Crude Enzyme Solution Research Articles

Expression and degradation function analysis of the key genes from Arthrobacter nitroquajacolicus HW08 in Lactococcus lactis for the degradation of swainsonine

In order to mitigate the adverse effects of madrassa poisoning disease on our livestock industry and to fully utilize the potential pasture resources, biodegradation of locoweed can remove swainsonine, the major toxic component of locoweed, so that the locoweed can be used as high-quality forage. Arthrobacter nitroquajacolicus HW08 can stably and efficiently degrade swainsonine. In this study, Lactococcus lactis, as a food-grade microorganism, was used as a vector to express four key degradation genes from A. nitroquajacolicus HW08. Subsequently, liquid chromatography was employed to evaluate the swainsonine-degrading performance. The crude enzyme solution extracted from the L. lactis strain transformed with the ethanol dehydrogenase gene A1R6C3 degraded 323.4 μg of swainsonine in 24 h at 30 ℃. The crude enzyme solutions from the L. lactis strains transformed with the genes encoding glutathione synthase, esterase/acyl hydrolase, and glycosyltransferase did not show any degradation ability for swainsonine when being used alone but degraded about 140.5 μg of swainsonine when being used in mixture. The findings will help the clinical promotion of swainsonine-degrading engineering strains and provide new research ideas for the prevention and treatment of swainsonine poisoning in animals and the detoxification and utilization of locoweed.

Enhanced extracellular production of maltotetraose amylase from Pseudomonas saccharophila in Bacillus subtilis through regulatory element optimization

Maltotetraose amylase (Mta) catalyzes the hydrolysis of amylaceous polysaccharides into maltotetraose, which is an important functional sugar used in the food industry. However, the lack of efficient expression systems for recombinant Mta has hindered its scale-up production and application. In this study, a codon-optimized mta gene from Pseudomonas saccharophila was efficiently produced in Bacillus subtilis by optimizing the regulatory elements. First, a plasmid library containing 173 different signal peptide sequences placed upstream of mta gene was constructed, and transformed into B. subtilis strain WB800N(amyEΔ1) for high-throughput screening. The signal peptide yhcR was found to significantly enhance the secretion of Mta, reaching an activity of 75.4 U/mL in the culture medium. After optimization of the promoters, the Mta activity was further increased to 100.3 U/mL using a dual-promoter PHpaIIPamyE. Finally, the carbon sources and nitrogen sources for recombinant Mta production were optimized, yielding a highest Mta activity of 288.9 U/mL under the optimal culture conditions. The crude enzyme solution containing recombinant Mta produced a highest maltotetraose yield of 70.3% with 200 g/L of maltodextrin as the substrate. Therefore, the present study have demonstrated a high yield of Mta produced in B. subtilis, laying the foundation for large-scale Mta production and application.

Effect of ultrasound on the activity and structure of polyphenol oxidase purified from mango (Mangifera indica cv. "Xiaotainong")

This study separated, purified, and identified mango polyphenol oxidase (PPO) in Xiaotainong mango, and the inactivation effect and possible inactivation mechanism of mango PPO by ultrasound (US) was investigated. The purified mango PPO, identified as Alfonso mango chloroplast PPO (NCBI number: XP:044462061.1), exhibited specific enzyme activity of 178,192 U/mg, with a yield of 1.2%, which was 9.85-fold higher than crude enzyme solution. Bioinformatics analysis of XP_044462061.1 indicated that the PPO consisted of a stable hydrophilic protein with a molecular weight of 66.77 kDa, an amino acid number of 593, and a structural domain similar to the TYROSINASE_1 (PS00497, tyrosinase) CuA and TYROSINASE_2 (PS00498; tyrosinase) CuB binding regions. The result showed that increased US treatment intensity and time better facilitated PPO inactivation, with a higher US intensity performing better than extended treatment time. US changed and disrupted the secondary and endogenous hydrophobic structures of the mango PPO, altered the exogenous hydrophobicity, and caused PPO molecule aggregation, ultimately leading to PPO inactivation. This article provided a theoretical basis for the application of US technology for processing mango products.

Low frequency magnetic field assisted production of acidic protease by Aspergillus niger.

