scholarly journals Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

2012 ◽  
Vol 56 (11) ◽  
pp. 5678-5686 ◽  
Author(s):  
Mei Li ◽  
Benjamin C. Conklin ◽  
Magdalena A. Taracila ◽  
Rebecca A. Hutton ◽  
Marion J. Skalweit
Keyword(s):  
Clavulanic Acid ◽  
Susceptibility Testing ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
Oxyanion Hole ◽  
Content Type ◽  
Class A ◽  
Inhibitor Resistance ◽  
Structural Probes ◽  
Variant Residue

ABSTRACTAmbler position 105 in class A β-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine β-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed thatEscherichia coliDH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all β-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, ≤2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increasedKm. Clavulanic acid and penemKivalues were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.

scholarly journals Inhibitor Resistance in the KPC-2 β-Lactamase, a Preeminent Property of This Class A β-Lactamase

2009 ◽  
Vol 54 (2) ◽  
pp. 890-897 ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Christopher R. Bethel ◽  
Anne M. Distler ◽  
Courtney Kasuboski ◽  
Magdalena Taracila ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Active Site ◽  
Susceptibility Testing ◽  
Enzyme Inactivation ◽  
Side Chain ◽  
Attractive Alternative ◽  
Active Site Residues ◽  
Class A ◽  
Mechanism Of Inhibition ◽  
Inhibitor Resistance

ABSTRACT As resistance determinants, KPC β-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type β-lactamases are often identified as resistant to standard β-lactam-β-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to β-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (k cat/k inact ratios, where k inact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp 2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06 ± 0.01 μM, and Km of penem 2, 0.006 ± 0.001 μM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (k cat/k inact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (k inact/Km = 6.5 ± 0.6 μM−1 s−1). We conclude that KPC-2 is unique among class A β-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type β-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current β-lactamase inhibitors.

scholarly journals A Novel Extended-Spectrum β-Lactamase, SGM-1, from an Environmental Isolate of Sphingobium sp.

2013 ◽  
Vol 57 (8) ◽  
pp. 3783-3788 ◽  
Author(s):  
Toni L. Lamoureaux ◽  
Viktoria Vakulenko ◽  
Marta Toth ◽  
Hilary Frase ◽  
Sergei B. Vakulenko
Keyword(s):  
Amino Acid ◽  
Clavulanic Acid ◽  
Amino Acid Sequence Identity ◽  
Susceptibility Testing ◽  
Environmental Isolate ◽  
Significant Activity ◽  
Content Type ◽  
Sequence Identity ◽  
Class A ◽  
Extended Spectrum

ABSTRACTSGM-1 is a novel class A β-lactamase from an environmental isolate ofSphingobiumsp. containing all of the distinct amino acid motifs of class A β-lactamases. It shares 77 to 80% amino acid sequence identity with putative β-lactamases that are present on the chromosome of allSphingobiumspecies whose genomes were sequenced and annotated. Thus, SGM-type β-lactamases are native to this genus. Antibiotic susceptibility testing classifies SGM-1 as an extended-spectrum β-lactamase, conferring the highest level of resistance to penicillins. Although SGM-1 contains the conserved cysteine residues characteristic of class A carbapenemases, it does not confer resistance to the carbapenem antibiotics imipenem, meropenem, or doripenem but does increase the MIC of ertapenem 8-fold. SGM-1 hydrolyzes penicillins and the monobactam aztreonam with similar catalytic efficiencies, ranging from 105to 106M−1s−1. The catalytic efficiencies of SGM-1 for cefoxitin and ceftazidime were the lowest (102to 103M−1s−1) among the cephalosporins tested, while the catalytic efficiencies against all other cephalosporins varied from about 105to 106M−1s−1. SGM-1 exhibited measurable but not significant activity toward the carbapenems tested. SGM-1 also showed high affinity for clavulanic acid, tazobactam, and sulbactam (Ki< 1 μM); however, only clavulanic acid significantly reduced the MICs of β-lactams.

scholarly journals Method-Dependent Epidemiological Cutoff Values for Detection of Triazole Resistance in Candida and Aspergillus Species for the Sensititre YeastOne Colorimetric Broth and Etest Agar Diffusion Methods

