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Hyaluronan (HA), a natural high molecular weight polysaccharide, has extensive applications in cosmetology and medical treatment. Hyaluronan-degrading enzymes (Hyals) act as molecular scissors that cleave HA by breaking the glucosidic linkage. Hyals are present in diverse organisms, including vertebrates, invertebrates and microorganisms, and play momentous roles in biological processes. In recent years, microbial Hyals (mHyals) have gained considerable attention for their exceptional performance in the production and processing of HA. Moreover, the applications of mHyals have been greatly extended to various biomedical fields. To explore the potential applications of mHyals, a thorough comprehension is imperative. In this context, this review systematically summarizes the sources, structures, mechanisms and enzymatic properties of mHyals and discusses their biological functions in host invasion, disease development, and regulation of intestinal flora. Furthermore, versatile applications inspired by their biological functions in medicine development, molecular biology, and industrial biotechnology are comprehensively reviewed. Finally, prospects are presented to emphasize the importance of exploration, expression and characterization of mHyals and the necessity of excavating their potential in biotechnological fields. [ABSTRACT FROM AUTHOR]

Hyaluronate lyase (HA lyase) has potential in the industrial processing of hyaluronan. In this study, HylP, an HA lyase from Streptococcus pyogenes phage (SPB) was successfully expressed in Bacillus subtilis. To improve the extracellular enzyme activity of HylP in B. subtilis, signal peptide engineering systematic optimization was carried out, and cultured it from shake flasks and fermenters, followed by purification, characterization, and analysis of degradation products. The results showed that the replacement of the signal peptide increased the extracellular enzyme activity of HylP from 1.0 × 104 U/mL to 1.86 × 104 U/mL in the shake flask assay, and using a 20 L fermenter in a batch fermentation process, the extracellular enzyme activity achieved the level of 1.07 × 105 U/mL. HylP exhibited significant thermal and pH stability in the temperature range of 40 °C and pH range of 4–8, respectively. The enzyme showed optimum activity at 40 °C and pH 6, with significant activity in the presence of Na+, Mg2+, and Co2+ ions. Degradation analysis showed that HylP efficiently degraded hyaluronan as an endonuclease, releasing unsaturated disaccharides. These comprehensive findings underscore the substantial industrial potential of HylP for hyaluronan processing applications, offering valuable insights into enzyme characterization and optimization of expression for potential industrial utilization. [ABSTRACT FROM AUTHOR]

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi‐epitope chimeric antigen (ChA) and screening two classes of protein‐assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin‐binding protein A of Glaesserella parasuis. Our engineered strain, which co‐expresses P17 tagged‐ChA and endogenous chaperones groEL‐ES, yields a 0.346 g/L highly soluble ChA with the property of HPS‐positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5‐L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications. [ABSTRACT FROM AUTHOR]

Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue–derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering. The recombinant hyaluronidase was first expressed under the control of a constitutive promoter PGCW14. Then, two endoplasmic reticulum–related secretory pathways were engineered to improve the secretion capability of the recombinant strain. Signal peptide optimization suggested redirecting the protein into co-translational translocation using the ost1-proα signal sequence improved the secretion level by 20%. Enhancing the co-translational translocation by overexpressing signal recognition particle components further enhanced the secretory capability by 48%. Then, activating the unfolded protein response by overexpressing a transcriptional factor ScHac1p led to a secreted hyaluronidase activity of 4.06 U/mL, which was 2.1-fold higher than the original strain. Finally, fed-batch fermentation elevated the production to 19.82 U/mL. The combined engineering strategy described here could be applied to enhance the secretion capability of other proteins in yeast hosts. Key points: • Improving protein secretion by enhancing co-translational translocation in P. pastoris was reported for the first time. • Overexpressing Hac1p homologous from different origins improved the rhPH-20 secretion. • A 4.9-fold increase in rhPH-20 secretion was achieved after fermentation optimization and fed-batch fermentation. [ABSTRACT FROM AUTHOR]

Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue–derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering. The recombinant hyaluronidase was first expressed under the control of a constitutive promoter PGCW14. Then, two endoplasmic reticulum–related secretory pathways were engineered to improve the secretion capability of the recombinant strain. Signal peptide optimization suggested redirecting the protein into co-translational translocation using the ost1-proα signal sequence improved the secretion level by 20%. Enhancing the co-translational translocation by overexpressing signal recognition particle components further enhanced the secretory capability by 48%. Then, activating the unfolded protein response by overexpressing a transcriptional factor ScHac1p led to a secreted hyaluronidase activity of 4.06 U/mL, which was 2.1-fold higher than the original strain. Finally, fed-batch fermentation elevated the production to 19.82 U/mL. The combined engineering strategy described here could be applied to enhance the secretion capability of other proteins in yeast hosts. Key points: • Improving protein secretion by enhancing co-translational translocation in P. pastoris was reported for the first time. • Overexpressing Hac1p homologous from different origins improved the rhPH-20 secretion. • A 4.9-fold increase in rhPH-20 secretion was achieved after fermentation optimization and fed-batch fermentation. [ABSTRACT FROM AUTHOR]

