Novel protein glutaminase for plant based beverages

Abstract

The present invention relates to protein glutaminases and their use in the manufacture of beverages. More particularly, the present invention relates to methods of adding texture and viscosity to non-dairy milk beverages such as oat milk.

Description

[0001] NB42152-WO-PCT[3]

[0002] TITLE

[0003] NOVEL PROTEIN GLUTAMINASE FOR PLANT BASED BEVERAGES

[0004] TECHNICAL FIELD

[0005] The present invention relates to protein glutaminases and their use in the manufacture of beverages. More particularly, the present invention relates to methods of adding texture and viscosity to non-dairy milk beverages such as oat milk.

[0006] REFERENCE TO A SEQUENCE LISTING

[0007] The contents of the electronic submission of the text file Sequence Listing, named “NB42152WOPCT3_SequenceListing” was created on September 12, 2024, and is 37 KB in size, which is hereby incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Human consumption of plant-based protein in place of animal protein has grown in recent years and become almost common place in the food industry. Plant-based protein requires fewer natural resources, reduces the risk of pathogen contamination during production and is regarded as a healthier diet. However, the physical and chemical properties of plant protein need to be adapted, depending on the application. There is a high demand for non-dairy milk and yogurt. Plant based dairy alternatives require an acceptable texture and mouthfeel. Moreover, the protein content of plant-based milk alternatives must be similar to their dairy counterparts. In this regard, plant proteins generally have poor solubility in aqueous solutions due to having been denatured via the process of preparing the plant protein from its source.

[0010] To alleviate these problems, various chemical modifications of plant protein have been investigated, such as mild acid treatment, anion-catalyzed deamidation, dry heating under mild alkaline conditions, and thermal treatment. But side reactions are prevalent and result NB42152-WO-PCT[3] in poor quality plant protein. In particular, protein hydrolyzation results in low molecular weight peptides which can negatively impact taste.

[0011] There is, thus, a continuing need for methods of improving plant protein properties to provide acceptable plant-based milk products.

[0012] SUMMARY OF THE INVENTION

[0013] In an aspect of the present invention, a method is presented for making a plant-based beverage having improved texture and improved precipitation rate, the method having the steps of: providing a plant protein in an aqueous medium; and contacting the plant protein with a protein glutaminase corresponding to a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NOT, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO: 26 or a protein glutaminase active fragment thereof to provide the beverage having improved texture and improved precipitation rate as compared with a plant-based beverage prepared without the protein glutaminase, wherein said improved texture comprises increased thickness and / or increased mouthfeel and said improved precipitation rate is less than 50%.

[0014] Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0015] Optionally, the protein glutaminase is a polypeptide having at least 85% sequence identity to SEQ ID NOT, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NB42152-WO-PCT[3]

[0016] NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NQ:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0017] Optionally, the protein glutaminase is a polypeptide having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NQ:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0018] Optionally, the protein glutaminase is a polypeptide having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form NB42152-WO-PCT[3] of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0019] Optionally, the protein glutaminase is a polypeptide having at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0020] Optionally, the protein glutaminase is a polypeptide having at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0021] Optionally, the protein glutaminase is a polypeptide according to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, NB42152-WO-PCT[3]

[0022] SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide according to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0023] Optionally, the protein glutaminase is present in amount from about 0.005 mg per 100 ml to about 15 mg per 100 ml. Optionally, the protein glutaminase is present in an amount from about 0.03 mg per 100 ml to about 12.5 mg per 100 ml.

[0024] Optionally, the improved texture is increased thickness. Optionally, the thickness is increased by 30, 50, 70, 90, 100, 110 or 120% or more.

[0025] Optionally, the improved texture is increased mouthfeel. Optionally, the mouthfeel is increased by 30 50, 70, 90, 100, 110 or 120% or more.

[0026] Optionally, the precipitation rate of the beverage is less than 40, 30, 20, 10 or 5%.

[0027] Optionally, the plant protein is wheat gluten, soy, soybean, pea, com or oat protein.

[0028] Optionally, the plant protein is hydrolyzed or partially hydrolyzed prior to contact with the protein glutaminase.

[0029] Optionally, the improved plant-based beverage is a calf or piglet milk replacer.

[0030] Optionally, the improved plant-based beverage is a non-dairy milk or drinkable yogurt.

[0031] Optionally, the method has the further step of adding a culture to produce a non-dairy yogurt or acidified milk product.

[0032] Optionally, the method has the further step of incorporating the improved beverage into a non-dairy cheese.

[0033] In another aspect of the instant invention, a method is presented of preparing a stabilized flavoured milk-based beverage having the steps of: a. providing a flavoured milk mix comprising milk and a non-milk protein component; and b. contacting said flavoured milk mix with a protein glutaminase corresponding to a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:4, NB42152-WO-PCT[3]

[0034] SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof to provide the stabilized flavoured milk beverage.

[0035] Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: I I, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0036] Optionally, the protein glutaminase is a polypeptide having at least 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0037] Optionally, the protein glutaminase is a polypeptide having at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the NB42152-WO-PCT[3] protein glutaminase is a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0038] Optionally, the protein glutaminase is a polypeptide having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0039] Optionally, the protein glutaminase is a polypeptide having at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:1, NB42152-WO-PCT[3]

[0040] SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0041] Optionally, the protein glutaminase is a polypeptide having at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11 , SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0042] Optionally, the protein glutaminase is a polypeptide according to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide according to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0043] Optionally, the protein glutaminase is present in amount from about 0.005 mg per 100 ml to about 15 mg per 100 ml. Optionally, the protein glutaminase is present in an amount from about 0.03 mg per 100 ml to about 12.5 mg per 100 ml.

[0044] Optionally, the non-milk protein component is cocoa.

[0045] In another aspect of the present invention, a protein glutaminase is presented having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NB42152-WO-PCT[3]

[0046] NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0047] Optionally, the protein glutaminase is a polypeptide having at least 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: I, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0048] Optionally, the protein glutaminase is a polypeptide having at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 90% NB42152-WO-PCT[3] sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 1 1 , SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0049] Optionally, the protein glutaminase is a polypeptide having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0050] Optionally, the protein glutaminase is a polypeptide having at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25. NB42152-WO-PCT[3]

[0051] Optionally, the protein glutaminase is a polypeptide having at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 1 , SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0052] Optionally, the protein glutaminase is a polypeptide according to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Optionally, the protein glutaminase is a polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Optionally, the protein glutaminase active fragment is a mature form of a polypeptide according to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0053] In another aspect of the present invention, an isolated polynucleotide is presented having a nucleic acid sequence encoding an isolated polypeptide as described above.

