica near Candida fragi, C. cidri near Pichia capsulata, and C. pomicola in the Pichia holstii clade.     

<Emphasis Type="Italic">Ogataea saltuana</Emphasis> sp. nov., a novel methanol-assimilating yeast species

Four ascosporulating strains of an undescribed methanol-assimilating yeast species were isolated from forest habitats in Hungary. Three were recovered from rotten wood and one from leaves of a sessile oak (Quercus petraea). An additional isolate of the undescribed species sharing similar phenotypic characters with the above-noted strains was recovered from the gut of an unidentified beetle collected from under the bark of a coniferous tree in Bulgaria. A closely related, but somewhat divergent strain was recovered from insect frass in a Ponderosa pine (Pinus ponderosa) collected in New Mexico, USA. Analysis of the D1/D2 sequences of the LSU rRNA gene placed the new species in the Ogataea clade. The ITS and the D1/D2 LSU sequences of the rRNA gene repeats were compared for the above-noted strains and that of the type strain of Ogataea zsoltii, the closest neighbour among currently recognized Ogataea species. Their relatedness was investigated by parsimony network analysis as well. As a result of the sequence analysis, it was concluded that the six strains isolated from tree associated habitats represent a single new yeast species. Ogataea saltuana sp. nov. is proposed to accommodate these strains. The type strain NCAIM Y.01833^T (CBS 10795^T, NRRL Y-48448^T) was recovered from rotten wood of Scotch pine (Pinus silvestris) in Hungary. The GenBank accession number for the D1/D2 domain nuclear large subunit rRNA gene sequence of strain NCAIM Y.01833^T (CBS 10795^T, NRRL Y-48448^T) is EU327033. The MycoBank number of the new species is MB 519966.     

Solid-state fermentation production of tetracycline by <Emphasis Type="Italic">Streptomyces</Emphasis> strains using some agricultural wastes as substrate

The ability of Streptomyces sp. OXCI, S. rimosus NRRL B2659, S. rimosus NRRL B2234, S. alboflavus NRRL B1273 S. aureofaciens NRRL B2183 and S. vendagensis ATCC 25507 to produce tetracycline using some local agricultural wastes as solid state media, were assessed. The wastes employed include peanut (groundnut) shells, corncob, corn pomace and cassava peels. Bacillus subtilis ATCC 6633 was used to assay antimicrobial activity. All the strains produced tetracycline in a solid-state fermentation process containing peanut (groundnut) as the carbohydrate source. Streptomyces sp. OXC1 had the highest ability for tetracycline production with peanut shells as the substrate in solid fermentation (13.18 mg/g), followed by S. vendagensis ATCC 25507 (11.08 mg/g), S.rimosus NRRL B1679 (8.46 mg/g), S. alboflavus NRRL B1273 (7.59 mg/g), S. rimosus NRRL B2234 (6.37 mg/g), S. aureofaciens NRRL B2183 (4.27 mg/g). Peanut (groundnut) shells were the most effective substrate (4.36 mg/g) followed by corncob (2.64 mg/g), cassava peels (2.16 mg/g) and corn pomace (1.99 mg/g). The composition of the peanut (groundnut) shell medium optimal for tetracycline production were peanut shells 100 g, organic nitrogen (peanut meal) 10 g, (NH ₄)₂ SO₄ 1 g, KH₂ PO ₄ 0.5 g, CaCO₃ > 0.5 g, NaCl 0.5 g, MgSO₄ · 7H₂ O 0.5 g, soluble starch 10 g, peanut oil 0.25 ml with initial moisture content of 65–68%, and initial pH 5.3–6.3. Substrate (1 g dry weight) was inoculated with 1.0 × 10 ⁸ conidia per ml and incubated at 28–31 °C for 5–7 days, producing 13.18 mg/g of total tetracycline. Tetracycline detection started on day 3 and attained its maximum level on day 5.     

Lipase‐catalyzed kinetic resolution of novel antitubercular benzoxazole derivatives

Novel benzoxazole derivatives were synthesized, and their antitubercular activity against sensitive and drug‐resistant Mycobacterium tuberculosis strains (M. tuberculosis H₃₇Rv, M. tuberculosis sp. 210, M. tuberculosis sp. 192, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium avium, and Mycobacterium kansasii) was evaluated. The chemical step included preparation of ketones, alcohols, and esters bearing benzoxazole moiety. All racemic mixtures of alcohols and esters were separated in Novozyme SP 435‐catalyzed transesterification and hydrolysis, respectively. The transesterification reactions were carried out in various organic solvents (tert‐butyl methyl ether, toluene, diethyl ether, and diisopropyl ether), and depending on the solvent, the enantioselectivity of the reactions ranged from 4 to >100. The enzymatic hydrolysis of esters was performed in 2 phase tert‐butyl methyl ether/phosphate buffer (pH = 7.2) system and provided also enantiomerically enriched products (ee 88‐99%). The antitubercular activity assay has shown that synthesized compounds exhibit an interesting antitubercular activity. Racemic mixtures of alcohols, (±)‐4‐(1,3‐benzoxazol‐2‐ylsulfanyl)butan‐2‐ol ((±)‐ 3a ), (±)‐4‐[(5‐bromo‐1,3‐benzoxazol‐2‐yl)sulfanyl]butan‐2‐ol ((±)‐ 3b ), and (±)‐4‐[(5,7‐dibromo‐1,3‐benzoxazol‐2‐yl)sulfanyl]butan‐2‐ol ((±)‐ 3c ), displayed as high activity against M. scrofulaceum, M. intracellulare, M. fortuitum, and M. kansasii as commercially available antituberculosis drug‐Isoniazid. Moreover, these compounds exhibited twice higher activity toward M. avium (MIC 12.5) compared with Isoniazid (MIC 50).