Journal of bioscience and bioengineering最新文献

{"title":"Beneficial base substitutions in Escherichia coli fucO gene for enhancement of glycolic acid production","authors":"Mayu Nemoto ,&nbsp;Wataru Muranushi ,&nbsp;Chen Shuting ,&nbsp;Yusuke Saito ,&nbsp;Daisuke Sugimori ,&nbsp;Miwa Yamada","doi":"10.1016/j.jbiosc.2024.06.007","DOIUrl":"10.1016/j.jbiosc.2024.06.007","url":null,"abstract":"<div><p>Microbial production of glycolic acid (GA) from ethylene glycol is extensively used in a variety of industries because ethylene glycol is not only an inexpensive raw material but also the main component of industrial wastes. In this study, we produced GA from ethylene glycol using <em>Escherichia coli</em> overexpressing the endogenous 1,2-propanediol oxidoreductase (<em>fucO</em>) and lactaldehyde dehydrogenase (<em>aldA</em>) genes. To increase GA productivity, we screened a random mutant library generated using an error-prone polymerase chain reaction of <em>fucO</em> and obtained FucO mutants MF2-9 and MF6-9 with enhanced GA production in Lysogeny Broth medium containing 800 mM ethylene glycol. MF2-9 contained three amino acid substitutions (D23E, E222K, and G363S) and two synonymous mutations (coding DNA [c.] 93G &gt; A and c.1131T &gt; C) in <em>fucO</em>. MF6-9 contained one amino acid substitution (L377H) in FucO. An amino acid substitution (L377H) and a single synonymous mutation (c.1131T &gt; C) in <em>fucO</em> contributed to the enhancement in GA production. Notably, cell lysates from <em>E. coli</em> harboring a synonymous mutation (c.1131T &gt; C) or amino acid substitution (L377H) in <em>fucO</em> showed that only AldA activity was 1.3-fold higher than that of the cell lysate from <em>E. coli</em> harboring the wild-type <em>fucO</em>. We confirmed that c.1131T &gt; C and L377H mutations increased <em>aldA</em> expression in <em>E. coli</em>. Analysis of mRNA levels and simulation of mRNA stabilization indicated that base substitutions at positions c.1130T, which corresponds to L377H amino acid substitution, and c.1131T increased <em>aldA</em> expression due to partial destabilization of the mRNA. These findings will be useful for the large-scale microbial production of GA from industrial waste.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 4","pages":"Pages 301-307"},"PeriodicalIF":2.3,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141855567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation of aflatoxin biosynthetic inhibitor from Chondrostereum purpureum mushroom culture filtrate","authors":"Yuya Matsuno ,&nbsp;Naoki Endo ,&nbsp;Kotomi Ueno ,&nbsp;Atsushi Ishihara","doi":"10.1016/j.jbiosc.2024.07.003","DOIUrl":"10.1016/j.jbiosc.2024.07.003","url":null,"abstract":"<div><p>Aflatoxins (AFs) are highly toxic mycotoxins produced by the fungi, <em>Aspergillus flavus</em> and <em>Aspergillus parasiticus</em>. AFs pose severe health risks owing to their acute toxicity and carcinogenic properties. The control of AF contamination remains significantly challenging despite the extensive efforts toward controlling it. Here, we investigated the potential of mushroom extracts as a source of AF biosynthetic inhibitors. The <em>A. parasiticus</em> mutant strain, NFRI-95, that accumulates an AF biosynthesis intermediate, norsolorinic acid, was used in the bioassay to detect the inhibitory activity against AF biosynthesis. The screening of 195 mushroom extracts revealed that the culture filtrate extract of <em>Chondrostereum purpureum</em> exhibited strong inhibitory activity against AF biosynthesis. Next, large-scale culturing of <em>C. purpureum</em> was performed to isolate the compounds accounting for the inhibitory activity. The culture filtrate was extracted with ethyl acetate, after which the active compound was isolated by silica gel column chromatography and preparative high performance liquid chromatography (HPLC). The active compound was identified as cyclo(Val–Pro) by spectroscopic analyses. Further, four stereoisomers of cyclo(Val–Pro) were synthesized by the condensation of the <em>N</em>-Boc derivatives of <span>d</span>- and <span>l</span>-valine with the methyl esters of <span>d</span>- and <span>l</span>-proline. The naturally isolated compound was identified as cyclo(<span>l</span>-Val-<span>l</span>-Pro) by comparing its retention time with those of synthetic compounds by chiral HPLC analysis and CD spectra. The IC₅₀ value of cyclo(L-Val-L-Pro) was 2.4 mM, whereas the LD, DL, and DD isomers exhibited weaker activities, with IC₅₀ values of &gt;5 mM.