Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g−1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.

•This was the first time that the gene treY of MTSase was expressed in Pichia pastoris..•In this paper, the highest level of thermostable MTSase expression was achieved.Two genes encoding the maltooligosyltrehalose synthase (MTSase) from Sulfolobus acidocaldarius ATCC 33909 were synthesized: one (designated E-treY) using codons preferred by Escherichia coli and the other (designated P-treY) using codons preferred by Pichia pastoris. E-treY was inserted into pET-24a(+), and the resulting plasmid was used to transform E. coli BL21(DE3). P-treY was inserted into pPIC3.5 K and pGAPZA, and the resulting plasmids were used to transform P. pastoris KM71. The maximum MTSase activity expressed by these E. coli and P. pastoris strains reached 31.6 and 133.0 U mL−1, respectively. The purified MTSases displayed the same pH (pH 5.5) and temperature (75 °C) optima. Both had a half-life of 12 h at 75 °C. The recombinant P. pastoris KM71/pPIC3.5K-treY strain producing the highest enzyme activity was used to optimize the fermentation conditions in a 3.6-L bioreactor. Fermentation for 96 h under optimal conditions yielded an MTSase activity of 747.4 U mL−1.

In this study, the impact of cutinases from Thermobifida fusca and Fusarium solani pisi on the deinking of old newsprint were evaluated for the first time. When repulped old newsprint was treated with 8 U/g of T. fusca cutinase at pH 8 and 60 \(^{\circ }\)C for 30 min, the brightness of the deinked papers reached 42.01%. With 8 U/g F. solani cutinase at pH 8.5 and 35 \(^{\circ }\)C for 30 min, the brightness reached 41.62%. These brightness values are higher than that achieved using chemical deinking by 5.13 and 4.38%, respectively. These brightness values were also superior to that achieved using commercial lipase in this study and those previously reported using cellulases, hemicellulases, and laccase, which were higher than that achieved using chemical deinking by 2–4%. The mechanical properties of the deinked paper, including tensile index, tear index, and burst index, were also measured. The results showed that the two cutinases had different effects on the paper fibers. In summary, both T. fusca and F. solani pisi cutinases were able to remove ink from old newsprint more efficiently than alkaline chemistry or other enzymes. This study provides a potential strategy to deink efficiently old newsprint using cutinase.

•Short-chain aliphatic esters were synthesized using Thermobifida fusca cutinase.•The highest ester yield ever reported, 99.2%, was achieved.•This process displayed strong tolerance for water content and acid concentration.•T. fusca cutinase exhibited wide selectivity for a series of acids and alcohols.Short-chain aliphatic esters are commonly used as fruit flavorings in the food industry. In this study, Thermobifida fusca (T. fusca) cutinase was used for the synthesis of aliphatic esters, and the maximum yield of ethyl caproate reached 99.2% at a cutinase concentration of 50 U/ml, 40 °C, and water content of 0.5%, representing the highest ester yield to date. The cutinase-catalyzed esterification displayed strong tolerance for water content (up to 8%) and acid concentration (up to 0.8 M). At substrate concentrations ⩽0.8 M, the ester yield remained above 80%. Moreover, ester yields of more than 98% and 95% were achieved for acids of C3–C8 and alcohols of C1–C6, respectively, indicating extensive chain length selectivity of the cutinase. These results demonstrate the superior ability of T. fusca cutinase to catalyze the synthesis of short-chain esters. This study provides the basis for industrial production of short-chain esters using T. fusca cutinase.

Addition of MgCl2 to the culture medium has been found to dramatically increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis. The specific activity of the pullulanase obtained from medium supplemented with MgCl2 was also higher than that obtained in culture medium without added magnesium ions. In this work, the mechanism of this increase was studied. When cultured in medium without added magnesium ions, B. choshinensis mainly produced a thermolabile, inactive form of pullulanase. The addition of magnesium ions led to the production of a thermostable, active form of pullulanase. Circular dichroism assays revealed considerable differences in secondary structure between the active and inactive pullulanase forms. Transmission electron microscopy suggested that magnesium ion addition inhibits the shedding of cell wall protein (HWP) layers from the cell surface. Quantitative real-time PCR showed that magnesium ion addition represses transcription of HWP. Because the pullulanase gene and HWP have identical promoters, pullulanase gene transcription was also inhibited. These results suggest that when pullulanase is expressed slowly, it tends to fold into an active form.

In this study, the pullulanase gene from Bacillus deramificans was efficiently expressed in Brevibacillus choshinensis. The optimal medium for protein expression was determined through a combination of single-factor experiments and response surface methodology. The initial pH of the medium and the culture temperature were optimized. The pullulanase yield increased 10.8-fold through medium and condition optimization at the shake-flask level. From the results of these experiments, the dissolved oxygen level was optimized in a 3-L fermentor. Under these optimized conditions, the pullulanase activity and the specific pullulanase productivity reached 1005.8 U/mL and 110.5 × 103 U/g dry cell weight, respectively, with negligible intracellular expression. The Brevibacillus choshinensis expression system has proven to be valuable for the extracellular production of pullulanase.

