Purification and characterization of an alkaline protease from <Emphasis Type="Italic">Micrococcus</Emphasis> sp. isolated from the South China Sea

Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0–11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn²⁺. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.     

Study on application of polyenzyme method to offal of Harengula zunasi

The new polyenzyme method for making gravy fromHarengula zunasi offal involves protein enzymolysis with flavorase after proper alkaline and neutral protease levels were established by orthogonal trials to select the best hydrolytic conditions for processing offal with alkaline and neutral protease. The conditions for the polyenzyme method were pH of 7.0, temperature of 50°C, alkaline and neutral protease concentration of 1.5% respectively, hydrolysis time of 120 min, and flavorase concentration of 2.0%, for 60 min. The new gravy-making technology yields a nutritious and delicious gravy containing 40.3% of total essential amino acids, with delicious amino acids Glu, Asp, Gly, Ala, Pro and Ser comprising 49.5%, total and amino nitrogen being respectively 1.9 and 1.1 g/100 g (amino acid nitrogen being 61.0% of total nitrogen), The polyenzyme method was used to make 14.8% protein gravy fromHarengula zunasi offal. In addition, inorganic elements, the phosphorus content is the highest. This project was aided financially by the Guangdong Science and Technology Office (No. 2KM06002S).     

罗非鱼鱼鳞胶制备降血压胶原肽的工艺及酶解物活性

以罗非鱼鱼鳞胶为原料,制备对血管紧张素转换酶(ACE)有抑制活性的降血压肽。以水解度(DH)及ACE抑制率为指标,筛选水解鱼鳞胶的最适酶种,并对其水解工艺条件进行优化;采用超滤技术对所制备的降血压肽进行分离纯化,比较不同组分的ACE抑制率,测定高ACE抑制率组分的氨基酸组成,并运用MALDI-TOF-MS法分析其分子质量分布。结果表明,复合蛋白酶是水解鱼鳞胶最适酶种,在50℃、pH7.5、料液质量体积比1∶5、酶与底物质量比(mE/mS)1.5%条件下,酶解6 h,酶解液的ACE抑制作用最强,其水解度DH 73%,ACE的抑制率76.15%。酶解液经超滤分离所获得的分子质量小于2 000 u的组分对ACE抑制率最高,达83.15%;该组分的氨基酸主要含有Gly和Pro,含量分别为22.04%和11.40%;该组分分子质量范围为1 300~2 000 u。     

Study on Application of Polyenzyme Method to Offal of Harengula zunasi

The new polyenzyme method for making gravy from Harengula zunasi offal involves protein enzymolysis with flavorase after proper alkaline and neutral protease levels were established by orthogonal trials to select the best hydrolytic conditions for processing offal with alkaline and neutral protease. The conditions for the polyenzyme method were pH of 7.0, temperature of 50℃ , alkaline and neutral protease concentration of 1.5% respectively, hydrolysis time of 120 min, andflavorase concentration of 2.0% , for 60 min. The new gravy-making technology yields a nutritious and delicious gravy containing 40.3 % of total essential amino acids, with delicious amino acids Glu, Asp, Gly, Ala, Pro and Ser comprising 49.5% , total and amino nitrogen being respectively1.9 and 1.1 g/100 g (amino acid nitrogen being 61.0% of total nitrogen), The polyenzyme method was used to make 14.8% protein gravy from Harengula zunasi offal. In addition, in inorganic elements, the phosphorus content is the highest.     

鲢鱼降血压肽的酶解条件

以鲢鱼蛋白为原料,采用5种不同复合蛋白酶(P1,P2,P3,P4,P5)进行酶解,综合研究了复合生物酶酶量、酶解时间、pH、酶解温度等对鲢鱼蛋白酶解降血压肽ACE活性抑制效果的影响。结果表明,复合酶P1酶解降血压肽对ACE抑制率经体外检测为88.08%,具有最佳的酶解效果。对应最佳条件为:酶量25 IU/mL,酶解时间2 h,pH7.0,酶解温度60℃。     

Cloning,expression and characterization of a lipase gene from marine bacterium <Emphasis Type="Italic">Pseudoalteromonas lipolytica</Emphasis> SCSIO 04301

A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 kDa. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and pH value of Lip1233 were 45°C and 8.0, respectively. It retained more than 70% of original activity after being incubated in pH ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45°C, but was unstable when the temperature was above 55°C. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30% (v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M NaCl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40°C. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.     

Effects of temperature, pH and NaC1 on protease activity in digestive tract of young turbot, Scophthalmus maximus

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish＇s internal organs. The optimal activity in the fish＇s stomach was at pH of 2.2, while that in the intestinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40℃ in stomach, intestine and hepatopancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an opposite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.     