Acid protease is widely used in industries such as food processing and feed additives. In the study, low frequency magnetic field (LF-MF) as an aid enhances acid protease production by Aspergillus niger (A. niger). The study assessed mycelial biomass, the enzymic activity of the acidic protease and underlying mechanism. At low intensities, alternating magnetic field (AMF) is more effective than static magnetic fields (SMF). Under optimal magnetic field conditions, acid protease activity and biomass increased by 91.44% and 16.31%, as compared with the control, respectively. Maximum 19.87% increase in enzyme activity after magnetic field treatment of crude enzyme solution in control group. Transcriptomics analyses showed that low frequency alternating magnetic field (LF-AMF) treatment significantly upregulated genes related to hydrolases and cell growth. Our results showed that low-frequency magnetic fields can enhance the acid protease production ability of A. niger, and the effect of AMF is better at low intensities. The results revealed that the effect of magnetic field on the metabolic mechanism of A. niger and provided a reference for magnetic field-assisted fermentation of A. niger.

Molecular characterization revealed the role of thaumatin-like proteins of Rhizoctonia solani AG4-JY in inducing maize disease resistance.

The gene family of thaumatin-like proteins (TLPs) plays a crucial role in the adaptation of organisms to environmental stresses. In recent years, fungal secreted proteins (SP) with inducing disease resistance activity in plants have emerged as important elicitors in the control of fungal diseases. Identifying SPs with inducing disease resistance activity and studying their mechanisms are crucial for controlling sheath blight. In the present study, 10 proteins containing the thaumatin-like domain were identified in strain AG4-JY of Rhizoctonia solani and eight of the 10 proteins had signal peptides. Analysis of the TLP genes of the 10 different anastomosis groups (AGs) showed that the evolutionary relationship of the TLP gene was consistent with that between different AGs of R. solani. Furthermore, it was found that RsTLP3, RsTLP9 and RsTLP10 were regarded as secreted proteins for their signaling peptides exhibited secretory activity. Prokaryotic expression and enzyme activity analysis revealed that the three secreted proteins possess glycoside hydrolase activity, suggesting they belong to the TLP family. Additionally, spraying the crude enzyme solution of the three TLP proteins could enhance maize resistance to sheath blight. Further analysis showed that genes associated with the salicylic acid and ethylene pathways were up-regulated following RsTLP3 application. The results indicated that RsTLP3 had a good application prospect in biological control.

Construction of a Synthetic Microbial Community for Enzymatic Pretreatment of Wheat Straw for Biogas Production via Anaerobic Digestion.

Biological pretreatment is a viable method for enhancing biogas production from straw crops, with the improvement in lignocellulose degradation efficiency being a crucial factor in this process. Herein, a metagenomic approach was used to screen core microorganisms (Bacillus subtilis, Acinetobacter johnsonii, Trichoderma viride, and Aspergillus niger) possessing lignocellulose-degrading abilities among samples from three environments: pile retting wheat straw (WS), WS returned to soil, and forest soil. Subsequently, synthetic microbial communities were constructed for fermentation-enzyme production. The crude enzyme solution obtained was used to pretreat WS and was compared with two commercial enzymes. The synthetic microbial community enzyme-producing pretreatment (SMCEP) yielded the highest enzymatic digestion efficacy for WS, yielding cellulose, hemicellulose, and lignin degradation rates of 39.85, 36.99, and 19.21%, respectively. Furthermore, pretreatment of WS with an enzyme solution, followed by anaerobic digestion achieved satisfactory results. SMCEP displayed the highest cumulative biogas production at 801.16 mL/g TS, which was 38.79% higher than that observed for WS, 22.15% higher than that of solid-state commercial enzyme pretreatment and 25.41% higher than that of liquid commercial enzyme pretreatment. These results indicate that enzyme-pretreated WS can significantly enhance biogas production. This study represents a solution to the environmental burden and energy use of crop residues.

Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 UmL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 UmL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.

One-Step Purification and Immobilization of Glycosyltransferase with Zn-Ni MOF for the Synthesis of Rare Ginsenoside Rh2.