Previously, nitrile hydratase (NHase) from Corynebacterium nitrilophilus was obtained and showed potential in polyacrylonitrile (PAN) fibre modification. In the present study, the modification conditions of C. nitrilophilus NHase on PAN were investigated. In the optimal conditions, the wettability and dyeability (anionic and reactive dyes) of PAN treated by C. nitrilophilus NHase reached a similar level of those treated by alkali. In addition, the chemical composition and microscopically observable were changed in the PAN surface after NHase treatment. Meanwhile, it revealed that cutinase combined with NHase facilitates the PAN hydrolysis slightly because of the ester existed in PAN as co-monomer was hydrolyzed. All these results demonstrated that C. nitrilophilus NHase can modify PAN efficiently without textile structure damage, and this study provides a foundation for the further application of C. nitrilophilus NHase in PAN modification industry.

A xylanase (XynA) was purified from the culture medium of Streptomyces sp. FA1, which was previously isolated from a bamboo retting system. XynA had a molecular mass of 43 kDa, displayed maximal activity at pH 5.5, retained 41% of its maximal activity at pH 11.0, and was stable over a wide pH range (3.0 ∼ 11.0). Purified XynA was subjected to peptide mass fingerprinting, which led to the cloning of the xynA gene. The xynA gene, which encodes a mature protein of 436 amino acids, was heterologously expressed in E. coli BL21(DE3). The activity in the culture medium could reach 213.5 U/mL, which was 11.2-fold higher than that produced by Streptomyces sp. FA1. BLAST searching revealed that full-length XynA shares less than 90% identity with most of its homologues, whereas amino acids 48-436 of the enzyme share 97% identity with an open reading frame encoding a putative full-length mature xylanase from Streptomyces tendae. The truncated xynA gene, xynA48-436, was cloned and expressed in E. coli, however, no xylanase activity could be detected in the culture medium. Based on these results, it is suggested that XynA is a new member of glycoside hydrolases family10 with exceptional catalytic efficiency at alkaline pH.

In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349 U ml−1, 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60 °C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8 h under optimal conditions, which included 400 g l−1 inulin, an enzyme concentration of 40 U g−1 substrate, 50 °C and pH 6.0, the inulooligosaccharide yield was 91 %. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.

•The CBM binding sites of cutinase–CBMCenAfusion protein was firstly modified.•The mutants of W68L and W68Y exhibited enhanced activity toward PET fiber.•The model of binding between the CBM and PET was proposed.•The mechanism of enhanced enzymatic activity toward PET upon mutation was explored.In the present study, cutinase–CBMCenA fusion protein was genetically modified in the carbohydrate-binding module (CBM) binding sites, by site-directed mutagenesis, to enhance its activity toward polyethylene terephthalate (PET) fiber. The effects of tryptophan at particular positions of CBMCenA on the binding and hydrolysis of polyester substrate were investigated by replacing each of Trp14, Trp50 and Trp68 with leucine or tyrosine, respectively. All the mutants were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that the mutants displayed similar thermostability and pH stabilities in response to the native enzyme. Furthermore, W68L and W68Y, among all the mutants, exhibited significant improvement in binding and catalytic efficiency (1.4–1.5 fold) toward PET fiber when compared to that of the native enzyme. The enhanced binding and hydrolytic activity might be a result of creating new hydrogen bond or hydrophobic interaction between the enzyme and PET fiber.

Galacto-oligosaccharides (GOS), an important class of functional food, are commonly produced from lactose using β-galactosidase. In the present study, β-galactosidase (LacS) from Sulfolobus solfataricus P2 was cloned and site-directed mutagenesis was performed to obtain two mutants, F359Q and F441Y. All of the wild-type enzyme and mutants were expressed in Escherichia coli BL21(DE3) and purified to homogeneity. The enzymatic properties and optimal condition for transglycosylation reaction of the enzymes were investigated in detail. Under their individual optimal conditions, yields of GOS could reach 50.9% for wild-type enzyme, 58.3% for F359Q, and 61.7% for F441Y. In addition, the potential mechanism for this enhancement was analysed.Highlights► β-Galactosidase (LacS) from Sulfolobus solfataricus P2 was expressed in E. coli. ► Transglycosylation capability of LacS was enhanced by sited-directed mutagenesis. ► The mechanism of the enhancement of GOS production by mutants was explored.