酶解蓝圆鲹鱼肉蛋白制备小分子肽的工艺研究

以蓝圆鲹为原料,酶解蓝圆鲹鱼肉蛋白制备小分子肽。运用响应面试验设计优化蓝圆鲹蛋白酶解的工艺条件,探讨了酶种类和添加量、液固比、酶解温度、酶解时间对多肽提取率的影响。结果表明:选用木瓜蛋白酶,添加量为0.10%,液固比2∶1,酶解温度54.4℃,酶解时间3.18 h下,多肽提取率为32.35%。     

Characterization of proteases from <Emphasis Type="Italic">Planomicrobium</Emphasis> sp. L-2 isolated from the gastrointestinal tract of <Emphasis Type="Italic">Octopus variabilis</Emphasis> (Sasaki)

A crude protease produced from Planomicrobium sp. L-2 is described, and its effectiveness as an additive in liquid detergent evaluated. We isolate the protease-producing Planomicrobium sp. L-2 from the gastrointestinal tract of Octopus variabilis. At least three caseinolytic protease clear bands were observed in zymogram analysis. The crude alkaline protease was highly tolerant of a pH range from 7.0 to 9.0, and temperatures to 50°C after incubation for 1 h. Proteolytic enzymes were stable towards three surfactants (5% Tween 80, 1% Triton X-100 and 0.05% SDS) and an oxidizing agent (1% hydrogen peroxide), in addition to being highly stable and compatible with popular commercial laundry powered detergent brands available in China. Our study demonstrates the potential these proteases have for development into novel classes of detergent additive. This study also suggests that the gastrointestinal tract of Octopus variabilis may be a rich source of commercially valuable strains of enzyme.     

Alkaline Protease Production by a Strain of Marine Yeasts

Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45℃. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min^- 1 and agitation speed 150 r min^-1 . Under the optimal conditions, 623.1 Umg^-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.     

Purification and characterization of 2-haloacid dehalogenase from marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.     

Comparative study on the protease inhibitors from fish eggs

The protease inhibitor was purified from five different fish eggs. The molecular weights of Pacific herring, chum salmon, pond smelt, glassfish, and Alaska pollock egg protease inhibitors were 120, 89, 84.5, 17, and 16.8kDa, respectively. The specific inhibitory activity of glassfish egg protease inhibitor was the highest followed by those of Pacific herring and Alaska pollock in order. The specific inhibitory activity and purity of glassfish egg protease inhibitor were 19.70 U mg^- 1 protein and 164.70 folds of purification, respectively. Glassfish egg protease inhibitor was reasonably stable at 50 - 65℃ and pH 8, which was more stable at high temperature and pH than protease inhibitors from the other fish species. Glassfish egg protease inhibitor was noncompetitive with inhibitor constant （Ki） of 4.44 nmol L^-1     

Optimum production and characterization of an acid protease from marine yeast <Emphasis Type="Italic">Metschnikowia reukaufii</Emphasis> W6b

The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin⁻¹. Under the optimal conditions, 72.5 UmL⁻¹ of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.     

The selection of alkaline protease-producing yeasts fi＇om marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains (HN3.11, N11b, YF04C and HN4.9) capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, Issatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N11b was 10.0. The optimal temperature for protease activity was 45°C for strains HN3.11, N11b, and YF04C, and 50°C for strain HN4.9. After digestion of shrimp (Penaeus vannamei) protein and spirulina (Arthospira platensis) protein with the four crude alkaline proteases, the filtrate from spirulina (Arthrospira platensis) powder digested by the crude alkaline protease of strain HN3.11 was found to have the highest antioxidant activity (61.4%) and the highest angiotensin I converting enzyme (ACE)-inhibitory activities (68.4%). The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.     

Effects of different enzymatic hydrolysis methods on the bioactivity of peptidoglycan in Litopenaeus vannamei

