Optimization of Cephalosporin C Acylase Expression in Escherichia coli by High-Throughput Screening a Constitutive Promoter Mutant library.

Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.

Effect of shaking speed on immobilization of cephalosporin C acylase: Correlation between protein distribution and properties of the immobilized enzymes.

During enzyme immobilization, enzyme activity and protein distribution are affected by various factors such as enzyme load, temperature, and pH. In general, two types of protein distribution patterns (heterogeneous or homogeneous) are observed inside a porous carrier, owing to differences in preparation parameters. During the immobilization of a fusion protein (CCApH) of cephalosporin C acylase (CCA) and pHluorin (a pH-sensitive mutant of green fluorescent protein), different shaking speeds induced obvious differences in protein distribution on an epoxy carrier, LX-1000EPC. Enzyme immobilization with a homogeneous distribution pattern was observed at a low shaking speed (120 rpm) with an operational stability of 10 batches at 37°C. The operational stability of an immobilisate with heterogeneous protein distribution prepared at a high shaking speed (200 rpm) was six batches. Given the pH-sensitive characteristics of pHluorin in the fusion protein, the intraparticle pH of CCApH immobilisates during catalysis was monitored using confocal laser scanning microscopy. The microenvironmental pH of the immobilisate with heterogeneous protein distribution sharply decreased by about 2 units; this decrease in the pH may be detrimental to the life-span of immobilized CCA. Thus, this work demonstrates the good operational stability of pH-sensitive proton-forming immobilized enzymes with homogeneous protein distribution.

Single-gene regulated non-spore-forming Bacillus subtilis: Construction, transcriptome responses, and applications for producing enzymes and surfactin.

Bacillus subtilis, a spore-forming industrial bacterium, is widely used for production of enzymes and valuable chemicals. The spore-formation, however, always results in remarkably reduced cell-density, thereby reducing product yield. Here, we constructed different non-spore-forming B. subtilis mutants via single-gene regulation. During the three spore-forming stages: signal sensing, transduction, and sporulation, we found that deleting only a single gene of sporulation, i.e. spo0A, spoIIIE, and spoIVB, can completely block the spore generation. Interestingly, the engineered non-sporulating mutants exhibited physiological heterogeneity and distinct synthetic capabilities. The spo0A-null spore-free mutant displayed remarkably high enzyme production capacity, such as 194% enhance amylase production. However, the spoIVB-null non-spore-forming mutant was especially efficient in producing secondary metabolites, such as surfactin; its flask titer increased significantly to 16.7 g/L, with the overexpression and Leu addition strategy. Our results offer a new strategy for re-modeling B. subtilis to further improve its fermentation efficiency and application.

A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH.

Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.

High-Throughput Screening of T7 Promoter Mutants for Soluble Expression of Cephalosporin C Acylase in E. coli.

Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.

Advances in acrylamide bioproduction catalyzed with Rhodococcus cells harboring nitrile hydratase.

Acrylamide is an important bulk chemical used for producing polyacrylamide, which is widely applied in diverse fields, such as enhanced oil recovery and water treatment. Acrylamide production with a superior biocatalyst, free-resting Rhodococcus cells containing nitrile hydratase (NHase), has been proven to be simple but effective, thereby becoming the main method adopted in industry to date. Under the harsh industrial conditions, however, NHase-containing Rhodococcus cells in a natural state are prone to deactivation. Thus, multiple genetic strategies able to evolve recombinant Rhodococcus biocatalysts at either the enzyme or cell level have been reported. While most of the methods on enzyme engineering concentrate on NHase stability enhancement by strengthening the flexible sites, Rhodococcus cell engineering with various methods can enhance both the NHase activity and stability as well. Developing some new types of reactors, especially the microreactor, is also an effective way to improve the hydration process efficiency. Compared with the conventional stirred tank reactor, the membrane dispersion microreactor can enhance the heat and mass transfer in the hydration process with Rhodococcus cells as biocatalysts, thereby significantly improving the productivity of the acrylamide bioproduction process.

Affinity-binding immobilization of D-amino acid oxidase on mesoporous silica by a silica-specific peptide.