[0054] In another aspect of the present invention, a recombinant expression vector is presented having the isolated polynucleotide as described above.

[0055] In another aspect of the present invention, a host cell is presented having the recombinant expression vector as described above. NB42152-WO-PCT[3]

[0056] BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0057] SEQ ID NO:1 is the protein sequence of CspGltl precursor protein.

[0058] SEQ ID NO:2 is the protein sequence of CspGltl predicted mature protein.

[0059] SEQ ID NO:3 is the DNA sequence of synthetic / optimized full length CspGltl DNA.

[0060] SEQ ID NO:4 is the protein sequence of CnaGltl precursor protein.

[0061] SEQ ID NO:5 is the protein sequence of CnaGltl predicted mature protein.

[0062] SEQ ID NO:6 is the DNA sequence of synthetic / optimized full length CnaGltl DNA.

[0063] SEQ ID NO:7 is the protein sequence of CjoGltl precursor protein.

[0064] SEQ ID NO:8 is the protein sequence of CjoGltl predicted mature protein.

[0065] SEQ ID NO:9 is the DNA sequence of synthetic / optimized full length CjoGltl DNA.

[0066] SEQ ID NO: 10 is the protein sequence of MetGlt8 precursor protein.

[0067] SEQ ID NO:11 is the protein sequence of MetGlt8 predicted mature protein.

[0068] SEQ ID NO: 12 is the DNA sequence of synthetic / optimized full length MetGlt8 DNA.

[0069] SEQ ID NO: 13 is the protein sequence of CinGltl precursor protein.

[0070] SEQ ID NO: 14 is the protein sequence of CinGltl predicted mature protein.

[0071] SEQ ID NO: 15 is the DNA sequence of synthetic / optimized full length CinGltl DNA.

[0072] SEQ ID NO: 16 is the protein sequence of ClaGltl precursor protein.

[0073] SEQ ID NO: 17 is the protein sequence of ClaGltl predicted mature protein.

[0074] SEQ ID NO: 18 is the DNA sequence of synthetic / optimized full length ClaGltl DNA.

[0075] SEQ ID NO: 19 is the protein sequence of CspGlt8 precursor protein.

[0076] SEQ ID NO:20 is the protein sequence of CspGlt8 predicted mature protein.

[0077] SEQ ID NO:21 is the DNA sequence of synthetic / optimized full length CspGlt8 DNA.

[0078] SEQ ID NO:22 is the protein sequence of CurGltl precursor protein.

[0079] SEQ ID NO:23 is the protein sequence of CurGltl predicted mature protein.

[0080] SEQ ID NO:24 is the DNA sequence of synthetic / optimized full length CurGltl DNA.

[0081] SEQ ID NO:25 is the protein sequence of CprGltl precursor protein.

[0082] SEQ ID NO:26 is the protein sequence of CprGltl predicted mature protein. NB42152-WO-PCT[3]

[0083] SEQ ID NO:27 is the DNA sequence of synthetic / optimized full length CprGltl DNA.

[0084] DETAILED DESCRIPTION OF THE INVENTION

[0085] Definitions

[0086] The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three- letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e. , N— >C).

[0087] The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0088] A “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

[0089] An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0090] Reference to the wild-type polypeptide is understood to include the mature form of the polypeptide. A “mature” polypeptide or variant, thereof, is one in which a signal sequence is absent, for example, cleaved from an immature or full length form of the NB42152-WO-PCT[3] polypeptide during or following expression of the polypeptide.

[0091] A “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0092] In addition to the specific amino acid sequences and polynucleotides mentioned herein, the present invention encompasses variants, homologues, derivatives and fragments thereof.

[0093] The term "variant" is used to mean a nucleotide sequence or amino acid sequence which differs from a wild-type sequence.

[0094] For example, a variant may include substitutions, insertions, deletions, truncations, transversions and / or inversions at one or more position(s) relative to a wild-type sequence. Variants can be made using methods known in the art for example site scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis and directed- evolution as well as using recombinant methods well known in the art. Polynucleotide sequences encoding variant amino acid sequences may readily be synthesized using methods known in the art.

[0095] In some aspects, the variant is a naturally occurring nucleotide sequence or amino acid sequence which differs from a wild-type sequence. For example, the variant may be a natural genetic variant.

[0096] In some aspects, the variant is an engineered variant. For example, the variant may be engineered by recombinant methods.

[0097] The protein sequences of the instant invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic NB42152-WO-PCT[3] acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0098] Conservative substitutions may be made, for example according to the Table below.

[0099] Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other as set forth in Table 1.

[0100] Table 1.

[0101] The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0102] Replacements may also be made by synthetic amino acids (e.g. unnatural amino acids) include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl- phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, B-alanine*, L-a- amino butyric acid*, L-g-amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic NB42152-WO-PCT[3] acid*, 7-amino heptanoic acid*, L-methionine sulfone**, L-norleucine*, L-norvaline*, p- nitro-L-phenylalanine*, L-hydroxyproline*, L- thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)*, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (l,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L- diaminopropionic acid#and L-Phe (4-benzyl)*.

[0103] The notation * has been utilized for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilized to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

[0104] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or b-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue’s nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[0105] The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and / or the addition of acridine or poly lysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.

[0106] The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein. NB42152-WO-PCT[3]

[0107] Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries or genomic DNA libraries made from other animal species and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.

[0108] Variants and strain / species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants / homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.

[0109] The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

[0110] Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterized sequences. This may be useful where for example silent codon sequence changes are required to optimize codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

[0111] The present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology and recombinant DNA, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the NB42152-WO-PCT[3] literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology : DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

[0112] As used herein, “percent (%) sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the

[0113] CLUSTAL W algorithm are:

[0114] Gap opening penalty: 10.0

[0115] Gap extension penalty: 0.05

[0116] Protein weight matrix: BLOSUM series

[0117] DNA weight matrix: IUB

[0118] Delay divergent sequences %: 40

[0119] Gap separation distance: 8

[0120] DNA transitions weight: 0.50

[0121] List hydrophilic residues: GPSNDQEKR

[0122] Use negative matrix: OFF

[0123] Toggle Residue specific penalties: ON

[0124] Toggle hydrophilic penalties: ON

[0125] Toggle end gap separation penalty: OFF

[0126] Deletions are counted as non-identical residues, compared to a reference sequence.

[0127] Deletions occurring at either terminus are included. For example, a variant with five amino NB42152-WO-PCT[3] acid deletions of the C-terminus of the mature 617 residue polypeptide would have a percent sequence identity of 99% (612 / 617 identical residues x 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having “at least 99% sequence identity” to a mature polypeptide.