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 4","pages":"Pages 308-313"},"PeriodicalIF":2.3,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141788128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolated hair bacteria reveal different isolation possibilities under various conditions","authors":"Azusa Yamada ,&nbsp;Yuri Nishi ,&nbsp;Mei Noguchi ,&nbsp;Kota Watanabe ,&nbsp;Mugihito Oshiro ,&nbsp;Kenji Sakai ,&nbsp;Yukihiro Tashiro","doi":"10.1016/j.jbiosc.2024.06.003","DOIUrl":"10.1016/j.jbiosc.2024.06.003","url":null,"abstract":"<div><p><span><span>Microorganisms are assumed to inhabit various environments and organisms, including the human body. The presence of more than 700 bacterial species on scalp hair has been reported through </span>rRNA gene<span> amplicon<span> analysis. However, the biological properties of bacteria on the scalp hair (hair bacteria) and their functions are poorly understood as few hair bacteria have been isolated from hair in previous studies. This study aimed to isolate hair bacteria using standard media under 24 different conditions (including medium components, component concentrations, gelling agents, and atmospheric environments). Furthermore, we evaluated the possibility of isolating strains under these isolation conditions and examined the carbon metabolic ability of several predominantly isolated strains. A total of 63 bacterial species belonging to 27 genera were isolated from hair under 24 isolation conditions. The predominant bacterial species isolated from human hair in this study showed different carbon metabolic capabilities than those of the reference strains. In addition, isolation possibility was newly proposed to systematically evaluate the number of isolation conditions that could cultivate a bacterial species. Based on isolation possibility, the isolates were categorized into groups with a high number of isolation conditions (e.g., ≥25%; such as </span></span></span><em>Staphylococcus</em>) and those with a low number (e.g., ≤25%; such as <span><em>Brachybacterium</em></span>). These findings indicate the existence of easily isolated microorganisms and difficultly isolated microorganism from human hair.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 4","pages":"Pages 290-300"},"PeriodicalIF":2.3,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141734182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effect of humic acid on aerobic denitrification by Achromobacter sp. strain GAD-3","authors":"Mengyao Gui","doi":"10.1016/j.jbiosc.2024.06.008","DOIUrl":"10.1016/j.jbiosc.2024.06.008","url":null,"abstract":"<div><p><span>Humic acid<span> (HA), a common natural organic matter, could affect conventional anoxic denitrification. Aim of this study was to investigate effect of HA on the process of aerobic denitrification in </span></span><span><span>Achromobacter</span></span> sp. GAD-3, an aerobic denitrifying strain. The findings demonstrated that an increase in HA concentrations (≥5 mg L⁻¹) promoted the aerobic denitrification process (excluding N₂O reduction), manifesting as higher rates of nitrate removal (6.67–11.1 mg L⁻¹ h⁻¹) and lower levels of nitrite accumulation (30.2–20.7 mg L⁻¹<span><span>). This was attributed to the increased electron transfer activities and denitrifying </span>reductase activities (including NAR, NIR and NOR) facilitated by HA. Accordingly, the expression of denitrification genes such as </span><span><em>napA</em></span>, <em>cnorB</em>, and <em>nirS</em> was enhanced by HA. Nonetheless, the <em>nosZ</em> gene and N₂OR activity underwent suppression by HA, which was accountable for N₂<span>O emission. It is crucial to understand the HA mechanism towards aerobic denitrifiers<span> for wastewater treatment plants to enhance nitrogen removal.