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
A. Espinel-Ingroff ◽  
J. Turnidge ◽  
A. Alastruey-Izquierdo ◽  
F. Botterel ◽  
E. Canton ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Species Complex ◽  
Susceptibility Testing ◽  
Yeast Species ◽  
Sensu Stricto ◽  
Wild Type ◽  
Pichia Kudriavzevii ◽  
Cutoff Value ◽  
Content Type ◽  
Clavispora Lusitaniae

ABSTRACT Although the Sensititre Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole, and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method/agent-dependent) 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex, 157 C. guilliermondii (Meyerozyma guilliermondii), 676 C. krusei (Pichia kudriavzevii), 298 C. lusitaniae (Clavispora lusitaniae), 911 C. parapsilosis sensu stricto, 3,691 C. parapsilosis species complex, 36 C. metapsilosis, 110 C. orthopsilosis, 1,854 C. tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A. flavus, 130 A. nidulans, 233 A. niger, and 302 A. terreus complex isolates. SYO/Etest MICs for 282 confirmed non-wild-type (non-WT) isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2 and CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast species and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for epidemiological cutoff value (ECV) definition were pooled, and we proposed SYO ECVs for S. cerevisiae and 9 yeast and 3 Aspergillus species and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 µg/ml for C. albicans and the Etest itraconazole ECV of 2 µg/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as, overall, the SYO appears to perform better for susceptibility testing of yeast species and the Etest appears to perform better for susceptibility testing of Aspergillus spp. Further evaluations should be conducted with more Candida mutants.

scholarly journals EUCAST Determination of Olorofim (F901318) Susceptibility of Mold Species, Method Validation, and MICs

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Karin Meinike Jørgensen ◽  
Karen M. T. Astvad ◽  
Rasmus Krøger Hare ◽  
Maiken Cavling Arendrup
Keyword(s):  
Fusarium Solani ◽  
Susceptibility Testing ◽  
Wild Type ◽  
Preparation Methods ◽  
Content Type ◽  
Microtiter Plates ◽  
Upper Limits ◽  
Limited Variation

ABSTRACT Olorofim is a novel antifungal agent with in vitro activity against Aspergillus and some other molds. Here, we addressed technical aspects for EUCAST olorofim testing and generated contemporary MIC data. EUCAST E.Def 9.3.1 testing was performed comparing two plate preparation methods (serial dilution in medium [serial plates] versus predilution in DMSO [ISO plates]), two lots of olorofim, visual (visual-MIC) versus spectrophotometer (spec-MIC) reading, and four polystyrene plates using 34 to 53 Aspergillus isolates from five genera. Subsequently, olorofim MICs were compared to itraconazole, voriconazole, posaconazole, and amphotericin B MICs for 298 clinical mold isolates (2016 to 2017). Wild-type upper limits (WT-UL) were determined following EUCAST principles for epidemiologic cutoff value (ECOFF) setting. Olorofim median MICs comparing serial plates and ISO plates were identical (25/36 [69%]) or one dilution apart (11/36 [31%]). Interperson agreement for visual-MICs was 92% to 94%/100% for ≤1/≤2 dilutions, respectively. The visual-MIC values across tested microtiter plates and olorofim lots revealed only discrete differences (≤1 dilution lower for treated plates). No single spec-MIC criterion was applicable to all species. Olorofim MICs were low against 275 Aspergillus species isolates (modal MIC, 0.06 mg/liter; MIC range, < 0.004 to 0.25 mg/liter) and three dermatophytes (MICs 0.03 to 0.06 mg/liter). MICs against Fusarium were diverse, with full inhibition of F. proliferatum (MIC, 0.016), 50% growth inhibition of Fusarium solani at 1 to 2 mg/liter, and no inhibition of F. dimerum. Olorofim displayed potent in vitro activity against most mold isolates and was associated with limited variation in EUCAST susceptibility testing.

scholarly journals Alternative Biotransformation of Retinal to Retinoic Acid or Retinol by an Aldehyde Dehydrogenase from Bacillus cereus

