Purification and comparison of soluble and membrane-bound polyphenol oxidase from potato (Solanum tuberosum) tubers.

Enzymatic browning greatly affects the quality of potato products. Polyphenol oxidase (PPO) is the enzyme mainly responsible for potato enzymatic browning. PPO has soluble polyphenol oxidase (sPPO) and membrane-bound polyphenol oxidase (mPPO) forms. In this study, the properties of sPPO and mPPO were investigated in potato tubers. The molecular weight of potato sPPO and mPPO were estimated to be 69 kDa in the form of homodimers in vivo. The mass spectrometry results showed that the purified sPPO and mPPO protein in potato tubers was mainly tr|M1BMR6 (Uniprot). The optimum pH for sPPO and mPPO was 6.5, and the optimum temperatures were 20 and 30 °C, respectively. The Michaelis constant (Km) and maximum unit enzyme activity (Vmax) of sPPO were 6.08 mM and 2161 U/S when catechol was used as the substrate, whereas those of mPPO were 2.95 mM and 2129.53 U/S, respectively. The mPPO had stronger affinity to the substrate catechol than sPPO, whereas pyrogallic acid was stronger affinity for sPPO. Ascorbic acid and sodium sulfite were inhibitors of sPPO and mPPO, respectively. After understanding the different binding states of polyphenol oxidase, different inhibitors and treatment methods can be used to treat the enzyme according to different enzymatic properties, so as to achieve a greater degree of Browning control. These results will provide a theoretical basis for regulating PPO activity to reduce enzymatic browning during potato processing.

Degradation of 2,4-DCP by immobilized laccase on modified biochar carrier.

Rape straw was used as the raw material for the biochar in this study, which was then changed using acid, alkali, and magnetic techniques. The laccase was attached using the adsorptions-crosslinking process, and the three modified biochars served as the carriers. The ideal circumstances for laccase immobilization were explored, and both biochar and immobilized laccase's characteristics were examined. The removal of 2,4-dichlorophenol (2,4-DCP) by immobilized laccase from modified biochar and its degradation products were researched. The main conclusions are as follows: the optimal concentration of glutaraldehyde (GLU) was 4%, and the pH was four, and the enzyme dosage was 1.75 mg/mL for the immobilized laccase of acid-modified biochar (SBC@LAC). The optimal concentration of GLU was 5%; the pH was four, and the enzyme dosage was 2 mg/mL for immobilized laccase from alkali-modified biochar (JBC@LAC). The optimal concentration of GLU was 5%; the pH was four, and the enzyme dosage was 1.75 mg/mL for immobilized laccase from magnetically modified biochar (CBC@LAC). SEM images could show the changes in the surface morphology of biochar caused by three modification methods. The BET results demonstrated that acid and magnetic modification increased the specific surface area of biochar, and alkali modification mainly expanded the pore size of biochar. FT-IR and XRD showed that modification and laccase loading had little effect on the structure of biochar. The stability of immobilized laccase was better than that of free laccase in acid-base, heat, and storage. Among the three modified biochar immobilized laccases, JBC@LAC showed the best acid-base stability and thermal stability, and the relative enzyme activity changed the least when pH and temperature conditions changed. The storage stability of SBC@LAC is the best. After 30 days of storage, the relative enzyme activity is still 83%. The removal rates of 2,4-DCP were 57, 99, and 63%, respectively, by SBC@LAC, JBC@LAC, and CBC@LAC. After five reuses, the removal rates of 2,4-DCP by SBC@LAC, JBC@LAC and CBC@LAC were 26, 42, and 27%, respectively. The intermediate products of 2,4-DCP degradation by immobilized laccase were p-phenol, p-benzoquinone and maleic acid.

Efficient Secretory Production of Lytic Polysaccharide Monooxygenase BaLPMO10 and Its Application in Plant Biomass Conversion.

