A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the kcat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (Ki, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.

A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The Km and kcat values of GSxyn for ρNPX were 0.48 mM and 36.64 s−1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The Km and kcat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s−1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The Km and kcat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s−1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.

A novel esterase gene, estB, was cloned from the marine microorganism Alcanivoraxdieselolei B-5(T) and overexpressed in E. coli DE3 (BL21). The expressed protein EstB with a predicted molecular weight of 45.1 kDa had a distinct catalytic triad (Ser211-Trp353-Gln385) and the classical consensus motif conserved in most lipases and esterases Gly209-X-Ser211-X-Gly213. EstB showed very low similarity to any known proteins and displayed the highest similarity to the hypothetical protein (46 %) from Rhodococcusjostii RHA1. EstB showed the optimal activity around pH 8.5 and 20 °C and was identified to be extremely cold-adaptative retaining more than 95 % activity between 0 and 10 °C. The values of kinetic parameters on p-NP caproate (Km, Kcat and Kcat/Km) were 0.15 mM, 0.54 × 103 s−1 and 3.6 × 103 s−1 mM−1, respectively. In addition, EstB showed remarkable stability in several studied organic solvents and detergents of high concentrations with the retention of more than 70 % activity after treatment for 30 min. The cold activity and its tolerance towards organic solvents made it a promising biocatalyst for industrial applications under extreme conditions.

A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25 %) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39 % activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93 % activity even at 4 M NaCl. The catalytic efficiency (kcat/Km) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg−1 s−1 (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl2. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.

An esterase gene, est10, was identified from the genomic library of a deep-sea psychrotrophic bacterium Psychrobacterpacificensis. The esterase exhibited the optimal activity around 25 °C and pH 7.5, and maintained as high as 55.0 % of its maximum activity at 0 °C, indicating its cold adaptation. Est10 was fairly stable under room temperatures, retaining more than 80 % of its original activity after incubation at 40 °C for 2 h. The highest activity was observed against the short-chain substrate p-nitrophenyl butyrate (C4) among the tested p-nitrophenyl esters (C2–C16). It was slightly activated at a low concentration (1 mM) of Zn2+, Mg2+, Ba2+, Ca2+, Cu2+, Fe3+, urea and EDTA, but was inhibited by DTT and totally inactivated by PMSF. Interestingly, increased salinity considerably stimulated Est10 activity (up to 143.2 % of original activity at 2 M NaCl) and stability (up to 126.4 % after incubation with 5 M NaCl for 6.5 h), proving its salt tolerance. 0.05 and 0.1 % Tween 20, Tween 80, Triton X-100 and CHAPS increased the activity and stability of Est10 while SDS, CTAB had the opposite effect. Est10 was quite active after incubation with several 30 % organic solvents (methanol, DMSO, ethanediol) but exhibited little activity with 30 % isopropanol, ethanol, n-butanol and acetonitrile.

Vegetative insecticidal proteins (VIPs), which were produced by Bacillus thuringiensis during its vegetative growth stage, display a broad insecticidal spectrum to Lepidoptera larvae. Sequence alignment of the Vip3A-type indicates that three cysteine residues were conserved in Vip3A-type proteins. To determine whether these conserved cysteine residues contributed to the insecticidal activity, the three residues were respectively substituted with serine in the Vip3Aa7 protein by site-directed mutagenesis. Bioassays using the third instar larvae of Plutella xylostella showed that the toxicity of C401S and C507S mutants were completely abolished. To find out the inactivity reason of mutants, three mutants and the wild-type Vip3Aa7 were treated with trypsin. The results indicated that the C507S mutant was rapidly cleaved and resulted in decrease of the 62 kDa toxic core fragment. These results indicated that the replacement of the Cys507 with a Ser507 caused decrease in C507S resistance against trypsin degradation. It is suggesting a possible association between insecticidal activity and trypsin sensitivity of Vip3A proteins. This study serves a guideline for the study of Vip3A protein structure and active mechanism.