</span></span></p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 4","pages":"Pages 338-344"},"PeriodicalIF":2.3,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141727289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preparation of microcapsules and evaluation of their biocontrol efficacy","authors":"","doi":"10.1016/j.jbiosc.2024.05.007","DOIUrl":"10.1016/j.jbiosc.2024.05.007","url":null,"abstract":"<div><p>In this study, a combination of <span><span>Serratia</span><em> nematophila</em></span> L2 and <span><em>Bacillus</em><em> velezensis</em></span> W24 was used to biocontrol <span><span>Sclerotinia sclerotiorum</span></span>. When the mixed ratio of L2 to W24 was 1:1, the inhibition rate on the growth of <em>S. sclerotiorum</em><span><span> was 88.1 %. To gain a large number of bacteria, the culture medium and conditions were optimized. When the medium formula involved molasses (8.890 g/L), </span>soy<span><span> peptone (6.826 g/L), and NaCl (6.865 g/L), and the culture conditions were 32 °C, inoculum 4%, rotation speed 200 rpm, and pH 7, the maximum amounts of bacterial cells obtained. In order to prepare microcapsules, spray drying conditions were optimized. These conditions included the soluble starch concentration of 30 g/100 mL, the inlet air temperature of 160 °C, and the feed flow rate of 450 mL/h. Under these optimized conditions to prepare microcapsules, the mixed strain (L2 and W24) exhibited a </span>survival rate of 93.9 ± 0.9% and a viable bacterial count of 6.4 × 10</span></span>¹² cfu/g. In addition, microcapsules (GW24Ms) which contained strains L2 and W24 had good storage stability. In the pot experiment, GW24Ms could effectively reduce the disease of soybean plants and the control effect was 88.4%. Thus, the microbial agent represents a promising biocontrol solution for managing <span><em>Sclerotinia</em></span> in soybean.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 4","pages":"Pages 328-337"},"PeriodicalIF":2.3,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141599922","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploring gingerol glucosides with enhanced anti-inflammatory activity through a newly identified α-glucosidase (ArG) from Agrobacterium radiobacter DSM 30147","authors":"","doi":"10.1016/j.jbiosc.2024.06.004","DOIUrl":"10.1016/j.jbiosc.2024.06.004","url":null,"abstract":"<div><p><span>Gingerols are phenolic biomedical compounds found in ginger (</span><span><span>Zingiber officinale</span></span>) whose low aqueous solubility limits their medical application. To improve their solubility and produce novel glucosides, an <em>α</em>-glucosidase (glycoside hydrolase) from <span><span>Agrobacterium radiobacter</span></span> DSM 30147 (<em>Ar</em>G) was subcloned, expressed, purified, and then confirmed to have additional <em>α</em>-glycosyltransferase activity. After optimization, the <em>Ar</em>G could glycosylate gingerols into three mono-glucosides based on the length of their acyl side chains. Compound <strong>1</strong> yielded 63.0 %, compound <strong>2</strong> yielded 26.9 %, and compound <strong>3</strong><span> yielded 4.37 %. The production yield of the gingerol glucosides optimally increased in 50 mM phosphate buffer (pH 6) with 50 % (w/v) maltose and 1000 mM Li</span>⁺ at 40 °C for an 24-h incubation. The structures of purified compound <strong>1</strong> and compound <strong>2</strong> were determined as 6-gingerol-5-<em>O</em>-<em>α</em>-glucoside (<strong>1</strong>) and novel 8-gingerol-5-<em>O</em>-<em>α</em>-glucoside (<strong>2</strong><span><span>), respectively, using nucleic magnetic resonance and mass </span>spectral analyses. The aqueous solubility of the gingerol glucosides was greatly improved. Further assays showed that, unusually, 6-gingerol-5-</span><em>O</em>-<em>α</em>-glucoside had 10-fold higher anti-inflammatory activity (IC₅₀ value of 15.3 ± 0.5 μM) than 6-gingerol, while the novel 8-gingerol-5-<em>O</em>-<em>α</em>-glucoside retained 42.7 % activity (IC₅₀ value of 106 ± 4 μM) compared with 8-gingerol. The new <em>α</em>-glucosidase (<em>Ar</em>G) was confirmed to have acidic <em>α</em>-glycosyltransferase activity and could be applied in the production of <em>α</em>-glycosyl derivatives. The 6-gingerol-5-<em>O</em>-<em>α</em>-glucoside can be applied as a clinical drug for anti-inflammatory activity.