Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution‑guided mutagenesis

Streptomyces griseus trypsin(SGT)is a bacteria-sourced trypsin that could be potentially applied to industrial insulin productions.However,SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine residue.In this study,by engineering theαsignal peptide in Pichia pastoris,we increased the SGT amidase activity to 67.91 U mL^(−1)in shake flask cultures.Afterwards,we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S,V177I and E180M with increased catalytic efficiencies.On this basis,we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL^(−1)in shake flasks and 2506 U mL^(−1)in 3-L fed-batch cultures.Moreover,single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5,which was 2 times better than the animal-sourced trypsin.

Microbial specific carboxylation of (–)-huperzine A by Streptomyces griseus

In a scale-up microbial transformation of（–）-huperzine A（1） by Streptomyces griseus CACC 200300, a specific carboxylated derivative（2） was yielded together with two known hydroxylated metabolites. The structure of 2 was characterized as 16-carboxyl huperzine A on the basis of IR, HRMS and NMR spectroscopic data analysis.

Microbial Hydroxylation of Quinovic Acid Glycoside by Streptomyces griseus ACCT 13273

Microbial transformation of a triterpene glycoside, quinovic acid 3-O-β-D-quinovopyranoside by Streptomyces griseus ACCT 13273 was achieved with the aim of generating new bio-active derivatives. Preparative-scale biotransformation with standard two-stage fermentation protocol afforded a new oxidized metabolite, as a result of the hydroxylation of the methyl group at C-30, which was difficult to achieve by chemical means. The metabolite was elucidated based on extensive NMR and high-resolution mass spectral analyses.

Effect of toasting grain silages from field peas(Pisum sativum)and field beans(Vicia faba)on in vitro gas production,methane production,and post-ruminal crude protein content

Legume grains such as field peas and field beans can be produced on a local level,and may be reliable sources of dietary protein and energy apart from common soybean and rapeseed meals.In ruminants,protein,starch,and carbohydrates from peas and field beans are fermented in large part before reaching the small intestine.The objective of this study was to evaluate the effects of a combination of ensiling and hydro-thermic treatment(i.e.,toasting at 160℃for 30 min)of grains of peas and field beans on the concentrations of post-ruminal crude protein(PRCP)and rumen-undegraded protein(RUP).Moreover,24-h gas production and methane production were measured.For this,an in vitro batch culture system with ruminal fluid from sheep was used.Rumen-undegraded protein was determined using the Streptomyces griseus protease test.Scanning electron micrographs were used to visualize morphological changes of starch granules and their joint matrices in peas and field beans after ensiling,toasting,or a combination of both.Native pea grains contained crude protein(CP)at 199 g/kg DM,PRCP at 155 g/kg DM at a ruminal passage rate of 0.08/h(Kp8),RUP at 33 g/kg DM at Kp8,and starch at 530 g/kg DM.Native field beans contained CP at 296 g/kg DM,PRCP at 212 g/kg DM at Kp8,RUP at 54 g of/kg DM at Kp8,and starch at 450 g/kg DM.The PRCP did not considerably differ among native and treated peas or field beans.Especially in the peas,RUP at Kp8 increased after ensiling by 10 g/kg DM(i.e.,30%;P<0.05).Toasting increased RUP(Kp8)in ensiled peas by another 28%(P<0.05).Toasting had no effect on PRCP or RUP when the peas or field beans were not ensiled before.Gas and methane production were not affected by any treatment,and scanning electron micrographs did not reveal structural changes on the starches doubtless of any treatment.Protein seemed to be more affected by treatment with ensiled+toasted peas than with ensiled+toasted field beans,but starches and other carbohydrates from both legumes remained unaffected.

Nitrogen fractionation of certain conventional-and lesser-known by-products for ruminants

Dietary proteins for ruminants are fractionated according to solubility, degradability and digestibility. In the present experiment, 11 vegetable protein meals and cakes used in ruminant nutrition were included with a main focus on determining various nitrogen(N) fractions in vitro. Total N(N×6.25) content varied from 22.98%(mahua cake) to 65.16%(maize gluten meal), respectively. Guar meal korma contained the lowest and rice gluten meal had the highest acid detergent insoluble nitrogen(ADIN; N×6.25). Boratephosphate insoluble N(BIN, N×6.25) and Streptomyces griseus protease insoluble N(PIN; N×6.25) were higher(P<0.01) in maize gluten meal than in other feeds, whereas groundnut cake and sunflower cake had lower(P < 0.01) BIN, and PIN, respectively. Available N, calculated with the assumption that ADIN is indigestible, was maximum in guar meal korma and minimum in rice gluten meal. Furthermore, rapid and slowly degradable N(N × 6.25) was found to be higher(P < 0.01) in groundnut cake and coconut cake, respectively. Intestinal digestion of rumen undegradable protein, expressed as percent of PIN, was maximum in guar meal korma and minimum in rice gluten meal. It was concluded that vegetable protein meals differed considerably in N fractions, and therefore, a selective inclusion of particular ingredient is needed to achieve desired level of N fractions to aid precision N rationing for an improved production performance of ruminants.