A novel biosensor strain (Escherichia coli ALM403) that responded to N-acyl homoserine lactone (AHL) was constructed using a luxR-Plux cassette as a regulatory sequence and β-mannanase as a reporter gene. Dinitrosalicylic acid method was used to detect the response of the sensor strain to N-acyl homoserine lactone. By investigating the response to a range of concentrations of N-β-oxooctanoyl-l-homoserine lactone (OOHL), it was demonstrated that the expression of mannanase in E. coli ALM403 could be greatly enhanced by OOHL and resulted in an assayable phenotype. A high-throughput screening approach was developed to isolate AHL-degrading microorganisms, and a marine Halomonas sp. S66-4 showing a marked AHL-degrading ability was successfully isolated. In conclusion, the bioassay system provided a simple and efficient approach to isolate AHL-degrading bacteria.

Bacillus thuringiensis insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIPs) have been widely used as a kind of safe bio-insecticides. A problem that has been of concern worldwide is how to improve their insecticidal activities. In this study, to determine the synergism between VIPs and ICPs effect on insecticidal activity, a construct that produces a chimeric protein of the Vip3Aa7 and the N terminus ofCry9Ca, named V3AC9C, was expressed in Escherichia coli BL21 cells. In additional experiments, the V3AC9C chimeric protein, the single Vip3Aa7, and the single N terminus of Cry9Ca were treated with trypsin. SDS–PAGE showed that the V3AC9C could be processed into two single toxins. Bioassays tested on third instar larvae of Plutella xylostella showed that the toxicity of the chimeric protein was markedly better than either of the single toxins. Interestingly, the toxicity of the chimeric protein was 3.2-fold higher than a mixture of the Vip3Aa7 and Cry9Ca toxins (mass ratio of 1:1). The synergism factor (SF) of chimeric protein containing Vip3Aa7 and Cry9Ca was calculated to be 4.79. The SF in mixture of toxins is only 1.46. Hence, the effect was more than the sum of the Vip3Aa7 and Cry9C activities. Analysis of the protein’s solubility showed that the Vip3Aa7 helped the N terminus of Cry9Ca to dissolve in an alkaline buffer. It was concluded that the increase in the toxicity of the V3AC9C chimeric protein over the constituent proteins mainly resulted from this increase in solubility. These results lay a foundation for the development of a new generation of bio-insecticides and multi-gene transgenic plants.

By constructing a genomic library, an endoglucanase gene (cel9P) was cloned from Paenibacillus sp. BME-14 which was isolated from the sea. It had an open-reading frame of 1,629 bp, encoding a peptide of 542-amino acid residue with a calculated molecular mass of 60 kDa. The enzyme showed the highest amino acid identity of 52% with other known endoglucanases and had a C-terminal catalytic domain belonging to the glycosyl hydrolases family 9. The optimum pH and temperature for enzymatic activity was pH 6.5 and 35 °C. The metal ions of Ca2+, Mg2+, and Mn2+ had a positive effect on the activity while Hg2+, Cu2+, and EDTA had a negative effect. Notably, Cel9P had 65% of the maximal activity at 5 °C. Based on the special characteristic of Cel9P, it had a potential significance for study of cold-active mechanism and industry applications.

A β-glucosidase gene designated gluc3m was cloned through construction of a genomic library of Martelella mediterranea 2928. The gluc3m consisted of 2,496 bp and encoded a peptide of 832 amino acids that shared the greatest amino acid similarity (59%) with a β-glucosidase of family 3 glycoside hydrolase from Agrobacterium radiobacter K84. The optimum reaction temperature and pH of Gluc3M were 45 °C and 8.0, respectively. The Km and Vmax for p-nitrophenyl-β-d-glucopyranoside were 0.18 mg/ml and 196.08 µmol/min/mg enzyme, respectively. Gluc3M was found to be highly alkali stable, retaining 80% of its maximum enzymatic activity after treatment with pH 11.0 buffers for 24 h. Furthermore, the activity of Gluc3M improved remarkably in the presence of univalent metal ions, whereas it was inhibited in the presence of divalent ions. Gluc3M also exhibited significant activities toward various substrates including pNPGlu, pNPGal, salicin, and konjac powder. It is important to note that Gluc3M is a cold-active enzyme that showed over 50% of the maximum enzymatic activity at 4 °C. SWISS-MODEL revealed that the amino acids near the conserved domain SDW of Gluc3M contributed to the cold-active ability. Based on these characteristics, Gluc3M has the potential for use in additional studies and for industrial applications.