Lytic polysaccharide monooxygenases (LPMOs) can oxidatively break the glycosidic bonds of crystalline cellulose, providing more actionable sites for cellulase to facilitate the conversion of cellulose to cello-oligosaccharides, cellobiose and glucose. In this work, a bioinformatics analysis of BaLPMO10 revealed that it is a hydrophobic, stable and secreted protein. By optimizing the fermentation conditions, the highest protein secretion level was found at a IPTG concentration of 0.5 mM and 20 h of fermentation at 37 °C, with a yield of 20 mg/L and purity > 95%. The effect of metal ions on the enzyme activity of BaLPMO10 was measured, and it was found that 10 mM Ca2+ and Na+ increased the enzyme activity by 47.8% and 98.0%, respectively. However, DTT, EDTA and five organic reagents inhibited the enzyme activity of BaLPMO10. Finally, BaLPMO10 was applied in biomass conversion. The degradation of corn stover pretreated with different steam explosions was performed. BaLPMO10 and cellulase had the best synergistic degradation effect on corn stover pretreated at 200 °C for 12 min, improving reducing sugars by 9.2% compared to cellulase alone. BaLPMO10 was found to be the most efficient for ethylenediamine-pretreated Caragana korshinskii by degrading three different biomasses, increasing the content of reducing sugars by 40.5% compared to cellulase alone following co-degradation with cellulase for 48 h. The results of scanning electron microscopy revealed that BaLPMO10 disrupted the structure of Caragana korshinskii, making its surface coarse and poriferous, which increased the accessibility of other enzymes and thus promoted the process of conversion. These findings provide guidance for improving the efficiency of enzymatic digestion of lignocellulosic biomass.

Insect α-Amylases and Their Application in Pest Management.

Amylase is an indispensable hydrolase in insect growth and development. Its varied enzymatic parameters cause insects to have strong stress resistance. Amylase gene replication is a very common phenomenon in insects, and different copies of amylase genes enable changes in its location and function. In addition, the classification, structure, and interaction between insect amylase inhibitors and amylases have also invoked the attention of researchers. Some plant-derived amylase inhibitors have inhibitory activities against insect amylases and even mammalian amylases. In recent years, an increasing number of studies have clarified the effects of pesticides on the amylase activity of target and non-target pests, which provides a theoretical basis for exploring safe and efficient pesticides, while the exact lethal mechanisms and safety in field applications remain unclear. Here, we summarize the most recent advances in insect amylase studies, including its sequence and characteristics and the regulation of amylase inhibitors (α-AIs). Importantly, the application of amylases as the nanocide trigger, RNAi, or other kinds of pesticide targets will be discussed. A comprehensive foundation will be provided for applying insect amylases to the development of new-generation insect management tools and improving the specificity, stability, and safety of pesticides.

Biochemical characterization and elucidation of the action mode of a GH16 family κ-carrageenase for efficient preparation of carrageenan oligosaccharides.

κ-Carrageenan oligosaccharides have a variety of biological activities. Degradation of κ-carrageenan by κ-carrageenase leads to degradation products with different degrees of polymerization (DPs). A novel gene (CecgkA) encoding a new κ-carrageenase was cloned from Colwellia echini and heterologously expressed in Escherichia coli BL21 (DE3). The enzyme is 1104 bp in length, encodes 367 amino acid residues and has a molecular weight of 41.30 kDa. Multiple alignment analysis showed that CeCgkA belongs to the glycoside hydrolase (GH16) family and has the highest homology with the κ-carrageenase of Rhodopirellula maiorica SM1, with 58% homology. The CeCgkA showed maximum activity (453.15 U/mg) at pH 8.0 and 35 °C. Determination of biochemical properties showed that CeCgkA was a thermal recovery enzyme, and 51.6% of the initial enzyme activity was recovered by immediately placing the sample at 35 °C for 60 min after enzymatic inactivation by boiling for 10 min. K+, Na+, and EDTA had an activating effect on the enzyme activity, while Ni2+, Cu2+, and Zn2+ inhibited the activity of the enzyme. In addition, TLC and ESI-MS analysis showed that the maximum recognition unit of CecgkA was decasaccharide and that the main degradation products were disaccharides, tetrasaccharides and hexasaccharides, indicating that the enzyme is an endo-type carrageenase.

Tuning Local Coordination Environments of Manganese Single-Atom Nanozymes with Multi-Enzyme Properties for Selective Colorimetric Biosensing.

