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Alginate lyase is an attractive biocatalyst that can specifically degrade alginate to produce oligosaccharides, showing great potential for industrial and medicinal applications. Herein, an alginate-degrading strain HB236076 was isolated from Sargassum sp. in Qionghai, Hainan, China. The low 16S rRNA gene sequence identity (<98.4%), ANI value (<71.9%), and dDDH value (<23.9%) clearly indicated that the isolate represented a potential novel species of the genus Vibrio . The genome contained two chromosomes with lengths of 3,007,948 bp and 874,895 bp, respectively, totaling 3,882,843 bp with a G+C content of 46.5%. Among 3482 genes, 3332 protein-coding genes, 116 tRNA, and 34 rRNA sequences were predicted. Analysis of the amino acid sequences showed that the strain encoded 73 carbohydrate-active enzymes (CAZymes), predicting seven PL7 (Alg1-7) and two PL17 family (Alg8, 9) alginate lyases. The extracellular alginate lyase from strain HB236076 showed the maximum activity at 50 °C and pH 7.0, with over 90% activity measured in the range of 30-60 °C and pH 6.0-10.0, exhibiting a wide range of temperature and pH activities. The enzyme also remained at more than 90% of the original activity at a wide pH range (3.0-9.0) and temperature below 50 °C for more than 2 h, demonstrating significant thermal and pH stabilities. Fe 2+ had a good promoting effect on the alginate lyase activity at 10 mM, increasing by 3.5 times. Thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI-MS) analyses suggested that alginate lyase in fermentation broth could catalyze sodium alginate to produce disaccharides and trisaccharides, which showed antimicrobial activity against Shigella dysenteriae , Aeromonas hydrophila , Staphylococcus aureus , Streptococcus agalactiae , and Escherichia coli . This research provided extended insights into the production mechanism of alginate lyase from Vibrio sp. HB236076, which was beneficial for further application in the preparation of pH-stable and thermo-stable alginate lyase and alginate oligosaccharides.

Human TPH2 ( hTPH2) catalyzes the rate-limiting step in CNS serotonin biosynthesis. We characterized a single-nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild-type enzyme). The S41Y polymorphism decreased cyclic AMP-dependent protein kinase A-mediated phosphorylation ~ 50% relative to wild-type hTPH2, suggesting that the S41Y mutation may disrupt the post-translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y-transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression. [ABSTRACT FROM AUTHOR]

In this study, SBA-15 was modified by halogen & haloalkanes and later used to immobilize lipases. The hydrolysis activity and the glycerolysis performance of the immobilized lipases was carefully studied. Highest activity of the immobilized Candida antarctica lipase B (CALB), Lipase from Aspergillus oryzae (AOL), Lecitase® Ultra (LU) and lipase from Rhizomucor miehei (RML) was respectively at 5577, 12000, 2822 and 11,577 U/g; in addition, the highest activity was obtained from the lowest or moderate lipase loading, at 25.73, 90.72, 89.52 and 30.56 mg/g respectively. The mechanism of lipase immobilization was studied and it was through interfacial activation. The halogen & haloalkanes modification of SBA-15 afforded considerable glycerolysis activity for diacylglycerols (DAG) preparation. CALB@SBA-15-CH 2 CH 2 (CF 2 ) 5 CF 3 and CALB@SBA-15-CH 2 CH 2 (CF 2 ) 7 CF 3 were suitable for DAG production, they both exhibited good reusability in glycerolysis reaction, with 117.36% and 93.06% of their initial glycerolysis activity retained respectively after ten cycles of reuse. The relationships between temperature with triacylglycerols (TAG) conversion were lnV 0  = 3.13-3.07/T and lnV 0  = 7.90-4.64/T respectively for CALB@SBA-15-CH 2 CH 2 (CF 2 ) 5 CF 3 and CALB@SBA-15-CH 2 CH 2 (CF 2 ) 7 CF 3 ; in addition, their activation energy (Ea) was respectively at 25.50 and 38.54 kJ/mol., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Thermal activity and stability are important characteristics for proteases applied in the detergent, pharmaceutical, food, and other green industries. With the intent to discover thermostable novel proteases, we constructed a fosmid metagenomic library from marine sediments in the East China Sea and isolated a clone endowed with high proteolytic activity from this library. Sequence analysis of the positive subclones allowed the identification of a coding region of 1254 bp related to protease activity. The unrooted phylogenetic tree and alignment results revealed that the sequence might be derived from Anaerolineaceae bacterium and encodes a new member of the peptidase S8A subfamily with the typical catalytic triad Asp 119 /His 150 /Ser 325 . The fusion protein, named pF1AL2, was expressed in Escherichia coli and showed a molecular weight of 35 kDa. pF1AL2 was active in the pH range of 5.0-11.0 with an optimal pH at 10.0 and had high stability under alkaline conditions, retaining more than 95% of its activity after 24 h at pH 11.0. The optimal temperature of pF1AL2 was 80 °C, and it retained nearly 80% of its activity after 6 h at 70 °C, showing great thermal activity and stability. In addition, the enzyme had great salt tolerance (the residual activity when kept in 3 M NaCl was 40%). Its thermal activity and stability, along with its halotolerance and pH-tolerance, indicate the high potential value of pF1AL2 in industrial applications. The exploitation of pF1AL2 could lay the foundation for the development and utilization of proteases with special features from marine resources by a metagenomic strategy. KEY POINTS: • A novel protease, pF1AL2, from marine sediments, was screened out by a metagenomic strategy. • The protease pF1AL2 analyzed in silico, cloned, and characterized. • pF1AL2 had an optimal temperature of 80 °C and retained nearly 80% of activity after 6 h at 70 °C. • pF1AL2 had great tolerance for high-temperature and acid, alkaline, and high salt environments.