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 3","pages":"Pages 218-224"},"PeriodicalIF":2.3,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141599921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-penetrating activity of a short-chain ε-poly-l-α-lysine","authors":"","doi":"10.1016/j.jbiosc.2024.06.006","DOIUrl":"10.1016/j.jbiosc.2024.06.006","url":null,"abstract":"<div><p>Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly-<span>l</span>-α-lysine consisting of 25–35 <span>l</span>-α-lysine residues (ε-PαL_25-35) and ε-poly-<span>l</span>-β-lysine consisting of <span>l</span>-β-lysine residues (ε-PβL_4-13), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5–14 <span>l</span>-α-lysine residues (ε-PαL_5-14) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL_5-14<span> and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL</span>_5-14-FAM, with HeLa cells. Unlike ε-PαL_25-35-FAM, ε-PαL_5-14-FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (&gt;50 μM) was required for the internalization events. ε-PαL_5-14 has a chain length almost equal to that of the membrane permeable ε-PβL_4-13, which can enter cells at low concentrations. Considering that the basicity of the β-amino group is higher than that of α-amino acid at physiological pH, ε-PβL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PβL would be more advantageous for cellular internalization of cargo proteins than ε-PαL_25-35.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 3","pages":"Pages 249-253"},"PeriodicalIF":2.3,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141590422","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biochemical characterization of l-asparagine synthetase from Streptococcus thermophilus and its application in the enzymatic synthesis of β-aspartyl compounds","authors":"","doi":"10.1016/j.jbiosc.2024.06.001","DOIUrl":"10.1016/j.jbiosc.2024.06.001","url":null,"abstract":"<div><p><span>β-Aspartyl compounds, such as β-aspartyl hydroxamate (serine racemase inhibitor), β-aspartyl-</span><span>l</span>-lysine (moisture retention), and β-aspartyl-<span>l</span><span>-tryptophan (immunomodulator) are physiologically active compounds. There is limited literature on the development of effective methods of production of β-aspartyl compounds. In this study, we describe the biochemical characterization of asparagine synthetase (AS) from </span><span><em>Streptococcus</em><em> thermophilus</em></span> NBRC 13957 (StAS) and the enzymatic synthesis of β-aspartyl compounds using StAS. Recombinant StAS was expressed in <span><em>Escherichia coli</em></span><span> BL21(DE3) and it displayed activity towards hydroxylamine<span><span>, methylamine, </span>ethylamine<span>, and ammonia, as acceptors of the β-aspartyl moiety. StAS exhibited higher activity toward hydroxylamine and ethylamine as acceptor substrates compared with the enzymes from </span></span></span><span><span>Lactobacillus delbrueckii</span></span> NBRC 13953, <span><span>Lactobacillus reuteri</span></span> NBRC 15892, and <em>E. coli</em><span>. The coupling of the synthesis of β-aspartyl compounds by StAS with an ATP-regeneration system using polyphosphate kinase from </span><span><span>Deinococcus</span><em> proteoliticus</em></span> NBRC 101906 displayed an approximately 2.5-fold increase in the production of β-aspartylhydroxamate from 1.06 mM to 2.53 mM after a 76-h reaction.