Enzyme immobilization is widely used for large-scale industrial applications. However, the weak absorption through physical methods limits the recovery ability. Here, affinity-binding immobilization of enzymes was explored using a silica-specific affinity peptide (SAP) as a fusion tag to intensify the binding force between the enzyme and mesoporous silica (MPS) carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme. The optimal screened SAP (LPHWHPHSHLQP) was selected from a M13 phage display peptide library and fused to the C-terminal of DAAO to obtain fused DAAOs with one, two and three SAP tags, respectively. The activity of DAAO-SAP-MPS was superior comparing with DAAO-2SAP-MPS and DAAO-3SAP-MPS; meanwhile DAAO-SAP-MPS shows 36% higher activity than that of DAAO-MPS. Fusion with one SAP improved the thermal stability with a 10% activity increase for immobilized DAAO-SAP-MPS compared to that of DAAO-MPS at 50 °C for 3 h. Moreover, the activity recovery of immobilized DAAO-SAP-MPS was 25% higher in operation stability assessment after six-batch conversions of cephalosporin to glutaryl-7-amino cephalosporanic acid than that of DAAO-MPS.

Application of ammonium bicarbonate buffer as a smart microenvironmental pH regulator of immobilized cephalosporin C acylase catalysis in different reactors.

In a stirred tank reactor, during catalysis with immobilized cephalosporin C acylase (CCA), the microenvironmental pH dropped to 7.2 in a nonbuffered system (with the pH maintained at 8.5 by adding alkali) due to the existence of diffusional resistance. Moreover, the immobilized CCA only catalyzed five batch reactions, suggesting that the sharp pH gradient impaired the enzyme stability. To buffer the protons produced in the hydrolysis of cephalosporin C by CCA, phosphate and bicarbonate buffers were introduced. When CCA was catalyzed with 0.1 M ammonium bicarbonate buffer, no obvious gradient between the bulk solution and intraparticle pH was detected, and the catalysis of 15 batch reactions was achieved. Accordingly, with 0.2 M ammonium bicarbonate buffer in a packed bed reactor, the immobilized CCA exhibited continuous catalysis with high conversion rates (≥95%) for 21 days. Reactions with ammonium bicarbonate buffer showed significant increases in the stability and catalytic efficiency of the immobilized CCA in different reactors compared to those in nonbuffered systems.

Antisense RNA-Based Strategy for Enhancing Surfactin Production in Bacillus subtilis TS1726 via Overexpression of the Unconventional Biotin Carboxylase II To Enhance ACCase Activity.

The antisense RNA (asRNA) strategy is commonly used to block protein expression and downregulate the contents of metabolites in several microorganisms. Here, we show that the asRNA strategy can also be used to block gfp expression in Bacillus subtilis TS1726, which could further be utilized in the identification of new genes and functions. Via application of this strategy, biotin carboxylase II encoded by yngH (GeneID 939474) was identified to play a more significant role in maintaining acetyl-CoA carboxylase (ACCase) activity and enhancing surfactin synthesis compared to those of other ACCase subunits. The yngH gene was then overexpressed in the engineered strain B. subtilis TS1726(yngH). The surfactin titer of TS1726(yngH) increased to 13.37 g/L in a flask culture, representing a 43% increase compared to that of parental strain TS1726. This strategy opens the door to achieving large-scale production and broad application of surfactin.

Improving stress tolerance and cell integrity of Rhodococcus ruber by overexpressing small-shock-protein Hsp16 of Rhodococcus.

Rhodococcus species have been successfully used as cell catalysts for valuable chemicals production due to their well-characterized resistance to harmful factors. An understanding of how they respond to stress is of great interest, which will enable the identification of engineering strategies for further improving their resistance and maintaining cell integrity and viability. Here, we assessed the transcriptome response of R. ruber TH3 to heat shock. Approximately, 376 genes were up-regulated in heat-shocked TH3. Among all the up-regulated functional genes, the small heat-shock-protein (Hsp16) with maximal enhanced transcript (463-fold) was identified, and its function was investigated. Results showed that overexpressed Hsp16 has no significant promotive effect on stress tolerance of in-cell enzyme. Interestingly, compared to the control TH3, a little fewer pores and folds on the surface of TH3(Hsp16) and more intact TH3(Hsp-GFP) cells under AM treatment were observed by SEM and LCSM, respectively. Moreover, survival test showed that more (about 501-700) TH3(Hsp16) colonies were observed while only 1-100 TH3 colonies after 50% AM treatment, and this trend is also found in high-temperature cultivation experiments. These results indicate that Hsp16 does great contributions to preventing cell leakage, maintaining cell integrity and viability of R. ruber under stress conditions.

