Safety evaluation of the food enzyme chymosin from the genetically modified Kluyveromyces lactis strain CHY.

The food enzyme chymosin (EC 3.4.23.4) is produced with the genetically modified Kluyveromyces lactis strain CHY by DSM Food Specialties B.V. It is intended to be used in milk processing for cheese production and for production of fermented milk products. Dietary exposure was estimated to be up to 0.69 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. The production strain contains multiple copies of known antimicrobial resistance genes and consequently, it does not fully fulfil the requirements for the qualified presumption of safety (QPS) approach to safety assessment. However, considering the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. As no other concerns arising from the microbial source and its subsequent genetic modification or from the manufacturing process have been identified, the Panel considered that toxicological tests were not needed for the assessment of this food enzyme. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure, although unlikely, cannot be excluded, particularly for individuals sensitised to cedar pollen allergens. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Raw walnut kernel: A natural source for dietary proteases and bioactive proteins.

Walnut kernels are health-promoting nuts, which are mainly attributed to polyunsaturated fatty acids, phenolics, and phytosterols. However, the information concerning benefits of walnut proteins are limited. In this study, endopeptidases, aminopeptidases, carboxypeptidases, superoxide dismutases, catalases, and phospholipases with respective relative abundance of 2.730, 1.728, 0.477, 3.148, 0.743, and 0.173‰ were identified by liquid chromatography tandem mass spectrometry. These endogenous proteases exhibited activity in a broad pH range of 2-6.5, and optimal at pH 4.5 and 50 °C. Aspartic endopeptidases were predominant endopeptidases, followed by cysteine ones. There were two types of aspartic endopeptidases, one (not inhibited by pepstatin A) exerted activity at pH 2-3 and the other (inhibited by pepstatin A) optimal at pH 4.5. Carboxypeptidases were optimal at pH 4.5, and aminopeptidases exerted activity at pH near 6.5. These endogenous proteases assisted the digestion of walnut proteins, and soaking, especially peeling, greatly improved the in vitro digestibility.

Endopeptidases, exopeptidases, and glutamate decarboxylase in soybean water extract and their in vitro activity.

The proteolytic activity of some soybean endogenous proteases have been clarified in the previous studies, but the information concerning the roles of these proteases and some other unknown ones during soybean processing are scarce. Herein, 16 endopeptidases, 13 exopeptidases, 24 inhibitors (two serpin-ZX and one subtilisin inhibitor firstly identified), and one glutamate decarboxylase were identified in the soybean water extract by the liquid chromatography tandem mass spectrometry analysis. Amongst the identified endopeptidases, just the aspartic endopeptidases (optimal at pH 2.5-3 and 35-45 °C) showed the detectable proteolytic activity by the tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and protease inhibitor assay analyses, whereas serine, cysteine, and metallo- endopeptidases (except P34 probable thiol protease) did not. Free amino acid analysis showed that the exopeptidases and glutamate decarboxylase were optimal at pH 6 and 45 °C, and by 6 h incubation, the free amino acids and γ-aminobutyric acid almost doubled.

Sesame water-soluble proteins fraction contains endopeptidases and exopeptidases with high activity: A natural source for plant proteases.

Recently, the interest in the plant proteases has greatly increased. However, only a few of proteases are isolated from the hugely produced oilseeds for the practical utilizations. In this study, the raw sesame milk prepared from peeled sesame seeds was separated into floating, skim, and precipitate fractions by centrifugation. The predominant aspartic endopeptidases and serine carboxypeptidases, which exerted high synergetic activity at pH 4.5-5 and 50-60 °C, were identified in the skim by the liquid chromatography tandem mass spectrometry, Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protease inhibitor assay, trichloroacetic acid-nitrogen soluble index (TCA-NSI), and free amino acid analyses. By incubating the mixture (protein content, 2%) of skim and precipitate at pH 4.5 and 50 °C for 6 h, the TCA-NSI and free amino acids achieved to 38.42% and 3148 mg/L, respectively. Moreover, these proteases efficiently degraded the proteins from soybean, peanut, and bovine milk.