A novel random mutagenesis strategy was developed by combining sodium bisulfite modification with polymerase chain reaction (PCR). This method introduced the predominant substitution of GC to AT, meaning that it was more suitable for mutagenesis of GC-rich genes and helped to decrease the GC content of target DNA. Mutation efficiency correlated with modification time and different mutation frequency could easily be obtained by controlling modification time. The results indicated that this method could yield a desired and adequate frequency of random mutation to the DNA of interest, especially GC-rich genes, and provided a powerful tool for directed molecular evolution.

Bacillus subtilis endo-β-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).

•A novel aroA gene was identified from deep-sea bacteria, Alcanivorax sp. L27.•AroAA.sp exhibited activity between pH 5.5 and pH 8.0 and low temperatures.•The KM for PEP and IC50 [glyphosate] of AroAA.sp were investigated.•Site-directed mutagenesis was performed to increase the activity of AroAA.sp.•The specific activities of the wild-type and mutant AroAA.sp were determined.The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.

Lipolytic enzymes with unique physico-chemical characteristics are gaining more attention for their immense industrial importance. In this study, a novel lipolytic enzyme (Est11) was cloned from the genomic library of a marine bacterium Psychrobacter pacificensis. The enzyme was expressed in Escherichia coli and purified to homogeneity with molecular mass of 32.9 kDa. The recombinant Est11 was able to hydrolyze short chain esters (C2–C8) and displayed an optimum activity against butyrate ester (C4). The optimal temperature and pH were 25 °C and 7.5, respectively. Est11 retained more than 70% of its original activity at 10 °C, suggesting that it was a cold-active esterase. The enzyme was highly active and stable at high concentration of NaCl (5 M). Further, incubation with ethanol, isopropanol, propanediol, DMSO, acetonitrile, and glycerol rendered remarkable positive effects on Est11 activity. Typically, even at the concentration of 30% (v/v), ethanol, DMSO, and propanediol increased Est11 activity by 1.3, 2.0, and 2.4-folds, respectively. This new robust enzyme with remarkable properties like cold-adaptability, exceptional tolerance to salt and organic solvents provides us a promising candidate to meet the needs of some harsh industrial processes.

•Heterologous expression of a novel glyA gene from the marine bacterium Alcanivorax sp.•Biochemical characterization of the AdSHMT encoded by the recombinant glyA gene.•Studies on fermentation time-AdSHMT activity relationship.•AdSHMT as a promising tool in enzymatic production of l-serine.A novel glyA gene from the marine bacterium Alcanivorax sp. was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant glyA encodes a polypeptide of 418 amino acids, which was designated as AdSHMT that shows the highest identity (70%) with a SHMT from Shewanella algae. The purified enzyme showed a single band at about 45 kDa by SDS-PAGE analysis. It was found that AdSHMT exhibited the maximal activity at 50 °C and pH 7.0. The Km, Vmax, and Kcat values of AdSHMT against dl-threo-3-phenylserine were calculated to be 0.097 mol/L, 3.255 μmol/min/mg and 2.451/s, respectively. More importantly, RP-HPLC detection showed that the AdSHMT achieved an 88.37% molecular conversion rate in catalyzing glycine to l-serine, with the final concentration of l-serine being 353.15 mM in the reaction at 35 °C and 22nd hour when the initial concentration of the substrate (glycine) was 0.399 M. The molecular conversion rate of the AdSHMT from the Alcanivorax sp. was 1.26-fold that of the EcSHMT from the E. coli, which is currently applied in industrial production. Therefore, AdSHMT has the potential for industrial applications due to its high enzymatic conversion rate.Download full-size image