Collagen is a kind of high macromolecular protein with unique tissue distribution and distinctive functions in the body. At present, most collagen products are extracted from the tissues and organs of mammals or marine fish. However, this method exhibits several disadvantages, including low efficiency and serious waste generation, which makes it difficult to meet the current market demand. With the rapid development of synthetic biology and the deepening of high-density fermentation technology, the collagen preparation by biosynthesis strategy emerges as the times require. Co-expression with the proline hydroxylase gene can solve the problem of non-hydroxylated collagen, but the yield may be affected. Therefore, improving the expression through molecular modification and dynamic regulation of synthesis is an entry point for future research. Due to the defects in certain properties of the natural collagen, modification of properties would be benefit for meeting the requirements of practical application. In this paper, in-depth investigations on recombinant expression, fermentation, and modification studies of collagen are conducted. Also, it summarizes the research progress of collagen in food, medicine, and beauty industry in recent years. Furthermore, the future development trend and application prospect of collagen are discussed, which would provide guidance for its preparation and application. [ABSTRACT FROM AUTHOR]

Nicotinamide mononucleotide (NMN), a naturally occurring biologically active nucleotide, mainly functions via mediating the biosynthesis of NAD+. In recent years, its excellent pharmacological activities including anti-aging, treating neurodegenerative diseases, and protecting the heart have attracted increasing attention from scholars and entrepreneurs for production of a wide range of formulations, including functional food ingredients, health care products, active pharmaceuticals, and pharmaceutical intermediates. Presently, the synthesis methods of NMN mainly include two categories: chemical synthesis and biosynthesis. With the development of biocatalyst engineering and synthetic biology strategies, bio-preparation has proven to be efficient, economical, and sustainable methods. This review summarizes the chemical synthesis and biosynthetic pathways of NMN and provides an in-depth investigation on the mining and modification of enzyme resources during NMN biosynthesis, as well as the screening of hosts and optimization of chassis cells via metabolic engineering, which provide effective strategies for efficient production of NMN. In addition, an overview of the significant physiological functions and activities of NMN is elaborated. Finally, future research on technical approaches to further enhance NMN synthesis and strengthen clinical studies of NMN are prospected, which would lay the foundation for further promoting the application of NMN in nutrition, healthy food, and medicine in the future. Key points: • NMN supplementation effectively increases the level of NAD+. • The chemical and biological synthesis of NMN are comprehensively reviewed. • The impact of NMN on the treatment of various diseases is summarized. [ABSTRACT FROM AUTHOR]

Collagen, the highest content protein in the body, has irreplaceable biological functions, and it is widespread concerned in food, beauty, and medicine with great market demand. The gene encoding the recombinant type III human-like collagen α1 chain fragment was integrated into P. pastoris genome after partial amino acids were substituted. Combined with promoter engineering and high-density fermentation technology, soluble secretory expression with the highest yield of 1.05 g L−1 was achieved using two-stage feeding method, and the purity could reach 96% after affinity purification. The determination of N/C-terminal protein sequence were consistent with the theoretical expectation and showed the characteristics of Gly-X-Y repeated short peptide sequence. In amino acid analysis, glycine shared 27.02% and proline 23.92%, which were in accordance with the characteristics of collagen. Ultraviolet spectrum combined with Fourier transform infrared spectroscopy as well as mass spectrometry demonstrated that the target product conformed to the characteristics of collagen spectrums and existed as homologous dimer and trimer in the broth. This work provided a sustainable and economically viable source of the recombinant type III human-like collagen. [ABSTRACT FROM AUTHOR]