2016 ◽  
Vol 82 (13) ◽  
pp. 3940-3946 ◽  
Author(s):  
Seung-Hye Hong ◽  
Ho-Phuong-Thuy Ngo ◽  
Hyun-Koo Nam ◽  
Kyoung-Rok Kim ◽  
Lin-Woo Kang ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Bacillus Cereus ◽  
Aldehyde Dehydrogenase ◽  
Catalytic Efficiency ◽  
No Oxidation ◽  
Wild Type ◽  
Biotechnological Production ◽  
Oxidation Activity ◽  
Content Type ◽  
Bacterial Enzyme

ABSTRACTA novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified inBacillus cereus. The amino acid sequence of ALDH fromB. cereus(BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)+. We newly found thatBcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues inBcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD+converted 400 μM all-trans-retinal to 210 μM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 μM all-trans-retinal to 20, 90, and 40 μM all-trans-retinol, respectively. These results indicate thatBcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore,BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol.IMPORTANCEAlthough mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)+as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid byBcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 inBcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

scholarly journals Implications of the EUCAST Trailing Phenomenon inCandida tropicalisfor theIn VivoSusceptibility in Invertebrate and Murine Models

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
K. M. T. Astvad ◽  
D. Sanglard ◽  
E. Delarze ◽  
R. K. Hare ◽  
M. C. Arendrup
Keyword(s):  
Susceptibility Testing ◽  
Galleria Mellonella ◽  
Growth Control ◽  
Resistant Isolate ◽  
Murine Models ◽  
Wild Type ◽  
Content Type ◽  
Gradual Loss

ABSTRACTCandida tropicalisisolates often display reduced but persistent growth (trailing) over a broad fluconazole concentration range during EUCAST susceptibility testing. Whereas weak trailing (<25% of the positive growth control) is common and found not to impair fluconazole efficacy, we investigated if more pronounced trailing impacted treatment efficacy. Fluconazole efficacy against two weakly (≤25% growth), two moderately (26% to 50% growth), and one heavily (>70% growth) trailing resistant isolate and one resistant (100% growth) isolate were investigatedin vitroandin vivo(in aGalleria mellonellasurvival model and two nonlethal murine models).CDR1expression levels andERG11sequences were characterized. The survival in fluconazole-treatedG. mellonellawas inversely correlated with the degree of trailing (71% to 9% survival in treatment groups). In mice, resistant and heavily trailing isolates responded poorly to fluconazole treatment.CDR1expression was significantly higher in trailing and resistant isolates than in wild-type isolates (1.4-fold to 10-fold higher). All isolates exhibitedERG11wild-type alleles. Heavily trailing isolates were less responsive to fluconazole in allin vivomodels, indicating an impact on fluconazole efficacy.CDR1upregulation may have contributed to the observed differences. Moderately trailing isolates responded less well to fluconazole in larvae only. This confirms clinical data suggesting fluconazole is effective against infections with such isolates in less severely ill patients and supports the current 50% growth endpoint for susceptibility testing. However, it is still unclear if the gradual loss of efficacy observed for moderately trailing isolates in the larva model may be a reason for concern in selected vulnerable patient populations.

scholarly journals APX001A In Vitro Activity against Contemporary Blood Isolates and Candida auris Determined by the EUCAST Reference Method

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Anuradha Chowdhary ◽  
Karen M. T. Astvad ◽  
Karin Meinike Jørgensen
Keyword(s):  
Susceptibility Testing ◽  
Yeast Species ◽  
Reference Method ◽  
Common Species ◽  
Drug Candidate ◽  
Its Sequencing ◽  
Wild Type ◽  
Candida Auris ◽  
Content Type