[0128] In accordance with the instant invention, proteins, including enzymes, of the present invention exist in multiple forms. Proteins of the instant invention may be clipped or trimmed (i.e. , removing amino acids) from the N-terminus and / or the C-terminus, resulting in a shorter protein. Proteins of the instant invention can also have internal deletions. Shorter proteins as described herein can have higher activity or lower activity than longer counterparts. Without being bound by theory, as used herein the term “pre-pro-protein” is a protein, including an enzyme, which has an N-terminal signal peptide that targets the protein for secretion. A pre-pro-protein is sometimes referred to herein as “full length” or “full length protein”. The N-terminal signal peptide is cleaved off in the endoplasmic reticulum to yield a “pro-protein”. A pro-protein, as used herein, is shorter in length than the full length protein (it is missing the signal peptide) but longer than the mature protein. In general, a pro-protein is inactive or less active than the mature protein. A pro-protein can be activated or converted to a more active mature form by post-translational modification such as N- or C- terminal clipping. A pro-protein which is an enzyme may be called a “proenzyme” or a “zymogen.” The clipped active protein (derived from the pro-protein) is also referred to herein as the mature protein. It is to be noted that the above terms are used for convenience and are not meant to override or determine the activities of a protein of the instant invention. It is also to be noted that any particular protein of the instant invention can have more than one variant described by the same term.

[0129] All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference.

[0130] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary NB42152-WO-PCT[3] skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0131] Other definitions are set forth below.

[0132] Production of enzymes

[0133] The enzymes of the present invention can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material (e.g., a whole-cell broth) having an enzyme can be obtained following secretion of the enzyme into the cell medium. Optionally, the enzyme can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final enzyme. Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae or Trichoderma reesei. Other host cells include bacterial cells, e.g., Bacillus subtilis or B. licheniformis, as well as Streptomyces, E. coli.

[0134] Vectors

[0135] A DNA construct comprising a nucleic acid encoding an enzyme can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding phospholipase can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.

[0136] The vector may be any vector that can be transformed into and replicated within a host cell. For example, a vector comprising a nucleic acid encoding an enzyme can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional phospholipase. Host cells that serve as expression hosts can include filamentous fungi, for example. The Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. NB42152-WO-PCT[3]

[0137] See FGSC, Catalogue of Strains, University of Missouri, at www.fgsc.net (last modified January 17, 2007). A representative vector is pJG153, a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et al. (June 2011) Applied Environ. Microbiol. 77: 3916-22. pJG153can be modified with routine skill to comprise and express a nucleic acid encoding a phospholipase.

[0138] A nucleic acid encoding an enzyme can be operably linked to a suitable promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing the transcription of the DNA sequence encoding a phospholipase, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, A. niger acid stable a-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding an enzyme is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris A0X1 or A0X2 promoters, cbhl is an endogenous, inducible promoter from Trichoderma reesei. See Liu et al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene cbhl ) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0139] The coding sequence can be operably linked to a signal sequence. The DNA encoding NB42152-WO-PCT[3] the signal sequence may be the DNA sequence naturally associated with the phospholipase gene to be expressed or from a different Genus or species. A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence is the cbhl signal sequence that is operably linked to a cbhl promoter.

[0140] An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, poly adenylation sequences operably linked to the DNA sequence encoding a variant phospholipase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

[0141] The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBl 10, pE194, pAMBl, and pIJ702.

[0142] The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xx.vC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91 / 17243.

[0143] Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of phospholipase for subsequent enrichment or purification. Extracellular secretion of phospholipase into the culture medium can also be used to make a cultured cell material comprising the isolated phospholipase.

[0144] The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene NB42152-WO-PCT[3] or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the phospholipase to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the phospholipase is operably linked to the control sequences in proper manner with respect to expression.

[0145] The procedures used to ligate the DNA construct encoding a phospholipase, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art see, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nded., Cold Spring Harbor, 1989, and 3rded., 2001).

[0146] Transformation and Culture of Host Cells

[0147] An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an enzyme according to the instant invention. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

[0148] Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus; lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis\ Lactobacillus sp. including NB42152-WO-PCT[3]

[0149] Lactobacillus reuterr, Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.

[0150] A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of & Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma sp. can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. An enzyme expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type phospholipase. The type and / or degree of glycosylation may impart changes in enzymatic and / or biochemical properties.

[0151] It may be advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh.2, egll, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art. NB42152-WO-PCT[3]

[0152] Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding an enzyme is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques.

[0153] The preparation of Trichoderma sp. for transformation, for example, may involve the preparation of protoplasts from fungal mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53-56. The mycelia can be obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall, resulting in protoplasts. The protoplasts are protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually, the concentration of these stabilizers varies between 0.8 M and 1.2 M, e.g., a 1.2 M solution of sorbitol can be used in the suspension medium.

[0154] Uptake of DNA into the host Trichoderma sp. strain depends upon the calcium ion concentration. Generally, between about 10-50 mM CaCh is used in an uptake solution. Additional suitable compounds include a buffering system, such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene glycol. The polyethylene glycol is believed to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.

[0155] Usually, transformation of Trichoderma sp. uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 105to 107 / mL, particularly NB42152-WO-PCT[3]

[0156] 2xlO6 / mL. A volume of 100 pL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCh) may be mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is useful to add about 0.25 volumes to the protoplast suspension. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Patent No. 6,022,725.

[0157] As used herein, Protein Identification (“JGI PID”) numbers for native Trichoderma genes reference Version 2 of the Trichoderma reesei QM6a genome sequence assembly generated by the Department of Energy Joint Genome Institute (JGI). (The Genome Portal of the Department of Energy Joint Genome Institute, Grigoriev et al., Nucleic Acids Res 2012 Jan;40(Database issue):D26-32. doi: 10.1093 / nar / gkr947). The JGI assembled Scaffold sequences and annotated genes have also been deposited in GeneBank (The National Center for Biotechnology) under the nucleotide accession numbers GL985056.1 through GL985132.1.

[0158] Expression

[0159] A method of producing an enzyme of the instant invention may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0160] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of a phospholipase. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes e.g., as described in catalogues of the American Type Culture Collection).

[0161] An enzyme secreted from the host cells can be used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the NB42152-WO-PCT[3] expression of a phospholipase. Fermentation may, therefore, he understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed- batch, or solid-state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the phospholipase to be expressed or isolated. The term “spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g. , enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.

[0162] An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like. The polynucleotide encoding an enzyme in a vector can be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may in particular comprise promoters.