•A novel phosphinothricin N-acetyltransferase gene, repat, from the marine bacterium was isolated.•RePAT can utilize both l-phosphinothricin and l-methionine sulfoximine.•RePAT is a cold-adaptive enzyme.Phosphinothricin (PPT) is a kind of non-selective, environmentally friendly herbicide. PPT-tolerance genes are vital in both plant biotechnology as selectable markers and the development of transgenic herbicide-resistant crops. However, there are no other well-identified and commercially available PPT-resistance genes for use in plant genetic engineering besides two PPT N-acetyltransferase genes, which known as pat and bar derived from Streptomyces sp. Here, we isolated a novel PPT N-acetyltransferase gene from PPT-resistant marine bacteria, Rhodococcus sp. strain YM12. The gene, designated as RePAT, encoded a protein (RePAT) of 162 amino acids, which showed 37% identity with that of PAT proteins. Key kinetic constants of RePAT were determined (Km = 0.076 mM, Kcat = 131 min−1) using PPT as a substrate, the enzyme retained considerable activity at pH 8.0 and had an optimum temperature of 35 °C. Interestingly, it possessed over 50% of its maximal activity at temperature conditions between 0 and 10 °C, suggesting that this enzyme is able to protect crop against PPT injury in cold environment. These results illustrated that RePAT could be a new resource for herbicide detoxification by transgenic crops.Download full-size image

BackgroundGlycine oxidase (GO), a type of d-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study, we have characterized a new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking.ResultsThe results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between β-18 and α-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme.ConclusionAsn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure–function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application.

•A novel gene encoding a serine protease BVSP was cloned from deep sea bacterium.•Detailed enzymatic characteristics of the enzyme were studied.•Ca2+ strongly enhanced the activity of BVSP.•The enzyme has the potential to control cardiovascular diseases.Based on a genomic library constructed, a novel alkaline serine protease gene (Bvsp) (963 bp) was cloned from a marine bacterium Bacillus vallismortis, encoding 320 amino acid residues with a deduced molecular mass of 34.4 kDa. Amino acid sequence analysis found that Bvsp shared highest identity (72%) to a previously reported protease. The Bvsp enzyme showed the optimal activity at pH 6.5 and 54 °C, and was stable over pH 6–10 and 40–60 °C. The activity of the enzyme could be activated by metal ions such as Ca2+, Mg2+, Zn2+ and Ba2+, especially, in the presence of 30 mmol l−1 Ca2+, reaching 5100 U mg−1, 13 fold that of the control. In addition, Bvsp could degrade directly on cross-linked fibrin at an activity of 3863 U mg−1, it is not a plasminogen activator. Bvsp could also digest Aα- and Bβ-chains readily, but the γ-chain of fibrinogen slowly. Therefore Bvsp may have the potential to control cardiovascular diseases.Download full-size image

•The EPSPS from a recently identified actinomycete species, Isoptericola variabilis was characterized.•The I. variabilis EPSPS displayed a sequence and similarity to glyphosate sensitive class I EPSPSs.•The I. variabilis EPSPS displayed functional and enzymatic similarity to natural glyphosate tolerant class II EPSPSs.The 5-enolpyruvylshikimate-3-phosphate synthases (EPSPSs) encoded by aroA genes are traditionally divided into two classes, and only the class II EPSPS is naturally insensitive to herbicide glyphosate. Here, a novel EPSPS from Isoptericola variabilis, designated as I. variabilis EPSPS, was characterized. Its aroA gene contained 1374 bp with 77% of GC contents, encoding a peptide of 458 amino acids. The EPSPS carried a highly conserved region involved in substrate bindings in EPSPSs and displayed a high sequence and structure similarity to class I EPSPS. However, this EPSPS had high naturally glyphosate-tolerant capability and enzymatic kinetics constants similar to that of class II EPSPSs. Escherichia coli AB2829 harboring the aroA gene grew well in medium containing 150 mM glyphosate. The purified EPSPS had the highest enzymatic activity at pH 8.0 and 30 °C, and retained its activity at pH 6–10 and 20–60 °C. This enzyme had a Km value of 28 μM for substrate phosphoenolpyruvate and 111 μM for shikimate-3-phosphate, respectively, with a rather high Ki value for glyphosate. Cations increased substrate affinity but slightly reduced glyphosate tolerance. The I. variabilis EPSPS may be used for generating transgenic glyphosate-tolerant plants and dissecting mechanisms underlying the glyphosate tolerance.Download full-size image