Single-atom nanozymes (SAzymes) are promising in next-generation nanozymes, nevertheless, how to rationally modulate the microenvironment of SAzymes with controllable multi-enzyme properties is still challenging. Herein, we systematically investigate the relationship between atomic configuration and multi-enzymatic performances. The constructed MnSA -N3 -coordinated SAzymes (MnSA -N3 -C) exhibits much more remarkable oxidase-, peroxidase-, and glutathione oxidase-like activities than that of MnSA -N4 -C. Based on experimental and theoretical results, these multi-enzyme-like behaviors are highly dependent on the coordination number of single atomic Mn sites by local charge polarization. As a consequence, a series of colorimetric biosensing platforms based on MnSA -N3 -C SAzymes is successfully built for specific recognition of biological molecules. These findings provide atomic-level insight into the microenvironment of nanozymes, promoting rational design of other demanding biocatalysts.

Characterization of a recombinant 10-linoleic acid hydratase from Lactiplantibacillus plantarum ZS2058 and biosynthesis of 10- hydroxy-cis-12-octadecenoic acid.

BACKGROUND: 10-Hydroxy-cis-12-octadecenoic acid (10-HOE, 10-OH C18:1), an emerging functional fatty acid, has anti-fungal and anti-inflammatory effects. 10-HOE is synthesized by bacterial 10-linoleic acid hydratase (10-LHT) with linoleic acid as the substate. However, the characterization of 10-LHT and its targeted synthesis of 10-HOE have been rarely reported. In this study, the recombinant 10-LHT from Lactiplantibacillus plantarum ZS2058 was characterized, and the biocatalysis of 10-HOE using crude enzyme was optimized. RESULTS: The recombinant 10-LHT catalyzed the conversion of linoleic acid (C18:2) to 10-HOE as identified using gas chromatography-mass spectrometry (GC-MS). It showed a molecular weight of about 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and was a flavin adenine dinucleotide (FAD)-dependent enzyme. The activity of 10-LHT was optimal at pH 6.5 and 25 °C, and it was pH-stable but thermo-sensitive. The optimal condition for the 10-HOE biosynthesis using crude enzyme was 5 g L-1 linoleic acid (C18:2), 148.0 U mL-1 10-LHT, 0.05 mmol L-1 FAD, 2% methanol and 100 mmol L-1 sodium chloride at 25 °C and pH 6.5. A conversion yield of 47.8 ± 1.5% and the corresponding 10-HOE concentration of 2.4 ± 0.1 g L-1 were achieved at 48 h under the optimal reaction conditions. CONCLUSION: This work achieved the highest conversion yield of 10-HOE with the highest substrate concentration, and provides some useful information for the industrial production of 10-HOE. © 2021 Society of Chemical Industry.

Characterization and rational modification of aspartate 4-decarboxylase from Acinetobacter radioresistens for the production of l-alanine.

Enzymatic synthesis of l-alanine has the advantages of less byproducts, strong stereoselectivity, and high catalytic efficiency. Aspartate 4-decarboxylase (ASD) is used industrially in DL-aspartic acid resolution and l-alanine production because it catalyzes the decarboxylation of l-aspartic acid. In this study, the ASD gene from Acinetobacter radioresistens (ArASD) was cloned, and its enzymatic properties were analyzed. ArASD is a dodecamer and has the highest enzyme activity ever reported to date. The optimal conditions for ArASD catalysis are 50°C and pH 4.5. Site-directed mutagenesis was used to improve ArASD stability under acidic conditions to compensate for its weak acid resistance, and the variant N35D with higher catalytic ability was obtained. The conversion by N35 recombinant cells of l-aspartic acid to l-alanine was 92.5% at pH 4.5% and 99.9% at pH 6.0, whereas that of the wild-type recombinant cells was 29.7% and 31.4%, respectively. Aspartase from Escherichia coli (AspA) was employed with ArASD to construct a dual-enzyme system that catalyzes fumaric acid to l-alanine, and the conversion reached 97.1% using recombinant cells harboring the dual-enzyme system. This study explored the enzymatic properties of ArASD and an effective strategy for the acidic resistance modification of ASD. Moreover, the strain expressing the ArASD variant and AspA engineered in this study has great potential application for the l-alanine production industry, especially in the case of high optical purity requirements.

Thermostable lipases and their dynamics of improved enzymatic properties.