Effects of phosphatidylinositol-3 kinase/serine threonine kinase pathway on expression of beta-site amyloid precursor protein cleaving enzyme-1 in the hippocampus neurons / 中华神经科杂志

Objective To investigate the effect of phosphatidylinesitol-3 kinase/serine threonine kinase (PI3K/Akt) signaling pathway on expression of beta-site amyloid precursor protein cleaving enzyme-1 (BACE1) in the hippocampus neurons of rat brain. Methods Forty SD rats were randomly divided into 4 groups: blank control group, sham-operated group, insulin group and wortmannin group. Insulin or the specific inhibitor of PI3K, wortmannin was injected into hippocampus neurons to activate or inhibit the signaling pathway in insulin group or wortmannin group, respectively. Immunoprecipitation and Western blot were used to analyze the proteins levels of PI3K/Akt and BACE1. Results In insulin treatment group,among the proteins downstream of signaling pathway, expression of Akt increased (0. 952±0.060 vs 0.835±0.029,t=4.9150, P=0.0001), phospho-Akt set473 increased (0.800±0.075 vs 0.657± 0.025,t=4.5598, P=0.0002), phospho-GSK-3α decreased (0.604±0.062 vs 0.726±0.041, t= 3.5871, P=0.0018 ), and the expression of mature BACE1 and β-CTF significantly decreased. In wortmannin group, the expression of Akt and phospho-Akt ser473 were inhibited; phospho-GSK-3α increased ; mature BACEI (1.004±0.096) and β-CTF (1.031±0.048) increased (t=11.5980, P= 0.0000 and t =4.2194, P =0.0004, respectively). Conclusions PI3K/Akt signaling pathway might effect the expression of BACE1, in which impaired signaling pathway may cause the amyloid precursor protein to be easily processed by BACE1, and thus involves the pathology of Alzheimer' s disease.

Expression of Secretory Aspartyl Proteinase in Human Vulvovaginal Candidiasis / 中华皮肤科杂志

Objective To investigate the differential expression of secretory aspartyl proteinase in patients with vaginal C.albicans infection and in asymptomatic candidal carriers.Methods Secretory aspartyl proteinase expression was determined with reverse transcription-PCR in vaginal specimens taken from 10 asymptomatic candidal carriers,14 patients with vulvovaginal candidiasis (VVC) and 10 patients with recurrent VVC (RVVC).Results SAP2 and SAP4 to SAP6 subfamily were detected in both candidal carriers and patients with vaginal candidiasis.SAP1 and SAP3 transcripts were not observed in 10 asymptomatic candidal carriers.All 9 SAP genes were differently expressed in VVC and RVVC patients.SAP1 and SAP3 transcripts were frequently amplified in some patients.Conclusion It is shown that C.albicans infection is associated with differential expression of individual SAP genes,which may be involved in the pathogenesis of vaginal candidiasis.

Effect of hypoxia and reoxygenation on the expression of a disintegrin and metalloprotease10,?-amyloid precursor protein cleaving enzyme mRNA in SH-SY5Y cells / 中华神经科杂志

Objective To study the effects of hypoxia and reoxygenation on expression of a disintegrin and metalloprotease10(ADAM10),?-amyloid precursor protein cleaving enzyme(BACE1) mRNA in SH-SY5Y cells.Methods SH-SY5Y cells were grouped into hypoxic and control groups.The hypoxic cells were incubated in hypoxic condition(95%N_2,5%CO_2) for 12 hours and 24 hours,and then cells were reoxygenated for 0 hours,12 hours and 24 hours.The expression of ADAM10,BACE1 mRNA were tested respectively at different time points after reoxygenation by RT-PCR.Control groups were incubated in normal conditions,seeded and treated at the same time with the hypoxic cells.Results The expression of ADAM10 mRNA was down-regulated by 19.8%,41.4% and 64.6%(P=0.005,0.038,0.001) at different time after reoxygenation with 12 hours hypoxia and down-regulated by 30.1%,75.9% and 86.5%(P=0.009,0.005,0.043)after reoxygenation with 24 hours hypoxia.The expression of BACE1 mRNA was up-regulated by 31.5% and 35.1%(P=0.028,0.005)only at 12 hours and 24 hours points after reoxygenation with 24 hours hypoxia.Conclusion Hypoxia and reoxygenation might alter the expression of ADAM10 and BACE1,which demonstrates that the vascular factors should make the amyloid precursor protein easy to be processed by ?-secrease pathway,thus to involve the pathology of Alzheimer's disease.

Expression of Candida albicans Secreted Aspartyl Proteinase in Acute Vaginal Candidiasis / 中华皮肤科杂志

Objective To analyze the expression of secreted aspartyl proteinases (Sap) in human vaginal infection in vivo. Methods Vaginal swabs were collected from 9 healthy volunteers and 20 patients with vaginal candidiasis, and the expression of Sap1-6 was evaluated by reverse-transcriptase polymerase chain reaction using specific primer sets. Results The Sap2 and Sap5 were the most common genes expressed in the patients with vaginal candidiasis. The expression Sap3 and Sap4 was detected in all subjects. All six Sap genes were simultaneously expressed in some patients with vaginal candidiasis. Conclusion The data shows that the Sap genes may be involved in the pathogenesis of vaginal candidiasis.