Background: New strategies are urgently needed to deal with the growing problem of multidrug-resistant bacterial pathogens. As the natural viruses against bacteria, recently, bacteriophages have received particular attention. Here, we identified and characterized a novel peptidoglycan hydrolase named MMPphg by decoding the complete genome sequence of Meiothermus bacteriophage MMP17, which was isolated in Tengchong hot spring in China and contains a circular genome of 33,172 bp in size and a GC content of 63.4%., Findings: We cloned the MMPphg gene, overproduced and purified the phage lytic protein, which contains a highly conserved M23 metallopeptidase domain and can be activated by Mg 2+ and Zn 2+ . MMPphg is capable of withstanding temperatures up to 70 °C, and preserved more than 80% of its activity after a 30 min treatment between 35 and 65 °C. More interestingly, by disrupting bacterial cells, MMPphg exhibits surprising antimicrobial activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains such as Escherichia coli O157, Staphylococcus aureus and Klebsiella pneumonia., Conclusions: In the current age of mounting antibiotic resistance, these results suggest the great potential of MMPphg, the gene product of bacteriophage MMP17, in combating bacterial infections and shed light on bacteriophage-based strategies to develop alternatives to conventional antibiotics for human or veterinary applications.

Fungal endo-β-1,4-xylanases (endo-xylanases) can hydrolyze xylan into xylooligosaccharides (XOS), and have potential biotechnological applications for the exploitation of natural renewable polysaccharides. In the current study, we aimed to screen and characterize an efficient fungal endo-xylanase from 100 natural humus-rich soil samples collected in Guizhou Province, China, using extracted sugarcane bagasse xylan (SBX) as the sole carbon source. Initially, 182 fungal isolates producing xylanases were selected, among which Trichoderma sp. strain TP3-36 was identified as showing the highest xylanase activity of 295 U/mL with xylobiose (X2) as the main product when beechwood xylan was used as substrate. Subsequently, a glycoside hydrolase family 11 endo-xylanase, TXyn11A, was purified from strain TP3-36, and its optimal pH and temperature for activity against beechwood xylan were identified to be 5.0 and 55 °C, respectively. TXyn11A was stable across a broad pH range (3.0-10.0), and exhibited strict substrate specificity, including xylan from beechwood, wheat, rye, and sugarcane bagasse, with K m and V max values of 5 mg/mL and 1250 μmol/mg min, respectively, toward beechwood xylan. Intriguingly, the main product obtained from hydrolysis of beechwood xylan by TXyn11A was xylobiose, whereas SBX hydrolysis resulted in both X2 and xylotriose. Overall, these characteristics of the endo-xylanase TXyn11A indicate several potential industrial applications.