Thermal stability is one of the most desirable characteristics in the search for novel lipases. The search for thermophilic microorganisms for synthesising functional enzyme biocatalysts with the ability to withstand high temperature, and capacity to maintain their native state in extreme conditions opens up new opportunities for their biotechnological applications. Thermophilic organisms are one of the most favoured organisms, whose distinctive characteristics are extremely related to their cellular constituent particularly biologically active proteins. Modifications on the enzyme structure are critical in optimizing the stability of enzyme to thermophilic conditions. Thermostable lipases are one of the most favourable enzymes used in food industries, pharmaceutical field, and actively been studied as potential biocatalyst in biodiesel production and other biotechnology application. Particularly, there is a trade-off between the use of enzymes in high concentration of organic solvents and product generation. Enhancement of the enzyme stability needs to be achieved for them to maintain their enzymatic activity regardless the environment. Various approaches on protein modification applied since decades ago conveyed a better understanding on how to improve the enzymatic properties in thermophilic bacteria. In fact, preliminary approach using advanced computational analysis is practically conducted before any modification is being performed experimentally. Apart from that, isolation of novel extremozymes from various microorganisms are offering great frontier in explaining the crucial native interaction within the molecules which could help in protein engineering. In this review, the thermostability prospect of lipases and the utility of protein engineering insights into achieving functional industrial usefulness at their high temperature habitat are highlighted. Similarly, the underlying thermodynamic and structural basis that defines the forces that stabilize these thermostable lipase is discussed. KEY POINTS: • The dynamics of lipases contributes to their non-covalent interactions and structural stability. • Thermostability can be enhanced by well-established genetic tools for improved kinetic efficiency. • Molecular dynamics greatly provides structure-function insights on thermodynamics of lipase.

Enzymes of Polyphosphate Metabolism in Yeast: Properties, Functions, Practical Significance.

Inorganic polyphosphates (polyP) are the linear polymers of orthophosphoric acid varying in the number of phosphate residues linked by the energy-rich phosphoanhydride bonds. PolyP is an essential component in living cells. Knowledge of polyP metabolizing enzymes in eukaryotes is necessary for understanding molecular mechanisms of polyP metabolism in humans and development of new approaches for treating bone and cardiovascular diseases associated with impaired mineral phosphorus metabolism. Yeast cells represent a rational experimental model for this research due to availability of the methods for studying phosphorus metabolism and construction of knockout mutants and strains overexpressing target proteins. Multicomponent system of polyP metabolism in Saccharomyces cerevisiae cells is presented in this review discussing properties, functioning, and practical significance of the enzymes involved in the synthesis and degradation of this important metabolite.

Identification, expression and enzyme activity of the group III sPLA2 s in Cyprinus carpio L.

Five group III secreted phospholipase (pla2g3s) homologous genes located on different linkage groups were identified from common carp (Cyprinus carpio), which we named Ccpla2g3a1, Ccpla2g3a2, Ccpla2g3b, Ccpla2g3c1 and Ccpla2g3c2. The five genes encode 530, 525, 461, 752 and 753 amino acids, respectively. Sequence analysis showed that the Ccpla2g3as contain seven exons and the others contain four exons. Synteny analysis of fish pla2g3s indicated that pla2g3a and pla2g3b were from the same ancestor gene, and Ccpla2g3a1, Ccpla2g3a2, Ccpla2g3c1 and Ccpla2g3c2 were from the specific genome duplication of common carp. Due to the significant variation of the pla2g3bs from common carp and zebrafish (Danio rerio), they formed a separate group in the phylogenetic tree. The tissue distributions of Ccpla2g3s coincided with their expression profiles during the embryo stages. The expression levels of Ccpla2g3as and Ccpla2g3cs were low at the embryo stages, and they were abundant in the liver and brain, respectively, whereas the expression of Ccpla2g3b was high at 0.5 h after fertilization and in the ovary. We obtained three soluble recombinant proteins of the bee venom-like PLA2 (BVLP) from Ccpla2g3 and evaluated their PLA2 enzyme properties. The optimum pHs of MBP-a1-BVLP, MBP-b-BVLP and MBP-c1-BVLP were 7.5, 7.0 and 8.0, respectively, and specific activities were 7.68 ± 0.66, 4.155 ± 0.158 and 1.93 ± 0.05 U µmol-1 , respectively. The Kd for Ca2+ of MBP-b-BVLP was the lowest (2.6 µM), whereas the values for both MBP-a1-BVLP and MBP-c1-BVLP were about 15 µM. The Km values of three proteins ranged from 31.9 to 41.91 µM.