Uridine diphosphate (UDP)-glucosyltransferases (UGTs) have received increasing attention in the field of ginsenoside Rh2 conversion. By harnessing the metal chelation between transition metal ions and imidazole groups present on His-tagged enzymes, a specific immobilization of the enzyme within metal-organic frameworks (MOFs) is achieved. This innovative approach not only enhances the stability and reusability of the enzyme but also enables one-step purification and immobilization. Consequently, the need for purifying crude enzyme solutions is effectively circumvented, resulting in significant cost savings during experimentation. The use of immobilized enzymes in catalytic reactions has shown great potential for achieving higher conversion rates of ginsenoside Rh2. In this study, highly stable mesoporous Zn-Ni MOF materials were synthesized at 150 °C by a solvothermal method. The UGT immobilized on the Zn-Ni MOF (referred to as UGT@Zn-Ni MOF) exhibited superior pH adaptability and thermal stability, retaining approximately 76% of its initial activity even after undergoing 7 cycles. Furthermore, the relative activity of the immobilized enzyme remained at an impressive 80.22% even after 45 days of storage. The strong specific adsorption property of Zn-Ni MOF on His-tagged UGT was confirmed through analysis using polyacrylamide gel electrophoresis. UGT@Zn-Ni MOF was used to catalyze the conversion reaction, and the concentration of rare ginsenoside Rh2 was generated at 3.15 μg/mL. The results showed that Zn-Ni MOF is a material that can efficiently purify and immobilize His-tagged enzyme in one step and has great potential for industrial applications in enzyme purification and ginsenoside synthesis.

Cross-linked enzyme aggregates of xylanase, XynR8(N58D), for effective degradation of untreated lignocellulosic biomass

Enzymatic degradation of biomass is preferred over chemical methods for environmental protection. However, in most cases, a chemical pretreatment is still required to help improve enzyme accessibility of the biomass. Therefore, it is necessary to develop highly efficient enzymes for those reasons and to save costs accordingly. Costs can be further reduced if the enzymes are recycled. XynR8(N58D) is an engineered and highly active xylanase originating from the rumen fungus Neocallimastix patriciarum. In this study, XynR8(N58D) was extracellularly over-expressed by Pichia pastoris. The resulting crude enzyme solution, with a purity of XynR8(N58D) of over 80%, was used to prepare the cross-linked enzyme aggregates (CLEA) of XynR8(N58D). Specific activity of the crude enzyme solution was 11,599.39 IU/mg, and that of CLEA was 6129.42 IU/mg. The immobilization yield and the immobilization efficiency were 62.68 ± 16.20% and 84.31 ± 3.11%, respectively. The thermostability, stress tolerance, and shelf life of CLEA were significantly better than those of the free enzyme. The free enzyme and CLEA both effectively decomposed insoluble xylan and autoclaved corncob powders. Therefore, XynR8(N58D) made into CLEA not only maintained high activity, but also improved its stability and permitted its reuse or recyclability. Both forms of the enzyme can effectively degrade lignocellulosic biomass without relying on chemical pretreatment to reduce recalcitrance of the biomass structure. The application of XynR8(N58D) and its CLEA to biomass degradation can help establish an eco-friendly and more cost-effective process.

Expression and application of aflatoxin degrading enzyme gene in Pichia pastoris.

In this study, three aflatoxin degrading enzyme genes, tv-adtz, arm-adtz and cu-adtz, were heterologously expressed in Pichia pastoris. The protein expression of the enzyme solution was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the results showed that specific protein bands were detected and the target genes were successfully integrated into Pichia pastoris. The enzyme activities and detoxification efficiency of TV-ADTZ, Arm-ADTZ and Cu-ADTZ crude enzyme solutions were detected, and the highest enzyme activities were up to 3.57, 4.30, and 2.41 UmL-1 , and the highest degradation rates were up to 45.58%, 60.0% and 34.21%, respectively. Arm-ADTZ with the best degradation effect was selected and designed for detoxification application experiments to test its detoxification efficiency of AFB1 in aqueous phase and in the process of moldy ground corn and preparation of DDGS, respectively, and the degradation rates reached 78.94%, 56.48%, and 24.31% after 24h of reaction, respectively. Thus, it can be seen that the aflatoxin-degrading enzyme gene was successfully integrated into Pichia pastoris and secreted for expression, and the expressed product could effectively degrade AFB1 .