[0163] Host cells may be cultured under suitable conditions that allow expression of a phospholipase. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNT™ (Promega) rabbit reticulocyte system.

[0164] An expression host also can be cultured in the appropriate medium for the host, under NB42152-WO-PCT[3] aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g., from about 25 °C to about 75°C (e.g., 30°C to 45°C), depending on the needs of the host and production of the desired phospholipase. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g., from 24 to 72 hours). Typically, the culture broth is at a pH of about 4.0 to about 8.0, again depending on the culture conditions needed for the host relative to production of a phospholipase.

[0165] Methods for Enriching and Purifying enzymes

[0166] Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare an enzyme polypeptide-containing solution.

[0167] After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.

[0168] It is desirable to concentrate an enzyme polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions requires increased incubation time in order to collect the enriched or purified enzyme precipitate.

[0169] The enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and / or ultrafiltration.

[0170] The enzyme solution is concentrated into a concentrated enzyme solution until the enzyme activity of the concentrated phospholipase polypeptide-containing solution is at a desired level.

[0171] Concentration may be performed using, e.g., a precipitation agent, such as a metal NB42152-WO-PCT[3] halide precipitation agent. Metal halide precipitation agents include but are not limited to alkali metal chlorides, alkali metal bromides and blends of two or more of these metal halides. Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide and blends of two or more of these metal halides. The metal halide precipitation agent, sodium chloride, can also be used as a preservative.

[0172] The metal halide precipitation agent is used in an amount effective to precipitate a phospholipase. The selection of at least an effective amount and an optimum amount of metal halide effective to cause precipitation of the enzyme, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, after routine testing.

[0173] Generally, at least about 5% w / v (weight / volume) to about 25% w / v of metal halide is added to the concentrated enzyme solution, and usually at least 8% w / v. Generally, no more than about 25% w / v of metal halide is added to the concentrated enzyme solution and usually no more than about 20% w / v. The optimal concentration of the metal halide precipitation agent will depend, among others, on the nature of the specific phospholipase polypeptide and on its concentration in the concentrated enzyme solution.

[0174] Another alternative way to precipitate the enzyme is to use organic compounds. Exemplary organic compound precipitating agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of the organic compound precipitation agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound and metal halide, may be carried out sequentially or simultaneously.

[0175] Generally, the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds. The organic NB42152-WO-PCT[3] compound precipitation agents can be, for example, linear or branched alkyl esters of 4- hydroxybenzoic acid, wherein the alkyl group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds. Exemplary organic compounds are linear alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6 carbon atoms, and blends of two or more of these organic compounds. Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid and blends of two or more of these organic compounds can also be used. Additional organic compounds also include but are not limited to 4- hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN), which also are both preservative agents. For further descriptions, see, e.g. , U.S. Patent No. 5,281,526.

[0176] Addition of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature, phospholipase concentration, precipitation agent concentration, and time of incubation.

[0177] The organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by means of the metal halide precipitation agent. The selection of at least an effective amount and an optimum amount of organic compound precipitation agent, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, in light of the present disclosure, after routine testing.

[0178] Generally, at least about 0.01% w / v of organic compound precipitation agent is added to the concentrated enzyme solution and usually at least about 0.02% w / v. Generally, no more than about 0.3% w / v of organic compound precipitation agent is added to the concentrated enzyme solution and usually no more than about 0.2% w / v.

[0179] The concentrated polypeptide solution, containing the metal halide precipitation agent, and the organic compound precipitation agent, can be adjusted to a pH, which will, of necessity, depend on the enzyme to be enriched or purified. Generally, the pH is adjusted at a level near the isoelectric point of the phospholipase. The pH can be adjusted at a pH in a NB42152-WO-PCT[3] range from about 2.5 pH units below the isoelectric point (pl) up to about 2.5 pH units above the isoelectric point.

[0180] The incubation time necessary to obtain an enriched or purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of enzyme, and the specific precipitation agent(s) and its (their) concentration. Generally, the time effective to precipitate the enzyme is between about 1 to about 30 hours; usually it does not exceed about 25 hours. In the presence of the organic compound precipitation agent, the time of incubation can still be reduced to less about 10 hours and in most cases even about 6 hours.

[0181] Generally, the temperature during incubation is between about 4°C and about 50°C. Usually, the method is carried out at a temperature between about 10°C and about 45°C (e.g., between about 20°C and about 40°C). The optimal temperature for inducing precipitation varies according to the solution conditions and the enzyme or precipitation agent(s) used.

[0182] The overall recovery of enriched or purified enzyme precipitate, and the efficiency with which the process is conducted, is improved by agitating the solution comprising the enzyme, the added metal halide and the added organic compound. The agitation step is done both during addition of the metal halide and the organic compound, and during the subsequent incubation period. Suitable agitation methods include mechanical stirring or shaking, vigorous aeration, or any similar technique.

[0183] After the incubation period, the enriched or purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further enrichment or purification of the enzyme precipitate can be obtained by washing the precipitate with water. For example, the enriched or purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents.

[0184] During fermentation, an enzyme polypeptide accumulates in the culture broth. For the isolation, enrichment, or purification of the desired phospholipase, the culture broth is NB42152-WO-PCT[3] centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme enrichment or purification. In one embodiment, the cell-free broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation-precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column and eluted to recover the enzyme-active fraction. For further enrichment or purification, a conventional procedure such as ion exchange chromatography may be used.

[0185] Enriched or purified enzymes can be made into a final product that is either liquid (solution, slurry) or solid (granular, powder).

[0186] Description of the Preferred Embodiments

[0187] In an aspect of the present invention, a method is presented for making a plant-based beverage having improved texture and improved precipitation rate, the method having the steps of: providing a plant protein in an aqueous medium; and contacting the plant protein with a protein glutaminase comprising a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof to provide the beverage having improved texture and improved precipitation rate as compared with a plant-based beverage prepared without the protein glutaminase, wherein said improved texture comprises increased thickness and / or increased mouthfeel and said improved precipitation rate is less than 50%.

[0188] Preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. More preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0189] In other preferred embodiments, the protein glutaminase is a polypeptide having at least NB42152-WO-PCT[3]

[0190] 85% sequence identity to SEQ TD NO:1 , SEQ TD NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID N0:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0191] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0192] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, NB42152-WO-PCT[3] the protein glutaminase is a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0193] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0194] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NQ:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25. NB42152-WO-PCT[3]

[0195] In other preferred embodiments, the protein glutaminase is a polypeptide according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NOd l, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NQ:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide according to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0196] Preferably, the protein glutaminase is present in amount from about 0.005 mg per 100 ml to about 15 mg per 100 ml. More preferably, the protein glutaminase is present in an amount from about 0.03 mg per 100 ml to about 12.5 mg per 100 ml.