Production of ß-glucanase and protease from Bacillus velezensis strain isolated from the manure of piglets.

In this study, a strain producing ß-glucanase and protease, identified as Bacillus velezensis Y1, was isolated from the manure of piglet. We attempted to produce ß-glucanase and protease after optimization of various process parameters with the submerged fermentation. The effects of each factor on producing ß-glucanase and protease were as follows: temperature > time > pH > loaded liquid volume. The properties of the ß-glucanase showed that the most suitable reaction temperature was 65 °C and pH was 6.0. However for protease optimum reaction temperature was 50 °C, and pH was 6.0. The amplified PCR fragments of ß-glucanase and protease were 1434 bp containing an open reading frame of 1413 bp encoding a protein with 444 amino acids and 1752 bp containing an open reading frame of 1521 bp encoding a protein with 506 amino acids, respectively. So, the study demonstrated a viable approach of using newly identified B. velezensis Y1 strain for the maximum yield of two industrially important enzymes.

Effect of Concentrated Salts Solutions on the Stability of Immobilized Enzymes: Influence of Inactivation Conditions and Immobilization Protocol.

This paper aims to investigate the effects of some salts (NaCl, (NH4)2SO4 and Na2SO4) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH4)2SO4 generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.

Archives of microbiology: screening of pectinase-producing bacteria from citrus peel and characterization of a recombinant pectate lyase with applied potential.

Pectinase is widely used in numerous industrial fields, including the food, wine, and paper industries. In this work, seven bacteria were isolated from orange peel and their pectinase production activity was assayed. One bacterium (OR-B2) identified as a Bacillus sp. showed the highest enzyme activity towards others. A gene encoding a pectate lyase designed as PelB-B2 in this work was amplified and heterogeneous expressed in E.coli. PelB-B2 was defined as a member of the PelB pectate lyase family after phylogenic tree analysis. 3D model of PelB-B2 was constructed by SWISS-MODEL and PelB-B2 showed conserved para-ß structure. After inducing culture and purified by Ni-affinity chromatography, the properties of the purified PelB-B2 were assayed. Optimal pH and temperature for PelB-B2 was pH 8.0 and 50 °C, respectively. PelB-B2 showed excellent pH stability and thermostability. It was stable within pH range 3.0-11.0 and retained more than 51% activity after incubation at 40 °C, 50 °C, or 60 °C for 1 h. Furthermore, we determined that PelB-B2 was a Ca2+-dependent pectinase and the pectin extracted from citrus was the benefit substrate for PelB-B2. The Km and Vmax of PelB-B2 were 1.64 g/L and 232.56 mol/(L min), respectively. The OR-B2 can be a new resource for pectinase production and the PelB-B2 has potential for industrial application. 7 bacteria were isolated from orange peel, namely OR-B1 to OR-B7 and their pectinase activities were assayed. One pectate lyase belongs to PelB family was cloned from OR-B2 and heterogeneous expressed in E. coli. Purified PelB-B2 was further studied with its properties. Effects of pH, temperature, chemicals, substrate on the enzyme activity were assayed and the enzyme kinetic was also measured.

Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense.

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus Stemphylium lucomagnoense. The laccase-encoding gene SlLac2 from S. lucomagnoense was cloned for heterologous expression in Aspergillus niger D15#26 for protein production in the extracellular medium of around 30 mg L-1. The extracellular recombinant enzyme SlLac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%. SlLac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of SlLac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate, SlLac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min. SlLac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt.

Preparation of high-quality sunflower seed protein with a new chlorogenic acid hydrolase from Aspergillus niger.

OBJECTIVE: To investigate the biochemical and enzymatic properties of chlorogenic acid hydrolase (ChlH) from Aspergillus niger SD14.721 and its applicability in sunflower seed protein processing. RESULTS: The ChlH with two identical subunits (97 kDa) was highly stable. Its optimal temperature and pH were determined as 60 °C and pH 7.0. The Km towards chlorogenic acid (CGA) was 1.85 µM. Based on its N-terminal sequence (AVDSVDAIFA), the purified ChlH appeared to be a new chlorogenic acid hydrolase. When applied in sunflower seed protein extraction, ChlH removed 99.13% of CGA in sunflower seed pastes, thus the colour of sunflower seed protein (SSP) changed from green to grey and its visual acceptance improved. Meanwhile, the solubility, water absorption capacity, and emulsification stability of SSP were increased 48.39%, 59.32% and 22.92%, respectively. CONCLUSIONS: A new ChlH was obtained and its feasibility as a CGA-removal tool to obtain high quality SSP was demonstrated.