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 3","pages":"Pages 206-211"},"PeriodicalIF":2.3,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141563469","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic heat based specific growth rate estimators: Does the choice of estimation model influence the state of bioprocesses?","authors":"","doi":"10.1016/j.jbiosc.2024.05.014","DOIUrl":"10.1016/j.jbiosc.2024.05.014","url":null,"abstract":"<div><p>Accurate and reliable estimation of specific growth rate (<span><math><mrow><mi>μ</mi></mrow></math></span><span><span><span><span>) in real-time is pivotal for reliable process monitoring of a bioprocess and subsequent implementation of advanced control strategies. </span>Gibbs free energy dissipation is imminent for any biological system, and the </span>metabolic heat </span>flow measurements (calorimetry) formed the basis for estimating </span><span><math><mrow><mi>μ</mi></mrow></math></span>. However, the rationale behind selecting a suitable <span><math><mrow><mi>μ</mi></mrow></math></span> estimator model based on calorimetric perspective remains unexplored. The present investigation addresses the notion behind the selection of an appropriate estimator for <span><math><mrow><mi>μ</mi></mrow></math></span> and the assessment of the estimator models was illustrated using different types of energy metabolism, namely, high exothermic and low exothermic processes. The results indicated that the <span><math><mrow><mi>μ</mi></mrow></math></span> values from the instantaneous heat flow estimator significantly deviated (10-fold higher) from the offline values for highly exothermic process. Notably, the cumulative heat-based estimator accurately estimated <span><math><mrow><mi>μ</mi></mrow></math></span> values on both types of energy metabolism with performance metrics &lt;0.005 h⁻¹.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 3","pages":"Pages 239-248"},"PeriodicalIF":2.3,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141563470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bilirubin oxidase expression and activity enhancement from Myrothecium verrucaria in Aspergillus species","authors":"","doi":"10.1016/j.jbiosc.2024.06.002","DOIUrl":"10.1016/j.jbiosc.2024.06.002","url":null,"abstract":"<div><p>We constructed a new <span><span>Aspergillus</span></span><span> expression vector<span><span> (pSENSU2512nid) under the control of the enolase promoter with 12 </span>tandem repeats of </span></span><em>cis</em><span>-acting elements (region III) and the heat shock protein 12 (</span><em>Hsp12</em><span><span><span>) 5′ untranslated region (UTR). </span>Bilirubin </span>oxidase (EC: 1.3.3.5) from </span><span><span>Myrothecium</span><em> verrucaria,</em></span><span><span> which catalyzes the oxidation<span> of bilirubin to </span></span>biliverdin, was overexpressed in </span><span><span>Aspergillus oryzae</span></span> and <em>A</em>. <em>niger</em><span>. The productivity was estimated to be approximately 1.2 g/L in the culture broth, which was approximately 6-fold higher than that of recombinant bilirubin oxidase (BOD) expressed in </span><span><span>Pichia pastoris</span></span> (<span><em>Komagataella</em><em> phaffii</em></span><span><span>). BOD was purified using hydrophobic interaction chromatography, followed by </span>ion exchange chromatography. The specific activity of the purified BOD against 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate was 57.6 U/mg and 66.4 U/mg for </span><em>A. oryzae</em> and <em>A. niger</em>, respectively. <span>l</span>-Ascorbic acid (4 mM) addition and storage under deoxygenated conditions for 3–7 d increased the specific activity of these <span><em>Aspergillus</em></span><span>-expressed BODs approximately 2.3-fold (154.1 U/mg). The BOD specific activity was enhanced by incubation at higher temperature (30–50 °C). Further characterization of the enzyme<span> catalytic efficiency revealed that the </span></span><em>K</em>_m value remained unchanged, whereas the <em>k</em>_cat value improved 3-fold. In conclusion, this high-level of BOD expression meets the requirements for industrial-level production. Additionally, we identified an effective method to enhance the low specific activity during expression, making it advantageous for industrial applications.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 3","pages":"Pages 212-217"},"PeriodicalIF":2.3,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}