[0197] Preferably, the improved texture is increased thickness. Preferably the thickness is increased by 30, 50, 70, 90, 100, 110 or 120% or more.

[0198] In other preferred embodiments, the improved texture is increased mouthfeel. Preferably, the mouthfeel is increased by 30 50, 70, 90, 100, 110 or 120% or more.

[0199] Preferably, the precipitation rate of the beverage is less than 40, 30, 20, 10 or 5%.

[0200] Preferably, the plant protein is, wheat gluten, soy, soybean, pea, corn or oat protein.

[0201] Preferably, the plant protein is hydrolysed or partially hydrolyzed prior to contact with the protein glutaminase.

[0202] Preferably, the improved plant-based beverage is a calf or piglet milk replacer.

[0203] Preferably, the improved plant-based beverage is a non-dairy milk or drinkable yogurt.

[0204] Preferably, the method has the further step of adding a culture to produce a non-dairy yogurt or acidified milk product.

[0205] In other preferred embodiments, the method has the further step of incorporating the improved beverage into a non-dairy cheese. NB42152-WO-PCT[3]

[0206] In another aspect of the instant invention, a method is presented of preparing a stabilized flavoured milk-based beverage having the steps of: a. providing a flavoured milk mix comprising milk and a non-milk protein component; and b. contacting said flavoured milk mix with a protein glutaminase corresponding to a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID O:13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof to provide the stabilized flavoured milk beverage.

[0207] Preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. More preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0208] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25. NB42152-WO-PCT[3]

[0209] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0210] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NQ:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0211] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the NB42152-WO-PCT[3] protein glutaminase is a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0212] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0213] In other preferred embodiments, the protein glutaminase is a polypeptide according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NQ:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide according to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25. NB42152-WO-PCT[3]

[0214] Preferably, the protein glutaminase is present in amount from about 0.005 mg per 100 ml to about 15 mg per 100 ml. More preferably, the protein glutaminase is present in an amount from about 0.03 mg per 100 ml to about 12.5 mg per 100 ml.

[0215] Preferably, the non-milk protein component is cocoa.

[0216] In another aspect of the present invention, a protein glutaminase is presented having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. Preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NQ:20, SEQ ID NO:23 or SEQ ID NO:26. More preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0217] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NQ:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25. NB42152-WO-PCT[3]

[0218] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

[0219] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NQ:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0220] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the NB42152-WO-PCT[3] protein glutaminase is a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0221] In other preferred embodiments, the protein glutaminase is a polypeptide having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

[0222] In other preferred embodiments, the protein glutaminase is a polypeptide according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NQ:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof. More preferably, the protein glutaminase is a polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26. Still more preferably, the protein glutaminase active fragment is a mature form of a polypeptide according to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25. NB42152-WO-PCT[3]

[0223] In another aspect of the present invention, an isolated polynucleotide is presented having a nucleic acid sequence encoding an isolated polypeptide as described above.

[0224] In another aspect of the present invention, a recombinant expression vector is presented having the isolated polynucleotide as described above.

[0225] In another aspect of the present invention, a host cell is presented having the recombinant expression vector as described above.

[0226] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference. To the extent the content of any citation, including website or accession number may change with time, the version in effect at the filing date of this application is meant. Unless otherwise apparent from the context any step, element, aspect, feature of embodiment can be used in combination with any other.

[0227] EXAMPLES

[0228] EXAMPLE 1

[0229] Cloning and expression of protein glutaminase

[0230] Protein glutaminase genes were identified from either public database (NCBI or UniProt,) or internal genome collections. The gene CprGltl described by Shotaro Yamaguchi et al was used as a benchmark. See Yamaguchi, S., Jeenes, D.J. and Archer, D.B. Protein-glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues in proteins. Purification, characterization and gene cloning. Eur. J. Biochem. 268 (5), 1410-1421 (2001). Generally, coding sequences of the protein glutaminases (without signal peptide) were codon-optimized based on Bacillus subtilis codon preference and cloned into p2JM vector (Vogtentanz, Protein Expr Purif. 55:40-52, 2007). The aprE promoter and signal peptide were used for transcription and secretion of protein NB42152-WO-PCT[3] glutaminase. The constructed vector was subjected to rolling-circle amplification and transformed into a suitable Bacillus subtilis host. The transformants were screened on LB plate supplementing 5 ppm chloramphenicol. Single colony was inoculated into 20 mL LB medium with 5 ppm chloramphenicol and subsequently transferred to an enriched semidefined media based on MOPS buffer, with urea, micronutrients, glucose and soy tone for robust cell growth. Clarified culture supernatant was harvested by centrifugation, concentrated and used for protein purification. All protein glutaminases were purified using standard procedures known in the art, including hydrophobic interaction chromatography, ion / cation exchange chromatography and size-exclusion chromatography, etc. The fractions containing the protein of interest were analyzed by SDS-PAGE and activity assay described below. Samples of purified protein were stored in 20 mM Tris-HCl pH7.5 40% glycerol at 20 °C until usage.

[0231] Table 1. Sequence list of protein glutaminases NB42152-WO-PCT[3]

[0232] EXAMPLE 2

[0233] Substrate specificity of protein glutaminase

[0234] 1% (w / v) of casein sodium salt, 1% (w / v) soybean peptide (local company), 1% (w / v) soy protein (Danisco SUPRO), 1% (w / v) pea protein (Danisco TURPRO), 1% (w / v) zein (Sigma, # Z3625), 1% (w / v) hydrolyzed oat protein (local company) were prepared in 50 mM NaPi buffer (pH 7.0) respectively. 3 pL of enzyme dilution (10 ppm) was transferred into a 96-well microtiter plate (Coming 3641) containing 147 pL of the substrate. The plate was incubated at 50 °C for 60 min with shaking (950 rpm) in an iEMS shaker (Thermofisher). After incubation, the reaction mixture was centrifuged at 3,700 rpm and the supernatant was collected to test the amount of free ammonium.