Core element characterization of Rhodococcus promoters and development of a promoter-RBS mini-pool with different activity levels for efficient gene expression.

To satisfy the urgent demand for promoter engineering that can accurately regulate the metabolic circuits and expression of specific genes in the Rhodococcus microbial platform, a promoter-ribosome binding site (RBS) coupled mini-pool with fine-tuning of different activity levels was successfully established. Transcriptome analyses of R. ruber TH revealed several representative promoters with different activity levels, e.g., Pami, Pcs, Pnh, P50sl36, PcbiM, PgroE and Pniami. ß-Galactosidase (LacZ) reporter measurement demonstrated that different gene expression levels could be obtained with these natural promoters combined with an optimal RBS of ami. Further use of these promoters to overexpress the nitrile hydratase (NHase) gene with RBSami in R. ruber THdAdN produced different expression levels consistent with the transcription analyses. The -35 and -10 core elements of different promoters were further analyzed, and the conserved sequences were revealed to be TTGNNN and (T/C)GNNA(A/C)AAT. By mutating the core elements of the strong promoters, Pnh and Pami, into the above consensus sequence, two even stronger promoters, PnhM and PamiM, were obtained with 2.2-fold and 7.7-fold improvements in transcription, respectively. Integrating several strategies, including transcriptome promoter screening, -35 and -10 core element identification, core element point-mutation, RBS optimization and diverse reporter verification, a fine-tuning promoter-RBS combination mini-pool with different activity levels in Rhodococcus strains was successfully established. This development is significant for broad applications of the Rhodococcus genus as a microbial platform.

Tuning and elucidation of the colony dimorphism in Rhodococcus ruber associated with cell flocculation in large scale fermentation.

Prevention of cell flocculation in large-scale fermentation is of great importance for most industrial microbes. Using Rhodococcus ruber TH3 as a model strain, we revealed that the undesired cell flocculation in a fermenter was associated with the colony dimorphism phenomenon, and it only occurred in the rough-type of cells (R-TH3) instead of the smooth-type of cells (S-TH3). By analyzing the transcriptome differences of R-TH3 and S-TH3, six representative genes with significantly upregulated transcription in S-TH3 were selected and overexpressed in R-TH3. The colony morphotypes of the six engineered strains changed to different extents, in which overexpressions of three lipid metabolism-related proteins LM1, LM2, and LM3 tuned the colony morphotype from rough to almost as smooth as in S-TH3. SEM observation confirmed the cell surface difference of the engineered strains from R-TH3. Their cell surface hydrophobicity also reduced, and the cell sedimentation behaviors were consequently changed as expected. Using R-TH3/LM1 as the representative of the engineered bacteria, fatty acids of the cell envelopes were measured. Fatty acid contents of S-TH3, R-TH3/LM1, and R-TH3 were 27.21, 24.10, and 22.24%, respectively. Among all the fatty acids, stearic acid binding to hydrophilic extracellular polysaccharides (EPS) in Rhodococcus showed significant differences among the cells. The EPS contents of S-TH3, R-TH3/LM1, and R-TH3 were 191, 163, and 137 mg/g cells. Hence, the hydrophilicity of the S-TH3 cells was mainly due to the EPS in the outermost layer of the cells. Increase of fatty acids especially stearic acid results in the increase of the bound EPS, finally bringing about the hydrophilicity enhancement.

In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters.

Surfactin-family lipopeptides are green biosurfactants with substantial industrial potential. The major problem prohibiting surfactin use is the low titer of the wild producer, Bacillus subtilis. Using transcriptomic analysis, four strong promoters, PgroE, Pcdd, PrplK, and PsspE, were identified and cloned from the genome of B. subtilis THY-7, a novel surfactin producer that has been identified from soil with a 0.55 g/L surfactin titer. An optimal promoter, PgroE, was selected to replace the native THY-7 surfactin synthase (SrfA) promoter through single-cross homologous recombination; however, the resulting engineered strain containing the PgroE substitution did not synthesize surfactin. The sucrose-inducible promoters PsacB and PsacP were then substituted in place of PsrfA, and the resulting engineered strains produced 1.09 and 0.22 g/L surfactin, respectively. An artificial, sucrose-inducible Pg1 promoter was produced through fusion of the PgroE and PsacB ribonucleic antiterminator (RAT), and the engineered strain containing the Pg1-substitution produced a surfactin titer of 1.44 g/L. An artificial IPTG-inducible promoter, Pg2, was constructed from a PgroE-lacO fusion and then substituted for the chromosomal PsrfA locus, and the surfactin titer of the resulting THY-7/Pg2-srfA increased to 5.98 g/L. The driving capacity of Pg2 was further improved by the inclusion of two point mutations in the -35 and -10 regions to produce the novel promoter Pg3. Pg3 exhibited super-strong activity as measured by lacZ reporter gene overexpression (approximately 3000 U). The Pg3-substitution strain THY-7/Pg3-srfA produced up to 9.74 g/L surfactin in a 5 L fermentor. The maximum productivity was 0.30 g/L/h, and the greatest yield reached 0.14 g surfactin/g sucrose. Biotechnol. Bioeng. 2017;114: 832-842. © 2016 Wiley Periodicals, Inc.