•Swine manure was converted into C. megacephala larvae in a pilot scale plant.•Larvae drying method was established to improve the properties of feedstock oil.•The oil components from C. megacephala larvae fed swine manure was reported.•Properties of C. megacephala larvae biodiesel met the EN 14214 standard.Swine manure may cause environmental pollution and resource waste if not handled properly on pig farms. In this paper, the technology for pig manure biodegradation and biodiesel production using Chrysomya megacephala (Fabricius) is described. About 700 kg of fresh pig manure (73% moisture) can be converted within one week into 18.2 kg dried larvae biomass containing about 21.11% oil in a pilot plant. The properties of the oil extracted from the larvae meal treated with three different drying methods were compared, indicating that the drying method may affect the properties of feedstock oil. The acid value (1.9 mg KOH/g), iodine value (86.3 gI/100 g), melt point (3.1 °C) and peroxide value (0.08 meq/kg) of the oil extracted from the larvae treated with both boiling water and oven-drying were superior to the values of the larvae treated with either oven-drying or sun-drying directly. The main fatty acids of the swine manure C. megacephala larvae oil were found to be composed of palmitic acid (36.91%), oleic acid (27.67%), palmitoleic acid (10.89%) and linoleic acid (9.49%). Most of the properties of the biodiesel converted from the feedstock oil by alkaline-catalyst transesterification met the EN 14214 standard in terms of density (0.89 g/cm3), viscosity (5.1 mm2/s), ester content (96.6%), flash point (138 °C), cetane number (56), water content (0.02%) and acid value (0.28 mg KOH/g). This study suggests that the swine manure-grown C. megacephala larvae could be a feasible feedstock for a large-scale biodiesel production.

•A novel gene encoding an α-amylase was cloned from marine bacterium.•The enzyme had a salinity-tolerance range of 0–5 M NaCl with a salt optimum at 2 M.•Enzyme thermostability was improved by 53.6-fold in the presence of 2 M NaCl.•The enzyme was stable at pH 10.0 and 11.0 after 5-day pre-incubation at 25 °C.A novel gene (amyZ2) encoding an alkali-stable and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda (MCCC 1A01486) was cloned and expressed in Escherichia coli. The gene (1446 bp) encodes a polypeptide with a predicted N-terminal signal peptide consisting of 24 amino acids and a mature α-amylase of 457 amino acids (AmyZ2). The estimated molecular mass of AmyZ2 was 50 kDa by SDS-PAGE. AmyZ2 belongs to glycoside hydrolase family 13 and shares the highest identity (45%) with the characterized α-amylase from Pyrocoocus woesei (PDB id: 1MWO). The purified enzyme showed the maximum activity at 50 °C, pH 7.0, and retained approximately 143 and 126% initial activity after 5 days of incubation (25 °C) at pH 10.0 and 11.0, respectively, indicating its alkali stability. In addition, it had a salinity-tolerance range of 0–5 M NaCl with a salt optimum at 2 M (138% initial activity), retaining more than 130% initial activity at 0.5–2.5 M and about 100% activity at 3.5 M NaCl. AmyZ2 was relatively stable in 0–4 M NaCl and its thermostability was significantly improved by the pre-incubation of enzyme solution with 2 M NaCl at 50 °C for 3 h, which led to a 53.6-fold increase in the residual activity. The Km and kcat values of AmyZ2 were 11.71 mg ml−1 and 1449.43 s−1, and the catalytic efficiency (kcat/Km) was 123.78 ml mg−1 s−1. AmyZ2 exhibited a specific activity of 1662.4 U/mg toward soluble starch. The salt-tolerance and extreme alkali-stability of AmyZ2 suggests its potential applications in harsh industrial processes.Download full-size image