[0235] Free ammonium was quantified by spectrofluorimetric method using OPA / NAC reagent. The OPA / NAC reagent was prepared freshly by mixing 1 volume of reagent A (200 mM o- phthalaldehyde in ethanol), 1 volume of reagent B (200 mM N-acetyl-L-cysteine and 20 mM NB42152-WO-PCT[3] tris(2-carboxyethyl) phosphine hydrochloride in 1 M HO) and 18 volumes of buffer (0.1 M sodium borate buffer, pH 10.5). 5 pL of sample or standard solution (ammonium chloride) was transferred into a 96- well microtiter plate (Coming 3605) containing 50 LIL of OPA / NAC reagent. The plate was shaken for 5 min and fluorescence intensity was then recorded with excitation and emission wavelengths set at / .ex = 415 nm / Zem = 485 nm using a microplate reader (Molecular devices, SpectraMax ID3). Concentrations in samples were determined using the linear calibration curves obtained with standards. The ammonium amount in the substrate was subtracted from the amount of each reaction mixture. Table 2 showed the substrate specificity results of protein glutaminase. The PI results indicated CspGltl, CnaGltl, CjoGltl, CinGltl and MetGlt8 have better deamidating activity on all substrates tested than CprGltl.

[0236] Table 2: Substrate specificity of protein glutaminase

[0237] EXAMPLE 3 pH profile of protein glutaminase

[0238] With casein sodium salt as the substrate, the pH profile was studied in 25 mM NB42152-WO-PCT[3]

[0239] NaAc / Glycine / HEPES buffer with different pH values at 2, 3, 5, 6, 7, 8, 9, and 10. To initiate the assay, 45 pL of 50 mM NaAc / Glycine / HEPES buffer with a specific pH was first mixed with 45 pL of 20 mM casein sodium salt in a 96-well microtiter plate and preincubated at 50 °C for 5 min, followed by the addition of 10 pL of water diluted enzyme or Milli-Q water (the blank control). The absorbance from a control, in which the enzyme solution was replaced by an equal volume of Milli-Q water, was subtracted from the absorbance of each reaction mixture. The reaction was performed and analyzed as described in Example 2. The resulting values were converted to percentages of relative activity by defining the maximal activity at the optimal pH as 100%. The pH profile results are summarized in Table 3. These enzymes show broad pH range, retaining >60% activity within pH5-10.

[0240] Table 3: pH profile of protein glutaminase

[0241] EXAMPLE 4

[0242] Temperature profile of protein glutaminase NB42152-WO-PCT[3]

[0243] The temperature profile of protein glutaminase was determined by measuring the activity of enzyme at temperature gradient from 30 °C to 80 °C in Thermo Cycler (T100, Bio-Rad) using the same substrate as described in Example 3. The absorbance from a control, in which the enzyme solution was replaced by an equal volume of Milli-Q water, was subtracted from the absorbance of each reaction mixture. The reaction was performed and analyzed as described in Example 2. The resulting values were converted to percentages of relative activity by defining the maximal activity at the optimal temperature as 100%. The temperature profile results were summarized in Table 4. The data suggested that optimal temperature of CinGltl, ClaGltl, CspGlt8, and CurGltl was 60 °C, and the optimal temperature of CnaGltl, CjoGltl, MetGlt8, and CprGltl was 64°C. Besides, CspGltl had the highest optimal temperature which was 70 °C.

[0244] Table 4. Temperature profile of protein glutaminase

[0245] EXAMPLE 5

[0246] Determination of degree of deamidation NB42152-WO-PCT[3]

[0247] The degree of deamidation refers to the amount of ammonia released by enzymatic deamidation of a protein relative to that released by complete deamidation of the same protein (maximum releasable ammonia). The technique used is based on ammonia measurements, as described in Example 2, and has been modified from Horstmann et al. 2017. See Horstmann, G., Ewert, J., Stressler, T., & Fischer, L. (2019). A novel protein glutaminase from Bacteroides / / c / cogcnc.v-characterization and comparison. Appl Microbiol Biotechnol and Ruidan Qu, Tian Dai, Jiajing Wu, et al. (2022). The characteristics of proteinglutaminase from an isolated Chryseobacterium cucumeris strain and its deamidation application. Front. Microbiol. 13: 969445. doi: 10.3389 / fmicb.2022.969445

[0248] The amount of ammonia released by protein glutaminase was determined by taking aliquots from reaction mixtures of protein with protein glutaminase, centrifugating the sample for 10 min at 14680 rpm and measuring ammonia in supernatant as described in Example 2. For determination of maximum releasable ammonia, 200 uL of a time zero aliquot was mixed with 200 uL 4 M HC1 in an Eppendorf tube. The tube was incubated in a Thermomixer at 95 °C, 300 rpm for 3 hours. After incubation the sample was cooled down and centrifuged for 10 min at 14680 rpm. The supernatant was appropriately diluted, and ammonia content was determined using the technique described in Example 2.

[0249] The degree of deamidation is calculated as the ratio between the enzymatically released ammonia concentration and the maximum releasable ammonia concentration. It is expressed in percentage.

[0250] EXAMPLE 6

[0251] Oat beverage with protein glutaminase

[0252] The effect of protein glutaminase was tested in an oat beverage system. Therefore, 50 g of oat protein (Poatein, Latmannen, Stockholm, Sweden) was dispersed in 450 mL of water. The final solution had a protein content of approximately 5%. The dispersion was divided into aliquots with 50 mL each. Half of the aliquots were heated at 95 °C for 10 min (preheating), the remaining aliquots were not heated. Subsequently, CspGltl was added to the NB42152-WO-PCT[3] heated and non-heated batches at 2 - 130 ppm enzyme protein relative to the protein content of the slurry. All samples were incubated for 2h at 50°C and subsequently heated at 95 °C for 15 minutes, to inactivate the enzyme. For reference, batches without enzyme were treated in the same manner as the batches containing the enzyme. All samples were stored at 5°C until further analyses.

[0253] A rotational rheological test was employed to evaluate the viscosity of the produced samples. Flow curves were obtained with an Anton Paar MCR302 rheometer (Anton Paar GmbH, Ostfildern, Germany) using an ST22-4V-40 vane geometry for alu 20 cups. Samples were filled into C-CC27 alu cups and stored at 5 °C for at least 5 hours before analysis. The shear rate intervals applied to the samples were 0.1-350 s’1, which defines the up-curve, and the reverse operation explains the down-curve (350-0.1 s’1). The value of the measuring point duration was selected to be at least as long as the value of the reciprocal shear rate, which is valid for the up-curve. The tests were performed under a constant temperature of 10 °C, and each sample was analyzed in duplicate. A water bath was connected to the rheometer to ensure isothermal conditions. The apparent viscosity was assessed from the flow curves, which is appropriate for fluids where the ratio of shear stress to shear rate varies with the shear rate. The apparent viscosity was extracted at either shear rate 1 1.6 Hz or 350 Hz. The apparent viscosity extracted at shear rate 11.6 Hz indicated the sensory perception “mouthfeel” of the sample. The apparent viscosity extracted at shear rate 350 Hz correlates to the “thickness”.