The α-glucosidase (AGL) from Aspergillus niger has been applied to produce isomaltooligosaccharides. In the present study, various factors which affect the yield of recombinant AGL, produced by engineered Pichia pastoris, were investigated. The expression level reached 5.5 U ml−1 in bioreactor after optimization of parameters of initial induction cell density, induction temperature and methanol concentration. In addition, it was found that coexpression of protein disulfide isomerase (PDI) inhibited the growth of the engineered P. pastoris strains and had an adverse effect on the production of AGL, while codon optimization of native A. niger α-glucosidase encoding gene (aglu) resulted in a significant enhancement of enzyme production, which reached 10.1 U ml−1. We believe that yield of AGL is increased by codon optimization as a result of enhanced translation efficiency as well as more stable mRNA secondary structure. In contrast, PDI coexpression under the control of alcohol oxidase promoter (PAOX1) seems to be less efficient in helping disulfide bond formation in AGL while probably induce unfolded protein response, which further leads to cell apoptosis and increased protein degradation.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme that degrades starch and starch related glucans into cyclodextrins (CDs) by intramolecular transglycosylation reaction. The biochemical activity of recombinant CGTase from Anaerobranca gottschalkii for the yield and product specificity of cyclodextrins was investigated in the presence of organic solvents. Compared with the control of starch bioconversion, addition of various organic solvents generally increased the total CD and product specificity by affecting product inhibition and/or intermolecular transglycosylation reaction. The highest conversion (45 %) of starch to CDs was obtained in the presence of ethanol, while the simultaneous addition of two organic solvents, decanol-ethanol, comparatively showed a reduced total yield of 39 %. Despite this, the highest product ratio of 91 % α-CD, and 64 % β-CD was obtained in the presence of decanol and cyclohexane respectively. The effect of organic solvents on the yield and specificity of CD was attributed mainly to their effect on product inhibition and transglycosylation reaction. Although the use of two organic solvents showed almost a significant increase in total yield of CDs, it resulted in a comparatively lower specific product yield compared to their respective individual effect. Generally, normal enzyme activity was favoured at higher temperature of 65 °C, but the addition of organic solvents, in most cases, was found to decrease the bioconversion. Thus, the preferred optimal condition was reduced to 40 °C, where the maximal conversion of starch to CDs in general and α-CD in particular was achieved.

Secretion of cytoplasmic expressed proteins into culture medium has significant commercial advantages in large-scale production of proteins. Our previous study demonstrated that the membrane permeability of Escherichia coli could be significantly improved when Thermobifida fusca cutinase, without a signal peptide, was expressed in cytoplasm. This study investigated the extracellular production of other recombinant proteins, including both secretory and cytosolic proteins, with co-expression of cutinase. When the secretory enzymes, xylanase and α-amylase, were co-expressed with cutinase, the culture period was shortened by half, and the productivity was 7.9 and 2.0-fold to that of their individual control without co-expression, respectively. When the normally cytosolic proteins, xylose isomerase and trehalose synthase, were co-expressed with cutinase, more than half of the target proteins were “secreted” into the culture medium. Moreover, by using β-galactosidase to detect membrane leakage, the improved secretion of the above model proteins was confirmed not to be due to cell lysis. The study provides a novel strategy for enhancing extracellular secretion of recombinant proteins in E. coli.

Self-assembling amphipathic peptides (SAPs) are a category of peptides that have unique sequences with alternating hydrophobic and hydrophilic residues that can spontaneously assemble into ordered nanostructures. In this study, we investigated the potential of fusion technique with SAPs to improve the thermal stability of lipoxygenase (LOX) from Pseudomonas aeruginosa. Six SAPs were individually fused to the N terminus of the LOX that resulted to the SAP–LOX fusions with approximately 2.3- to 4.5-fold enhanced thermal stability at 50 °C. The specific activities of the SAP–LOX fusions were also increased by 1.0- to 2.8-fold as compared with the wild-type LOX. This is the first report on the improvement of the thermal stability and specific activity of an enzyme by the fused SAPs, suggesting a simple technique to improve the catalytic properties of the recombinant enzymes by fusion expression.

Polyamidase was able to hydrolyze the amide bond of insoluble polymer. In the present study, a polyamidase from Nocardia farcinica CGMCCC4.1166 was cloned and expressed in E. coli BL21(DE3). The recombinant polyamidase was purified to homogeneity, through a combination of chromatography of anion exchange, and hydrophobic interaction. The purified enzyme was characterized in detail. The optimum temperature of the enzyme was 50°C, and it was stable below 40°C. The enzyme had an optimum pH of 8.0, with pH stability between pH 7.0 and 9.0. The enzyme does not need metal ion as cofactor. In addition, when the enzyme was utilized to hydrolyze polyamide, the monomeric product of adipic acid was verified by HPLC analysis; as well, the wettability and dyeability of polyamide fabric after enzyme treatment were significantly improved, which differed from those of its inactive S173A mutant, and the amidase from Rhodococcus pyridinivorans. Furthermore, the structural features near the active site of polyamidase, different from other amidases, were explored.

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PSAp) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PSAp would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PSAp in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PSAp is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PSAp follows Michaelis–Menten kinetics at 60 °C, with a KM for erythrose 4-phosphate of 280 μM, a KM for phosphoenolpyruvate of 891 μM, and a kcat of 1.0 s−1. None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PSAp. The structure of DAH7PSAp is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.Graphical abstractDownload full-size imageHighlights► We investigate the DAH7P synthase from Aeropyrum pernix, a primitive archaebacterium. ► We present the biochemical characterization and crystal structure of the enzyme. ► The enzyme activity is not regulated by metabolites from the shikimate pathway.