The effects of different hydrolysis methods on peptidoglycan(PG) were assessed in terms of their impact on the innate immunity and disease resistance of Pacific white shrimp,Litopenaeus vannamei.PG derived from Bifidobacterium thermophilum was prepared in the laboratory and processed with lysozyme and protease under varying conditions to produce several different PG preparations.A standard shrimp feed was mixed with 0.05% PG preparations to produce a number of experimental diets for shrimp.The composition,concentration,and molecular weight ranges of the soluble PG were analyzed.Serum phenoloxidase and acid phosphatase activity in the shrimp were determined on Days 6-31 of the experiment.The protective activity of the PG preparations was evaluated by exposing shrimp to white spot syndrome virus(WSSV).Data on the composition of the PG preparations indicated that preparations hydrolyzed with lysozyme for 72 h had more low-molecular-weight PG than those treated for 24 h,and hydrolysis by protease enhanced efficiency of hydrolysis compared to lysozyme.SDS-PAGE showed changes in the molecular weight of the soluble PG produced by the different hydrolysis methods.Measurements of serum phenoloxidase and acid phosphatase activity levels in the shrimp indicated that the PG preparations processed with enzymes were superior to the preparation which had not undergone hydrolysis in enhancing the activity of the two serum enzymes.In addition,the preparation containing more low-molecular-weight PG enhanced the resistance of the shrimp to WSSV,whereas no increased resistance was observed for preparations containing less low-molecular-weight PG.These findings suggest that the immunity-enhancing activity of PG is related to its molecular weight and that increasing the quantity of low-molecular-weight PG can fortify the effect of immunity enhancement.     

米曲霉中性蛋白酶特性的研究

研究了米曲霉中性蛋白酶的特性。结果表明,米曲霉中性蛋白酶的最佳提取方法为加入pH 7.2的缓冲液研匀,置恒温(30℃)培养箱中提取30 min,每10 min搅动1次,最后用4层纱布过滤。酶反应最适温度为55℃,最适pH值为7.2。NaCl和EDTA对其有一定的抑制作用。酶的热稳定性好,在45℃的温度下处理24 h后,酶活力仍有45.5%。米曲霉中性蛋白酶与培养物一起保存时,稳定性也较好,在4℃保存一个月后,酶活力几乎无变化。     

Preparation of κ-carra-Oligosaccharides with Microwave Assisted Acid Hydrolysis Method

A rapid method of microwave assisted acid hydrolysis was established to prepare κ-carra-oligosaccharides. The optimal hydrolysis condition was determined by an orthogonal test. The degree of polymerization(DP) of oligosaccharides was detected by high performance thin layer chromatography(HPTLC) and polyacrylamide gel electrophoresis(PAGE). Considering the results of HPTLC and PAGE, the optimum condition of microwave assisted acid hydrolysis was determined. The concentration of κ-carrageenan was 5 mg m L-1; the reaction solution was adjusted to p H 3 with diluted hydrochloric acid; the solution was hydrolyzed under microwave irradiation at 100 for 15℃ min. Oligosaccharides were separated by a Superdex 30 column(2.6 cm × 90 cm) using AKTA Purifier UPC100 and detected with an online refractive index detector. Each fraction was characterized by electrospray ionization mass spectrometry(ESI-MS). The data showed that odd-numbered κ-carra-oligosaccharides with DP ranging from 3 to 21 could be obtained with this method, and the structures of the oligosaccharides were consistent with those obtained by traditional mild acid hydrolysis. The new method was more convenient, efficient and environment-friendly than traditional mild acid hydrolysis. Our results provided a useful reference for the preparation of oligosaccharides from other polysaccharides.     

Screening and characterization of the alkaline protease isolated from PLI-1, a strain of Brevibacillus sp. collected from Indonesia’s hot springs

A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia’s hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70℃ and pH 8.0-9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL-1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70℃ and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.     

Properties of Klebsiella phage P13 and associated exopolysaccharide depolymerase

The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide(EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70℃ for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60℃ and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a double-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.     

Optimization of dilute acid hydrolysis of Enteromorpha

Acid hydrolysis is a simple and direct way to hydrolyze polysaccharides in biomass into fermentable sugars. To produce fermentable sugars effectively and economically for fuel ethanol, we have investigated the hydrolysis of Enteromorpha using acids that are typically used to hydrolyze biomass:H2SO4, HCl, H3PO4 and C4H4O4 (maleic acid). 5%(w/w) Enteromorpha biomass was treated for different times (30, 60, and 90 min) and with different acid concentrations (0.6, 1.0, 1.4, 1.8, and 2.2%, w/w) at 121°C. H2SO4 was the most effective acid in this experiment. We then analyzed the hydrolysis process in H2SO4 in detail using high performance liquid chromatography. At a sulfuric acid concentration of 1.8% and treatment time of 60 min, the yield of ethanol fermentable sugars (glucose and xylose) was high, (230.5 mg/g dry biomass, comprising 175.2 mg/g glucose and 55.3 mg/g xylose), with 48.6% of total reducing sugars being ethanol fermentable. Therefore, Enteromorpha could be a good candidate for production of fuel ethanol. In future work, the effects of temperature and biomass concentration on hydrolysis, and also the fermentation of the hydrolysates to ethanol fuel should be focused on.