The functional stability of soil enzymes is fundamental to the sustainability of soil biochemical processes and is affected by many environmental stressors. This study focused on the influences of long-term arsenic (As) contamination on soil enzyme functional stability: the resistance (ratio of the disturbed to control) and resilience (integrated recovery rate) of soil enzyme activities (β-glucosidase, urease, acid phosphatase, fluorescein diacetate (FDA) hydrolase) over 30 days incubation after an experimental heat disturbance (50 o C for 18 h). Results showed that the resistance of soil enzymes to heat disturbance differed among the enzyme types and followed the order: urease > β-glucosidase > acid phosphatase > FDA hydrolase. Urease activity was generally not affected and showed high stability against heat disturbance. The β-glucosidase activity recovered to the control level by 30 days, while 80% and 90% recovery on average occurred for acid phosphatase and FDA hydrolase, respectively. Long-term As contamination altered soil enzyme functional resistance and resilience to heat disturbance and resulted in three kinds of responses: (i) no apparent alteration (urease); (ii) moderate As contamination increased enzyme heat resistance (β-glucosidase); (iii) the resistance and resilience decreased with increasing As concentration (acid phosphatase and FDA hydrolase). The results demonstrated that different enzyme-catalytic biochemical processes have different functional stabilities under combined As and heat disturbance, and the negative changes in the soil enzyme activity led to losses in soil functions. Our study provides further evidence on the impacts of heavy metal/metalloid on soil enzyme functional stability in response to additional disturbance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

One novel protease sin3406-1 was identified from Streptomyces niveus SCSIO 3406, which was isolated from the deep sea of the South China Sea, and heterologously expressed in E. coli BL21(DE3). Protease sin3406-1 was further used as a green biocatalyst in the kinetic resolution of racemic ethyl-3-hydroxybutyrate. After careful process optimization, chiral product ethyl (S)-3-hydroxybutyrate was generated with an enantiomeric excess of over 99% and a conversion rate of up to 50% through direct hydrolysis of inexpensive racemic ethyl-3-hydroxybutyrate catalyzed by sin3406-1. Interestingly, protease sin3406-1 exhibited the same enantio-preference as that of esterase PHE21 during the asymmetric hydrolysis of the ester bonds of racemic ethyl-3-hydroxybutyrate. Through mutation studies and molecular docking, we also demonstrated that the four residues close to the catalytic center, S85, A86, Q87 and Y254, played key roles in both the hydrolytic activity and the enantioselectivity of protease sin3406-1, possibly through forming hydrogen bonds between the enzyme and the substrates. Deep-sea microbial proteases represented by sin3406-1 are new contributions to the biocatalyst library for the preparation of valuable chiral drug intermediates and chemicals through enzymatic kinetic resolution.

A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To enrich the genetic resource of microbial xylanases with high activity and stability under alkaline conditions, a xylanase gene (xynSL4) was cloned from Planococcus sp. SL4, an alkaline xylanase-producing strain isolated from the sediment of soda lake Dabusu. Deduced XynSL4 consists of a putative signal peptide of 29 residues and a catalytic domain (30-380 residues) of glycosyl hydrolase family 10, and shares the highest identity of 77% with a hypothetical protein from Planomicrobium glaciei CHR43. Phylogenetic analysis indicated that deduced XynSL4 is closely related with thermophilic and alkaline xylanases from Geobacillus and Bacillus species. The gene xynSL4 was expressed heterologously in Escherichia coli and the recombinant enzyme showed some superior properties. Purified recombinant XynSL4 (rXynSL4) was highly active and stable over the neutral and alkaline pH range from 6 to 11, with maximum activity at pH 7 and more than 60% activity at pH 11. It had an apparent temperature optimum of 70°C and retained stable at this temperature in the presence of substrate. rXynSL4 was highly halotolerant, retaining more than 55% activity with 0.25-3.0 M NaCl and was stable at the concentration of NaCl up to 4M. The enzyme activity was significantly enhanced by β-mercaptoethanol and Ca(2+) but strongly inhibited by heavy-metal ions and SDS. This thermophilic and alkaline- and salt-tolerant enzyme has great potential for basic research and industrial applications.