Microenvironmental pH changes in immobilized cephalosporin C acylase during a proton-producing reaction and regulation by a two-stage catalytic process.

Cephalosporin C acylase (CCA), a proton-producing enzyme, was covalently bound on an epoxy-activated porous support. The microenvironmental pH change in immobilized CCA during the reaction was detected using pH-sensitive fluorescein labeling. The high catalytic velocity of the initial stage of conversion resulted in a sharp intraparticle pH gradient, which was likely the key factor relating to low operational stability. Accordingly, a novel strategy for a two-stage catalytic process was developed to reduce the reaction rate of stage I at a low temperature to preserve enzymatic activity and to shorten the duration of catalysis at a high reaction temperature in stage II. The reaction using the two-stage catalytic process (10-37°C shift at 30min) showed significantly improved stability compared with that of the single-temperature reaction at 37°C (29 batches versus five batches, respectively) and a shorter catalytic period than the reaction at 10°C (40min versus 70min, respectively).

Ammonium acrylate biomanufacturing by an engineered Rhodococcus ruber with nitrilase overexpression and double-knockout of nitrile hydratase and amidase.

Rhodococcus ruber TH was selected as a parent strain to engineer for biomanufacturing of ammonium acrylate; the characteristics of this strain included accelerated growth rate, high cell tolerance and natively overexpressed nitrile hydratase (NHase). Transcriptome analysis revealed that the transcription levels of the native NHase, amidase and nitrilase were extremely high, moderate and extremely low, respectively. Through NHase-amidase double-knockout and amidase single-knockout, the engineered strains R. ruber THdAdN and R. ruber THdA were obtained for overexpression of a heterologous nitrilase from R. rhodochrous tg1-A6 using a urea-induced Pa2 promoter. The nitrilase activity toward substrate acrylonitrile in the engineered THdAdN(Nit) reached 187.0 U/mL at 42 h, threefold of that R. rhodochrous tg1-A6 and 2.3-fold of that of THdA(Nit). The optimal catalysis temperature and pH of the nitrilases in different cells exhibited no significant difference. Using the cells as catalysts, biomanufacturing of ammonium acrylate was performed under room temperature. When catalyzed by the engineered THdAdN(Nit), the titer and productivity of ammonium acrylate dramatically increased to 741.0 g/L and 344.9 g/L/h, which are the highest results reported to date.

Overproduction of the Escherichia coli Chaperones GroEL-GroES in Rhodococcus ruber Improves the Activity and Stability of Cell Catalysts Harboring a Nitrile Hydratase.

Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpEGroEL- GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases.

Directed Evolution and Mutant Characterization of Nitrilase from Rhodococcus rhodochrous tg1-A6.

In this paper, a molecularly directed evolution-based approach was applied to modify the nitrilase from Rhodococcus rhodochrous tg1-A6 for improving properties in catalyzing nitriles. In the process of error-prone polymerase chain reaction (PCR) with the wild-type nitrilase gene acting as the template, a library of the randomly mutated nitrilase gene was constructed. Since the pH value of catalyzing solution decreased when glycolonitrile was used as the substrate of nitrilase, a high-throughput strategy based on the color change of a pH-sensitive indicator was established for rapid screening of the mutated nitrilase. After three rounds of random mutation and screening about 5000 clones, a variant (Mut3) with 5.3-fold activity of the wild-type counterpart was obtained. Five amino acid substitutions (D27E, N97K, L246F, D108E, and S111R) were found in the variant Mut3. The properties of three mutated enzymes obtained in the three-round mutation were investigated. In the conversion of glycolonitrile, the variant (Mut2) accumulated the highest concentration of glycolic acid at 10.6 g l(-1), a much higher value than the wild type (3.2 g l(-1)).