ABSTRACT APX001A is the active moiety of the first-in-class drug candidate APX001. So far, most susceptibility testing studies have examined ≤30 isolates/species, and only one used the EUCAST method. Here, we investigated the in vitro activity of APX001A and five comparators against 540 candidemia and 122 C. auris isolates. Isolates (17 Candida and 3 yeast species) were identified using CHROMagar, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) and, when needed, internal transcribed space (ITS) sequencing. EUCAST E.Def 7.3.1 susceptibility testing included APX001A, amphotericin B, anidulafungin, micafungin, fluconazole, and voriconazole. Wild-type upper limits (WT-UL) were established following the EUCAST principles for epidemiological cutoff value setting for APX001A, allowing classification as wild type (WT) or non-WT. APX001A MIC50 values (mg/liter) were as follows: Candida albicans, Candida dubliniensis, and Candida tropicalis, 0.004 to 0.008; Candida parapsilosis and Candida auris, 0.016; Candida glabrata, 0.06; and Candida krusei, >0.5. APX001A MICs against the rare species varied from ≤0.0005 (C. pelliculosa) to >0.5 (Candida norvegensis). APX001A was equally or more active in vitro than the comparators against all species except C. krusei and C. norvegensis. Four isolates were APX001A non-WT; all were fluconazole resistant. A correlation was observed between APX001A and fluconazole MICs across all species except Candida guilliermondii and C. auris, and when comparing high and low fluconazole MIC isolates of C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, and C. auris. APX001A showed promising in vitro activity against most Candida and other yeast species, including C. auris, compared to five comparators. WT-UL were suggested for the common species, and a new and unexplained correlation to fluconazole susceptibility was observed.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals Multicenter Study of Method-Dependent Epidemiological Cutoff Values for Detection of Resistance in Candida spp. and Aspergillus spp. to Amphotericin B and Echinocandins for the Etest Agar Diffusion Method

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
A. Espinel-Ingroff ◽  
M. Arendrup ◽  
E. Cantón ◽  
S. Cordoba ◽  
E. Dannaoui ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
The United States ◽  
Fungal Isolate ◽  
Diffusion Method ◽  
Wild Type ◽  
Candida Spp ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Cutoff Values

ABSTRACTMethod-dependent Etest epidemiological cutoff values (ECVs) are not available for susceptibility testing of eitherCandidaorAspergillusspecies with amphotericin B or echinocandins. In addition, reference caspofungin MICs forCandidaspp. are unreliable.CandidaandAspergillusspecies wild-type (WT) Etest MIC distributions (microorganisms in a species-drug combination with no detectable phenotypic resistance) were established for 4,341Candida albicans, 113C. dubliniensis, 1,683C. glabrataspecies complex (SC), 709C. krusei, 767C. parapsilosisSC, 796C. tropicalis, 1,637Aspergillus fumigatusSC, 238A. flavusSC, 321A. nigerSC, and 247A. terreusSC isolates. Etest MICs from 15 laboratories (in Argentina, Europe, Mexico, South Africa, and the United States) were pooled to establish Etest ECVs. Anidulafungin, caspofungin, micafungin, and amphotericin B ECVs (in micrograms per milliliter) encompassing ≥97.5% of the statistically modeled population were 0.016, 0.5, 0.03, and 1 forC. albicans; 0.03, 1, 0.03, and 2 forC. glabrataSC; 0.06, 1, 0.25, and 4 forC. krusei; 8, 4, 2, and 2 forC. parapsilosisSC; and 0.03, 1, 0.12, and 2 forC. tropicalis. The amphotericin B ECV was 0.25 μg/ml forC. dubliniensisand 2, 8, 2, and 16 μg/ml for the complexes ofA. fumigatus,A. flavus,A. niger, andA. terreus, respectively. While anidulafungin Etest ECVs classified 92% of theCandida fksmutants evaluated as non-WT, the performance was lower for caspofungin (75%) and micafungin (84%) cutoffs. Finally, although anidulafungin (as an echinocandin surrogate susceptibility marker) and amphotericin B ECVs should identifyCandidaandAspergillusisolates with reduced susceptibility to these agents using the Etest, these ECVs will not categorize a fungal isolate as susceptible or resistant, as breakpoints do.

scholarly journals Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

2019 ◽  
Vol 85 (17) ◽  
Author(s):  
Zhu Jiang ◽  
Tengfei Niu ◽  
Xueqin Lv ◽  
Yanfeng Liu ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Efficiency ◽  
Molecular Engineering ◽  
Fine Tuning ◽  
Wild Type ◽  
Content Type ◽  
Chitosan Oligosaccharides ◽  
In The Wild ◽  
Secretory Production ◽  
Extracellular Activity

ABSTRACT Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5′ untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s−1 in the wild type to 7.56 μM s−1 in the mutant R157T. IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

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