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins from starch by an intramolecular transglycosylation reaction. Due to the increasing industrial application of cyclodextrins in many fields such as pharmacy, agriculture, biotechnology, food, environment and cosmetics, CGTases have attracted the attention of many scientific researches. Undoubtedly, due to its well-known genetic properties, simplicity and capacity to accommodate many foreign proteins, Escherichia coli remains the most widely used host for recombinant proteins production and thus for CGTases. Like all other proteins, CGTases are originally produced in the cytoplasm, but expressing them out into the periplasm or further to the culture media is preferred due to several advantages such as simplified downstream processing and high expression level which otherwise would be costly, complicated and time consuming. Since E. coli, other than some of its degradative enzymes and toxins, does not normally secrete proteins extracellularly, many strategies have been tried to overcome this drawback using the recombinant technologies. Unfortunately, oversecretion of the recombinant proteins most of the time results in the formation of inactive protein aggregates, called inclusion bodies, which result as a consequence of the burden caused by the methods meant to enhance the secretion. Thus, in this mini-review, the few but most commonly used strategies which offered a solution to the enhancement of extracellular secretion of CGTase in its native state are discussed.

The production of cyclodextrins (CDs) by cyclodextrin glycosyltransferase (CGTase) from Bacillus clarkii 7364 was studied. Forty-seven percent (w/w) conversion rate to γ-CD was obtained in the process performed by reacting 5 U per gram of starch CGTase with 15 % (w/v) soluble starch in 0.025 M sodium phosphate–NaOH buffer (pH 12) at 55 °C in the presence of 2 % (w/v) glycyrrhizic acid. Meanwhile, the ratio of γ:β-CD was 89:11, with negligible formation of α-CD. Under these conditions, there is a significant increase in overall production of CDs and a marked change in product selectivity for γ-CD. The possible mechanisms were discussed upon different product profiles with respect to the size and amount of CDs synthesized at different reaction conditions. The approach described here can be easily applied to an enzymatic process for the production of γ-CD on an industrial scale, and such high selectivity, at high conversions, is especially attractive from a commercial perspective.

High-level production of α-cyclodextrin glycosyltransferase (CGTase) is one of the key factors in α-cyclodextrin (CD) preparation. In the present study, a fed-batch fermentation strategy for high-cell-density cultivation of Escherichia coli and the extracellular production of recombinant α-CGTase from Paenibacillus macerans JFB05-01 was established. A combined feeding strategy based on both specific growth rate before induction and the amount of glycerol residues after induction was used to control cell growth, acetate production, and glycerol consumption. When induced by lactose, a feeding solution with complex nitrogen was found beneficial for α-CGTase production. In addition, different induction temperatures and induction points were investigated, and the results indicated that these factors played an important role in α-CGTase production. When induced at 25 °C and at a dry cell weight of 30 g L−1, the extracellular activity of α-CGTase could reach 275.3 U mL−1.

Escherichia coli is one of the most commonly used host strains for recombinant protein production. More and more research works on the production of recombinant protein indicate that extracellular production throughout a culture medium is more convenient and attractive compared to intracellular production. In present work, inducing temperature and isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration were investigated to decrease the formation of inclusion body and increase the amount of soluble recombinant cutinase initially. Enzyme activity in the culture medium reached to 118.9 U/ml at 64 h of culture, and no inclusion body was detected in cytoplasm under the inducement condition of 0.2 mM IPTG and 30°C. In addition, it was found that a large amount of cutinase had been accumulated in periplasm since 16-h cultivation under the same inducement condition. Therefore, glycine and surfactant sodium taurodeoxycholate (TDOC) were further used to promote the leakage of recombinant cutinase from periplasm. Supplied with 100 mM glycine and 1 mM TDOC, the amount of cutinase in periplasm decreased remarkably, and the activity in the culture medium reached to 146.2 and 149.2 U/ml after 54 h of culturing, respectively.

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Protein folding and disulfide bond formation were identified as rate-limited steps in recombinant protein secretion in yeast cells. For the production of β-glucosidase in Pichia pastoris, final β-glucosidase activity reached 1,749 U/mL after fermentation optimization in a 3 L bioreactor, while the specific activity decreased from 620 to 467 U/mg, indicating a potential protein misfolding. To solve this problem, protein disulfide isomerase, a chaperone protein which may effectively regulate disulfide bond formation and protein folding, was co-expressed with β-glucosidase. In the co-expression system, a β-glucosidase production level of 2,553 U/mL was achieved and the specific activity of the enzyme reached 721 U/mg, which is 1.54 fold that of the control.