Pichia pastoris has been recognized as an important platform for the production of various heterologous proteins in recent years. The strong promoter AOX1, induced by methanol, with the help of the α-pre-pro signal sequence, can lead to a high expression level of extracellular protein. However, this combination was not always efficient, as protein secretion in P. pastoris involves numerous procedures mediated by several cellular proteins, including folding assisted by endoplasmic reticulum (ER) molecular chaperones, degradation through ubiquitination, and an efficient vesicular transport system. Efficient protein expression requires the cooperation of various intracellular pathways. This article summarizes the process of protein secretion, modification, and transportation in P. pastoris. In addition, the roles played by the key proteins in these processes and the corresponding co-expression effects are also listed. It is expected to lay the foundation for the industrial protein production of P. pastoris. Key points: • Mechanisms of chaperones in protein folding and their co-expression effects are summarized. • Protein glycosylation modifications are comprehensively reviewed. • Current dilemmas in the overall protein secretion pathway of Pichia pastoris and corresponding solutions are demonstrated. [ABSTRACT FROM AUTHOR]

Keratinases can specifically degrade keratins, which widely exist in hair, horns, claws and human skin. There is a great interest in developing keratinase to manage keratin waste generated by the poultry industry and reusing keratin products in agriculture, medical treatment and feed industries. Degradation of keratin waste by keratinase is more environmentally friendly and more sustainable compared with chemical and physical methods. However, the wild-type keratinase-producing strains usually cannot meet the requirements of industrial production, and some are pathogenic, limiting their development and utilization. The main purpose of this study is to improve the catalytic performance of keratinase via directed evolution technology for the degradation of feathers. We first constructed a mutant library through error-prone PCR and screened variants with enhanced enzyme activity. The keratinase activity was further improved through fermentation conditions optimization and fed-batch strategies in a 7-L bioreactor. As a result, nine mutants with enhanced activity were identified and the highest enzyme activity was improved from 1150 to 8448 U/mL finally. The mutant achieved efficient biodegradation of feathers, increasing the degradation rate from 49 to 88%. Moreover, a large number of amino acids and soluble peptides were obtained as degradation products, which were excellent protein resources to feed. Therefore, the study provided a keratinase mutant with application potential in the management of feather waste and preparation of protein feed additive. [ABSTRACT FROM AUTHOR]

Keratin is a kind of natural polymer that is abundant in feathers, wool, and hair. Being one of the natural biomolecules, keratin has excellent biological activity, biocompatibility, biodegradability, favorable material mechanical properties, and natural abundance, which exhibit significant biological and biomedical application potentials. At present, the strategies commonly used for preparing keratin from hair, feathers, wool, etc. include physical, chemical, and enzymatic methods. The present article mainly reviews the structure, classification, preparation methods, and the main biological applications of keratin, and these applications cover wound healing, hemostasis, targeted release of tissue engineering drugs, and so on. It is expected to lay the foundations for its future in-depth investigations and wide applications of keratin biomaterials. Key points: • There are several pathways to prepare biologically active keratin from wool, feathers, and human hair, etc • Promoting blood coagulation by keratin is related to the adhesion and activation of platelets and the aggregation of fibrin • The biological applications of keratin, including wound healing and tissue engineering, are summarized [ABSTRACT FROM AUTHOR]

To improve nicotinic acid (NA) yield and meet industrial application requirements of sodium alginate-polyvinyl alcohol (SA-PVA) immobilized cells of Pseudomonas putida mut-D3 harboring nitrilase, inorganic materials were added to the SA-PVA immobilized cells to improve mechanical strength and mass transfer performance. The concentrations of inorganic materials were optimized to be 2.0% silica and 0.6% CaCO3. The optimal pH and temperature for SA-PVA immobilized cells and composite immobilized cells were both 8.0 and 45 °C, respectively. The half-lives of composite immobilized cells were 271.48, 150.92, 92.92 and 33.12 h, which were 1.40-, 1.35-, 1.22- and 1.63-fold compared to SA-PVA immobilized cells, respectively. The storage stability of the composite immobilized cells was slightly increased. The composite immobilized cells could convert 14 batches of 3-cyanopyridine with feeding concentration of 250 mM and accumulate 418 g ·L−1 nicotinic acid, while the SA-PVA immobilized cells accumulated 346 g L−1 nicotinic acid. [ABSTRACT FROM AUTHOR]