Study on the protein expression of CYP3A4 and CYP2C18

Background As a drug enzyme, CYP450 has important significance in clinical treatment and the combined use of drugs, but the current research has not yet achieved high efficiency and large amount of expression in vitro. Therefore, the expression of CYP450 in vitro has become a hot spot.Objective Study the in vitro high-efficiency expression and time optimization of CYP3A4 and CYP2C18. Methods (1) CYP3A4 and CYP2C18 yeast recombinants were induced, and 24h, 48h and 72h bacterial liquid were collected respectively, to obtain the crude enzyme solution at different time periods, and then the enzyme solution at different time periods was subjected to SDS-PAGE protein electrophoresis to obtain its target bands. The results of comparative analysis between the protein bands and the Marker were observed by observing the gel running results. Finally, the enzyme activity was determined by liquid chromatography. Results The expressed proteins of CYP3A4 and CYP2C18 yeast recombinants were successfully induced. The target bands were obtained by SDS-PAGE protein electrophoresis and the optimal expression time was analyzed. Finally, the activity of the products was verified by liquid chromatography. Conclusion CYP3A4 and CYP2C18 yeast recombinants were successfully expressed in this experiment, and the most abundant expression of CYP3A4 and CYP2C18 was found at 72h by comparing the position of electrophoresis bands. At the same time, it was also verified that the expressed products were active, and CYP3A4 had the highest activity at 48h, which could be used for the study of CYP450 metabolism in vitr.

Bioconversion of chitin into chitin oligosaccharides using a novel chitinase with high chitin-binding capacity

Chitin is the second largest renewable biomass resource in nature, it can be enzymatically degraded into high-value chitin oligosaccharides (CHOSs) by chitinases. In this study, a chitinase (ChiC8–1) was purified and biochemically characterized, its structure was analyzed by molecular modeling. ChiC8–1 had a molecular mass of approximately 96 kDa, exhibited its optimal activity at pH 6.0 and 50 °C. The Km and Vmax values of ChiC8–1 towards colloidal chitin were 10.17 mgmL−1 and 13.32 U/mg, respectively. Notably, ChiC8–1 showed high chitin-binding capacity, which may be related to the two chitin binding domains in the N-terminal. Based on the unique properties of ChiC8–1, a modified affinity chromatography method, which combines protein purification with chitin hydrolysis process, was developed to purify ChiC8–1 while hydrolyzing chitin. In this way, 9.36 ± 0.18 g CHOSs powder was directly obtained by hydrolyzing 10 g colloidal chitin with crude enzyme solution. The CHOSs were composed of 14.77–2.83 % GlcNAc and 85.23–97.17 % (GlcNAc)2 at different enzyme-substrate ratio. This process simplifies the tedious purification and separation steps, and may enable its potential application in the field of green production of chitin oligosaccharides.

Safety evaluation and whole genome sequencing for revealing the ability of Penicillium oxalicum WX-209 to safely and effectively degrade citrus segments

The microbial potential of Penicillium has received critical attention. The present research aimed to elucidate the efficacy of crude enzyme secreted from Penicillium oxalicum WX-209 in degrading citrus segments and evaluate the safety of the process. Results showed that citrus segment membranes gradually dissolved after treatment with the crude enzyme solution, indicating good degradation capability. No significant differences in body weight, food ingestion rate, hematology, blood biochemistry, and weight changes of different organs were found between the enzyme intake and control groups. Serial experiments showed that the crude enzyme had high biological safety. Moreover, the whole genome of P. oxalicum WX-209 was sequenced by PacBio and Illumina platforms. Twenty-five scaffolds were assembled to generate 36 Mbp size of genome sequence comprising 11 369 predicted genes modeled with a GC content of 48.33 %. A total of 592 genes were annotated to encode enzymes related to carbohydrates, and some degradation enzyme genes were identified in strain P. oxalicum WX-209. Data availabilityThe whole genome sequencing data in this study can be found in GenBank database of NCBI website with the accession number PRJNA 715321. The link: http://www.ncbi.nlm.nih.gov/bioproject/715321.

Whole-Cell Biocatalytic Production of Acetoin with an aldC-Overexpressing Lactococcus lactis Using Soybean as Substrate