Construction and improvement of industrial strains play a central role in the commercial development of microbial fermentation processes. l-tryptophan producers have usually been developed by classical random mutagenesis due to its complicated metabolic network and regulatory mechanism. However, in the present study, an l-tryptophan overproducing Escherichia coli strain was developed by defined genetic modification methodology. Feedback inhibitions of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (AroF) and anthranilate synthase (TrpED) were eliminated by site-directed mutagenesis. Expression of deregulated AroF and TrpED was achieved by using a temperature-inducible expression plasmid pSV. Transcriptional regulation of trp repressor was removed by deleting trpR. Pathway for L-Trp degradation was removed by deleting tnaA. l-phenylalanine and l-tyrosine biosynthesis pathways that compete with l-tryptophan biosynthesis were blocked by deleting their critical genes (pheA and tyrA). The final engineered E. coli can produce 13.3 g/l of l-tryptophan. Fermentation characteristics of the engineered strains were also analyzed.

The aglu of Aspergillus niger encodes the pro-protein of α-glucosidase, and the mature form of wild-type enzyme is a heterosubunit protein. In the present study, the cDNA of α-glucosidase was cloned and expressed in Pichia pastoris strain KM71. The activity of recombinant enzyme in a 3 L fermentor reached 2.07 U/mL after 96 h of induction. The recombinant α-glucosidase was able to produce oligoisomaltose. The molecular weight of the recombinant enzyme was estimated to be about 145 kDa by SDS−PAGE, and it reduced to 106 kDa after deglycosylation. The enzymatic activity of recombinant α-glucosidase was not significantly affected by a range of metal ions. The optimum temperature of the enzyme was 60 °C, and it was stable below 50 °C. The enzyme was active over the range of pH 3.0−7.0 with maximal activity at pH 4.5. Using pNPG as substrate, the Km and Vmax values were 0.446 mM and 43.48 U/mg, respectively. These studies provided the basis for the application of recombinant α-glucosidase in the industry of functional oligosaccharides.

The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 °C, and half-lives are approximately 8 h at 40 °C, 1.25 h at 45 °C and 0.5 h at 50 °C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca2+, Ba2+, and Zn2+ in a concentration-dependent manner, while it is dramatically inhibited by Hg2+. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.The cgt gene encoding α-cyclodextrin glycosyltransferase from Paenibacillus macerans was overexpressed extracellularly in Escherichia coli as a C-terminal His-tagged protein. The purified recombinant α-CGTase was detailedly characterized with respect to the cyclization activity.

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant α-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the α-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture.

The nature of amino acid residue 47 shows a clear discrimination between the different groups of cyclodextrin glycosyltransferase (CGTase). The effects of amino acid side chain at position 47 on cyclodextrin product specificity were investigated by replacing Lys47 in the CGTase from Paenibacillus macerans strain JFB05-01 with arginine, histidine, threonine, serine, or leucine. All of the mutations reduced α-cyclodextrin-forming activity, whereas significant increases in β-cyclodextrin-forming activity were achieved. Especially, the mutations of Lys47 into threonine, serine, or leucine converted P. macerans CGTase from α-type to β/α-type. As a result, all of the mutants displayed a shift in product specificity toward the production of β-cyclodextrin. Thus, they were more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhancement of β-cyclodextrin specificity might be due to weakening or removal of hydrogen-bonding interactions between the side chain of residue 47 and the bent intermediate specific for α-cyclodextrin formation.

Overexpression of recombinant genes in Escherichia coli and targeting recombinant proteins to the culture medium are highly desirable for the production of industrial enzymes. However, a major barrier is inadequate secretion of recombinant protein across the two membranes of E. coli cells. In the present study, we have attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine, as a medium supplement, could enhance the extracellular secretion of recombinant α-CGTase in E. coli. In the culture with glycine at the optimal concentration of 150 mM, the α-CGTase activity in the culture medium reached 23.5 U/mL at 40 h of culture, which was 11-fold higher than that of the culture in regular TB medium. A 2.3-fold increase in the maximum extracellular productivity of recombinant α-CGTase was also observed. However, further analysis indicated that glycine supplementation exerted impaired cell growth as demonstrated by reduced cell number and viability, increased cell lysis, and damaged cell morphology, which prevented further improvement in overall enzyme productivity. Significantly, Ca2+ could remedy cell growth inhibition induced by glycine, thereby leading to further increase in the glycine-enhanced extracellular secretion of recombinant α-CGTase. In the culture with 150 mM glycine and 20 mM Ca2+, both extracellular activity and maximum productivity of recombinant enzyme were 1.5-fold higher than those in the culture with glycine alone. To the best of our knowledge, this is the first article about the synergistic promoting effects of glycine and Ca2+ on the extracellular secretion of a recombinant protein in E. coli.

A major disadvantage of cyclodextrin production is the limited cyclodextrin product specificity of cyclodextrin glycosyltransferase (CGTase). Here, we described mutations of Asp372 and Tyr89 at subsite −3 in the CGTase from Paenibacillus macerans strain JFB05-01. The results showed that Asp372 and Tyr89 played important roles in cyclodextrin product specificity of CGTase. The replacement of Asp372 by lysine and Tyr89 by aspartic acid, asparagine, lysine, and arginine resulted in a shift in specificity towards the production of α-cyclodextrin, which was most apparent for the mutants D372K and Y89R. Furthermore, the changes in cyclodextrin product specificity for the single mutants D372K and Y89R could be combined in the double mutant D372K/Y89R, which displayed a 1.5-fold increase in the production of α-cyclodextrin, with a concomitant 43% decrease in the production of β-cyclodextrin when compared to the wild-type CGTase. Thus, the D372K and Y89R single and double mutants were much more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The enhanced α-cyclodextrin specificity of these mutants might be a result of stabilizing the bent conformation of the intermediate in the cyclization reaction.