A haloarchaeal strain G41 showing lipolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterizations along with 16S rRNA gene sequence analysis placed the isolate in the genus Haloarcula. Lipase production was strongly influenced by the salinity of growth medium with maximum in the presence of 20% NaCl or 15% Na2SO4. The lipase was purified to homogeneity with a molecular mass of 45 kDa. Substrate specificity test revealed that it preferred long-chain p-nitrophenyl esters. The lipase was highly active and stable over broad ranges of temperature (30-80 °C), pH (6.0-11.0), and NaCl concentration (10-25%), with an optimum at 70 °C, pH 8.0, and 15% NaCl, showing thermostable, alkali-stable, and halostable properties. Enzyme inhibition studies indicated that the lipase was a metalloenzyme, with serine and cysteine residues essential for enzyme function. Moreover, it displayed high stability and activation in the presence of hydrophobic organic solvents with log Pow ≥ 2.73. The free and immobilized lipases from strain G41 were applied for biodiesel production, and 80.5 and 89.2% of yields were achieved, respectively. This study demonstrated the feasibility of using lipases from halophilic archaea for biodiesel production.

The bark beetle Dendroctonus armandi is able to kill living Pinus armandi and has caused serious damage to pine forest in Northern China. As the most important symbiotic fungus of D. armandi, Leptographium qinlingensis plays an important role in the invasion process of the bark beetle. The laccase secreted by it are involved in lignin degradation to provide utilizable nutrition for D. armandi, and catalyze some biochemical reactions, causing the damages of tree tissue. In present study, the extracellular laccase of L. qinlingensis was purified by using the ammonium sulfate precipitation and DEAE-cellulose (DE-52) column chromatography. Furthermore, the effects of temperature, pH value and metal ions on it were investigated and characterized. The purified enzyme exerted its optimal activity with guaiacol. The catalytic efficiencies K(m) and V(max) determined for substrate guaiacol were 15.4 μM and 372.9 IU mg⁻¹, respectively. The optimum pH and temperature for the purified enzyme was 4.4 and 45 °C, respectively, with the highest enzyme specific activity of 7,000 IU mg⁻¹. Moreover, the metal ions, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺, Fe²⁺ and Cd²⁺, especially Hg²⁺, showed significantly inhibition effects on its activity. To understand the characteristics of this laccase might provide an opportunity and theoretical basis to promote integrated pest management of D. armandi.

Phospholipid hydrolysis, as the main stage and cause of phopholipid degradation, is generally attributed to phospholipases. In this study, the changes of phospholipase A₂ (PLA₂) and C (PLC) activities, neutral lipid, free fatty acids and phospholipids in dry-cured duck leg muscles during processing, were examined. The composition of free fatty acids and neutral lipids increased significantly (P<0.05) with extension of processing time while the phospholipids composition decreased. The PLA₂ and PLC activity decreased in the final product, but retained 83.70% and 86.78% of their initial activities, respectively. The relative activities of both PLA₂ and PLC highly correlated with the decline of phospholipids and the increase of free fatty acids. High correlations were also obtained between the relative activities of PLC and the increase of neutral lipid (P<0.01). All these results suggest that PLA₂ and PLC contribute to the degradation of intramuscular phospholipids during the processing of dry-cured duck., (Copyright © 2013. Published by Elsevier Ltd.)

Background: β-Galactosidases can be used to produce low-lactose milk and dairy products for lactose intolerant people. Although commercial β-galactosidases have outstanding lactose hydrolysis ability, their thermostability is low, and reaction products have strong inhibition to these enzymes. In addition, the β-galactosidases possessing simultaneously high thermostability and tolerance of galactose and glucose are still seldom reported until now. Therefore, identification of novel β-galactosidases with high thermostability and tolerance to reaction products from unculturable microorganisms accounting for over 99% of microorganisms in the environment via metagenomic strategy is still urgently in demand., Results: In the present study, a novel β-galactosidase (Gal308) consisting of 658 amino acids was identified from a metagenomic library from soil samples of Turpan Basin in China by functional screening. After being overexpressed in Escherichia coli and purified to homogeneity, the enzymatic properties of Gal308 with N-terminal fusion tag were investigated. The recombinant enzyme displayed a pH optimum of 6.8 and a temperature optimum of 78 °C, and was considerably stable in the temperature range of 40 °C - 70 °C with almost unchangeable activity after incubation for 60 min. Furthermore, Gal308 displayed a very high tolerance of galactose and glucose, with the highest inhibition constant K(i,gal) (238 mM) and K(i,glu) (1725 mM) among β-galactosidases. In addition, Gal308 also exhibited high enzymatic activity for its synthetic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG, 185 U/mg) and natural substrate lactose (47.6 U/mg)., Conclusion: This study will enrich the source of β-galactosidases, and attract some attentions to β-galactosidases from extreme habitats and metagenomic library. Furthermore, the recombinant Gal308 fused with 156 amino acids exhibits many novel properties including high activity and thermostability at high temperatures, the pH optimum of 6.8, high enzyme activity for lactose, as well as high tolerance of galactose and glucose. These properties make it a good candidate in the production of low-lactose milk and dairy products after further study.