[0254] Table 5: Results for shear stress at two different shear rates for oat samples with different doses of CspGltl. NB42152-WO-PCT[3]

[0255] *) “No heat” means that the enzyme was added to the oat protein slurry immediately before the incubation step. “Heat treated” means that the oat protein slurry was pre-heated to 95 °C for lOmin and then cooled to 50 °C, before enzyme addition and incubation.

[0256] It is seen from Table 5 that shear stress increases with increasing doses of CspGltl both at shear stress of 11 ,5 and 350 s'1. The results at 11,5 s'1can be interpreted as an increasingly thicker mouthfeel at increasing enzyme dose. The results at 350 s1can be interpreted as thickness during production or visual perception of thickness when stirring the product. This thickness parameter also increases with increasing enzyme dose. For all enzyme doses it is seen that shear stress is higher for samples that were heat treated before enzyme addition. This shows that the enzyme works more efficiently on heat treated oat protein. It is likely that the heat treatment opens the structure of the protein, making it more accessible to the enzyme.

[0257] Precipitation rate was determined by letting the samples sediment for 7 days at room temperature followed by measuring the height of the supernatant layer and the total height of the sample in mm. The precipitation rate was then calculated as the ratio of supernatant height over total height and expressed in % relative to the reference sample (no enzyme addition). The results are shown in Table 6. In this context, a low precipitation rate is synonymous with a high stability of the oat beverage.

[0258] Table 6: Results for precipitation rate of oat samples with different doses of CspGltl NB42152-WO-PCT[3]

[0259] *) Heat treated means that the oat protein slurry was pre heated to 95 °C for lOmin and then cooled to 50 °C before enzyme addition and incubation.

[0260] It is seen for both “no heat” and “heat treated” samples that precipitation rate declines with increasing CspGltl dose. Hence, regardless of heat treatment, the higher dose of CspGltl the more stable the oat beverage. For all enzyme doses, heat treatment has a positive influence on stability. Again, this can be explained by the oat protein becoming more open in its structure and, thereby, more accessible to the enzyme.

[0261] EXAMPLE 7

[0262] Comparing the effect of different protein glutaminases in oat beverage

[0263] Oat beverages were made according to the procedure described in example 6 but instead of different doses of one protein glutaminase, 3 different protein glutaminase’s were compared at a dose of 62,4 ppm relative to the amount of protein. Results on degree of deamidation, rheology and precipitation rate are seen in Table 7

[0264] Table 7: Data from comparison of oat beverages made with three different protein glutaminases. DD = Degree of deamidation

[0265] Relative to the reference sample, without enzyme addition, it is observed for all the protein glutaminases tested that: 1) degree of deamidation increases, 2) shear stress at both low and high shear increases and 3) precipitation rate decreases. In other words, the protein NB42152-WO-PCT[3] glutaminases cause an increase in the viscosity and stability of the oat beverages. Also, the increase in viscosity and stability correlates positively with the degree of deamidation. Most interestingly, CspGltl gives the highest degree of deamidation combined with the highest increase in viscosity and stability. Since all protein glutaminases were dosed at the same enzyme-protein to substrate level it follows that CspGltl is the most efficient of the three enzymes in generating these positive functional characteristics for oat beverages.

[0266] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

NB42152-WO-PCT[3]CLAIMSWhat is claimed is:

1. A method for making a plant-based beverage having improved texture and improved precipitation rate, said method comprising the steps of: providing a plant protein in an aqueous medium; and contacting the plant protein with a protein glutaminase comprising a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NOT, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NOTO, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof to provide the beverage having improved texture and improved precipitation rate as compared with a plant-based beverage prepared without the protein glutaminase, wherein said improved texture comprises increased thickness and / or increased mouthfeel and said improved precipitation rate is less than 50%.

2. The method of claim 1 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

3. The method of claim 1 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

4. The method of claim 1 wherein the protein glutaminase comprises a polypeptide having at least 85% sequence identity to SEQ ID NO:1 , SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:5,NB42152-WO-PCT[3]SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

5. The method of claim 4 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

6. The method of claim 4 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

7. The method of claim 4 wherein the protein glutaminase comprises a polypeptide having at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOT, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

8. The method of claim 7 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

9. The method of claim 7 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1 , SEQ ID NOT, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16,NB42152-WO-PCT[3]SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

10. The method of claim 7 wherein the protein glutaminase comprises a polypeptide having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.1 1. The method of claim 10 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

12. The method of claim 10 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:1, SEQ ID NON, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

13. The method of claim 10 wherein the protein glutaminase comprises a polypeptide having at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

14. The method of claim 13 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.NB42152-WO-PCT[3]15. The method of claim 13 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

16. The method of claim 13 wherein the protein glutaminase comprises a polypeptide having at least 99% sequence identity to SEQ ID NOT, SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NOT, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 1 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NOTO, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

17. The method of claim 16 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NOT, SEQ ID NOT, SEQ ID NO:8, SEQ ID NO:1 1, SEQ ID NO: 14, SEQ ID NO:17, SEQ ID NOTO, SEQ ID NO:23 or SEQ ID NO:26.

18. The method of claim 16 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NOT, SEQ ID NO:4, SEQ ID NOT, SEQ ID NOTO, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

19. The method of claim 16 wherein the protein glutaminase comprises a polypeptide according to SEQ ID NOT, SEQ ID NOT, SEQ ID NOT, SEQ ID NOT, SEQ ID NOT, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO 3, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NOTO, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.NB42152-WO-PCT[3]20. The method of claim 19 wherein the protein glutaminase comprises a polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

21. The method of claim 19 wherein the protein glutaminase active fragment is a mature form of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

22. The method of any of claims 1 to 21 wherein the protein glutaminase is present in amount from about 0.005 mg per 100 ml to about 15 mg per 100 ml.

23. The method of claim 22 wherein the protein glutaminase is present in an amount from about 0.03 mg per 100 ml to about 12.5 mg per 100 ml.

24. The method of any of claims 1 to 23 wherein the improved texture comprises increased thickness.

25. The method of claim 24 wherein the thickness is increased by 30% or more.

26. The method of claim 25 wherein the thickness is increased by 50% or more.

27. The method of claim 26 wherein the thickness is increased by 70% or more.

28. The method of claim 27 wherein the thickness is increased by 90% or more.

29. The method of claim 28 wherein the thickness is increased by 100% or more.

30. The method of claim 29 wherein the thickness is increased by 110% or more.NB42152-WO-PCT[3]31. The method of claim 30 wherein the thickness is increased by 120% or more.