Transglutaminase (TGase) is widely used in the food industry for improving protein properties by catalyzing the cross-linking of proteins. In Streptomyces, TGase is secreted as a zymogen, and an activation process has been observed in liquid culture. However, the activation mechanism remains unclear. In the present study, the TGase activation process in Streptomyces hygroscopicus was investigated by biochemical approaches. In a liquid culture, Pro-TGase was secreted and gradually was converted into active TGase during the growth period; however, in a cell-free system in which cells were removed from the liquid culture, TGase activation stalled unexpectedly. Subsequently, the TGase activation process was found to be inhibited by a TGase-activating protease inhibitor (TAPI). N-Terminal amino acid sequencing and a homology search of the purified TAPI revealed that it is a member of the Streptomyces subtilisin inhibitor (SSI) family. Furthermore, it was found that TAPI (0.1 mg/mL) decreased the surface tension of water from 72 to 60 mJ/m2 within 5 min, suggesting that it possesses surface activity. This is the first report that an SSI member functions as a surfactant protein. On the basis of these findings, a model for TAPI-regulated TGase activation process was proposed. This study provides novel insights into the TGase activation process in Streptomyces.

Cyclodextrins are cyclic α-1,4-glucans that are produced from starch or starch derivates using cyclodextrin glycosyltransferase (CGTase). The most common forms are α-, β-, and γ-cyclodextrins. This mini-review focuses on the enzymatic production, unique properties, and applications of γ-cyclodextrin as well as its difference with α- and β-cyclodextrins. As all known wild-type CGTases produce a mixture of α-, β-, and γ-cyclodextrins, the obtaining of a CGTase predominantly producing γ-cyclodextrin is discussed. Recently, more economic production processes for γ-cyclodextrin have been developed using improved γ-CGTases and appropriate complexing agents. Compared with α- and β-cyclodextrins, γ-cyclodextrin has a larger internal cavity, higher water solubility, and more bioavailability, so it has wider applications in many industries, especially in the food and pharmaceutical industries.

Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time. For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F. solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T. fusca cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca cutinases, they may have promising applications in related industries.

•β-Galactosidase production conditions (induction time, temperature, concentration of lactose, etc) were optimized in a 3-L fermentor.•The highest extracellular enzyme activity was 18.5-fold that of the original extracellular enzyme activity in 3-L fermenter.•Peak extracellular β-galactosidase activity reached 220.0 U mL−1.The β-galactosidase-catalyzed conversion of lactose to galactooligosaccharides offers an attractive approach for the efficient production of galactooligosaccharides. In this study, the β-galactosidase from Bacillus circulans was overexpressed in Escherichia coli BL21(DE3), and its extracellular secretion was characterized and optimized. Using initial parameters determined during fermentation tests in shake-flasks, extracellular expression was optimized in a 3-L fermentor. Three key parameters were studied: time of induction initiation, induction temperature, and inducer (lactose) feeding rate. Optimal extracellular β-galactosidase activity was observed when: induction was initiated when the optical density at 600 nm reached 40; (2) expression was induced at 37 °C; and (3) lactose was added at a constant feeding rate of 1.0 g L−1 h−1. Under optimal conditions, the extracellular activity reached 220.0 U mL−1, which represents 65.0% of the total β-galactosidase activity expressed. This high-level production of soluble β-galactosidase in a semisynthetic medium provides a model for the industrial production of β-galactosidase.Download high-res image (123KB)Download full-size image

•Novel α-cyclodextrin glycosyltransferases have increased activity and specificity.•Recent improvements in the enzymatic production of α-cyclodextrin are reviewed.•The use of α-cyclodextrin as a natural, soluble dietary fiber is discussed.This mini-review focuses on the unique properties, enzymatic production, and food applications of α-cyclodextrin, as well as its differences with β- and γ-cyclodextrins. The fermentative production of α-cyclodextrin glycosyltransferase (α-CGTase) is also discussed. More efficient processes for the production for α-cyclodextrin have been developed, including the use of α-CGTases with improved α-cyclodextrin specificity, the addition of appropriate complexing agents, and the simultaneous use of an α-CGTase with another amylase. Compared with other cyclodextrins, α-cyclodextrin has the smallest internal cavity and highest resistance to enzymatic hydrolysis, so it has special applications in food industry, especially as a natural, soluble dietary fiber.