•Directed evolution and H179 saturation mutagenesis of endo-β-1, 4-xylanase from Geobacillus stearothermophilus.•The catalytic efficiency of H179F was found to be 3.46-fold that of the wild-type.•H179F retained 7% enzyme activity after 1 h incubation at 80 °C, but no activities for the wild-type enzyme were observed.•When Histidine was substituted by tryptophan, arginine, methionine and proline, respectively, the enzyme lost activity.Endo-β-1, 4-xylanase was cloned from Geobacillus stearothermophilus 1A05583 by PCR. Enzymes with improved catalytic efficiency were obtained using error-prone PCR and a 96-well plate high-throughout screening system. Two variants 1-B8 and 2-H6 were screened from the mutant library containing 9000 colonies, which, when compared with the wild-type enzyme increased the catalytic efficiency (kcat/Km) by 25% and 89%, respectively, acting on beechwood xylan. By sequencing 1-B8 and 2-H6, an identical mutation point H179Y was detected and found to overlap in the active site cleft. Following the introduction of the remaining 19 amino acids into position 179 by site-saturation mutagenesis, the catalytic efficiency of H179F was found to be 3.46-fold that of the wild-type. When Whistidine was substituted by tryptophan, arginine, methionine or proline, the enzyme lost activity. Therefore, the position 179 site may play an important role in regulating the catalytic efficiency.

•EstLiu is a novel esterase cloned from the marine bacterium Zunongwangia profunda.•EstLiu belongs to a new family.•EstLiu could retain 75% activity at the freezing point of water.•EstLiu shows good tolerance against salt and organic solvents.A novel cold active esterase, EstLiu was cloned from the marine bacterium Zunongwangia profunda, overexpressed in E. coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography. The mature esterase EstLiu sequence encodes a protein of 273 amino acids residues, with a predicted molecular weight of 30 KDa and containing the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243. Although, EstLiu showed 64% similarity with the hypothetical esterase from Chryseobacterium sp. StRB126 (WP_045498424), phylogenetic analysis showed it had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family. The purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). EstLiu showed remarkable activity (75%) at 0 °Cand the optimal activity at pH 8.0 and 30 °C with good thermostability and quickened inactivation above 60 °C. EstLiu retained 81, 103, 67 and 78% of its original activity at 50% (v/v) in ethanol, isopropanol, DMSO and ethylene glycol, respectively. In the presence of Tween 20, Tween 80 and Triton X-100, EstLiu showed 88, 100 and 117% of relative activity. It is also co-factor independent. The high activity at low temperature and desirable stability in organic solvents and salts of this novel family esterase represents a good evidence of novel biocatalyst. Overall, this novel enzyme showed better activity than previously reported esterases in extreme reaction conditions and could promote the reaction in both aqueous and non-aqueous conditions, indicating its great potential for industrial applications.

•A novel gene encoding an α-galactosidase was cloned from deep sea bacterium.•Detailed enzymatic characteristics of the enzyme were studied.•Directed evolution of the enzyme was carried out by site-directed mutagenesis.•Two mutants with improved catalytic efficiency were obtained.A novel gene (BmelA) (1323 bp) encoding an α-galactosidase of 440 amino acids was cloned from the deep-sea bacterium Bacillus megaterium and the protein was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of about 45 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 4, with the highest identity (74%) to α-galactosidase Mel4A from Bacillus halodurans among the characterized α-galactosidases. The recombinant BmelA displayed its maximum activity at 35 °C and pH 8.5–9.0 in 50 mM Tris–HCl buffer, and could hydrolyze different substrates with the Km values against p-nitrophenyl-α-d-galactopyranoside (pNP-α-Gal), raffinose and stachyose being 1.02 ± 0.02, 2.24 ± 0.11 and 3.42 ± 0.17 mM, respectively. Besides, 4 mutants (I38 V, I38A, I38F and Q84A) were obtained by site-directed mutagenesis based on molecular modeling and sequence alignment. The kinetic analysis indicated that mutants I38 V and I38A exhibited a 1.7- and 1.4-fold increase over the wild type enzyme in catalytic efficiency (kcat/Km) against pNP-α-Gal, respectively, while mutant I38F showed a 3.5-fold decrease against pNP-α-Gal and mutant Q84A almost completely lost its activity. All the results suggest that I38 and Q84 sites play a vital role in enzyme activity probably due to their steric and polar effects on the predicted “tunnel” structure and NAD+ binding to the enzyme.