Background: Xylanases have attracted much attention owing to their potential applications. The applicability of xylanases, however, was bottlenecked by their low stabilities at high temperature or extreme pH. The purpose of this work was to enhance the thermostability of a mesophilic xylanase by N-terminal replacement., Results: The thermostability of AoXyn11, a mesophilic family 11 xylanase from Aspergillus oryzae, was enhanced by replacing its N-terminal segment with the corresponding one of EvXyn11(TS) , a hyperthermotolerant family 11 xylanase. A hybrid xylanase with high thermostability, NhXyn11⁵⁷, was predicted by molecular dynamics (MD) simulation. An NhXyn11⁵⁷-encoding gene, Nhxyn11⁵⁷, was then constructed as designed theoretically, and overexpressed in Pichia pastoris. The temperature optimum of recombinant NhXyn11⁵⁷ (re-NhXyn11⁵⁷) was 75 °C, much higher than that of re-AoXyn11. Both xylanases were thermostable at 65 and 40 °C, respectively. Additionally, the pH optimum and stability of re-NhXyn11⁵⁷ were 5.5 and at a range of 4.0-8.5. Its activity was not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²⁺ and Ag⁺., Conclusion: This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn11⁵⁷ a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes., (© 2013 Society of Chemical Industry.)

An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel β-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0 ~ 9.0 and at temperatures below 42 °C. The K m and V max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.

A halophilic isolate Thalassobacillus sp. LY18 producing extracellular amylase was isolated from the saline soil of Yuncheng Salt Lake, China. Production of the enzyme was synchronized with bacterial growth and reached a maximum level during the early stationary phase. The amylase was purified to homogeneity with a molecular mass of 31 kDa. Major products of soluble starch hydrolysis were maltose and maltotriose, indicating an α-amylase activity. Optimal enzyme activity was found to be at 70°C, pH 9.0, and 10 % NaCl. The α-amylase was highly stable over broad temperature (30-90°C), pH (6.0-12.0), and NaCl concentration (0-20 %) ranges, showing excellent thermostable, alkalistable, and halotolerant nature. The enzyme was stimulated by Ca(2+), but greatly inhibited by EDTA, indicating it was a metalloenzyme. Complete inhibition by diethyl pyrocarbonate and β-mercaptoethanol revealed that histidine residue and disulfide bond were essential for enzyme catalysis. The surfactants tested had no significant effects on the amylase activity. Furthermore, it showed high activity and stability in the presence of water-insoluble organic solvents with log P (ow) ≥ 2.13.

Through three chromatographic steps, a new thrombin-like enzyme (TLE), named TA-2, from the venom of the Chinese white-lipped green pitviper (Trimeresurus albolabris) has been isolated and purified to homogeneity. TA-2 was a single-chain glycoprotein with about 6% sugar, pI 3.9 and a molecular weight of 38.8 kD. Its N-terminal sequence (VVGGDECNIN) showed high sequence conformity with many other TLEs. In vitro, it coagulated bovine fibrinogen (108.6 NIH units/mg) and cleaved the Aα and Bβ chains of bovine fibrinogen-releasing fibrinopeptide A and B, but did not degrade bovine fibrin; displayed high stability at different temperature, pH, and presence of several divalent cations and inhibitors; also exhibited strong activity towards casein (192.3 units/mg) and high esterase activity upon Nα-p-tosyl-L-arginine methyl ester (11 units/mg); and behaved as a promoter to platelet aggregation induced by ADP or collagen. In vivo, TA-2 caused dose-dependent prolongation of bleeding time in mice, but had no hemorrhagic and edema-inducing activities even at high concentrations.