Cutinases (E.C. 3.1.1.74) belong to the α/β-hydrolase superfamily. They were initially discovered because they are secreted by fungi to hydrolyze the ester bonds of the plant polymer cutin. Since then, they have been shown to catalyze the hydrolysis of a variety of polymers, insoluble triacylglycerols, and low-molecular-weight soluble esters. Cutinases are also capable of catalyzing esterification and transesterification reactions. These relatively small, versatile, secreted catalysts have shown promise in a number of industrial applications. This review begins by describing the characteristics of cutinases, pointing out key differences among cutinases, esterases and lipases, and reviewing recent progress in engineering improved cutinases. It continues with a review of the methods used to produce cutinases, with the goal of obtaining sufficient quantities of material for use in industrial processes. Finally, the uses of cutinases in the textile industry are described. The studies presented here demonstrate that the cutinases are poised to become important industrial catalysts, replacing older technologies with more environmentally friendly processes.

•Thermostability of AmyMH is improved by mutagenesis.•The improved thermostability is caused by enhanced Ca2+ binding.•The mutants exhibit better activity and stability in the presence of the EDTA.•Improved thermostability without added Ca2+ is beneficial for starch liquefaction.The thermostability of the maltohexaose-forming α-amylase from Bacillus stearothermophilus (AmyMH) without added Ca2+ was improved through structure-based rational design in this study. Through comparison of a homologous model structure of AmyMH with the crystal structure of the thermostable α-amylase from Bacillus licheniformis, Ser242, which located at the beginning of fourth α-helix of the central (β/α)8 barrel was selected for mutation to improve thermostability. In addition, an amide-containing side chain (Asn193) and a loop in domain B (ΔIG mutation), which have been proven to be important for thermostability in corresponding position of other α-amylases, were also investigated. Five mutants carrying the mutations ΔIG, N193F, S242A, ΔIG/N193F, and ΔIG/N193F/S242A were generated and their proteins characterized. The most thermostable mutant protein, ΔIG/N193F/S242A, exhibited a 26-fold improvement in half-life at 95 °C compared to the wild-type enzyme without added Ca2+. Mutant ΔIG/N193F/S242A also exhibited substantially better activity and stability in the presence of the chelator EDTA, demonstrating enhanced Ca2+ binding. These results suggest that mutant ΔIG/N193F/S242A has potential for use in the industrial liquefaction of starch.

•The pelB-cutinase was extracellularly expressed in Escherichia coli.•The periplasmic and cytoplasmic fractions had phospholipid hydrolase activity.•The recombinant cells showed improved membrane permeability.•The secretion efficiency of inactive mutant significantly decreased.•The activity of pelB-cutinase plays a main role in its extracellular production.Our previous studies demonstrated that Thermobifida fusca cutinase is released into culture medium when expressed without a signal peptide in Escherichia coli, and this extracellular expression results from an enhanced membrane permeability caused by cutinase's phospholipid hydrolase activity. The present study investigated whether this phenomenon would also occur during the expression of cutinase fused to pelB signal peptide (pelB-cutinase). Secretion of fusion proteins of this type is generally believed to occur via type II secretion pathway. The results showed that when pelB-cutinase was expressed in a secB knockout strain, which has a defective type II secretion pathway, there was still a large amount of cutinase in the culture medium. Additional experiments confirmed that the periplasmic and cytoplasmic fractions of the expressing cells had hydrolytic activity toward phosphatidyl ethanolamine, and the recombinant cells showed correspondingly improved membrane permeability. All these phenomena were also observed in the parent E. coli strain. Moreover, the secretion efficiency of the inactive cutinase mutant was found to be significantly lower than that of pelB-cutinase in the parent E. coli. Based on these results, the phospholipid hydrolase activity of pelB-cutinase must play a larger role in its extracellular production than does type II secretion pathway.

•The Fusarium oxysporum putative lipase was identified as a PLB.•The PLB activity obtained (6503.8 U mL−1) is the highest reported to date.•The PLB reduced the phosphorus content of a crude oil from 75.88 ppm to 3.3 ppm.•This study provided insights into production and application of the F. oxysporum PLB.In this study, a gene encoding a putative lipase from Fusarium oxysporum was optimized via codon optimization and expressed in Pichia pastoris KM71. The gene product was identified as a phospholipase B (PLB). The engineered P. pastoris was further cultured in a 3.6-L bioreactor. After optimization of the induction conditions, this system produced 6.6 mg mL−1 protein and 6503.8 U mL−1 PLB activity in the culture medium. Efficient expression of this PLB in P. pastoris should reduce the costs of production and application. The purified enzyme, with a specific activity of 1170 U mg−1, was optimally active at pH 5.0 and 55 °C. The results of a degumming experiment performed using the recombinant PLB showed that the phosphorus content of a test oil was decreased from 75.88 ppm to 3.3 ppm in 2 h under optimal reaction conditions. This study provides a basis for the industrial use of F. oxysporum PLB in oil degumming applications.