Gene Cloning and Expression of Cellulase of Bacillus amyloliquefaciens Isolated from the Cecum of Goose.

A kind of bacteria secreting cellulase and showing probiotic attributes was isolated from the cecum of goose and identified as Bacillus amyloliquefaciens by analysis of 16S rRNA gene sequence and named as B. amyloliquefaciens S1. In vitro assays, the enzymatic activity of the strain was determined by the reducing-sugar method, and the proper culture conditions of producing cellulase and some properties of the cellulase were investigated. The cultural mixture of the bacteria had a high cellulase activity of 1.25 U/mL. In order to improve the utilization rate of the cellulase, some properties of the cellulase were studied. The best reaction pH of the enzymes was 7.0 and the optimum reaction temperature was 60°C. The enzyme was a kind of neutral cellulase that possessing strong resistance against heat and acidity. It showed high activity to absorbent cotton, soybean meal, and filter paper. Meanwhile, a gene encoding a kind of cellulase was cloned and prokaryotic expressed in Escherichia coli. The gene had 1500 bp in length, encoding a protein of 55 kDa, which was confirmed by SDS-PAGE and Western blotting. This study explored the possibility of degrading ability of bacteria with its probiotic attributes to enhance digestibility of the feed and gut health of animal. It also provided some basis for its further functional analysis and practical application as a microbial preparation for the breeding.

The optimization of fermentation conditions and enzyme properties of Stenotrophomonas maltophilia for protease production.

Intestinal bacteria play a significant physiological role in silkworms. Proteases secreted by intestinal microbes can promote the digestion of the nutrient by Bombyx mori and the absorption of mulberry leaves. Intestinal bacteria from Jingsong × Haoyue in the fourth larvae were isolated and purified to obtain high activity protease-producing bacteria. The morphology of the identified bacterial colony was examined by microscopy combined with the 16S rDNA method. The results showed that this bacterium was Gram negative and that it belonged to Stenotrophomonas maltophilia, which produces the proteases. To improve the utilization rate of these proteases, we studied the proper culture conditions for producing proteases, and we further studied the properties of the proteases that were produced. The results showed that the optimal enzyme-producing conditions were as follows: pH of 7.0, culture temperature of 35 °C, incubation time of 36 H, and outfit fluid amount of 60 mL per 100 mL. Meanwhile, the properties of the preliminary enzyme purification indicated that the best pH of the enzymes was 9.0 and the optimal reaction temperature was 50 °C. The enzymes are alkaline proteases that show satisfactory stability at 30 °C and pH 9.0. Consequently, it is suitable for the proteases secreted by S. maltophilia to play a bioactive role in the silkworm gut.

Purification and characterization of alginate lyase from locally isolated marine Pseudomonas stutzeri MSEA04.

An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).

Modulation of a Loop Region in the Substrate Binding Pocket Affects the Degree of Polymerization of Bacillus subtilis Chitosanase Products.

The hydrolytic products of chitosanase from Streptomyces avermitilis (SaCsn46A) were found to be aminoglucose and chitobiose, whereas those of chitosanase from Bacillus subtilis (BsCsn46A) were chitobiose and chitotriose. Therefore, the sequence alignment between SaCsn46A and BsCsn46A was conducted, revealing that the structure of BsCsn46A possesses an extra loop region (194N-200T) at the substrate binding pocket. To clarify the impact of this loop on hydrolytic properties, three mutants, SC, TJN, and TJA, were constructed. Eventually, the experimental results indicated that SC changed the ratio of chitobiose to chitotriose hydrolyzed by chitosanase from 1:1 into 2:3, while TJA resulted in a ratio of 15:7. This experiment combined molecular research to unveil a crucial loop within the substrate binding pocket of chitosanase. It also provides an effective strategy for mutagenesis and a foundation for altering hydrolysate composition and further applications in engineering chitosanase.