•A genomic library of a psychrotrophic bacterium Psychrobacter celer 3Pb1 was constructed.•Est12, a phylogenetically new esterase, was isolated from the genomic library.•The highest sequence identity of Est12 was only 77% with a hypothetical esterase.•Est12 was cold-active and remained 41% maximum activity at 0 °C.•Esterase activity was significantly increased by high salinity.A genomic library of a psychrotrophic Psychrobacter celer 3Pb1 was constructed and screened for lipolytic proteins, and a novel esterase Est12 was cloned and characterized. The esterase gene, est12, contained an open reading frame of 990 bp that encoded a protein of 329 amino acids with an estimated molecular mass of 35,150 Da. Est12 displayed the highest amino acid identity (77%) with a hypothetical esterase from Psychrobacter sp. PAMC 21119 (WP_010200623.1). Phylogenetic analysis suggested that the protein belonged to a new lipase/esterase family. Substrate specifity study showed that Est12 preferred short-chain p-nitrophenyl esters and was most active toward p-nitrophenyl butyrate. Est12 displayed the optimal activity at pH 7.5 and 35 °C, and remained 41% activity at 0 °C while being unstable at temperatures above 40 °C, indicating its cold-adaptation. Besides, Est12 was a salt-tolerant esterase as 4.5 M NaCl significantly declined Km from 0.069 to 0.033 mM and increased kcat from 4.20 to 9.21 s−1, resulting in the increased catalytic efficiency kcat/Km from 60.72 to 276.31 s−1 mM−1. The enzyme activity was also quite stable after 24 h incubation in 0–4.5 M NaCl solutions. In addition, Est12 was very active and stable in the presence of several detergents and organic solvents. This new cold-active and halotolerant esterase would be a potential candidate in industrial applications under extreme conditions (low temperatures, high salinity), and was valuable for studying other unknown esterases/lipases in this new family.Download full-size image

•Compared to the wild-type enzyme, several variants with 1.14- to 3.30-fold increased catalytic efficiency (kcat/Km) toward locust bean gum (LBG) were obtained.•The molecular modeling and the docking analysis of the substrate suggested the nature of the residue at the 267 site significantly affect the enzyme activity by regulating substrate binding affinity and product release.•The molecular modeling and the docking analysis of the substrate revealed that enhanced polar contacts or positive charges were more favorable at the 134 site.Pantoea agglomerans endo-β-1, 4-mannanase (Man26P) hydrolyzes hemicellulose by cleaving the internal bonds in the mannan-based chains. In order to obtain improved enzymes and understand the structure-function relationship better, Man26P was engineered using a modified DNA shuffling method and a 96-well plate high-throughput screening technology. Compared to the wild-type enzyme, two mutants Gly267Ser and His134Arg/Phe141Leu with 1.14- and 3.30-fold increased catalytic efficiency (kcat/Km) toward locust bean gum (LBG) were obtained from the mutant library containing 19,700 clones. Combined with site-directed mutagenesis, more substitutions were introduced into the 134 site, among which, His134Arg and His134Lys exhibited 2.81- and 2.75-fold increased catalytic efficiency versus the wild-type enzyme. And with an increase in the pKa values of the side chains of the mutated residues at the 134 site, a decrease was observed in their Km values against LBG. The molecular modeling and the docking analysis of the substrate suggest that the enhancement of polar contacts or positive charges at the 134 site contribute to the interaction of the enzyme with the substrate. Furthermore, the nature of the residue at the 267 site could also significantly affect the enzyme activity by regulating substrate binding affinity and product release. The study provides useful information for the directed evolution of mannanases.