Douchi is a traditional Chinese fermented soybean product, in which acetoin is a key flavor substance. Here, the α-acetolactate decarboxylase gene aldC was cloned from Lactiplantibacillus (L.) plantarum and overexpressed in Lactococcus (L.) lactis NZ9000 by nisin induction. The ALDC crude enzyme solution produced an enzyme activity of 35.16 mU. Next, whole cells of the recombinant strain NZ9000/pNZ8048-aldC were employed as the catalyst to produce acetoin in GM17 medium. An optimization experiment showed that an initial OD600 of 0.6, initial pH of 7.5, nisin concentration of 20 ng/mL, induction temperature of 37 °C and static induction for 8 h were the optimal induction conditions, generating the maximum acetoin production (106.93 mg/L). Finally, after incubation under the optimal induction conditions, NZ9000/pNZ8048-aldC was used for whole-cell biocatalytic acetoin production, using soybean as the substrate. The maximum acetoin yield was 79.43 mg/L. To our knowledge, this is the first study in which the aldC gene is overexpressed in L. lactis and whole cells of the recombinant L. lactis are used as a biocatalyst to produce acetoin in soybean. Thus, our study provides a theoretical basis for the preparation of fermented foods containing high levels of acetoin and the biosynthesis of acetoin in food materials.

Selective Immobilization of His-Tagged Enzyme on Ni-Chelated Ion Exchange Resin and Its Application in Protein Purification.

Ion exchange resins are suitable as carriers for immobilized enzymes because of their stable physicochemical properties, appropriate particle size and pore structure, and lower loss in continuous operation. In this paper, we report the application of the Ni-chelated ion exchange resin in the immobilization of His-tagged enzyme and protein purification. Acrylic weak acid cation exchange resin (D113H) was selected from four cationic macroporous resins that could chelate the transition metal ion Ni. The maximum adsorption capacity of Ni was ~198 mg/g. Phosphomannose isomerase (PMI) can be successfully immobilized on Ni-chelated D113H from crude enzyme solution through chelation of transition metal ions with the His-tag on the enzyme. The maximum amount of immobilized PMI on the resin was ~143 mg/g. Notably, the immobilized enzyme showed excellent reusability and maintained 92% of its initial activity with 10 cycles of catalytic reaction. In addition, PMI was successfully purified using an affinity chromatography column prepared by Ni-chelated D113H, which showed the potential for the immobilization and purification process to be realized in one step.

Function and safety evaluation of Staphylococcus epidermidis with high esterase activity isolated from strong flavor Daqu

This study was aimed at evaluating the safety of Staphylococcus epidermidis YTPW-4 isolated from strong flavor Daqu and its beneficial functional characteristics in Baijiu-making. The content of ethyl caproate catalyzed by crude enzyme solution of Staphylococcus epidermidis YTPW-140 was 583.09 mg/100 mL, and the activity of esterase was 2.13 U/mL. Vancomycin, erythromycin, and gentam were sensitive to the strain, which was negative for the causative gene, hemolytic activity, and biofilm formation capacity. In addition, the strain was selected for laboratory-scale fermentation using HS-SPME-GCMS and statistical analysis to confirm its role in the production of flavor compounds. Propyl-3-methylbutyrate, hexanoic acid, and ethyl acetate had higher contents after fermentation than other flavor compounds, at 530.40 μg/L, 471.07 μg/L, and 297.33 μg/L, respectively. There was also a small amount of ethyl hexanoate in these products. Esterase could increase the levels of short-chain free fatty acids, which were precursors of flavor compounds. Most importantly, the isolate maintained viability under simulated pit conditions, and was resistant to pH 3.5 and 8% ethanol. These results indicated that the strain had the potential to be used as a starter to improve the quality of Nongxiangxing baijiu.

Optimized production of keratinolytic proteases from Bacillus tropicus LS27 and its application as a sustainable alternative for dehairing, destaining and metal recovery

The present study describes the isolation and characterization of Bacillus tropicus LS27 capable of keratinolytic protease production from Russell Market, Shivajinagar, Bangalore, Karnataka, with its diverse application. The ability of this strain to hydrolyze chicken feathers and skim milk was used to assess its keratinolytic and proteolytic properties. The strain identification was done using biochemical and molecular characterization using the 16S rRNA sequencing method. Further a sequential and systematic optimization of the factors affecting the keratinase production was done by initially sorting out the most influential factors (NaCl concentration, pH, inoculum level and incubation period in this study) through one factor at a time approach followed by central composite design based response surface methodology to enhance the keratinase production. Under optimized levels of NaCl (0.55 g/L), pH (7.35), inoculum level (5%) and incubation period (84 h), the keratinase production was enhanced from 41.62 U/mL to 401.67 ± 9.23 U/mL (9.65 fold increase) that corresponds to a feather degradation of 32.67 ± 1.36% was achieved. With regard to the cost effectiveness of application studies, the crude enzyme extracted from the optimized medium was tested for its potential dehairing, destaining and metal recovery properties. Complete dehairing was achieved within 48 h of treatment with crude enzyme without any visible damage to the collagen layer of goat skin. In destaining studies, combination of crude enzyme and detergent solution [1 mL detergent solution (5 mg/mL) and 1 mL crude enzyme] was found to be most effective in removing blood stains from cotton cloth. Silver recovery from used X-ray films was achieved within 6 min of treatment with crude enzyme maintained at 40 °C.