32. The method of any of claims 1 to 31 wherein the improved texture comprises increased mouthfeel.

33. The method of claim 32 wherein the mouthfeel is increased by 30% or more.

34. The method of claim 33 wherein the mouthfeel is increased by 50% or more.

35. The method of claim 34 wherein the mouthfeel is increased by 70% or more.

36. The method of claim 35 wherein the mouthfeel is increased by 90% or more.

37. The method of claim 36 wherein the mouthfeel is increased by 100% or more.

38. The method of claim 37 wherein the mouthfeel is increased by 110% or more.

39. The method of claim 38 wherein the mouthfeel is increased by 120% or more.

40. The method of any of the preceding claims wherein the precipitation rate of the beverage is less than 40%.

41. The method of claim 40 wherein the precipitation rate of the beverage is less than 30%.

42. The method of claim 41 wherein the precipitation rate of the beverage is less than 20%.

43. The method of claim 42 wherein the precipitation rate of the beverage is less than 10%.NB42152-WO-PCT[3]44. The method of claim 43 wherein the precipitation rate of the beverage is less than 5%.

45. The method of any of the preceding claims wherein the plant protein is wheat gluten, soy, soybean, pea, corn or oat protein.

46. The method of claim any of claims 8 to 40 wherein the plant protein is hydrolysed or partially hydrolysed prior to contact with the protein glutaminase.

47. The method of any of the preceding claims wherein the improved plant-based beverage is a calf or piglet milk replacer.

48. The method of any of claims 1 to 46 wherein the improved plant-based beverage is a nondairy milk or drinkable yogurt.

49. The method of any of claims 1 to 46 comprising the further step of adding a culture to produce a non-dairy yogurt or acidified milk product.

50. The method of any of claims 1 to 46 comprising the further step of incorporating the improved beverage into a non-dairy cheese.

51. A method of preparing a stabilized flavoured milk-based beverage comprising the steps of: a. providing a flavoured milk mix comprising milk and a non-milk protein component; and b. contacting said flavoured milk mix with a protein glutaminase comprising a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NQ:20,NB42152-WO-PCT[3]SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof to provide the stabilized flavoured milk beverage.

52. The method of claim 51 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

53. The method of claim 51 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

54. The method of claim 51 wherein the protein glutaminase comprises a polypeptide having at least 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

55. The method of claim 54 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

56. The method of claim 54 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.NB42152-WO-PCT[3]57. The method of claim 54 wherein the protein glutaminase comprises a polypeptide having at least 90% sequence identity to SEQ ID NOT, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

58. The method of claim 57 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

59. The method of claim 57 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

60. The method of claim 57 wherein the protein glutaminase comprises a polypeptide having at least 95% sequence identity to SEQ ID NOT, SEQ ID NO:2, SEQ ID NOT, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NOT 1, SEQ ID NOT3, SEQ ID NOT4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

61. The method of claim 60 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NOT 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.NB42152-WO-PCT[3]62. The method of claim 60 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

63. The method of claim 60 wherein the protein glutaminase comprises a polypeptide having at least 98% sequence identity to SEQ ID NOT, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

64. The method of claim 63 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

65. The method of claim 63 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

66. The method of claim 63 wherein the protein glutaminase comprises a polypeptide having at least 99% sequence identity to SEQ ID NOT, SEQ ID NO:2, SEQ ID NOT, SEQ ID NO:5, SEQ ID NOT, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NOT 1, SEQ ID NOT3, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NOTO, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

67. The method of claim 66 wherein the protein glutaminase comprises a polypeptideNB42152-WO-PCT[3] having an amino acid sequence with at least 99% sequence identity to SEQ TD NO:2, SEQ TD NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

68. The method of claim 66 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

69. The method of claim 66 wherein the protein glutaminase comprises a polypeptide according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

70. The method of claim 69 wherein the protein glutaminase comprises a polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

71. The method of claim 69 wherein the protein glutaminase active fragment is a mature form of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 22 or SEQ ID NO:25.

72. The method of any of claims 51 to 71 wherein the protein glutaminase is present in amount from about 0.005 mg per 100 ml to about 15 mg per 100 ml.

73. The method of claim 72 wherein the protein glutaminase is present in an amount from about 0.03 mg per 100 ml to about 12.5 mg per 100 ml.NB42152-WO-PCT[3]74. The method of any of claims 51 to 73 wherein the non-milk protein component is cocoa.

75. An isolated polypeptide comprising a protein glutaminase having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NOT, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

76. The isolated polypeptide of claim 75 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

77. The isolated polypeptide of claim 75 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

78. The isolated polypeptide of claim 75 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NOT, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

79. The isolated polypeptide of claim 78 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ IDNB42152-WO-PCT[3]N0:2, SEQ ID N0:5, SEQ ID N0:8, SEQ ID NO:1 1 , SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

80. The isolated polypeptide of claim 78 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

81. The isolated polypeptide of claim 78 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

82. The isolated polypeptide of claim 81 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

83. The isolated polypeptide of claim 81 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:1, SEQ ID NON, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

84. The isolated polypeptide of claim 81 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10,NB42152-WO-PCT[3]SEQ ID NO:! !, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

85. The isolated polypeptide of claim 84 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

86. The isolated polypeptide of claim 84 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

87. The isolated polypeptide of claim 84 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOT, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

88. The isolated polypeptide of claim 87 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 98% sequence identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11 , SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

89. The isolated polypeptide of claim 87 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 98% sequenceNB42152-WO-PCT[3] identity to SEQ ID NOT , SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

90. The isolated polypeptide of claim 87 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NOT, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NOTO, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

91. The isolated polypeptide of claim 90 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NOT, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11 , SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NOTO, SEQ ID NO:23 or SEQ ID NO:26.

92. The isolated polypeptide of claim 90 wherein the protein glutaminase active fragment is a mature form of a polypeptide having an amino acid sequence with at least 99% sequence identity to SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NOTO, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

93. The isolated polypeptide of claim 90 wherein the protein glutaminase comprises a polypeptide having an amino acid sequence according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOT, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NOTO, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:26 or a protein glutaminase active fragment thereof.

94. The isolated polypeptide of claim 93 wherein the protein glutaminase comprises aNB42152-WO-PCT[3] polypeptide according to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11 , SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:26.

95. The isolated polypeptide of claim 93 wherein the protein glutaminase active fragment is a mature form of SEQ ID NO:1, SEQ ID NON, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO:25.

96. An isolated polynucleotide comprising a nucleic acid sequence encoding the isolated polypeptide of any of claims 75 to 95.

97. A recombinant expression vector comprising the isolated polynucleotide of claim 96.

98. A host cell comprising the recombinant expression vector of claim 97.

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