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by mutations in the subunits of the branched-chain α-ketoacid dehydrogenase (BCKD) complex. BCKD is a mitochondrial complex encoded by four nuclear genes (BCKDHA, BCKDHB, DBT, and DLD) and is involved in the metabolism of branched-chain amino acids (BCAAs). In this study, we investigated the DNA sequences of BCKDHA, BCKDHB and DBT genes for mutations in a Chinese newborn with the classic form of MSUD and predicted the associated conformational changes using molecular modeling. We identified two previously unreported mutations in the BCKDHB gene, R170H (c.509G>A) in exon 5 and Q346R (c.1037 A>G) in exon 9. In silico analysis of the two novel missense mutations revealed that the mutation R170H-β alters the spatial orientation with both Y195-β' and S206-α, which results in unstable β-β' assembly and an unstable K(+) ion binding loop of the α subunit, respectively; The Q346R mutation is predicted to disrupt the spatial conformation between Q346-β and I357-β', which reduces the affinity of the β-β' subunits. These results indicate that R170-β and Q346-β are crucial for the activity of the E1 component. These two novel mutations, R170H and Q346R result in the patient's clinical manifestation of the classic form of MSUD., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A metagenomic library of China Holstein cow rumen microbes was constructed and screened for novel gene cluster. A novel feruloyl esterase (FAE) gene was identified with a length of 789 bp and encoded a protein displaying 56% identity to known esterase sequences. The gene was functionally expressed in Escherichia coli BL21 (DE3), and the total molecular weight of the recombined protein was 32.4 kDa. The purified enzyme showed a broad specificity against the four methyl esters of hydroxycinnamic acids and high activity (259.5 U/mg) to methyl ferulate at optimum conditions (pH 8.0, 40 °C). High thermal and pH stability were also observed. Moreover, the enzyme showed broad resistance to proteases. FAE-SH1 can enhance the release of ferulic acid from wheat straw with cellulase, β-1,4-endoxylanase, β-1,3-glucanase, and pectase. These features suggest FAE-SH1 as a good candidate to enhance biomass degradation and improve the health effects of food and forage.

Marine-derived fungi are prone to produce structurally unique secondary metabolites, a considerable number of which display the promising biological properties and/or industrial applications. Among those, ligninolytic enzymes have attracted great interest in recent years. In this work, about 20 strains were isolated from sea mud samples collected in the East China Sea and then screened for their capacity to produce lignin-degrading enzymes. The results showed that a strain, named J63, had a great potential to secrete a considerable amount of laccase. Using molecular method, it was identified as an endophytic fungus, Pestalotiopsis sp. which was rarely reported as ligninolytic enzyme producer in the literature. The production of laccase by Pestalotiopsis sp. J63 was investigated under submerged fermentation (SF) and solid state fermentation (SSF) with various lignocellulosic by-products as substrates. The SSF of rice straw powder accumulated the highest level of laccase activity (10,700 IU/g substrate), whereas the SF of untreated sugarcane bagasse provided the maximum amount of laccase activity (2,000 IU/ml). The value was far higher than those reported by other reports. In addition, it produced 0.11 U/ml cellulase when alkaline-pretreated sugarcane bagasse was used as growth substrate under SF. Meanwhile, the growth of fungi and laccase production under different salinity conditions were also studied. It appeared to be a moderately halo-tolerant organism.

The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035U mg(-1) towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining >60% of its maximum activity at pH 3.0-7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The K(m) and V(max) values were 5.6 and 4.8mgmL(-1), and 2785 and 1608μmolmin(-1)mg(-1), respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96mgg(-1) reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas., Results: A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est&#95;p1), was successfully expressed in E. coli and purified. In a series of assays, Est&#95;p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est&#95;p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure., Conclusions: Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est&#95;p1 has potential application in flavor industries requiring hydrolysis of short chain esters.

Laccases are blue multicopper oxidases with potential applications in environmental and industrial biotechnology. In this study, a new bacterial laccase gene of 1.32 kb was obtained from a marine microbial metagenome of the South China Sea by using a sequence screening strategy. The protein (named as Lac15) of 439 amino acids encoded by the gene contains three conserved Cu(2+)-binding domains, but shares less than 40% of sequence identities with all of the bacterial multicopper oxidases characterized. Lac15, recombinantly expressed in Escherichia coli, showed high activity towards syringaldazine at pH 6.5-9.0 with an optimum pH of 7.5 and with the highest activity occurring at 45 °C. Lac15 was stable at pH ranging from 5.5 to 9.0 and at temperatures from 15 to 45 °C. Distinguished from fungal laccases, the activity of Lac15 was enhanced twofold by chloride at concentrations lower than 700 mM, and kept the original level even at 1,000 mM chloride. Furthermore, Lac15 showed an ability to decolorize several industrial dyes of reactive azo class under alkalescent conditions. The properties of alkalescence-dependent activity, high chloride tolerance, and dye decolorization ability make the new laccase Lac15 an alternative for specific industrial applications.