The membrane-bound gluconate dehydrogenase (mGADH) is a critical enzyme for 2-keto-d-gluconic acid (2KGA) production in Pseudomonas plecoglossicida JUIM01. The purified native flavin adenine dinucleotide-dependent mGADH (FAD-mGADH) was consisted of a gamma subunit, a flavoprotein subunit, and a cytochrome c subunit with molecular mass of ~ 27, 65, and 47 kDa, respectively. The specific activity of FAD-mGADH was determined as 90.71 U/mg at optimum pH and temperature of 6.0 and 35 °C. The Km and Vmax values of calcium d-gluconate were 0.631 mM and 0.734 mM/min. The metal ions Mg2+ and Mn2+ showed slight positive effects on FAD-mGADH activity. On the other hand, a 3868-bp-length gad gene cluster was amplified and expressed in Escherichia coli BL21(DE3). The recombinant protein showed the same molecular weight and enzyme activity as the native FAD-mGADH, which confirmed it as a FAD-mGADH encoding gene. The flavoprotein subunit and the cytochrome c subunit containing a putative FAD-binding motif and three possible heme-binding motifs concluded from alignment results of mGADHs. This study characterized the native and recombinant FAD-mGADH and would provide the basis for further genetic modification of Pseudomonas plecoglossicida JUIM01 with the intention of 2KGA productivity improvement. [ABSTRACT FROM AUTHOR]

Phosphatidylserine is widely used in food, health, chemical and pharmaceutical industries. The phospholipase D-mediated green synthesis of phosphatidylserine has attracted substantial attention in recent years. In this study, the phospholipase D was heterologously expressed in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum, respectively. The highest activity of phospholipase D was observed in C. glutamicum, which was 0.25 U/mL higher than these in B. subtilis (0.14 U/mL) and P. pastoris (0.22 U/mL). System engineering of three potential factors, including (1) signal peptides, (2) ribosome binding site, and (3) promoters, was attempted to improve the expression level of phospholipase D in C. glutamicum. The maximum phospholipase D activity reached 1.9 U/mL, which was 7.6-fold higher than that of the initial level. The enzyme displayed favorable transphosphatidylation activity and it could efficiently catalyze the substrates l-serine and soybean lecithin for synthesis of phosphatidylserine after optimizing the conversion reactions in detail. Under the optimum conditions (trichloromethane/enzyme solution 4:2, 8 mg/mL soybean lecithin, 40 mg/mL l-serine, and 15 mM CaCl2, with shaking under 40 °C for 10 h), the reaction process showed 48.6% of conversion rate and 1.94 g/L of accumulated phosphatidylserine concentration. The results highlight the use of heterologous expression, system engineering, and process optimization strategies to adapt a promising phospholipase D for efficient phosphatidylserine production in synthetic application. [ABSTRACT FROM AUTHOR]

The nitrilase-mediated green synthesis of nicotinic acid, an important reaction in synthetic organic chemistry, has attracted considerable attention in recent years. However, the application potential of nitrilase is hindered by several limitations, such as low catalytic efficiency and byproduct formation. In this study, the site-saturation mutagenesis of asparagine 40, phenylalanine 50, and glutamine 207 in recombinant nitrilase from Pseudomonas putida CGMCC3830 was conducted to improve the specificity of nitrilase for nicotinic acid. The resulting mutants, which contain the mutations N40G (asparagine→glycine), F50W (phenylalanine→tryptophan), and Q207E (glutamine→glutamic acid), produced higher nicotinic acid yields than the wild type. Furthermore, double and triple mutations were introduced, and four mutants that contained N40G/F50W, N40G/Q207E, F50W/Q207E, and N40G/F50W/Q207E were obtained and evaluated for their capacity to produce nicotinic acid. The double mutant F50W/Q207E and the triple mutant N40G/F50W/Q207E displayed the highest activity, which was nearly twofold higher than that of the wild type. The kinetics analysis of nicotinic acid synthesis with the mutant nitrilase revealed the higher catalytic capability of all the mutants than the wild type. These results provide new insights into the catalytic performance of P. putida nitrilase toward 3-cyanopyridine to promote nicotinic acid production. [ABSTRACT FROM AUTHOR]

An alginate lyase producing bacterial strain, Cobetia sp. WG-007, was isolated and identified from rotting seaweed. The alginate lyase, Aly-W02, was purified by procedures of ultrafiltration, Q-Sepharose Fast Flow, Phenyl Sepharose 6 Fast Flow, and Superdex-G100 with specific activity of 21,285.5 U/mg. Aly-W02 had an apparent molecular mass of 35 kDa. It exhibited maximum activity at 45 °C in 50 mM sodium phosphate buffer (pH 8.5). This alginate lyase was stable in the pH range of 6.0-8.5. Among the tested metal ions, the addition of K+, Na+, and Mg2+ ions can enhance the enzyme activities, while Ba2+, Ni+, Cu2+, Mn2+, Zn2+, Ag+, and ethylenediaminetetraacetic acid decreased the activities. It displayed high salt-tolerant ability; 0.8 M NaCl or 1.5 M KCl significantly enhanced the enzyme activity. Furthermore, Aly-W02 mainly released disaccharide, trisaccharide, and tetrasaccharid from alginate. It showed potential in producing low molecular weight alginate oligosaccharides. [ABSTRACT FROM AUTHOR]