Study on Arsenic Methyltransferase of <i>Cellulomonas</i> sp. K31 Expressed in Recombinant <i>E. coli</i>

This study aimed to facilitate efficient arsenic detoxification by inducing the microbial methylation of inorganic arsenic. Recombinant Escherichia coli strains K31 was transformed to overexpress arsenic methyltransferase, and the methylation of inorganic arsenic was evaluated using the enzyme extracted from the strain. To ensure continuous reactions by maintaining enzymatic activity, the extracted enzyme was immobilized in microcapsules (MC) to catalyze the methylation of inorganic arsenic. The total yield of methylated organic arsenic compounds in strain K31 was 57.0% (after 2 h of incubation at pH 6.5 and 40°C), of which trimethyl arsenic compounds (TMAC) accounted for 17.9%. When arsenic was methylated using MC prepared with the crude enzyme solution, the total yield of methylated organic arsenic compounds was 15.6%. The residual enzymatic activity was calculated to be 55.8%. Future studies should aim to increase the efficiency of conversion to TMAC, thus elevating the yield of organic arsenic compounds, by optimizing the conditions for enzyme immobilization in MC.

Efficient Adsorption and Extraction of Glutathione S-Transferases with Glutathione-Functionalized Graphene Oxide–Polyhedral Oligomeric Silsesquioxane Composite

Glutathione S-transferases (GSTs) are important type-II detoxification enzymes that protect DNA and proteins from damage and are often used as protein tags for the expression of fusion proteins. In the present work, octa-aminopropyl caged polyhedral oligomeric silsesquioxane (OA-POSS) was prepared via acid-catalyzed hydrolysis of 3-aminopropyltriethoxysilane and polymerized on the surface of graphene oxide (GO) through an amidation reaction. Glutathione (GSH) was then modified to GO-POSS through a Michael addition reaction to obtain a GSH-functionalized GO-POSS composite (GPG). The structure and characteristics of the as-prepared GPG composite were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), thermogravity analysis, and surface charge analysis. The specific binding interactions between glutathione and GST gave GPG favorable adsorption selectivity towards GST, and other proteins did not affect GST adsorption. The adsorption behavior of GST on the GPG composite conformed to the Langmuir isotherm model, and the adsorption capacity of GST was high up to 364.94 mg g-1 under optimal conditions. The GPG-based solid-phase adsorption process was applied to the extraction of GST from a crude enzyme solution of pig liver, and high-purity GST was obtained via SDS-PAGE identification.

Heterologous expression and characterization of an M4 family extracellular metalloprotease for detergent application.

Proteolytic enzymes stand out as the most widely employed category utilized in manufacturing industry. A new protease was separated from Planococcus sp.11815 strain and named as nprS-15615 in this research. The gene of this protease has not been reported, and its enzymatic properties have been studied for the first time. To enhance enzyme production, the Planococcus sp. protease gene was expressed in Bacillus licheniformis 2709. The expression level of nprS-15615 was observed under the control of regulatory elements PaprE. nprS-15615 protease activity reached 1186.24±32.87 U/mL after 48 hours of cultivation in shake flasks which was nearly four times the output of the original bacteria (291.38±25.73U/mL). The optimum temperature and pH of the recombinant protease were 30 ℃ and 8.0, respectively.The enzyme exhibited the highest capacity for hydrolyzing casein and demonstrated resilience towards a NaCl concentration of 10.0% (wt/v). Furthermore, in the presence of 0.5% surfactants, the recombinant protease activity can maintain above 75%, and with the existence of 0.5% liquid detergents, there was basically no loss of enzyme activity which indicated that nprS-15615 had good compatibility with surfactants and liquid detergents. In addition, npS-15615 performed well in the washing experiment, and the washing effect at 20 ℃ can be significantly improved by adding crude enzyme solution in the washing process.