A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading frame of 1,332 bp, cel5G, encoding an endo-β-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range of β-1,4-, β-1,3/β-1,4-, or β-1,3/β-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose. Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0 to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in the presence of pepsin and pancreatin. The K (m) and V (max) values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 μmol  min(-1) mg(-1), respectively. These characteristics indicate that Cel5G has potential for industrial use.

A strain F1 with high cellulase activity obtained from the deadwood stack was characterized as Ceriporia lacerate by examination of the general taxonomical characteristics and phylogenetic sequence analysis of rDNA ITS gene. The endoglucanase (EG) and filter paper cellulase (FPase) activities of the strain showed remarkable stability in the pH range of 4.0-7.0, and maintained about their maximal value of 76% and 50% after incubation at 70 degrees C for 6 h respectively. The strain grew particularly well with CMC-Na (1.0%) and yeast extract (0.4%) at 28 degrees C (pH 6.0) in flasks stirred at 150 x g for 6 days. Based on the thermostability and pH stability of cellulase, the strain appears to have potential in industrial applications and bioresource utilization.

One clone encoding glycoside hydrolases was identified through functional screening of a rumen bacterial artificial chromosome (BAC) library. Of the 68 open reading frames (ORFs) predicted, one ORF encodes a novel endo-β-1,4-xylanase with two catalytic domains of family GH43 and two cellulose-binding modules (CBMs) of family IV. Partial characterization showed that this endo-xylanase has a greater specific activity than a number of other xylanases over a wide temperature range at neutral pH and could be useful in some industrial applications.

A novel gene encoding an endoglucanase designated Cel5D was cloned from a marine bacterium Martelella mediterranea by genomic library. The gene had a 1,113 bp opening reading frame encoding a 371-amino-acid protein with a molecular mass of 40,508 Da and containing a putative signal peptide (41 amino acids). Cel5D had low similarity (48-51% identity) with other known endoglucanases and consisted of one single catalytic domain, which belonged to the glycosyl hydrolase family 5. The maximum activity of Cel5D was observed at 60 degrees C and pH 5.0. Cel5D displayed broad pH stability within the range of pH 3.0-11.0 and retained hydrolytic activity in the presence of a wide variety of metal ions and some chemical reagents. These characteristics suggest that the enzyme has considerable potential in industrial applications.

A feather-degrading bacterium was isolated from poultry decomposition feathers in China. The strain, named L1, showed significant feather-degrading activity because it grew and reproduced quickly on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen. According to the phenotypic characteristics and 16S rRNA profile, the isolate belongs to Stenotrophomonas maltophilia. Keratinase activity of the isolate was determined during cultivation on raw feathers at different temperatures and initial pH. Maximum growth and feather-degrading activity of the bacterium were observed at 40 degrees C and initial pH ranging from 7.5 to 8.0. The crude enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 chromatographic and ceramic hydroxyapatite (CHT) chromatographic. Its molecular mass estimated as 35.2 kDa in SDS-PAGE. The enzyme had an optimum activity at the pH was 7.8 and the temperature was 40 degrees C. The keratinase was wholly inhibited by a serine protease inhibitor, PMSF. Its activity was activated or inhibited by different metal ions. The keratinase activity of enzyme from strain L1 functioned on different keratins, such as feather, hair, wool, horn, and so on.

The demand for novel biocatalysts is increasing in modern biotechnology, which greatly stimulates the development of powerful tools to explore the genetic resources in the environment. Metagenomics, a culture independent strategy, provides an access to valuable genetic resources of the uncultured microbes. In this study, two novel esterase genes designated as estA and estB, which encoded 277- and 328-amino-acid peptides, respectively, were isolated from a marine microbial metagenomic library by functional screening, and the corresponding esterases EstA and EstB were biochemically characterized. Amino acid sequence comparison and phylogenetic analysis indicated that EstA together with other putative lipolytic enzymes was closely related to family III, and EstB with its relatives formed a subfamily of family IV. Site-directed mutagenesis showed that EstA contained classical catalytic triad made up of S146-D222-H255, whereas EstB contained an unusual catalytic triad which consisted of S-E-H, an important feature of the subfamily. EstA exhibited habitat-specific characteristics such as its high level of stability in the presence of various divalent cations and at high concentrations of NaCl. EstB displayed remarkable activity against p-nitrophenyl esters and was highly stable in 30% methanol, ethanol, dimethylformamide, and dimethyl sulfoxide, making EstB a potential candidate for industrial applications.