•The Fusarium oxysporum putative lipase was identified as a PLB.•The PLB activity obtained (6503.8 U mL−1) is the highest reported to date.•The PLB reduced the phosphorus content of a crude oil from 75.88 ppm to 3.3 ppm.•This study provided insights into production and application of the F. oxysporum PLB.In this study, a gene encoding a putative lipase from Fusarium oxysporum was optimized via codon optimization and expressed in Pichia pastoris KM71. The gene product was identified as a phospholipase B (PLB). The engineered P. pastoris was further cultured in a 3.6-L bioreactor. After optimization of the induction conditions, this system produced 6.6 mg mL−1 protein and 6503.8 U mL−1 PLB activity in the culture medium. Efficient expression of this PLB in P. pastoris should reduce the costs of production and application. The purified enzyme, with a specific activity of 1170 U mg−1, was optimally active at pH 5.0 and 55 °C. The results of a degumming experiment performed using the recombinant PLB showed that the phosphorus content of a test oil was decreased from 75.88 ppm to 3.3 ppm in 2 h under optimal reaction conditions. This study provides a basis for the industrial use of F. oxysporum PLB in oil degumming applications.

•Maltose production is enhanced by mutagenesis at acceptor binding subsite +2.•Transglycosylation activity is affected by acceptor binding site hydrophilicity.•Optimized conditions for maltose production determined using mutant W177S.•Decreased maltotriose content in the product benefits purification of maltose.Maltogenic amylases are used to decrease the maltotriose content of high maltose syrups. However, due to the interplay between the hydrolysis and transglycosylation activities of maltogenic amylases, the maltotriose contents of these syrups are still greater than that necessary for pure maltose preparation. In this study, the maltogenic amylase from Bacillus stearothermophilus was engineered to decrease its transglycosylation activity with the expectation that this would enhance maltose production. Site-directed mutagenesis was used to generate Trp 177 variants W177F, W177Y, W177L, W177N, and W177S. The transglycosylation activities of the mutant enzymes decreased as the hydrophilicity of the residue at position 177 increased. The mutant enzymes exhibited notable enhancements in maltose production, with a minimum of maltotriose contents of 0.2%, compared with 3.2% for the wild-type enzyme. Detailed characterization of the mutant enzymes suggests that the best of them, W177S, will deliver performance superior to that of the wild-type under industrial conditions.

•FruR was inactivated in l-tryptophan production strain Escherichia coli FB-04, forming FB-04(ΔfruR).•FB-04(ΔfruR) maintained higher vitality and growth efficiency.•FB-04(ΔfruR) showed substantially improved l-tryptophan production.•Knockout of fruR improved the efficiency of the carbon source utilization.•Knockout of fruR improved the levels of critical precursors and substrates for l-tryptophan biosynthesis by modulating the direction of carbon flow.The fructose repressor (FruR) affects carbon flux through the central metabolic pathways of Escherichia coli. In this study, l-tryptophan production in Escherichia coli FB-04 was improved by knocking out the fruR gene, thereby inactivating FruR. This fruR knockout strain, E. coli FB-04(ΔfruR), not only exhibited higher growth efficiency, it also showed substantially improved l-tryptophan production. l-tryptophan production by E. coli FB-04(ΔfruR) and l-tryptophan yield per glucose were increased by 62.5% and 52.4%, respectively, compared with the parent E. coli FB-04. Metabolomics analysis showed that the fruR knockout significantly enhances metabolic flow through glycolysis, the pentose phosphate pathway and the TCA cycle, increasing levels of critical precursors and substrates for l-tryptophan biosynthesis. These results indicate that fruR deletion should enhance l-tryptophan production and improve the efficiency of carbon source utilization independent of genetic background.

•A new critical position in P. macerans CGTase for AA-2G production is analyzed.•CGTase mutants were constructed to enhance AA-2G production.•Disproportionation activity is critical for AA-2G yield when using cyclodextrin as donors.The markedly stable l-ascorbic acid (L-AA) derivative 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) has been widely used in the fields of food, medicine, cosmetics, and husbandry. Cyclodextrin glycosyltransferase (CGTase) is considered suitable for the large-scale production of AA-2G. In this work, Paenibacillus macerans CGTase was used to produce AA-2G and the production was 13.5 g/l. An amino-acid sequence alignment of α-, β-, and α⁄β-CGTase indicated that the Phe at position 228 of P. macerans CGTase was different from the amino acids at this position in other CGTases (Met, Val, or Ile). In addition, the CGTases from Anaerobranca gottschalkii and Bacillus circulans 251, which have Val and Met at position 228, were shown to produce 28.9 and 35.7 g/l AA-2G, respectively, which verified the importance of this position for AA-2G synthesis. Subsequently, P. macerans CGTase mutants F228M and F228V were constructed and shown to produce 24.8 g/l and 24.0 g/l AA-2G, respectively, which are 84% and 78% higher than that of wild-type P. macerans CGTase, respectively. Kinetic analysis of AA-2G synthesis showed that affinities of the two mutants for L-AA and the catalytic efficiencies increased. Meanwhile, the mutants had lower cyclization activity but higher disproportionation activities, which is beneficial for AA-2G synthesis. All these results indicated that amino acid at position 228 of P. macerans CGTase is crucial to AA-2G synthesis.