3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag, Pb and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry. [ABSTRACT FROM AUTHOR]

Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed. [ABSTRACT FROM AUTHOR]

Microbial keratinase is a well-recognized enzyme that can specifically degrade insoluble keratins. A keratinase-producing bacterium was isolated from a duck ranch soil and identified as Acinetobacter sp. R-1 based on the biochemical characteristics and 16S rDNA gene sequencing. It showed high keratinase activity and low collagenase activity. The keratinase was purified to electrophoretic homogeneity with 6.69 % recovery, 2.68-fold purification and an estimated molecular weight of 25 kDa. Additionally, the keratinase showed optimal activity at 50 °C and pH11. Keratinase activity of Acinetobacter sp. significantly increased in the presence of Li, Na, and Ca, while it was completely inhibited by EDTA, indicating it was a metallo-keratinase. Moreover, the crude keratinase from Acinetobacter sp. R-1 could thoroughly depilate goat skin and simultaneously modify the wool surface, which indicated its applicable potential in leather and textile industries. [ABSTRACT FROM AUTHOR]

Comparative studies of immobilized and free cells of Gibberella intermedia CA3-1 in bioconversion of 3-cyanopyridine to nicotinic acid were performed. Entrapping method was chosen based on the advantages in enzymatic activity recovery, mechanical strength and preparation procedure. Four entrapment matrices were investigated and sodium alginate was screened to be the most suitable material. Maximal nitrilase activity of alginate immobilized cells was obtained under conditions of 2 % alginate, 0.6 % CaCl, 0.4 g cell/g alginate, 1.8 mm bead size. The immobilized cells showed excellent substrate tolerance even when the 3-cyanopyridine concentration was 700 mM. The half-lives of immobilized cells at 30, 40 and 50 °C were 315, 117.5 and 10.9 h, respectively, correspondingly 1.4, 1.6 and 1.7-fold compared with that of the free cells. Efficient reusability of immobilized cells up to 28 batches was achieved and 205.7 g/(g dcw) nicotinic acid was obtained with 80.55 % enzyme activity preserved. [ABSTRACT FROM AUTHOR]

Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L h. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far. [ABSTRACT FROM AUTHOR]

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag+, Hg2+, and excess substrate inhibited the nitrilase activity, whereas Fe2+, Mn2+, and Li+ improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only <5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production. [ABSTRACT FROM AUTHOR]

Nitrilase is one of the most important members in the nitrilase superfamily and it is widely used for bioproduction of commodity chemicals and pharmaceutical intermediates as well as bioremediation of nitrile-contaminated wastes. However, its application was hindered by several limitations. Searching for new nitrilases and improving their application performances are the driving force for researchers. Genetic data resources in various databases are quite rich in post-genomic era. Besides, more than 99 % of microbes in the environment are unculturable. Metagenomic technology and genome mining are thus becoming burgeoning areas and provide unprecedented opportunities for searching more useful novel nitrilases due to the abundance of already existing but unexplored gene resources, namely uncharacterized genome information in the database and unculturable microbes in the natural environment. These techniques seem to be innovative and highly efficient. This study reviews the current status and future directions of metagenomics and genome mining in nitrilase exploration. Moreover, it discussed their utilization in coping with the challenges for nitrilase application. In the next several years, with the rapid development of nitrile biocatalysis, these two techniques would be bound to attract increasing attentions and even become a dominant trend for finding more novel nitrilases. Also, this review would provide guidance for exploitation of other commercially important enzymes. [ABSTRACT FROM AUTHOR]

Microbial transformation of glycinonitrile into glycine by nitrile hydrolase is of considerable interest to green chemistry. A novel fungus with high nitrile hydrolase was newly isolated from soil samples and identified as Fusarium oxysporum H3 through 18S ribosomal DNA, 28S ribosomal DNA, and the internal transcribed spacer sequence analysis, together with morphology characteristics. After primary optimization of culture conditions including pH, temperature, carbon/nitrogen sources, inducers, and metal ions, the enzyme activity was greatly increased from 326 to 4,313 U/L. The preferred carbon/nitrogen sources, inducer, and metal ions were glucose and yeast extract, caprolactam, and Cu, Mn, and Fe, respectively. The maximum enzyme formation was obtained when F. oxysporum H3 was cultivated at 30 °C for 54 h with the initial pH of 7.2. There is scanty report about the optimization of nitrile hydrolase production from nitrile-converting fungus. [ABSTRACT FROM AUTHOR]