A novel high-throughput and quantitative method based on visible color shifts for screening Bacillus subtilis THY-15 for surfactin production.

A novel chromatic visible screening method using bromothymol blue (BTB) as a color indicator and cetylpyridinium chloride (CPC) as a mediator was constructed to obtain the high titer surfactin-producing strains. The reliability and quantification accuracy of color shift were also confirmed. Regular chromatic responses from faint yellow-green to dark green and bright blue reflected the different ranges of surfactin concentrations. Moreover, the quantitative accuracy of surfactin quantification in the range of 100-500 mg/L was verified by reverse-phase high-performance liquid chromatography (RP-HPLC) using different fermentation supernatant samples. Using this CPC-BTB method, a superior surfactin producer, Bacillus subtilis THY-15, was successfully screened. The producer's surfactin (Srf) titer reached 1240 mg/L. RP-HPLC analysis of THY-15 revealed four surfactin isoforms. As identified by amino acid analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, the isoforms of surfactin in fraction 1, 2 and 4 had the same circular peptide sequence of Glu-Leu-Leu-Val-Asp-Leu-Leu but different iso-C13, C14 and C15 fatty acid chains, but the isoform in fraction 3 possessed a special peptide sequence of Glu-Val-Leu-Leu-Asp-Leu-Val.

Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules.

In this work, cephalosporin C acylase (CA), a heterodimeric enzyme of industrial potential in direct hydrolysis of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA), was covalently immobilized on the aminated support LX1000-HA (HA) with two different protocols. The stability of CA adsorbed onto the HA support followed by crosslinking with glutaraldehyde (HA-CA-glut) was better than that of the CA covalently immobilized on the glutaraldehyde preactivated HA support (HA-glut-CA). The thermostabilization factors (compared with the free enzyme) of these two immobilized enzymes were 11.2-fold and 2.2-fold, respectively. In order to improve the stability of HA-CA-glut, a novel strategy based on postimmobilization modifying with aminated molecules was developed to take advantage of the glutaraldehyde moieties left on the enzyme and support. The macromolecules, such as polyethyleneimine (PEI) and chitosan, had larger effects than small molecules on the thermal stability of the immobilized enzyme perhaps due to crosslinking of the enzymes and support with each other. The quaternary structure of the CA could be much stabilized by this novel approach including physical adsorption on aminated support, glutaraldehyde treatment, and macromolecule modification. The HA-CA-glut-PEI20000 (the HA-CA-glut postmodified with PEI Mw = 20,000) had a thermostabilization factor of 20-fold, and its substrate affinity (Km = 14.3 mM) was better than that of HA-CA-glut (Km = 33.4 mM). The half-life of the immobilized enzymes HA-CA-glut-PEI20000 under the CPC-catalyzing conditions could reach 28 cycles, a higher value than that of HA-CA-glut (21 cycles).

Identification of lipopeptide isoforms by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC.

A three-stage linear gradient strategy using reverse-phase high-performance liquid chromatography (HPLC) was optimized for rapid, high-quality, and simultaneous purification of the lipopeptide isoforms of iturin, fengycin, and surfactin, which may differ in composition by only a single amino acid and/or the fatty acid residue. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was applied to detect the lipopeptides harvested from each reversed-phase HPLC peak. Amino acid analysis based on phenyl isothiocyanate derivatization was further used for confirmation of the amino acid species and molar ratio in a certain HPLC fraction. By this MALDI-TOF-MS/MS coupled with amino acid analysis, it was revealed that iturin at m/z 1,043 consists of a circular Asn-Tyr-Asn-Gln-Pro-Asn-Ser peptide and C14 ß-OH fatty acid. Surfactin homologs from Bacillus subtilis THY-7 at m/z 1,030, 1,044, 1,058, and 1,072 possess a circular Glu-Leu-Leu-Val-Asp-Leu-Leu peptide and the ß-OH fatty acid with a different length (C13-C16). Fengycin species at m/z 1,463 and 1,477 are homologs possessing the circular peptide Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile linked to a C16 or C17 γ-OH fatty acid, whereas fengycin at m/z 1,505 contains a Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile sequence with a Val instead of Ala at position 6. The method developed in this work provided an efficient approach for characterization of diverse lipopeptide isoforms from the iturin, fengycin, and surfactin families.