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn(2+), Mg(2+) or Ca(2+) increased AG-a activity, while Mn(2+), Cu(2+) or Ca(2+) increased AG-b activity. However, Ag(+), Hg(2+), Fe(3+), EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.

Four pepsinogens (PG-I, PG-II, PG-III(a), and PG-III(b)) were highly purified from the stomach of the freshwater fish mandarin fish (Siniperca chuatsi) by ammonium sulfate fractionation, anion exchange, and gel filtration. The molecular masses of the four purified PGs were 36, 35, 38, and 35 kDa, respectively. All the pepsinogens converted into their active form pepsins within a few minutes under pH 2.0. The optimum pH and temperature of the four enzymes were 3.0-3.5 and 45-50 degrees C, using hemoglobin as a substrate. The N-terminal amino acid sequences of PG-I and PG-II were determined to the 12th and 17th amino acid residues, respectively. Western blot analysis using antisea bream polyclonal antibodies cross reacted with PG-I, PG-II, and PG-III(b) while no cross reaction with PG-III(a) was detected, suggesting the diversity of pepsinogens in fish.

A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe-SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80 degrees C and retained 50% activity after heat treatment at 95 degrees C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe(2+) was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.

Thirty-eight high lipase activity strains were isolated from soil, seawater, and Brassica napus. Among them, a novel organic solvent tolerant bacterium (strain M36) was isolated from the seawater in Jiangsu, China. Isolate M36 was able to grow at high concentration of benzene or toluene up to 40% (vol/vol), and later identified as Staphylococcus saprophyticus by biochemical test and 16s ribosomal DNA sequence. No work on Staphylococcus producing lipase with organic solvent tolerance has been reported so far. The lipase of strain M36 whose activity in liquid medium was 42 U mL(-1) at 24-h incubation time was stable in the presence of 25% (vol/vol) p-xylene, benzene, toluene, and hexane.

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55 degrees C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS-PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), beta-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bbeta-chains and Aalpha-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.

Aims: A xylanase from the newly isolated thermophilic fungus, Thermomyces lanuginosus CAU44, was characterized and evaluated for its suitability in bread making., Methods and Results: Xylanase was purified 3.5-fold to homogeneity with a recovery yield of 32.8%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of c. 25.6 kDa. The purified xylanase had an optimum pH of 6.2, and it was stable over pH 5.6-10.3. The optimal temperature of xylanase was 75 degrees C and it was stable up to 65 degrees C at pH 6.2. Study was further carried out to investigate the effect of the purified xylanase on the properties of wheat bread and its staling during storage., Conclusions: The purified xylanase from T. lanuginosus CAU44 was stable up to 65 degrees C and had a broad pH range. The presence of thermostable xylanase during bread making led to an improvement of the specific bread volume and better crumb texture. Besides, addition of xylanase provided an anti-staling effect., Significance and Impact of the Study: The xylanase from the newly isolated Thermomyces lanuginosus CAU44 shows great promise as a processing aid in the bread-making industry.

An isolation procedure, consisting of ion exchange chromatography on CM-Sepharose, affinity chromatography on Affi-gel blue gel, and fast protein liquid chromatography on Mono S, was utilized to purify a base-nonspecific, heterodimeric ribonuclease (RNase) with diverse activities from roots of the sanchi ginseng Panax notoginseng. The RNase is unique in that it consists of two different nonglycoprotein subunits with a molecular weight of 27 and 29 kDa, respectively. The latter subunit is characterized by an N-terminal sequence showing remarkable similarity to that of the bitter gourd RNase. The Panax notoginseng RNase demonstrates potent RNase and translation-inhibitory activities. In addition, it exhibits antiproliferative activity toward leukemia L1210 cells and antifungal activity against Physalospora piricola and Coprinus comatus. Its RNase activity is not heat-resistant, unlike most RNases which are thermostable., (Copyright 2001 Academic Press.)